Developmental and Inherited Liver Disease


atresia, ciliopathy, cystic fibrosis, disorders of bile acid synthesis and transport, porphyria, carbohydrate metabolism, mitochondrial disorders


  • Chapter Contents

  • Diagnostic approach to histology 113

    • Neonatal and early infantile liver disease 113

    • Postneonatal, childhood and adulthood presentation 118

  • Neonatal hepatitis 119

    • Histopathological features 120

  • Biliary atresia 121

    • Classification and aetiopathogenesis 121

    • Pathological features at surgical intervention 124

    • Pathology of intrahepatic changes 126

  • Paucity of intrahepatic bile ducts 129

    • Alagille syndrome (arteriohepatic dysplasia) 129

    • Nonsyndromic duct paucity disorders 131

  • Bile duct anomalies, congenital dilations and ductal plate malformation disorders 131

    • Choledochal cyst 132

    • Hereditary fibropolycystic disease (ductal plate malformation) 133

  • Cystic fibrosis 141

  • Hereditary disorders of bile acid synthesis and bilirubin metabolism 143

    • Primary disorders of bile acid synthesis 143

    • Disorders of bile canalicular transporters 146

    • Other diseases causing intrahepatic cholestasis 150

    • Hereditary defects of bilirubin metabolism 151

  • Disorders of porphyrin metabolism 153

    • Porphyria cutanea tarda 154

    • Erythropoietic protoporphyria 155

  • Disorders of carbohydrate metabolism and related conditions 156

    • Glycogen storage diseases (glycogenoses) 156

    • Myoclonus epilepsy, Lafora type (Lafora disease) 161

    • Galactosaemia 162

    • Hereditary fructose intolerance 163

  • Disorders of glycoprotein and glycolipid metabolism 164

    • Mucopolysaccharidoses 164

    • Aspartylglucosaminuria 166

    • α-Mannosidosis 166

    • Fucosidosis 167

    • Mucolipidoses 167

    • Congenital disorders of glycosylation (carbohydrate-deficient glycoprotein syndrome) 168

  • Endoplasmic reticulum storage diseases 169

    • α1-Antitrypsin deficiency 169

    • α1-Antichymotrypsin deficiency 173

    • Afibrinogenaemia and hypofibrinogenaemia 174

    • Antithrombin III deficiency 175

  • Disorders of amino acid metabolism 175

    • Hereditary tyrosinaemia type 1 175

    • Congenital hyperammonaemia syndromes and urea cycle disorders 177

    • Cystinosis 177

    • Homocystinuria (cystathionine β-synthase deficiency) 179

  • Disorders of lipoprotein and lipid metabolism 180

    • Abetalipoproteinaemia 180

    • Familial hypobetalipoproteinaemia 180

    • Familial high-density lipoprotein deficiency (Tangier disease) 181

    • Familial hypercholesterolaemia 181

    • Wolman disease and cholesteryl ester storage disease 181

    • Gangliosidoses 183

    • α-Galactosidase A deficiency (Fabry disease) 186

    • Sulphatide lipidosis (metachromatic leucodystrophy) 187

    • Ceramidase deficiency (Farber lipogranulomatosis) 189

    • Glycosyl ceramide lipidosis (Gaucher disease) 190

    • Sphingomyelin–cholesterol lipidosis (Niemann–Pick disease) 192

  • Peroxisomal disorders 194

    • Peroxisomal biogenesis disorders 195

    • Disorders with a single peroxisomal functional deficiency 196

  • Mitochondrial cytopathies and related conditions 196

    • Genetic mitochondrial disorders 198

    • Multiple complex deficiencies related to nuclear translation factor genes 198

    • Fatty acid oxidation disorders 199

    • Histological diagnosis of mitochondriopathies 199

  • Disorders of copper metabolism 200

    • Wilson disease (hepatolenticular degeneration) 200

    • Indian childhood cirrhosis 206

    • Endemic Tyrolean infantile cirrhosis 207

  • Primary immunodeficiencies 207

    • Chronic granulomatous disease 207

    • Common variable immunodeficiency 208

    • Liver disease in X-linked hyper-IgM syndrome 208

  • Histiocytic and haemophagocytic syndromes 209

    • Familial haemophagocytic lymphohistiocytosis (familial haemophagocytic reticulosis) 209

    • Haemophagocytic lymphohistiocytosis 210

    • Langerhans cell histiocytosis 211

    • Sinus histiocytosis with massive lymphadenopathy (Rosai–Dorfman disease) 214

  • Miscellaneous disorders 215

    • Neonatal lupus erythematosus 215

    • Shwachman–Diamond syndrome 215

    • Familial and genetic nonalcoholic steatohepatitis and cirrhosis 215

    • Congenital total lipodystrophy 216

    • Down syndrome 216

    • Reye syndrome 216

    • Kawasaki disease 217

    • Aarskog–Scott syndrome 219

    • Leprechaunism (Donohue, Rabson–Mendenhall syndrome) 219

    • Albinism (Chediak–Higashi, Hermansky–Pudlak syndromes) 220

  • Anatomical anomalies 220

    • Gross abnormalities of liver 220

    • Congenital anomalies of biliary tract 222

Developmental and inherited disorders affecting the liver typically present in childhood but may affect individuals of any age. The effect of inherited metabolic disorders on the liver may be considered primary , caused by the accumulation of a metabolite resulting from an enzyme defect (e.g. sphingomyelin in Niemann–Pick disease), or secondary , when the major changes in the liver are the result of a primarily extrahepatic defect (e.g. steatosis of the liver secondary to pancreatic insufficiency in Shwachman syndrome). Likewise, as the list of identified inherited metabolic disorders continues to expand, many have significant but indirect relevance to the liver. For example, liver histology is usually normal in primary hyperoxaluria while the kidneys and other organs may be irreparably damaged; however, despite medical management, dialysis and repetitive kidney transplants, cure is only obtained with a liver transplant. In other inherited disorders, the liver disease may remain asymptomatic until precipitous acute liver failure develops; the classic example is Wilson disease. This similarly occurs with developmental disorders; choledochal cyst may be diagnosed by prenatal sonography, or it may not become evident until late in childhood. In many cases the clinical diagnosis of an inherited disorder is not clinically evident, and consequently, diagnosis becomes the responsibility of the histopathologist, who may save not only the life of the patient, but that of other family members as well. Advances in molecular genetics constantly improve our understanding of the biological basis of many metabolic diseases. Prenatal diagnosis is available for more families.

Many metabolic disorders manifest similar morphological findings despite having diverse aetiopathogenesis. Numerous diverse disorders are characterized neonatally by cholestasis and giant cell transformation of liver cells, such as α1-antitrypsin deficiency, Niemann–Pick disease type C and bile salt export pump (BSEP) deficiency, also known as progressive familial intrahepatic cholestasis type 2. Steatosis is one of the most frequent abnormalities, either alone (as in the urea cycle disorders, homocystinuria, lipoprotein disorders, the mitochondrial cytopathies and Shwachman syndrome) or in combination with other changes such as cholestasis, pseudogland formation and fibrosis (e.g. in galactosaemia, hereditary tyrosinaemia type 1 and hereditary fructose intolerance). Furthermore, neutral lipid may be stored in combination with other metabolites such as cholesterol (e.g. in Wolman disease or cholesterol ester storage disease) or glycogen (as found in glycogen storage disease types I and III). Many disorders of lipid metabolism are expressed morphologically by ‘foam’ cells (as in Niemann–Pick disease types A and B and the gangliosidoses). The ultimate diagnosis depends on the clinical and laboratory data and identification of the specific enzyme defect. Histological changes may not lead to a specific diagnosis, but characteristic findings add to the phenotypic expression of individual disorders and guide selection of further investigations, in particular enzymatic assays and genomic studies. Accordingly, the following discussion includes the major clinical, laboratory and genetic features—the context in which histological changes should be evaluated.

The histopathologist has access to many special stains and techniques, both at the light microscopic and ultrastructural levels. The best all-round fixative for light microscopy remains 10% buffered formalin; however, some metabolic diseases (e.g. the mucopolysaccharidoses, cystinosis and the glycogen storage diseases) require special fixatives to prevent leaching of water-soluble metabolites. Special stains for lipid, cholesterol and sphingomyelin must be performed on frozen sections cut from formalin-fixed or fresh material since routine processing will extract the lipid material from the cells. The histopathologist must be familiar with the many special stains that can be used on formalin-fixed and routinely processed material: uroporphyrin, haemosiderin, copper, copper-binding protein, bile, lipofuscin, lipomelanin (the pigment in the Dubin–Johnson syndrome), carbohydrates, mucopolysaccharides and other substances. Immunohistochemical stains are useful in a number of diseases, such as for identifying catalase in patients with peroxisomal diseases or the presence of α1-antitrypsin, α1-antichymotrypsin or fibrinogen in eosinophilic globules in various storage diseases. The repertoire of specific antibodies working on paraffin sections is continuously broadening, including antibodies reacting with various bile canalicular transporters such as the BSEP and multidrug-resistance protein 3 (MDR3); such antibodies can be commercially sourced and are readily available in specialist centres. Special microscopy should be utilized whenever necessary. The porphyrins can be demonstrated by their autofluorescence in frozen sections made from unfixed hepatic biopsy or autopsy material. Polarizing microscopy is especially useful for the identification of various crystals such as cholesterol, cystine, calcium oxalate, uroporphyrin and protoporphyrin.

Transmission electron microscopy (TEM) is very important in the diagnosis of many inherited metabolic diseases. According to Phillips et al., the findings are diagnostic in α1-antitrypsin deficiency, Farber disease, glycogen storage diseases types II and IV, hereditary fructose intolerance, Gaucher disease, metachromatic leucodystrophy, the gangliosidoses, Dubin–Johnson syndrome, erythropoietic protoporphyria, Wilson disease, Zellweger syndrome and infantile Refsum disease. In other disorders, electron microscopy (EM), although not diagnostic, can help to categorize the disease (e.g. as a glycogenosis, phospholipidosis or oligosaccharidosis) or to suggest the correct diagnosis, as in progressive familial intrahepatic cholestasis type 1 (PFIC1) disease, arteriohepatic dysplasia and cholesterol ester storage disease. The role of scanning electron microscopy (SEM) is much more limited. Whether TEM will continue to serve a critical role in histological diagnosis of genetic/metabolic liver diseases, given the wide-ranging and cost-effective advances in immunohistochemical and molecular methods, is currently an area of debate.

In summary, histopathological studies contribute significantly to the diagnosis of metabolic disorders. Selection of appropriate pathological studies must be made before liver biopsy, to ensure appropriate fixation and handling of samples. Prebiopsy consultation with a histopathologist, and at times with laboratory staff of a centre dealing with highly specialized techniques, may preclude the loss of diagnostic information. In general, a portion of the biopsy core at least 5 mm in length should be snap-frozen using optimal cutting temperature (OCT) compound, which does not interfere with most enzyme assays and allows subsequent frozen sectioning for microscopy. Ideally, a few bits (1 mm 3 ) should be immersed in a fixative appropriate for EM. Tissue freezing and EM fixative, in addition to usual fixation and paraffin processing, should be applied to all biopsy specimens taken from children or older patients in whom a metabolic disorder is suspected.

Diagnostic approach to histology

Neonatal and early infantile liver disease

Many different disorders manifest with conjugated hyperbilirubinaemia in neonates ( Table 3.1 ). Some are caused by direct hepatocellular injury, and others by defects in hepatocyte bile formation or physical obstruction to bile flow. The distinction between primarily hepatocellular versus primarily hepatobiliary injury is critical for clinical management. The primary histological differential diagnosis for infantile conjugated bilirubinemia disorders is therefore between obstructive and nonobstructive causes.

Table 3.1

Classification of infantile conjugated bilirubinaemia disorders (neonatal hepatitis syndrome) with major histological and diagnostic investigations

Categories Specific diseases/causes Comments Liver histology Diagnostic investigations
Infection Toxoplasmosis (congenital) NSH (calcifications) Maternal infection/IgM-specific Abs
PCR on amniotic fluid
Rubella (congenital) NSH IgM-specific Abs
Cytomegalovirus (CMV) (congenital) NSH ‘owl-eye’ nuclear inclusions Urine for viral culture, IgM Abs, PCR
Herpes simplex virus (HSV) (congenital) Acute-pattern neonatal liver failure Necrotizing hepatitis/viral inclusions (IHC) Liver biopsy
Viral culture (scrapings from skin vesicles)
Syphilis (congenital) Diffusely fibrosing hepatitis Standard test, VDRL, fluorescent treponema Abs
Human herpesvirus 6 (HHV6) Acute-pattern neonatal liver failure Necrotizing hepatitis/viral inclusions (rare) Serology, PCR
Herpes zoster virus (HZV) NSH Serology, PCR
Hepatitis B virus (HBV) (mainly vertical) Acute-pattern neonatal liver failure Severe hepatitis Mother’s serum eAg positive (or negative due to precore mutant)
Hepatitis C virus (HCV) (mainly vertical) Rarely cause of NHS Screening of infants born to HCV+ mothers by RT-PCR
Human immunodeficiency virus (HIV) (vertical) Rarely cause of NHS NSH (opportunistic infections, in particular CMV) Anti-HIV, CD4 count
Parvovirus 19 infection Chronic-pattern neonatal liver failure NSH, marked haemopoiesis, siderosis, perisinusoidal fibrosis, few GC Severe anaemia
IgM Abs, PCR
Syncytial giant cell hepatitis (?paramyxovirus) NSH, prominent syncytial GC (EM paramyxovirus-like inclusions) Liver ultrastructure (no supportive serology)
Enteric viral sepsis (echoviruses, coxsackieviruses, adenoviruses) Acute-pattern neonatal liver failure NSH GC/cholestasis Appropriate serology, viral culture or direct fluorescent assay
Bacterial infection (extrahepatic or sepsis) Acute-pattern neonatal liver failure Nonspecific hepatitis/cholestasis (ductular bile casts) Blood, urine or CNS culture
Listeriosis NSH, focal necrosis granulomas Listeria isolation from blood, CSF or liver
Tuberculosis Caseating granulomas (acid-fast bacilli) High index of suspicion
Mantoux test
Structural Biliary atresia (BA) Biliary features: loose portal fibroplasia, ductular reaction and cholestasis including ductular bile plugs/GC (15%); DPM-like (20%) Acholic stools/liver histology
No excretion on hepatobiliary scan
Choledochal cyst Differentiate from BA Biliary features Ultrasound, cholangiography
Caroli disease/syndrome Biliary features/DPM Ultrasound, cholangiography
Choledocholithiasis Differentiate from BA Biliary features Ultrasound, cholangiography
Neonatal sclerosing cholangitis Biliary features/periductal fibrosis inconstant Cholangiography
Extrahepatic biliary hypoplasia (‘hair-like bile duct syndrome’) Differentiate from BA Biliary features Cholangiography
Spontaneous perforation of common bile duct Differentiate from BA Biliary features Imaging
Bile-stained ascites (paracentesis)
Nonsyndromic duct paucity (idiopathic) Paucity of intrahepatic bile ducts Liver biopsy
Alagille syndrome Differentiate from BA Paucity of intrahepatic bile ducts
Extrahepatic syndromic features
Identifiable bile ducts ± mild ductular reaction occasionally seen in early biopsy High serum cholesterol
Liver biopsy
JAG1 mutations (20p) or Notch2 (1p13)
Metabolic genetic (see Chapter 4 ) α1-Antitrypsin deficiency Differentiate from BA Variable; biliary features mimicking BA, duct paucity or NSH (DPAS not diagnostic before 8–12 weeks of age); GC rare; periportal steatosis Serum α1-antitrypsin concentration
α1-Antitrypsin phenotype (PI type)
Cystic fibrosis Differentiate from BA Steatosis, cholestasis, biliary features (focal fibrosis)
Cholangiolar eosinophilic casts
Sweat chloride, extrahepatic complications
Gene mutation (7q31.2–CFTR protein)
Galactosaemia Acute- or chronic-pattern neonatal liver failure Steatosis, biliary features, fibrosis
Severe parenchymal damage and loss
Later cirrhosis (now rare)
Galactose-1–6-phosphate uridyltransferase assay
Erythrocyte galactose-1-phosphate level
Tyrosinaemia, type 1 Acute- or chronic-pattern neonatal liver failure Severe parenchymal injury and loss, steatosis, GC, regenerative nodules cell dysplasia; fibrosis, cirrhosis Elevated serum tyrosine, phenylalanine, methionine/elevated succinylacetone in urine
FAA activity in fibroblasts or lymphocytes
Gene mutation (15q23–25)
Hereditary fructosaemia Chronic-pattern neonatal liver failure Steatosis, biliary rosettes, GC, fibrosis, later cirrhosis Liver biopsy (enzyme analysis)
Gene mutation (9q22)
Glycogen storage disease, type IV Early perinatal variant, very rare Eosinophilic (‘ground-glass’) cytoplasmic inclusions, PAS positive, diastase resistant (DPAS+) (amylopectin-like) Liver biopsy
Brancher enzyme in liver, white blood cells or cultured fibroblasts
Niemann–Pick, type A Hepatocyte and macrophage storage (lipidic, microvesicular/foamy) Sphingomyelinase assay (peripheral blood cells or liver)
Niemann–Pick, type C Chronic-pattern neonatal liver failure Hepatocytic/macrophage storage as type A, but few cells; biliary features Storage cells in bone marrow aspirate
Cultured fibroblasts, cholesterol esterification studies
Wolman disease Cholesterol ester stored mainly in macrophages (cholesterol crystals), neutral lipids in hepatocytes Liver biopsy
Lysosomal acid lipase activity
Gaucher disease Macrophage storage (foamy striated cytoplasm), variable perisinusoidal fibrosis Liver biopsy
Acid β-glucosidase activity (white blood cells or cultured fibroblasts)
FIC-1 deficiency (PFIC type 1) Bland cholestasis, mild disease Low-GGT cholestatic syndrome
EM: granular bile (‘Byler bile’) Liver biopsy (EM)
Genomic mutation (18q21–22)
Bile salt export pump (BSEP) deficiency (PFIC type 2) Severe parenchymal injury, ballooned (cholate-static) hepatocytes, GC, absent canalicular BSEP (IHC staining) Low-GGT cholestatic syndrome
Liver biopsy
Gene mutation (2q24)
Multidrug-resistant protein 3 (MDR3) deficiency (PFIC type 3) Biliary features; progressive fibrosis; absent canalicular MDR3 (IHC staining) High-GGT cholestatic syndrome
Histology, IHC
Gene mutation (7q21)
North American Indian familial cholestasis Bland cholestasis Gene mutation (16q22/cirhin)
Later progressive fibrosis
Aagenaes syndrome Very rare Cholestasis Cholestatic syndrome
Lymphoedema (lower limb)
Primary disorders of bile acid synthesis: Resemble PFIC type 2, but canalicular Low-GGT cholestatic syndrome
BSEP present (IHC) Urine and plasma bile acids
3β-hydroxy-Δ 5 -C 27 -steroid dehydrogenase/isomerase deficiency
Δ 4 −3-oxosteroid 5β-reductase deficiency
Arthrogryposis, renal dysfunction and cholestasis (ARC) Variable cholestasis Ichthyosis, recurrent infection, failure to thrive
Fanconi-like renal tubular dysfunction; arthrogryposis may not be evident
Gene mutation ( VPS33B on 15q26)
Peroxisomal disorders (e.g. Zellweger syndrome) Variable Dysmorphic features
Cholestasis, fibrosis, haemosiderosis Very-long-chain fatty acid study/red cell plasmalogens
Absence of peroxisomes on EM
X-linked adrenoleukodystrophy Chronic-pattern neonatal liver failure Absent or reduced peroxisomes on EM Dysmorphic features (less striking than Zellweger)
Perinatal haemochromatosis Chronic-pattern neonatal liver failure Severe parenchymal injury and loss; GC, haemosiderin pigment in hepatocytes High serum ferritin, TIBC
Iron accumulation in heart and/or pancreas on computed tomography or magnetic resonance imaging
Liver biopsy/lip biopsy for extrahepatic iron storage (accessory salivary glands)
Mitochondrial DNA depletion syndrome Chronic-pattern neonatal liver failure Microvesicular steatosis, oxyphilic cells, siderosis, cirrhosis Lactic acidosis hypoglycaemia
Abnormally low ratio of mtDNA/nDNA in tissue
Mitochondrial anomalies (EM)
Citrullinaemia, type II Mainly Asian descent Cholestasis, steatosis, siderosis Gene mutation ( SLC25A13 )
Adenosine deaminase deficiency Very rare
Panhypopituitarism (septo-optic dysplasia) NS hepatitis, no distinctive features Low cortisol, TSH and T4
Hypothyroidism NSH, cholestasis High TSH titre, low T4, free T4 and T3
Genetic (gross chromosomal abnormalities) Trisomy 18 Associated with biliary atresia Biliary features Karyotype
Cat-eye syndrome Associated with biliary atresia Biliary features Karyotype
Trisomy 21 Chronic-pattern neonatal liver failure (rare); associated biliary atresia (occasional) Fibrosing hepatitis with leukaemoid cell infiltration Karyotype
Kabuki syndrome a,b Rarely associated with biliary atresia Biliary features Congenital anomalies, mental retardation
Neoplasia (see Chapter 15 ) Neonatal leukaemia Acute-pattern neonatal liver failure Leukaemic infiltration Peripheral blood, bone marrow aspirate
Neuroblastoma Acute-pattern neonatal liver failure Small cell tumour, rosettes Imaging
IHS, EM Urinary vanilmandelic and homovanillic acid
Langerhans cell histiocytosis Biliary features similar to sclerosing cholangitis; Langerhans cells (CD1a) inconstant Involvement of other systems (skin, bone, lung)
Haemophagocytic lymphohistiocytosis Chronic-pattern neonatal liver failure Haemophagocytic activity High level of macrophage-derived cytokines in serum
Elevated ferritin; elevated triglycerides
Toxic Total parenteral nutrition-associated cholestasis Differentiate from BA Biliary features, cholestasis
Drug-induced (via breast milk or other)
Vascular Budd–Chiari syndrome Rare Features of venous outflow block
Severe congestive heart failure Perivenular cell dropout/congestion
Perinatal/neonatal asphyxia Differentiate from BA Ischaemic necrosis
Immune Inspissated bile syndrome associated with ABO incompatibility Differentiate from BA Biliary features Coombs test
Neonatal lupus erythematosus NSH Maternal history, congenital heart block, skin rash (discoid lupus)
Anti-Ro–anti-La in liver tissue (IHC)
Anti-Ro and anti-La antibodies
Neonatal hepatitis with autoimmune haemolytic anaemia Acute or chronic pattern liver disease Prominent syncytial GC, necrosis, inflammation, fibrosis Coombs-positive haemolytic anaemia
Idiopathic Idiopathic neonatal hepatitis (seronegative) Differentiate from biliary atresia GC, parenchymal loss with stromal collapse of variable severity Liver biopsy
‘Le foie vide’ (infantile hepatic nonregenerative disorder) Chronic-pattern neonatal liver failure Total parenchymal cell dropout, scant ductular reaction Liver biopsy
Hardikar syndrome c Differentiate from biliary atresia, Alagille syndrome Biliary features Associated with cleft palate, pigmentary retinitis, hydronephrosis

Note: Acute-pattern neonatal liver failure denotes metabolic instability, coagulopathy and extremely elevated serum aminotransferases in a neonate; chronic-pattern neonatal liver failure denotes metabolic instability, coagulopathy, hypoalbuminaemia, near-normal serum aminotransferases in a neonate; conditions which require specific consideration vis-à-vis biliary atresia are denoted ‘Differentiate from biliary atresia’.

Abs, Antibodies; CNS, central nervous system; CSF, cerebrospinal fluid; DPM, ductal plate malformation; EM, electron microscopy; ERCP, endoscopic retrograde cholangiopancreatography; FAA, fumarylacetoacetase; GC, multinucleated giant cells; GGT, γ-glutamyltransferase; IHC, immunohistochemistry; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; NSH, nonspecific hepatitis; PAS, periodic acid-Schiff stain; PFIC, progressive familial intrahepatic cholestasis; RT PCR, reverse-transcriptase polymerase chain reaction; T3, triiodothyronine; T4, thyroxine; TIBC, total iron-binding capacity; TSH, thyroid-stimulating hormone; VDRL, Venereal Disease Research Laboratories.

a Selicorni A, Colombo C, Bonato S, et al. Biliary atresia and Kabuki syndrome: another case with long-term follow-up (letter). Am J Med Genet 2001;100:251.

b Van Haelst MM, Brooks AS, Hoogeboom J, et al. Unexpected life-threatening complications in Kabuki syndrome. Am J Med Genet 2000;94:170–3.

c Nydegger A, Van Dyck M, Fisher RA, et al. Hardikar syndrome: long term outcome of a rare genetic disorder. Am J Med Genet 2008;146A:2468–72.

Obstructive causes include biliary atresia (BA), choledochal cysts, neonatal sclerosing cholangitis, spontaneous perforation of the common bile duct, the inspissated bile syndrome specifically associated with cystic fibrosis, and Langerhans cell histiocytosis. A survey at King’s College Hospital of 1086 consecutive cases of neonatal conjugated hyperbilirubinaemia showed that BA constituted approximately one-third of all cases. Destruction and obliteration of the extrahepatic biliary tree of variable extent occurring early in embryonal development or perinatally is the cause of BA. Surgery is the only treatment option at present. Clinically, jaundice due to conjugated hyperbilirubinaemia, acholic stools, ‘nondraining’ hepatobiliary scan and ultrasonographic abnormalities of the biliary tree support a diagnosis of BA.

Portal expansion associated with oedema and fibroplasia, a variable mixed inflammatory cell infiltrate along with a ductular reaction, and cholangiolar bile plugs are the histological hallmarks of an obstructive pattern. In the lobule, giant cell transformation with multinucleation of hepatocytes and canalicular bile plugs are also present. These portal changes are typically seen at about 6 weeks of life in infants with BA. These may be very mild or absent in biopsies taken earlier than 6 weeks. Biopsies from neonates 2–4 days old have shown minimal portal changes, suggesting that the portal changes observed later are the result of the perinatal bile flow causing periductal leakage and consequent inflammation and fibrosis.

The typical obstructive pattern observed in BA can be simulated by other disorders, such as α1-antitrypsin deficiency (alpha-1AT), cystic fibrosis and MDR3 deficiency. Steatosis often accompanies the biliary changes in both α1-antrypsin deficiency and cystic fibrosis. Diastase-resistant periodic acid-Schiff (PAS)-positive globules are usually not evident in the first few months of life but may be demonstrated by immunohistochemistry (IHC) for α1-antitrypsin. If present, eosinophilic cholangiolar casts are a classic finding in liver biopsies from patients with cystic fibrosis. MDR3 deficiency is caused by a mutation of the ABCB4 gene, which encodes for the MDR3 protein, critical for the transport of phospholipids, and in particular phosphatidylcholine, into the bile. Without phosphatidylcholine, bile salts are not incorporated into micelles. The bile remains hydrophobic and damages the biliary epithelium, causing a cholangiopathy, without gross architectural changes to the bile ducts, which may nevertheless manifest histologically with an obstructive pattern. Conditions manifesting with an obstructive pattern simulating BA include total parenteral nutrition, sepsis and the so-called inspissated bile syndrome associated with ABO incompatibility.

Nonobstructive causes include diverse disorders causing hepatocellular injury. The portal changes previously described are not observed. The histological picture is dominated by the lobular changes, usually described with the term ‘neonatal hepatitis’. These consist of a combination of hepatocellular changes such as swelling, multinucleation, apoptosis, cholestasis, lobular and portal inflammation, extramedullary haematopoiesis and fibrosis. These features are described in more detail in the section on neonatal hepatitis. Hepatocellular injury can be very severe in some patients, resulting in confluent hepatocellular loss and parenchymal micronodular transformation or even massive hepatic necrosis. The clinical scenario is that of neonatal acute liver failure. There may be clues in the clinical presentation, biochemistry and/or histology pointing to a metabolic aetiology or even to a specific metabolic disorder.

A defect of bile acid metabolism including inborn errors of bile acid synthesis, and certain defects in bile formation due to defective bile canalicular transporters, previously called ‘progressive familial intrahepatic cholestasis’ (PFIC), caused by mutations of ATP8B1 (FIC1) or ABCB11 (BSEP) are usually suspected when conjugated hyperbilirubinaemia is associated with normal or near-normal serum levels of γ-glutamyltransferase (GGT).

Inborn errors of bile acid synthesis and BSEP deficiency are associated with marked hepatocyte disarray, hepatocyte multinucleation and a tendency to progressive fibrosis.

FIC1 deficiency caused by ATP8B1 mutations manifests histologically more mildly with bland cholestasis, in the form of pale canalicular bile plugs not accompanied by hepatocellular disarray or giant cell transformation. Other forms of low-GGT cholestasis exist. Severe low-GGT cholestasis has been described in patients with mutations in the tight junction protein 2 gene ( TJP2 ).

Steatosis associated with features of neonatal hepatitis is seen in galactosaemia, hereditary tyrosinaemia type 1, fructosaemia, mitochondrial disorders and citrullinaemia type 2 (citrin deficiency). Ground-glass inclusions are associated with the uncommon early manifestation of glycogen storage disease (GSD) type IV. Accumulation of lipidic substances in hepatocytes and portal or sinusoidal macrophages suggests a storage disorder such as Niemann–Pick disease type A or B, Wolman disease and Gaucher disease.

Histological features of neonatal and early infantile liver disease must be evaluated in the context of normal histological features at birth. These include two-cell-thick hepatic plates, extramedullary haemotopoiesis, and periportal siderosis and accumulation of copper and copper-binding protein. Portal tracts may be lacking interlobular bile ducts. Biliary development appears to progress from the hilum through to the periphery, and the biliary tree may not be fully developed at birth. Degree of hepatobiliary maturation needs to be taken into consideration in the differential diagnosis of cholestatic conditions.

Postneonatal, childhood and adulthood presentation

The clinical and histological manifestations of inherited and metabolic liver disorders in older children and adults are heterogeneous. They constitute a wide spectrum, which includes acute, subacute or chronic liver or multiorgan dysfunction, organomegaly, and systemic metabolic abnormalities such as hypoglycaemia, protein intolerance or metabolic acidosis. An external factor can exacerbate their course and make them manifest acutely. They can mimic other metabolic disorders, disorders of various other aetiologies or even the effect of medical treatment. Wilson disease is a typical example. Its histological picture ranges from steatosis resembling nonalcoholic fatty liver disease (NAFLD) to a chronic hepatitis similar to autoimmune hepatitis. The clarified appearance of hepatocytes in GSD is identical to that of poorly controlled diabetes in Mauriac syndrome.

Histological interpretation is usually required in patients with a known disorder (to confirm the diagnosis and estimate the extent of liver damage) or in patients in whom the clinical diagnosis is uncertain. A systematic approach ensures a comprehensive histological assessment. It can be divided into the following components: (1) assessment of structural changes caused by acute parenchymal injury or chronic disease and fibrosis; (2) changes affecting hepatocytes; (3) presence and pattern of cholestasis and (4) changes suggestive of reticuloendothelial storage. These four components are described in more detail next. Of note, in some metabolic inherited disorders, there may be minimal or no significant histological changes, sometimes in relation to metabolic fluctuations. With urea cycle defects, for example, the histological appearance of the liver parenchyma ranges from near-normal to steatosis or cytoplasmic clarification simulating GSD when ammonia levels are high. Liver tumours may be the first manifestation or may complicate the course of many metabolic and inherited disorders (e.g. α1-antitrypsin deficiency, GSD). Cystic transformation is a characteristic of some of the ductal plate malformation (ciliopathy) disorders.

Structural changes

Since metabolic and inherited disorders can present acutely, subacutely or chronically, their histological manifestations can be in the form of acute parenchymal injury or fibrosis, whose pattern, development and rate of progression may vary among different diseases, single-disease variants or individual patients. In some cases the lobular architecture can be entirely normal. For example, Wilson disease, hereditary tyrosinaemia type 1, α1-antitrypsin deficiency, mitochondrial disorders, inherited disorders associated with intrahepatic cholestasis and Niemann–Pick disease type C tend to progress more or less rapidly to advanced fibrosis. In cystic fibrosis and MDR3 deficiency the pattern of fibrosis is biliary, although these are highly disparate disorders. Type IV GSD progresses rapidly to cirrhosis, whereas certain other types of GSD do not. Bands of fibrous tissue associated with irregularly shaped parenchymal islands and containing bile ducts reminiscent of the ductal plate and scant portal vein branches are typical of congenital hepatic fibrosis.

Despite the presence of fibrosis, some metabolic disorders (e.g. Wilson disease, hereditary tyrosinaemia type 1) may present with fulminant liver failure. External factors such as infections or drugs may act as triggers. They manifest histologically with confluent parenchymal collapse, in some cases resulting in multiacinar loss or even massive hepatic necrosis, which may be superimposed to underlying fibrosis. Liver biopsy has a limited role in these cases because of its risks and proven limited contribution to diagnosis and clinical management. Mitochondrial disorders may present with neonatal liver failure or severe liver dysfunction later in infancy. Liver biopsy may play a role in confirming the diagnosis, noteworthy because multisystemic mitochondriopathies are not an indication for liver transplantation.

Changes affecting hepatocytes

Hepatocyte morphology is affected in many metabolic and inherited disorders. Changes can be evident on standard haematoxylin and eosin (H&E)-stained sections or manifest as cytoplasmic accumulations demonstrated by histochemical stains, IHC or EM. In some disorders, the hepatocyte morphology can be entirely normal.

Steatosis is a common change, caused by primary or secondary abnormalities of pathways for metabolism of sugars, fats and amino acids. Macrovesicular steatosis, defined as a single, large droplet displacing the hepatocyte nucleus, is seen in galactosaemia, fructosaemia, citrullinaemia type 2 (citrin deficiency), hypertriglyceridaemia, urea cycle defects and Wilson disease. Paediatric obesity and associated fatty liver disease may act as a confounding factor. Wilson disease should always be considered, even in obese children and in the absence of histochemically demonstrable copper or copper-binding protein. Poor nutrition (as in cystic fibrosis) or a metabolic crisis can contribute to its development, severity and distribution. Microvesicular steatosis, defined as fine cytoplasmic vacuolation around a centrally placed nucleus, is typically observed in fatty acid oxidation defects and other mitochondrial disorders. Histochemical techniques on frozen tissue or EM may be necessary to confirm that the changes noted are caused by accumulation of lipid. Micro- and macrovescicular macrovesicular steatosis can coexist and also can be associated with other hepatocyte changes, such as oncocytosis (mitochondriopathies), or clarification of the hepatocyte cytoplasm due to glycogen accumulation (GSD). Reye syndrome, first described in the 1960s and now very rare, was characterized clinically by encephalopathy and liver dysfunction after a viral illness and exposure to salicylates and histologically by microvesicular steatosis. Reye syndrome could be part of an idiosynchratic reaction to acute illness in individuals harbouring an underlying metabolic disorder.

Accumulation of glycogen in the hepatocyte cytoplasm results in a plant cell-like appearance. It is typically seen in the context of GSD. It simulates microvesicular steatosis, lysosomal storage and urea cycle disorders, and drug-induced expansion of the smooth endoplasmic reticulum. EM can help in the differential diagnosis. It is indistinguishable from the clarification of hepatocellular cytoplasm observed in patients with poorly controlled diabetes (Mauriac syndrome).

Ground-glass cytoplasmic inclusions are observed in a number of metabolic conditions, including GSD type IV myoclonic epilepsy (Lafora disease) and hypofibrinogenaemia, as well as in drug-induced hepatitis and chronic hepatitis B.

Diastase-resistant PAS-positive (D-PAS+) globules in periportal hepatocytes usually indicate α1-antitrypsin deficiency, and typically patients with the PI*Z allele, due to retention of the polymerized abnormal enzyme within the rough endoplasmic reticulum. These globules may represent an acute-phase phenomenon in patients with PI*M phenotype or when heterozygosity for a defective M variant has not been detected. They are seen in the liver of patients with PI*MZ but not with PI*S or PI*MS. Finer D-PAS+ inclusions have been described in association with α1-antichymotrypsin deficiency, and D-PAS-negative/α1-antitrypsin-negative globules may be found with antithrombin III deficiency.

Copper accumulation may be observed. Periportal copper and copper-binding protein deposits (rhodanine and orcein or analogue stains) and siderosis (Perls stain) are physiological up to approximately 4 months after birth. Accumulation of copper or copper-binding protein suggests a chronic cholestatic disorder, Wilson disease or copper toxicosis. In the early stage of Wilson disease, hepatocellular copper is intracytoplasmic rather than lysosomal and therefore not demonstrable histochemically.

Iron accumulation may be localized in hepatocytes or nonparenchymal cells. Siderosis of Kupffer and endothelial cells indicates secondary iron overload or ferroportin disease. Hepatocellular siderosis is a feature of genetic HFE haemochromatosis (see Chapter 4 ). Children with genetic haemochromatosis are asymptomatic. Juvenile haemochromatosis usually presents clinically in the second decade of life, mainly as a cardiac disorder. Siderosis may be observed in children with peroxisome disorders (e.g. Zellweger syndrome).

Siderosis in residual hepatocytes, pancreas, heart and salivary glands (demonstrated with a lip biopsy)—rather than in reticuloendothelial cells—of infants with neonatal liver failure due to massive necrosis characterizes the syndrome of neonatal (or perinatal) haemochromatosis. It is currently regarded for most, but not all, cases as a form of alloimmune injury to the fetal liver (gestational alloimmune liver disease). A similar clinical presentation occurs with ‘le foie vide’, characterized by massive loss of hepatic plates, but not cirrhosis. Its pathogenesis remains uncertain, apparently a failure of regenerative capacity.

Presence of cholestasis

Canalicular cholestasis in the absence of portal changes of large bile duct obstruction, cholate stasis or ductopenia is called ‘bland cholestasis’. It may be the only histological change in children, adolescents or adults presenting with pruritus with or without jaundice and with or without an external trigger such as an infection or hormonal imbalance (e.g. oral contraceptives). These symptoms may be caused by mutations of the same genes that cause bile canalicular transport defects ( ATP8B1 and ABCB11 ) in neonates, but in a milder form. IHC for canalicular enzymes, mutation analysis and exclusion of other causes (e.g. drugs, lymphoma) is part of the diagnostic workup. Lobular cholestasis accompanied by progressive bile duct loss and periportal deposits of copper and copper-binding protein is the histological picture of Alagille syndrome. A ductular reaction may be present at an early stage. A sclerosing cholangitis-type pattern is observed in MDR3 deficiency. ‘Cholate stasis’ refers to the changes affecting periportal hepatocytes, usually as a result of a chronic biliary process. Hepatocytes contain granules of copper and copper-binding protein and may be swollen with clarification of their cytoplasm. Cholestasis is not seen in uncomplicated congenital hepatic fibrosis, although septal ducts may contain bile. Histological changes are usually minimal in Gilbert syndrome and may consist simply of accumulation of lipofuscin. Dubin–Johnson syndrome is characterised by black Fontana-positive pigment in the hepatocyte cytoplasm, particularly in the perivenular region. Deficient expression of canalicular multispecific organic anion transporter (cMOAT, MRP2) is diagnostic.

Reticuloendothelial storage

Deficiency of enzymes (typically single-enzyme deficiency) involved in lipid or glycoprotein metabolism results in the accumulation of substances usually in the lysosomes of one or more cell types and one or more organs. These disorders are rare and often fatal in infancy, but some may remain undetected through adulthood.

The combination of cell type and organ involvement helps in identifying a specific disorder. For example, cholesterol ester storage disease affects the liver, Gaucher disease affects liver and reticuloendothelial organs, and mucopolysaccharidosis affects multiple organs including the central nervous system. The affected cell type in the liver is also an important clue to the underlying disorder. Both hepatocytes and reticuloendothelial cells, including Kupffer cells, portal macrophages and endothelial cells, are affected in acid lipase deficiency (Wolman disease and cholesterol ester storage disease). Sphingomyelin accumulation in Kupffer cells and hepatocytes is characteristic of Niemann–Pick disease types A and B.

In contrast, other disorders affect reticuloendothelial cells only and spare hepatocytes. In Niemann–Pick disease type C, accumulation of esterified cholesterol and other lipids affects very few Kupffer cells, often in a hepatitic fibrotic background. They can be easily overlooked as ceroid-laden macrophages in the context of a nonspecific hepatitic condition. In Gaucher disease, pale PAS-positive portal macrophages and Kupffer cells show a typical ‘crinkled paper’ appearance. In Fabry disease, Kupffer cells, portal macrophages and endothelial cells contain D-PAS+ globotriaosylceramide. Other lysosomal storage disorders associated with predominant accumulation in Kupffer cells include gangliosidoses, metachromatic leucodystrophy, Farber lipogranulomatosis and cystinosis. D-PAS and IHC for CD68 help in evaluating Kupffer cells. Again, portal or sinusoidal storage cells can easily be missed or interpreted as scavenging ceroid-laden macrophages, particularly in disorders associated with hepatitis and fibrosis.

Neonatal hepatitis

‘Neonatal hepatitis’ is a term that was coined for presumed viral infections of the liver in early infancy. It has become evident that disorders demonstrating neonatal hepatitis are by no means exclusively viral, or even infectious, in aetiology. Moreover, disease processes affecting mainly the infantile biliary tree can display parenchymal inflammation. In effect, neonatal hepatitis represents a clinical pattern of neonatal liver disease; thus the designation ‘neonatal hepatitis syndrome’ has merit despite the inherent vagueness of the term ‘syndrome’. Other diseases, such as galactosaemia, hereditary fructose intolerance, cystic fibrosis and the conditions discussed in relation to biliary atresia and paucity of the intrahepatic bile ducts, may also present with pathological changes in the liver resembling an infectious process. Giant cell transformation, a frequent histological component of neonatal hepatitis, has been seen in all cholestatic conditions in infancy, including pure haemolytic anaemias and endotoxic injury. Although clinical jaundice is not present in every case of neonatal hepatitis syndrome, conjugated hyperbilirubinaemia is invariably present. Therefore the entire spectrum of these diseases might best be called ‘infantile conjugated bilirubinaemia disorders’, a term which avoids the inherent disadvantages of each of the component terms of ‘neonatal hepatitis syndrome’. Table 3.1 shows a classification of infantile conjugated bilirubinaemia disorders (neonatal hepatitis syndrome). In the past 20 years the proportion of cases with no known aetiology has fallen substantially, from 50–60% to approximately 30% or less. Many of the disorders dissected out of the ‘idiopathic’ category are inherited metabolic diseases.

Nomenclature for neonatal liver disease is very problematic. The simplest term ‘neonatal jaundice’ may be confused with physiological jaundice in the newborn. The term ‘neonatal cholestasis’ is not precise because in the first 3–4 months of life, every infant has some degree of cholestasis physiologically. This physiological cholestasis occurs because mechanisms for uptake of bile acids and other organic anions by hepatocytes are immature and thus inefficient, leading to high concentrations of bile acids in blood. In addition, hepatocellular pathways for bile acid conjugation and biliary secretion are also immature, in part because bile canalicular transporters are also regulated developmentally. The circulating bile acid pool is contracted, and ileal uptake of bile acids is underdeveloped. The term ‘neonatal hepatitis’ is obviously imprecise because hepatic inflammation is not a feature of every condition. ‘Neonatal hepatitis syndrome’ emphasizes the uniformity of the clinical presentation and similarity of pathological findings, as well as the broad spectrum of causative disease processes. The term ‘infantile conjugated bilirubinaemia disorders’ (ICBRDs) not only identifies the definitional finding but conveys inclusion of a broad spectrum of infectious, metabolic, structural and other aetiologies. It suffers only from being linguistically unwieldy.

Numerous infections, usually congenital, are implicated in neonatal hepatitis syndrome, including cytomegalovirus (CMV), rubella virus, hepatitis B virus (HBV), herpes simplex virus (HSV), herpes zoster virus, coxsackievirus, echovirus, paramyxovirus, Toxoplasma and Treponema pallidum . An unusually high incidence of CMV infection (49%) was reported in a series of 45 cases from Taiwan. HSV, enteroviruses, adenovirus and HBV may cause neonatal liver failure characterised by an acute pattern of liver disease, with extremely elevated serum aminotransferases. In addition to genetic metabolic disorders, endocrine disorders may cause neonatal hepatitis syndrome. An association with hypopituitarism was reported in two infants by Herman et al., and later Sheehan et al. Immunological disorders may cause neonatal hepatitis syndrome; neonatal lupus erythematosus is most common. Most infants with the rare disorder known as ‘neonatal (or perinatal) haemochromatosis’ have an immunological disorder which results in neonatal liver failure with a chronic pattern (near-normal serum aminotransferases, profound coagulopathy, subnormal serum albumin), called ‘gestational alloimmune liver disease’ (GALD).

Rarely, a Coombs-positive haemolytic anaemia defines a severe form of giant cell hepatitis which rapidly progresses to cirrhosis or death. Early and sustained immunosuppressive therapy may control the disease in some patients. The liver lesion has been shown to recur in the allograft of the few cases transplanted. Infants with birth asphyxia may develop severe neonatal hepatitis syndrome. It may be caused by an ischaemic hepatitis. Conjugated hyperbilirubinaemia typically occurs at 1 week of age, lasts 3–4 months, and the hepatomegaly and liver tests return to normal by 1 year of age. Various hepatobiliary structural abnormalities, most importantly biliary atresia, are associated with the neonatal hepatitis syndrome. Furthermore, certain chromosomal defects predispose to the syndrome.

In approximately 30% of infants with conjugated hyperbilirubinaemia, no aetiology is found. The prognosis for this so-called idiopathic neonatal hepatitis is generally good, with mortality of 13–25%. In the study of Dick and Mowat, 2 of 29 patients with idiopathic neonatal hepatitis died, and only two others had signs of persisting liver disease. Overall, predictors of poor prognosis include persisting severe jaundice, acholic stools, prominent hepatomegaly, severe inflammation on liver biopsy and familial occurrence. Numerous inherited disorders causing the neonatal hepatitis syndrome have recently been described in terms of gene defect, such as the bile canalicular transporter disorders and bile acid synthesis disorders, and these had previously been classified as idiopathic neonatal hepatitis. Undoubtedly, other such disorders remain to be defined. A hereditary form with giant cell transformation and lymphoedema resulting from abnormal deep lymphatics has been reported, but the gene abnormality has not yet been determined. Another identified aetiology is adenosine deaminase deficiency , with recovery after enzyme replacement. Citrullinaemia type 2 due to deficiency of citrin can cause neonatal hepatitis syndrome (then also known as NICCD, neonatal intrahepatic cholestasis caused by citrin deficiency ); it may occur in ethnicities other than East Asian. ‘Le foie vide’, of which multiple cases have been identified, describes a severe neonatal liver disorder characterized by failure of hepatocellular regeneration, also manifested as chronic-pattern neonatal liver failure.

Histopathological features

Liver biopsy specimens are characterized by varying degrees of cholestasis (with or without pseudoglandular structures), giant cell transformation, ballooning, apoptotic bodies, extramedullary haemopoiesis, lobular and portal inflammation and progressive fibrosis in some cases. Unusually severe inflammation and hepatocellular damage may be found in α1-antitrypsin deficiency, hereditary tyrosinaemia type 1, Niemann–Pick disease type C, syncytial giant cell hepatitis, citrullinaemia type 2, primary disorders of bile acid synthesis (mainly δ 4 –3-oxosteroid-5β-reductase deficiency), BSEP deficiency (progressive familial intrahepatic cholestasis type 2) and idiopathic neonatal hepatitis. Associated macrovesicular steatosis favours a metabolic disorder. Confluent hepatocyte necrosis or loss with bridging collapse is seen in the rare patients presenting with acute-pattern neonatal liver failure or having a subacute clinical course associated with perinatal haemochromatosis, non-Wilsonian copper toxicosis or other metabolic disorders such as hereditary tyrosinaemia type 1. It may also occur occasionally with viral hepatitis, especially in infants born to mothers carrying the precore mutant of hepatitis B, and idiopathic neonatal hepatitis.

The pathological aspects of giant cell transformation, a frequent and often dominant finding in neonatal hepatitis, have been reviewed extensively. The change is seen throughout the parenchyma but is often more marked in the perivenular areas. The giant cells contain four or more nuclei, sometimes as many as 40 per cell, have poorly defined outlines and may be detached from other cells in the hepatic plate ( Figs 3.1 and 3.2 ). The cytoplasm of some giant cells may contain remnants of cell membranes. It is partially rarefied and often contains bile and/or haemosiderin. The cells may have more glycogen than normal hepatocytes and a greater activity of a variety of enzymes, such as glucose-6-phosphatase, acid phosphatase and succinic dehydrogenase. Death of the giant cells is associated with a neutrophilic inflammatory response ( Fig. 3.3 ). In severe forms, extensive bridging cell loss may divide the parenchyma into micronodules which are highlighted by a reticulin stain ( Fig. 3.4 A and B ). The number of giant cells decreases as patients grow older and are rare after age 1 year. Formation of giant cells is considered to be a characteristic change resulting from mitotic inhibition of the young, growing liver tissue by a number of agents, such as viruses, drugs and hereditary abnormalities, or from dissolution of cell membranes, as suggested by Craig and Landing, who first described this entity. Negative nuclear staining for cell proliferation markers and the demonstration of canalicular remnants using carcinoembryonic antigen (CEA) immunostaining support a fusion of rosette-forming hepatocytes as the likely mechanism of giant cell formation. Phillips et al. used the term ‘syncytial giant cell hepatitis’ to describe 10 patients (age range, 5 months to 41 years) with hepatitis characterized by giant multinucleated hepatocytes, usually containing up to 30 nuclei within a single large cell body. These large cells conformed to the reticular framework of the hepatic cords but in some cases crossed the sinusoids to form syncytial masses with adjacent cords. The authors concluded that these large cells were the product of fusion of hepatocytes (thus the term ‘syncytial’), most likely caused by paramyxovirus infection.

Figure 3.1

Neonatal hepatitis, idiopathic, with giant cell transformation (haematoxylin and eosin [H&E] stain).

Figure 3.2

The same case as illustrated in Fig. 3.1 . Detail of multinucleated giant hepatocyte. Periphery of cell (left) suggests fusion with several smaller cells. (H&E stain.)

Figure 3.3

The same case as illustrated in Figs 3.1 and 3.2 . Neutrophilic satellitosis of degenerated giant cell. (H&E stain.)

Figure 3.4

Severe neonatal hepatitis of undetermined cause. Extensive bridging parenchymal loss with concomitant reticulin collapse divides the parenchyma into minute nodules with prominent giant cell transformation. A, H&E stain; B, Gordon–Sweet reticulin.

Biliary atresia

Biliary atresia is one of the most important causes of severe neonatal liver disease. It is the major indication for liver transplantation in young children. Initially, the extrahepatic biliary tree is affected, evident as an obstructive picture both clinically and histopathologically; this is the defining lesion of this disorder. Biliary cirrhosis develops early in life. The children who survive infancy because of a successful Kasai portoenterostomy continue to have intrahepatic bile duct damage, which leads eventually to profound loss of small intrahepatic bile ducts and recurrent cholestasis due to bile duct paucity. Accordingly, this hepatobiliary disorder is now called simply biliary atresia , without specifying an extrahepatic (or intrahepatic) location. The term ‘biliary atresia’ (BA) is used in this section interchangeably with the former term ‘extrahepatic biliary atresia’ (EHBA). The term ‘intrahepatic biliary atresia’ to denote intrahepatic bile duct paucity has been obsolete for many years and should be abandoned. Additionally, biliary ‘atresia’ must be distinguished from biliary ‘agenesis’.

Classification and aetiopathogenesis

Approximately 30% of infants presenting with conjugated hyperbilirubinaemia in the neonatal period have BA, the overall incidence being approximately 1 in 6000 to 1 in 19,000 live births. There is no clear-cut racial predilection, although some ethnicities appear to have a higher incidence: African-Americans and Polynesians compared with Caucasian infants. BA is more common in girls than boys. Seasonal variation in the occurrence of this disease has been suggested in North American studies, although this pattern has not been confirmed in Europe or Japan. Unquestionably, multiple disease mechanisms can produce BA because it may occur as an isolated lesion or in association with various types of congenital structural abnormalities or specific chromosomal abnormalities. It can be conceptually useful to classify BA in two general patterns: ‘embryonic,’ occurring early in development and accounting for 15–35% of cases, and ‘perinatal/acquired’, comprising 65–85% of cases. This aetiopathogenic heterogeneity of BA was first delineated by a study of 237 children by Silveira et al. Forty-seven of the children (20%) had associated congenital anomalies (28 cardiovascular, 22 digestive and 19 splenic). The splenic malformations included 13 with polysplenia syndrome and two with asplenia. Karyotypic abnormalities were found in two of eight children studied. The investigators divided BA into four distinct subgroups, three involving a congenital form that could arise through a malformation, a disruption or a chromosomal abnormality, with the fourth attributable to agents active in the perinatal period (the acquired form).

Most infants have this perinatal/acquired pattern; their apparently normal biliary system has been subjected to a fibrosing inflammatory process late in gestation or shortly after birth. Discordance for BA in HLA identical twins supports a postnatal event being of primary importance in the pathogenesis of perinatal/acquired BA. By contrast, approximately 10–30% of infants with BA have extrahepatic congenital abnormalities such as polysplenia, left atrial isomerism, double-sided left lung, preduodenal portal vein, intestinal malrotation and/or congenital heart defects. Preduodenal portal vein is the result of a variation in the normal developmental pattern of the embryonic precursors of the portal vein, i.e. the right and left vitelline veins and their three anastomotic channels. These congenital defects are sometimes grouped as a ‘laterality complex’. Abnormalities of the spleen are not invariably present in infants who have other typical features of the laterality complex, and thus ‘splenic disorder’ does not define this association. The best currently available term for this category of BA is ‘biliary atresia with structural malformations’ (BASM).

A further subset of infants with BA, comprising 6–10% of patients overall, have damage to the biliary tree which produces cystic dilation ; these patients look as if they have a choledochal cyst. Exceptionally, the cystic change is confined to intrahepatic ducts. This type of BA, now termed ‘cystic’ BA (formerly called ‘correctable’ BA), may be identified on sonography of the fetus in approximately 40% of affected cases. It may represent yet another disease mechanism for BA, distinct from those represented by embryonic and perinatal BA, although cystic BA has been identified in patients with embryonic BA. Notably, some infants with BA have specific chromosomal abnormalities such as trisomy 17–18, Turner syndrome or cat-eye syndrome. BA has been reported with the Kabuki make-up syndrome, in one patient with Zimmermann–Laband syndrome and brachydactyly and in Martinas–Frias syndrome. Other genetic disorders may be associated with BA because familial occurrence has been reported, and in one case a woman who previously had BA gave birth to a daughter with BA. A lethal autosomal recessive syndrome with intrauterine growth retardation, once called intra- and extrahepatic biliary atresia, and oesophageal and duodenal atresia was reported in one family; description of the findings suggests agenesis of the extra- and intrahepatic ducts.

Viral infection in combination with a genetic predisposition to a robust or disordered inflammatory response may play a role in the development of perinatal/acquired BA. Chance occurrence of a viral infection during a limited period of susceptibility would explain the rarity of BA. No consensus exists as to which viruses are pre-eminent in such an aetiopathogenesis. The proposed viruses form a heterogeneous group, including both DNA and RNA viruses. Some recent observations suggest that viral infection is a secondary phenomenon. CMV infection is found in a high proportion of children with BA. Accordingly, a recent classification of BA specifies CMV association not only in the perinatal form, but also in the embryonic form and cystic variant. Tarr et al. found evidence for viral infection in 5 of 23 patients with BA. The diagnosis was based on histopathological evidence of CMV infection, serology (IgM antibodies) or culture. The detection of CMV infection by the polymerase chain reaction (PCR) is higher in neonatal hepatitis than in BA. In a set of identical twins both infected with CMV, one twin had BA and the other had neonatal hepatitis. Infants with BA and concurrent CMV infection may have a worse prognosis. CMV may elicit immune responses, which interfere with the action of regulatory T (Treg) cells. Reovirus 3 was suggested as a cause of BA and neonatal hepatitis on the basis of clinical and experimental studies, but this association was questioned by other investigators. Tyler et al. provided more compelling evidence for an aetiological association between BA and reoviruses, detecting reovirus RNA from hepatobiliary tissues of 55% of patients with BA and 78% of patients with choledochal cysts. Riepenhoff-Talty et al. suggested a possible relationship between group C rotavirus and BA. Subsequently, however, no evidence of group A, B or C rotaviruses was detected by PCR in BA. Human papillomavirus (HPV) has been detected in neonatal hepatitis and BA by nested PCR for DNA, but the role, if any, of HPV needs to be clarified.

Recent progress has focused on immune mechanisms in the pathogenesis of BA, especially perinatal/acquired BA. The envisioned disease mechanism is that during the perinatal period a viral infection occurs and targets the biliary epithelium and provokes an aberrant autoimmune injury to the bile ducts that persists long after the viral infection is gone. This proposed mechanism entails an active, ongoing immune response, which can be documented empirically, and it accounts for the conflicting reports of viral infection in BA as well as the absence of detectable ongoing viral infection in liver or biliary tissue from BA patients. Currently, it is seen as involving macrophages and dendritic cells primed as a result of the infection, followed by activation of natural killer (NK) cells, which then actuate biliary epithelial damage. Amplification of the immune response by T cells and proinflammatory cytokines then takes place. Molecular mimicry may play a role. This general disease mechanism is based on findings in human tissue from BA patients and also on disease models of BA. The hepatic inflammatory infiltrate in BA was found remarkable for evidence of lymphocyte activation. Studies in liver tissue from infants with BA showed a T-helper cell type 1 (Th1) form of cytokine expression pattern with CD4+ and CD8+ lymphocytes, CD68+ macrophages in portal tracts and increased interleukin-2 (IL-2), IL-12, interferon-γ (IFN-γ) and tumour necrosis factor alpha (TNFα). Determinants on the activated T cells are typical of an oligoclonal expansion, consistent with being evoked by a specific antigen.

The rarity of BA raises the possibility of genetic susceptibility in perinatal/acquired BA. Genomic studies of liver from infants with BA have shown upregulation of genes involved in regulating lymphocyte differentiation, mainly of those with Th1 commitment. Upregulated expression of IFN-γ and osteopontin was notable. Subsequently, upregulation of osteopontin expression in intrahepatic biliary epithelium was found to correlate with portal fibrosis and ductular reaction. A different genomic study, which included somewhat older patients, also found upregulation of genes involved in morphogenesis, cell signalling and regulation of gene transcription. Further studies suggested that the pattern of regulatory gene expression in perinatal/acquired BA is not equivalent to that in embryonic BA; however, these data also failed to show a pattern of gene expression relating to laterality genes in embryonic BA. Both forms of BA appear to induce a strong immunological response.

Genome-wide association studies have been used to identify possible susceptibility loci. In North American children, a locus at 2q37.3 was found to be GPC1, which encodes a regulator of Hedgehog signalling and inflammation. In Chinese children a locus at 10q24.2 was also identified as a possible susceptibility locus for BA; it proved to be ADD3, and this finding was confirmed in a North American series. A further study in North American children found a locus at 14q21.3, and this was identified as encoding ADP ribosylation factor-6 ( ARF6 ). These three proteins have been investigated in the zebrafish, where they play a role in biliary morphogenesis. Of note, GPC1 mediates fibroblast growth factor signaling, and ARF mediates epidermal growth factor signaling. XPNPEP1 , which is also found in the 10q24 region, is expressed as X-prolyl aminopeptidase P1 on biliary epithelial cells. XPNPEP1 is involved in metabolism of inflammatory mediators, but whether XPNPEP1 is a susceptibility locus remains uncertain.

Human leukocyte antigen (HLA) studies in BA might support a disease mechanism involving autoimmunity, but results to date are contradictory. One early study showed that infants with perinatal/acquired BA have a high prevalence of the HLA-B12 determinant compared both to normal controls and to infants with BA plus congenital anomalies ; haplotypes A9-B5 and A28-B35 were more common in infants with late-pattern (perinatal/acquired) BA. A subsequent study failed to confirm any characteristic HLA pattern in BA. However, additional studies have shown an association with HLA-DR2 and with HLA-B8 and HLA-DR3.

Further insight into the possible mechanism of perinatal/acquired BA has come from recent work in the Rhesus rotavirus (RRV) murine model of BA. It can be simulated in Balb/c-mice which have been infected with rotavirus. This model shares many features with the human disease. IFN-γ plays an important role in bile duct damage: knockout mice not expressing IFN-γ failed to incur severe duct damage after infection with RRV despite a brief hepatitis, whereas wild-type animals did; administration of recombinant IFN-γ abrogated the protective effect of not being able to produce IFN-γ. Certain chemokines may also contribute to biliary damage ; IL-12 seems to play a lesser role ; TNFα appears not to be involved. Recent observations implicate IL-17, which plays a role in various autoimmune disorders. In this mouse model, primed neonatal CD8 T cells appear capable of initiating damage to bile ducts. When T cells from RRV-disease mice were transferred into naive syngeneic severe combined immunodeficiency (SCID) mice, the recipients developed bile duct-specific inflammation without previous RRV infection. Some autoantibodies have been detected in this model (directed to α-enolase or vimentin). Thus the combination of observations in infants with BA and in this mouse model strongly suggests that a complex pattern of immune reactivity appears to be important in perinatal/acquire BA. Of interest, circulating markers of inflammation persist after surgical palliation with the Kasai portoenterostomy, although it is not clear why. In embryonic BA, other genes may play a more direct role.

Other theories have been proposed for the pathogenesis of BA. Landing first proposed that neonatal hepatitis, BA, infantile choledochal cyst and possibly some cases of ‘intrahepatic biliary atresia’ (meaning congenital duct paucity syndromes) are all manifestations of a single basic disease process that he named ‘infantile obstructive cholangiopathy’. He considered BA (and choledochal cyst) to be the result of an inflammatory rather than a maldevelopmental process and postulated that the most probable cause was a viral infection. Since the aetiopathogenesis of an important congenital bile duct paucity syndrome, namely, Alagille syndrome, has since been elucidated as genetic, this theory clearly requires modification. Nevertheless, as previously discussed, the role of viral infection, at least in the pathogenesis of perinatal/acquired BA, is an important focus of current research. Certain chromosomal abnormalities may give rise to complex syndromes, including altered immune reactivity, and thus predispose to hepatobiliary disease mediated by viral infection. Microchimerism has recently been proposed as part of the mechanism of hepatobiliary damage in BA ; it might initiate an immune response in the absence of viral infection. The various manifestations of infantile obstructive cholangiopathy may depend on the timing of the insult. Specifically, rats given the drug 1,4-phenylenediisothiocyanate during fetal life developed stenotic or atretic bile ducts caused by thickening and fibrosis, whereas those given the drug after birth had dilation of the ducts with inflammation. It is difficult to generalize from this rat model to human infants. BA with features of the ductal plate malformation (DPM) might reflect a different disease mechanism. Recent observations, however, have argued against the presence of DPM in BA as unique to patients with embryonic BA.

Whereas the pathogenesis of perinatal/acquired BA probably involves immunogenetic susceptibility and exposure to an instigating factor, such as viral infection, during a limited period of susceptibility, the aetiopathogenesis of embryonic BA appears to be much more diverse. An important subset has BASM (biliary atresia with structural malformations). Extrahepatic congenital abnormalities such as polysplenia, congenital heart defects and disturbed rotation of the intestines suggest an extensive and early developmental abnormality. Some infants with BASM show features of the ductal plate lesion on liver biopsy. This abnormal configuration of small bile ducts is attributed to disorganization in the fetal development of the biliary tree; failure of remodelling of the ductal plate leads to residual embryonic bile duct structures in this rather striking configuration. Finding the ductal plate lesion in extrahepatic BA is consistent with a destructive hepatobiliary process beginning early in gestation. Abnormal cilia have been reported in children with the polysplenia syndrome and BA. Although the association with abnormal cilia is unclear, ciliary function appears to be important in left/right asymmetry. There is a pathogenic role for multiple defects in the laterality sequence. Early studies focused on the inversion ( inv ) gene, one of three genes that control left/right asymmetry in the mouse. Beginning in early embryonic development, the liver is a predominant site for this gene expression. A transgenic mouse with recessive deletion of inv develops situs inversus and jaundice; the early fetal lesion is a complete obstruction with cystic change of the biliary tree. Few of the various genes which have been found mutated in human laterality disorders ( ZIC3 , CFC1 , LEFTYA , ACVR2B , NODAL ) have been investigated in BA; however, mutations in CFC1 and ZIC3 have been found in infants with BA and major laterality defects. Three children were described with ultrastructural abnormalities of the canalicular microvilli and no expression of villin; phenotypically they had BA without laterality complex or DPM.

The extrahepatic biliary tree in BA may be totally atretic, or the atresia may involve only proximal or distal segments. The intrahepatic bile ducts are gradually destroyed with progression of the disease. Most infants with BA have conjugated hyperbilirubinaemia from an early age, but clinical jaundice is not always apparent or appreciated. Indeed, in many infants, jaundice is initially physiological and merges with the jaundice of advancing liver disease. Infants typically have dark urine and pale stools, but the stools may retain enough colour to be falsely reassuring. The infants look well and generally gain weight adequately. At clinical presentation they have hepatomegaly and usually some degree of splenomegaly, unless polysplenia is present. The infant who presents with congenital heart disease and conjugated hyperbilirubinaemia requires intensive evaluation because the leading hepatic diagnoses will be BA or Alagille syndrome. Untreated BA rapidly progresses to hepatic fibrosis and cirrhosis with all the complications of portal hypertension, in addition to malnutrition and fat-soluble vitamin deficiency. The median age of death is 12 months if BA is not diagnosed and treated. Early diagnosis and treatment may be promoted by the use of stool colour cards for all infants at discharge from the newborn nursery.

Clinically, the differential diagnosis is the broad spectrum of disorders constituting the neonatal hepatitis syndrome presenting with conjugated hyperbilirubinemia (see Table 3.1 ). Congenital infection should be excluded, although CMV may be found along with BA. Systemic bacterial infection should be ruled out, including a silent urinary tract infection. Inherited metabolic diseases require specific attention, especially α1-antitrypsin deficiency, which can be associated with severe cholestasis and acholic stools and very rarely has been associated with BA. Cystic fibrosis can generate a duct lesion indistinguishable clinically from BA. These two conditions as well as galactosaemia may produce a histological picture closely resembling that of BA. Structural abnormalities of the extrahepatic biliary tree cause the clinical presentation similar to BA: choledocholithiasis, idiopathic perforation of the biliary tract, true choledochal cyst and extrahepatic biliary hypoplasia or ‘hair-like’ bile duct syndrome. Some infants with Alagille syndrome show ductular reaction, rather than duct paucity, on liver biopsy taken early in the course of the disease. Whether some infants with perinatal/acquired BA have an unusually slow progression of liver disease, sometimes called ‘BA in evolution’, is disputed, but these patients pose a diagnostic challenge and require repeated assessment.

Preoperative diagnosis relies on demonstrating the presence or absence of bile secretion in the intestine. Hepatic sonography may reveal a dilated extrahepatic biliary tree, consistent with distal cystic atresia, but it is unusual to find dilated intrahepatic bile ducts. Hepatobiliary scanning, using a technetium 99m-labelled iminodiacetic acid derivative such as DISIDA or PIPIDA, fails to demonstrate passage of the radiolabelled substance into the intestinal tract over a 24-hour period. This is the ‘nondraining hepatobiliary scan’. Although hepatobiliary scanning has high sensitivity, scanning may appear normal if performed very early in the disease process in late-pattern BA. Hepatobiliary scanning is informative if it shows that tracer, and thus bile, reaches the intestine; it is objective, recorded, and can be quantified. A negative or nondraining scan does not mean that the disorder is necessarily BA, because nondraining hepatobiliary scans may be found with severe idiopathic neonatal hepatitis, small duct paucity syndromes (e.g. Alagille syndrome), severe α1-antitrypsin deficiency or TPN-associated cholestasis. The role of endoscopic retrograde cholangiopancreatography (ERCP) remains controversial: ERCP is technically feasible in infants and may be useful in select cases. Percutaneous liver biopsy is essential and has high diagnostic specificity in the range of 60–95%, depending on the timing of the biopsy, adequacy of the specimen and expertise of the pathologist. In our experience, the majority of nondiagnostic biopsy specimens are taken within the first few weeks of life. In fact, incidental liver biopsy specimens taken at laparotomy for duodenal stricture within the first week of life in three infants with BA showed only trivial liver abnormalities. This may imply that although bile duct destruction is likely to have started in utero, the actual liver damage may not occur until the placenta no longer provides clearance of biliary products, in particular bile salts.

Pathological features at surgical intervention

Portoenterostomy (Kasai procedure) was introduced in 1959 and remains the only potentially corrective procedure for BA, other than liver transplantation. In this operation, the atretic biliary tree is resected, and bile drainage is re-established through a broad anastomosis at the end of an intestinal Roux-en- Y loop to the bare edge of the transected porta hepatis. The efficacy of the laparoscopic version of the Kasai portoenterostomy, introduced in the past 10 years, remains uncertain. Although meta-analysis has suggested that it is inferior to the traditional portoenterostomy, other reports suggest favourable results, partly dependent on local expertise. Extensive retrospective studies have shown that the prognosis for a good long-term result from the conventional Kasai procedure depends primarily on operation before 60 days of age and the absence of cholangitis. The potential exists, however, for reasonable medium-term survival in about one-third of infants coming to primary corrective surgery at 100 days or older. Most centres continue to favour the Kasai procedure as the first therapeutic option, rather than subjecting patients to immediate liver transplantation simply on the basis of age. The lack of significant fibrosis at operation may play a role in a good long-term outcome. Computerized quantification of fibrosis on liver biopsy obtained at portoenterostomy may discriminate between negligible fibrosis and sufficient fibrosis to portend a poor prognosis. One group suggested that histological features on the initial liver biopsy specimen can predict the success of portoenterostomy. Another group reported that the extent of ductular reaction, based on keratin 7 (K7) immunostaining, found in the liver biopsy specimen obtained at the Kasai procedure was predictive of native liver survival over the following year.

Histological studies of the extrahepatic bile ducts removed at surgery have been performed by several groups of investigators. In a study of 98 cases, Gautier and Eliot classified the biliary remnants into three types. In the first the duct is completely atretic, with few or no inflammatory cells in the surrounding connective tissue ( Fig. 3.5 A ). In the second type the duct is present as a cleft-like lumen lined by occasional cuboidal or low columnar epithelium which is variably necrotic, hyperplastic or focally absent ( Fig. 3.5 B and C ). The altered ducts are sometimes very numerous, usually having lumina of <50 µm; periluminal neutrophilic infiltration is characteristic; and cellular debris, and less often bile, may be found in the lumen. Epithelial necrosis is evident in ducts with a diameter exceeding 300 µm. The third type of biliary remnants consists of altered bile duct incompletely lined by columnar epithelium, in addition to numerous smaller epithelial structures ( Fig. 3.5 D). These histological types were evaluated by Gautier et al. at three levels: porta hepatis, junction of the cystic and common hepatic ducts and an intermediate level; completely atretic duct becomes increasingly more common from the porta hepatis to the junction of the hepatic duct and cystic ducts. This classification, which may help pathologists describe the changes observed in the biliary remnants removed at portoenterostomy, is of limited clinical significance because in individual cases, serial sectioning often shows atretic ducts alternating randomly with variably destroyed ducts. In addition, the numerous smaller structures intermingled with variably altered ducts are likely to represent anastomosing channels recruited from peribiliary glands, of which effectiveness in bypassing the atretic duct is uncertain; anastomoses between ramified peribiliary glands are well demonstrated in normal adult livers using injection techniques (see Chapter 9 ). Correlations between the size and number of residual ducts and establishment of bile flow after surgery have yielded conflicting results. Two groups of investigators believed that bile flow is most likely to occur when the diameter of the residual ducts exceeds 150 µm. However, another study of the extrahepatic biliary remnants of 204 cases of BA showed that the patterns of bile duct obliteration are not indicative of prognosis.

Figure 3.5

Biliary atresia. Transverse sections of biliary remnants removed at portoenterostomy. A, Atretic common hepatic duct showing luminal occlusion by vascular fibrous tissue with very few inflammatory cells. B, Distorted bile duct inconsistently lined by desquamated columnar epithelium and surrounded by fibroplasia with a light inflammatory cell infiltrate. C, Cleft-like lumen devoid of epithelial lining side by side with duct structures which may represent adjacent segments of the same ducts or hyperplastic peribiliary glands. D, Hilar region close to the surgical resection line showing numerous ducts or glandular structures set in a loose, mildly inflamed fibrous tissue. (H&E stain.)

Although the Kasai procedure is essentially a palliative operation, many children enjoy prolonged good health afterward, and approximately 20–25% of patients who undergo portoenterostomy will survive into adulthood without liver transplantation. Approximately 30–35% of patients drain bile but develop complications of cirrhosis and require liver transplantation before age 10 years. For the remaining patients, bile flow is inadequate after portoenterostomy and cirrhosis rapidly develops. Survival in BA with a functioning Kasai portoenterostomy but without orthotopic liver transplantation is 10–20% by age 20 years. In a recent survey of 271 patients, 23% were alive with their native liver 20 years after surgery, all but two having signs of cirrhosis; after age 20, two patients died of liver failure and 14 underwent, or were waiting for, a liver transplant. Women who are long-term survivors with BA and have not yet had a liver transplant may have a normal pregnancy, but in general such pregnancies must be treated as high risk because of complications from portal hypertension or hepatic decompensation. Liver transplantation has become the treatment of choice for infants and children in whom the Kasai portoenterostomy has failed. The safety and results of liver transplantation with the use of livers from living-related donors and cadaveric donors are excellent. One-year survival is >90%, with better results obtained under elective conditions and in children who weigh more than 10 kg. Recent data indicate that the overall survival at 10 years for all surgical treatment is 75–80% ; general health 10 years after paediatric liver transplantation is good, although renal impairment and cardiovascular disease may develop.

The macroscopic appearance of the liver in BA varies according to the stage of the disease. At first the liver is enlarged and dark green in colour, becoming finely nodular as cirrhosis develops ( Fig. 3.6 ). In untreated cases, the cirrhosis may take between 1 and 6 months from birth to develop. Dilated bile ducts filled with inspissated bile may be seen in sections of large portal areas ( Fig. 3.7 ). The cystically dilated bile ducts may resemble Caroli disease. They only occur after age 3 months and are not amenable to surgical drainage procedures. There may be portal lymphadenopathy. The median maximum node dimension in six cases studied by Hübscher and Harrison was 14 mm. These lymph nodes are brown in colour and full of pigment-laden macrophages. Livers removed at transplantation after an apparently successful Kasai procedure (loss of jaundice), but with subsequent development of cirrhosis and portal hypertension, are often coarsely nodular with areas of macronodular hypertrophy and broad intervening or peripherally located scars resembling the gross appearance of focal nodular hyperplasia ( Fig. 3.8 ).

Figure 3.6

Liver removed at transplantation 6 months after a failed Kasai procedure. Cirrhosis is characterized by small and dark-green parenchymal nodules.

Figure 3.7

Bisected liver from patient with biliary atresia. Multiple dilated bile ducts are filled with black bilirubin casts. Some of the ducts have thick fibrous walls. The hepatic parenchyma is cirrhotic with a periseptal distribution of the cholestasis ( right of the field).

Figure 3.8

Liver removed at transplantation 8 years after ‘successful’ Kasai procedure. Bisected specimen showing macronodular areas of re-expanded parenchyma ( centre of the field) with more fibrotic micronodular areas particularly at the periphery. Note the large fibrotic and seemingly stretched portal areas which contain well-identifiable bile duct branches ( yellow and light green ).

Pathology of intrahepatic changes

The histological features of BA include cholestasis, portal tract expansion by oedematous fibroplasia and periportal ductular reaction with the presence of bile plugs in dilated lumens of cholangioles that are distorted and often form an irregularly anastomosing network at the portal periphery ( Figs 3.9 and 3.10 ). Arterial branches are unusually prominent, and portal vein branches appear attenuated. Giant cell transformation of hepatocytes is seen in some cases ( Fig. 3.11 ) and may occasionally be prominent. Loose fibrosis is progressive and periportal/perilobular in location, with linkage of portal areas and eventual development of a secondary biliary cirrhosis ( Fig. 3.12 ). In a study of the extracellular and cellular components of the connective tissue matrix in BA, de Freitas et al. suggested that activation of a connective tissue cellular clone by the reactive ductules may be responsible for the portal fibroplasia. Ho et al. reported an arteriopathy (hyperplasia and hypertrophy) of the common hepatic artery and its peripheral branches supplying the entire biliary tree in 11 cases of biliary atresia. Thickening of the medial layer of small hepatic arteries may be present. Subsequent studies have indicated that the transcription factor HNF6 plays an important role in intrahepatic bile duct and arterial development. HNF6 and HNF1ß appear necessary for ductal plate formation. Large perihilar bile ducts may show ulceration with loss of epithelial lining, bile impregnation of the wall and bile sludge formation in the lumen. In addition to the severe cholestasis, the cirrhotic stage of BA is characterized by marked pseudoxanthomatous transformation, the presence of bile lakes, Mallory–Denk bodies and variable accumulation of copper and copper-associated protein in liver cells ( Figs 3.13 and 3.14 ). In one study, copper concentrations were increased in more than two-thirds of liver samples obtained during portoenterostomy and decreased in some patients after successful biliary drainage. However, copper deposition in liver is elevated in the first 2 months of life, and periportal deposition of copper on liver biopsy specimens does not discriminate extrahepatic from intrahepatic causes of cholestasis in early infancy. Acute and chronic inflammation is noted in portal/periportal areas in BA in both precirrhotic and cirrhotic stages, and bile duct degeneration and inflammation may be evident ( Fig. 3.15 ). The mononuclear infiltrate in portal and lobular areas of livers with end-stage BA is similar to normal adult liver and very different from that associated with autoimmune hepatitis or chronic HBV infection. It is particularly prominent in cases associated with CMV infection. The growth of large, perihilar regenerative nodules, probably as a consequence of functioning intrahepatic ducts in this region, may be important for maintaining biliary drainage after Kasai procedure. Bile lakes occur after age 3 months, by which time irreversible hepatic damage has occurred.

Figure 3.9

Biliary atresia. Liver biopsy performed at 3 weeks of age. Characteristic portal tract expansion by loose fibrous tissue containing irregularly anastomosing bile ductules at the periphery, some being dilated with inspissated bile in their lumen. (H&E stain.)

Figure 3.10

Biliary atresia. Cholangiolar reaction forming crescent-shaped profile reminiscent of the embryonal ductal plate. In our experience, this pattern may be observed irrespective of the patient having associated extrahepatic malformation. (H&E stain.)

Figure 3.11

Biliary atresia. Portal features of distal obstructive cholangiopathy are associated with giant cell transformation in the parenchyma. (H&E stain.).

Figure 3.12

Biliary atresia, cirrhotic transformation. Portoportal bridging septa made of oedematous fibrous tissue show a marginal ductular reaction and delineate small, irregularly shaped parenchymal nodules. (H&E stain.)

Figure 3.13

Biliary atresia. The same case as illustrated in Fig. 3.12 . Mallory–Denk bodies in ballooned periseptal hepatocytes. (H&E stain.)

Figure 3.14

Biliary atresia. Same case as illustrated in Figs 3.12 and 3.13 . Marked copper accumulation in liver cells (red granules). (Rhodanine stain.)

Figure 3.15

Biliary atresia. Inflamed and arteriole-rich, lemon-shaped fibrous tissue marks the site of a small, largely destroyed, intrahepatic bile duct. (H&E stain.)

Interlobular bile ducts become few in number as early as the fourth or fifth month after birth ( Fig. 3.16 ), and advanced duct loss accompanies progressive fibrosis by age 8 or 9 months. Activated hepatic stellate cells are responsible for increased collagen production. In addition to paucity of ducts, Raweily et al. identified concentric tubular ductal structures in 21.6% of cases of BA; these bore some resemblance to those seen in ductal plate malformations ( Fig. 3.17 ). Similar observations had been made earlier by Desmet and Callea, who hypothesized that this subgroup has more severe and rapidly progressive liver damage. Interestingly, children in this subgroup reveal a histopathological picture resembling that of congenital hepatic fibrosis 4 or 5 years after portoenterostomy ( Fig. 3.18 ; see also Fig. 3.17 ). The interlobular bile ducts continue to disappear, and cirrhosis can develop despite satisfactory bile drainage after portoenterostomy. The bile duct loss has been attributed to persistent interference with bile flow, recurrent cholangitis or continuation of the immune process causing the atresia. Detailed histopathological study of sequential liver specimens taken at Kasai operation, relaparotomy and/or transplantation has provided evidence that the bile duct loss is caused by an unpredictable and uneven obliteration of bile ducts in the porta hepatis during wound healing and scarring after portoenterostomy.

Figure 3.16

Biliary atresia. A portal area is bereft of bile ducts. There is periportal cholangiolar reaction with an associated neutrophilic response. (H&E stain.)

Figure 3.17

Biliary atresia. Liver resection 1 year after portoenterostomy. Pattern of fibrosis and reactive small bile ducts and ductules are reminiscent of congenital hepatic fibrosis. There were only minimal chronic cholestatic features in this liver. (Periodic acid-Schiff [PAS] stain after diastase digestion.)

Figure 3.18

Biliary atresia. Same liver as illustrated in Fig. 3.17 . Note absence of an interlobular bile duct and a pattern of ductular reaction reminiscent of a ductal plate malformation. (Masson trichrome stain.)

Ultrastructural degenerative changes affecting the intrahepatic bile ducts and ductules in BA have been described in detail by Ito et al., who found that the degree of obstruction of the lumen of these ducts appears to be an important determinant of prognosis following corrective surgery.

Malignant epithelial tumours of hepatobiliary origin rarely complicate biliary cirrhosis associated with biliary atresia, but both hepatocellular carcinoma and cholangiocarcinoma have been reported. Focal nodular hyperplasia after portoenterostomy has been reported in a few children with BA. Macroregenerative nodules may also develop.

Paucity of intrahepatic bile ducts

Paucity of the intrahepatic bile ducts has been reported in many conditions, either congenital or acquired, affecting all age groups, especially infants and children. The relevant finding is a reduction in the number of interlobular bile ducts, that is, in the small bile ducts within portal tracts. The normal ratio of small bile ducts per portal tract in full-term infants, children and adults is 0.9–1.8 ducts per tract. In duct paucity syndromes, this ratio is <0.5, given an adequate number of portal tracts (at least 10) examined on biopsy. Herman et al. have proposed the use of IHC for K7 and EMA to help calculate the ratio between interlobular bile ducts and neocholangioles, with a low ratio indicating bile duct paucity. Premature infants have a reduced number of small bile ducts per portal tract, and if they have duct paucity with cholestasis, it may be physiological. In addition, early biopsy specimens in a few cases of clinically undisputed paucity of the intrahepatic bile ducts have shown not only identifiable ducts, but significant ductular reaction as well.

Liver disorders with paucity of the intrahepatic bile ducts are generally divided into two groups: ‘syndromic’, which refers to Alagille syndrome, and ‘nonsyndromic’, which refers to all the rest of these diseases. The nonsyndromic duct paucity conditions include numerous diseases in which portal small duct paucity is associated with another identifiable disease. These include infection (congenital rubella or CMV infection), immune abnormality (graft-versus-host disease, liver allograft chronic rejection) and hepatotoxicity (from carbamazepine or amoxicillin–clavulanic acid), the latter two groups being generally referred to as ductopenia, formerly ‘vanishing bile duct syndromes’ (see Chapters 9 and 14 ). Various inherited metabolic diseases such as Zellweger syndrome, α1-antitrypsin deficiency and inborn errors of bile acid metabolism may display paucity of the intrahepatic ducts. Chromosomal defects, such as 45,XO Turner syndrome, trisomy 17–18, trisomy 21 and prune-belly syndrome, may have duct paucity. More importantly, the term nonsyndromic duct paucity may be used to refer to isolated, idiopathic paucity of interlobular bile ducts in infancy and childhood; this condition may be the same as idiopathic adulthood ductopenia. Finally, paucity of the intrahepatic ducts or ductopenia is frequently found as a late feature of certain chronic diseases, such as BA, primary sclerosing cholangitis, Langerhans cell histiocytosis and primary biliary cirrhosis (see Chapter 9 ).

Alagille syndrome (arteriohepatic dysplasia)

Patients in the syndromic group have Alagille syndrome (ALGS), also known as arteriohepatic dysplasia. Comprehensive descriptions have been reported by Alagille and colleagues and others. Two distinct genetic mechanisms are known to be responsible for ALGS. Most cases (ALGS1) are caused by mutations in JAGGED1 ( JAG1 , encoding the ligand for the Notch 1 receptor) on chromosome 20p12, and may be associated with a macroscopic deletion of the short arm of chromosome 20 in some patients or microdeletions of 20p in others. The pattern of genetic transmission is autosomal dominant due to haploinsufficiency or dominant negative effect ; gene penetrance is high, but expression is extremely variable. The reported incidence of arteriohepatic dysplasia as 1 in 70,000 live births was an underestimate, and it is actually 1 in 30,000. Approximately 50–70% of patients have new mutations, rather than genetic transmission within the family. Crosnier et al. found mutations of the JAG1 gene in 69 of 109 patients (63%) with ALGS, and transmission analysis showed a high frequency of sporadic cases (70%). Numerous mutations have now been defined.

The common clinical findings in Alagille syndrome include cholestatic liver disease caused by paucity of the intrahepatic bile ducts (94%); congenital heart disease, usually peripheral pulmonary stenosis, although complex congenital heart disease, usually right sided, may occur (92%); a typical facies (91%); posterior embryotoxon in the eye (80–93%) and butterfly-shaped vertebral arch deficits (40–67%). The facies of ALGS consists of an inverted triangle shape, slight hypertelorism, deep-set eyes, broad and rather prominent forehead, and beak-like nose. Although the specificity of the facies has been questioned, it is accepted as a typical finding, better appreciated in the actual clinical setting than in photographs. The facies is sometimes not evident in the first months of life, and in adults the facies is somewhat different from that described in children (longer face, rather coarse features, prominent forehead and comparatively small nose). Most patients have a systolic murmur related to stenosis of the pulmonary arterial system. More severe conditions include tetralogy of Fallot, pulmonary valve stenosis, aortic stenosis, ventricular septal defect, atrial septal defect, anomalous pulmonary venous return and complex problems involving a single right ventricle with pulmonary valve atresia. Up to 15% of patients may have life-threatening cardiac complications. In addition to posterior embryotoxon or Axenfeld anomaly, abnormal retinal pigmentation may be found.

Strabismus, ectopic pupil, and hypotrophic optic discs have also been reported. Optic disc drusen, which are calcified deposits in the extracellular space of the optic nerve head, often occur in Alagille syndrome. These can be found by ocular ultrasound examination and may facilitate the diagnosis. In addition to ‘butterfly vertebrae’ (vertebral sagittal cleft), other skeletal abnormalities include short distal phalanges and clinodactyly, and ALGS patients may be prone to bone fractures, beyond what might be attributed to chronic cholestatic liver disease.

Systems other than those in the cardinal criteria for the syndrome may be affected. Renal abnormalities may be prominent; renal dysplasia with or without cysts is most common. Renal tubular acidosis may be found, and vesicoureteral reflux is also a frequent finding. Renovascular hypertension may develop. Single horseshoe kidney has been reported. The most frequent histological finding is mesangiolipidosis, and other findings include tubulointerstitial nephropathy and membranous nephropathy. Nephrolithiasis may occur. Two children with ALGS and nephroblastoma were reported. Renal impairment in ALGS detected before liver transplantation does not improve spontaneously after liver transplant and mandates consideration of ‘renal-sparing’ immunosuppression. Systemic vascular disease appears to be more prevalent than originally appreciated. Several cases of moyamoya disease in association with ALGS have been reported. The propensity to intracranial bleeding found in the first few years of life in ALGS may be caused by abnormal intracranial vessels. Abnormalities in the large intra-abdominal vessels have been found, and these abnormalities may complicate liver transplantation. Skin changes relate mainly to formation of xanthomas, which regress after successful liver transplantation.

Abnormalities of the biliary tract include hypoplasia of the extrahepatic bile ducts, hypoplasia of the gallbladder and cholelithiasis. ERCP has demonstrated narrowing of the intrahepatic ducts, reduced arborization and focal areas of dilation, as well as narrowing of the extrahepatic ducts. Portal venous disease may also be present, specifically hypoplasia of the portal vein.

Many patients develop growth retardation before adolescence, especially if they have persisting clinical jaundice. Short stature is found in some affected children whose nutrition is normal. Although some display a degree of mental retardation, in general children with ALGS show a broad range of cognitive and intellectual ability. Almost all patients have pruritus, although it may be mild. They have elevated serum bile acids; cholic acid levels are greater than chenodeoxycholic acid levels. Variable hyperlipidaemia (sometimes severe, with xanthomas) is common. Treatments for the pruritus include cholestyramine, rifampicin or surgical diversion of bile flow.

In general, the prognosis is good for children whose jaundice resolves; however, approximately 25% of patients succumb in childhood to severe cardiac disease or progressive liver disease. The outcome in 92 patients in the series of Emerick et al. was as follows: the 20-year predicted life expectancy was 75% for all patients, 80% for those not requiring liver transplantation (LT) and 60% for those who required LT. LT has been reserved for patients with chronic liver failure, intolerable pruritus unresponsive to medical treatment and severe growth failure. LT for hepatic decompensation was necessary in 19 of 92 cases (21%), with 79% alive 1 year after transplantation; mortality was 17%. In the Bicêtre series of 163 patients followed over 40 years, 102 of 132 patients presenting with cholestatic jaundice in infancy remained jaundiced at the study endpoint, and one-third required LT; cirrhosis was found in some children with ALGS, but no neonatal hepatitis syndrome, and none underwent LT (two succumbing to end-stage liver disease before the advent of LT). Overall survival in this series was 62% at 20 years and seemed unaffected by availability of LT. Mortality is higher among patients who have more severe cardiac disease or intracranial bleeding, or who had previously undergone portoenterostomy. A retrospective study suggested that affected preschool-aged children with total serum bilirubin >111 µmol/L, serum conjugated bilirubin >77 µmol/L and serum cholesterol >13.5 mmol/L were likely to have a severe outcome, irrespective of actual JAG1 mutation. LT is an option for those in end-stage liver failure, or with severe chronic cholestasis or intractable pruritus; however, severity of cardiac and renal involvement, as well as intra-abdominal vascular abnormalities, must be evaluated as contributing to increased surgical risk.

Catch-up growth after LT is variable. Long-term complications of Alagille syndrome not requiring LT have included renal failure and intracranial bleeding or stroke. Hepatocellular carcinoma has been reported in children and adults with ALGS. Although rare, hepatic malignancy may occur in ALGS infants.

Histopathological aspects of Alagille syndrome are described in various reports and reviews. The major finding is absence of bile ducts from portal areas ( Figs 3.19 and 3.20 ). The ratio of interlobular bile ducts to the number of portal areas is between 0.0 and 0.4, compared with 0.9–1.8 in normal children. A reduced number of portal areas has been noted. The loss of bile ducts is progressive from early infancy to childhood. Cholangiodestructive lesions have been observed in infants between 3 and 6 months of age. The degree of cholestasis is variable in intensity and is especially prominent in the first 12 months of life. Immunohistochemical staining for endopeptidase (CD10) is typically absent from the canalicular surface in ALGS children. Endopeptidase, however, is not expressed physiologically until about 7 years of age, as well as during fibrosis progression in various liver disorders. Giant cell transformation of hepatocytes may be seen in early infancy. There is usually patchy pseudoxanthomatous change and accumulation of stainable copper and copper-associated protein in periportal hepatocytes. Copper accumulation has been demonstrated by quantitative methods in both syndromic and nonsyndromic types of paucity of the intrahepatic bile ducts. Periportal fibrosis is mild, and it may remain unchanged long-term, possibly due to the absence of ductular reaction, known to play a role in periportal fibrogenesis. Nonetheless, although progression to cirrhosis is rare, some patients do develop extensive fibrosis or cirrhosis ( Fig. 3.21 ). Portal inflammation and periportal ductular reaction, when present, are seen mainly in early infancy and can suggest the presence of distal duct obstruction.

Figure 3.19

Arteriohepatic dysplasia (Alagille syndrome). Two fused portal areas lack bile ducts. (H&E stain.)

Figure 3.20

The same case as illustrated in Fig. 3.19 . Portal area contains vessels but no ducts. There is patchy periportal fibrosis but no cholangiolar proliferation. (Masson trichrome stain.)

Figure 3.21

Arteriohepatic dysplasia. Micronodular cirrhosis. Note ductopenia, moderate septal inflammation and cholate stasis. (H&E stain.)

Ultrastructural studies of the intrahepatic bile ducts in Alagille syndrome have been reported. Bile canalicular changes are controversial. In one study of 12 biopsies from 10 patients, distinctive ultrastructural changes were noted. Bile pigment retention was found in the cytoplasm of liver cells, especially in lysosomes and in vesicles of the cis -Golgi, but only rarely in bile canaliculi or the immediate pericanalicular region. It was suggested that the basic defect in ALGS involves the bile secretory apparatus. The aetiopathogenesis of the cholangiodestructive lesions described histopathologically and ultrastructurally remains to be elucidated, but the possibility of ‘disuse atrophy’ has been raised by two groups of investigators. Recent studies suggest defective branching of intrahepatic bile ducts in the postnatal period.

Alagille syndrome is the first childhood disorder identified with a mutation in a ligand for a Notch protein. JAG1 encodes a ligand of Notch 1, one of a family of transmembrane proteins with epidermal growth factor (EGF)-like motifs. Notch proteins are highly conserved and have a role in determining cell fate during differentiation, especially in tissues where epithelial-mesenchymal interactions are important. Jagged/Notch interactions are known to be critical for determination of cell fates in early development. Notch 4 expression during embryogenesis is seen in endothelial cells of vessels forming the dorsal aorta, intersegmental vessels, cephalic vessels and the heart. The expression of Notch 1 and its ligand includes many of the organs potentially abnormal in ALGS. JAG1 plays an important role in embryogenesis of the heart, kidneys and blood vessels; in embryonic and fetal liver it is expressed in portal vascular tissue. Recent studies in mice indicate that Notch signalling regulates the remodelling of the ductal plate and bile duct morphogenesis, and that Jagged 1 plays an important role in this complex process. Jagged 1 on ductal plate and Notch 3 on portal tract mesenchyme and hepatic arterial endothelium interact for ductal plate remodelling and development of intrahepatic bile ducts. Mice with defects in murine Jagged 1 and Notch 2 expression have abnormalities similar to human ALGS; zebrafish with knockdowns of jagged ± notch genes have biliary, pancreatic, cardiac, renal and craniofacial developmental abnormalities; these studies suggest that Notch may promote biliary epithelial cell evolution from a bipotential precursor cells. In humans, mutations in JAG1 result in truncated and inactive proteins; since residual gene expression cannot compensate, there is haploinsufficiency. Dose of Notch ligands is critical and may contribute to the clinical diversity of ALGS. No clear relationship between genotype and phenotype has been found, but the Delta/Serrate/Lag-2 (DSL) domain in the JAG1 protein may influence the severity of liver disease.

A second genetic basis for Alagille syndrome (ALGS2) has been reported. It accounts for a very small proportion of cases. Children with mutations in the gene encoding Notch 2 (found on chromosome 1p13) have a clinical disorder similar to conventional Alagille syndrome, notably with intrahepatic duct paucity leading to cholestatic liver disease; however, the typical facies and skeletal abnormalities were much less common. Frequency of renal involvement is similar to that in ALGS1. Notch 2 appears to have an important role in bile duct development.

Nonsyndromic duct paucity disorders

In contrast to patients with Alagille syndrome, those with nonsyndromic bile duct paucity do not have a constellation of extrahepatic features. Children with nonsyndromic bile duct paucity are supposed to have a less favourable outlook than children with ALGS. They present with persistent cholestasis and severe pruritus. Growth retardation is common. No associated aetiological agent, defined genetic factors or congenital anomalies have been found in this group, except for one study of 10 patients with a high rate of consanguinity. A chronic cholestatic disease with duct paucity, called ‘idiopathic adulthood ductopenia,’ has been described in adults. Most of these patients are young adults, although patients over age 60 have been reported. Nonsyndromatic paucity of bile ducts in infancy and idiopathic adulthood ductopenia may be related diseases. The outlook for younger patients with idiopathic adulthood ductopenia is poor; approximately 50% succumb to progressive liver disease or require transplantation. Hepatocellular carcinoma has been reported in a woman with intrahepatic biliary hypoplasia, apparently distinct from ALGS.

A histopathological study of 17 children with nonsyndromic paucity of bile ducts was reported by Kahn et al. Before 90 days of age there was paucity of ducts and periportal fibrosis as well as nonspecific parenchymal changes (cholestasis, giant cell transformation, perisinusoidal fibrosis and haematopoiesis). After 90 days the duct paucity and fibrosis persisted but cholestasis was mild or no longer apparent. Kahn et al. suggested that the paucity in nonsyndromic cases may result from a primary ductal insult with destruction and disappearance of the ducts. The differential diagnosis of paucity of the intrahepatic bile ducts in children and adults has been reviewed by West and Chatila.

Bile duct anomalies, congenital dilations and ductal plate malformation disorders

Congenital dilations of the bile ducts are classified into five types, both extrahepatic and intrahepatic:

  • Type I: a dilation of the common bile duct which may present three anatomical variations: (a) large saccular; (b) small localized; (c) diffuse fusiform

  • Type II: diverticulum of the common bile duct or the gallbladder

  • Type III: choledochocoele

  • Type IV: multiple intrahepatic and extrahepatic dilations (Caroli disease)

  • Type V: fusiform intrahepatic and extrahepatic dilations

Types I and IV account for the majority of reported cases, although types IV and V may prevail in the Far East, where the disease occurs more frequently. Type IV cysts are more frequent in adults than in children. Although this classification remains in common use, its value has been questioned by Visser et al., who consider the distinction between types I and IV arbitrary; they suggest that the term ‘choledochal cyst’ should be reserved for congenital dilation of the extrahepatic and intrahepatic bile ducts, with other forms referred to by their name, e.g. choledochocoele and bile duct diverticulum. Caroli disease, assimilated to type IV in Hadad classification, is not clearly related to ‘choledochal cyst’, given its common association with both congenital hepatic fibrosis and fibrocystic lesions in the kidney and its distinct morphological features (see next). Hukkinen et al. have observed a considerable increase in the incidence of cholechal malformation in Finland.

Choledochal cyst

The classic clinical triad of pain, a mass in the right upper quadrant and jaundice occurs in less than a third of patients with a choledochal cyst. In children, jaundice is the most common presentation, while in adults the signs and symptoms are those of ascending cholangitis. In the early years of life, cholestasis is usually associated with cystic dilation of the common bile duct (CBD) and accounts for 2% of infants presenting with cholestasis. Up to 60% of choledochal cysts are diagnosed before age 10 years, but diagnosis can be made at any age, and some cases may present for the first time at as late as the eighth decade. Several cases have been diagnosed antenatally. About 80% of the patients are female. Differences in presentation between children and adults with choledochal cysts have been emphasized in two large series. The preoperative diagnosis can be made in the majority of patients by cholangiographic studies, ultrasonography (US) and isotope scanning. Dynamic magnetic resonance cholangiopancreatography (MRCP), including secretin stimulation, contributes to the understanding of the pathophysiology.

Complications include perforation, liver abscesses, stone formation, secondary biliary cirrhosis, pancreatitis, amyloidosis and carcinoma of the biliary tree. Regression of biliary cirrhosis following drainage of a choledochal cyst has been reported. One patient presented with anaemia secondary to bleeding from erosions of the duodenal mucosa between the ampullary sphincter and the sphincters of the CBD and pancreatic duct. Biliary tract anomalies reported in association with a choledochal cyst include double CBD, double gallbladder, absent gallbladder, annular pancreas, biliary atresia or stenosis; stenoses of the intrahepatic bile ducts and most frequently, anomalies of the pancreaticobiliary junction. In a series of 104 choledochal cysts from Japan, 25% of patients were found to have coexisting biliary anomalies. Differences between (1) isolated choledochal cysts and (2) choledochal cysts associated with biliary atresia have been noted by US. In the former the cysts are larger, intrahepatic ducts are dilated and the gallbladder is not atretic as compared to those with choledochal cysts and biliary atresia. In general, the apparent choledochal cyst associated with BA is actually proximal duct dilation associated with focal atresia of the distal CBD, so-called correctable or cystic atresia.

Maljunction of the pancreaticobiliary ductal system (common channel) remains the most plausible aetiopathogenic mechanism for choledochal cysts, which is supported by experimental studies. The lesion is defined as a junction of the pancreatic and bile ducts located outside the duodenal wall, usually forming an extremely long common channel. The anomaly allows pancreatobiliary regurgitation because hydrostatic pressure within the pancreatic duct is usually higher than that in the CBD. As a result, high pancreatic enzyme levels are found in the bile. Common channels may occur without bile duct dilation and lead to primarily gallbladder rather than biliary complications, including malignancy. In this situation, prophylactic cholecystectomy might be sufficient, whereas biliary complications and the risk of cholangiocarcinoma underpin the need for radical surgical resection in cases associated with choledochal cysts.

Reovirus-3 RNA sequences have been recovered from resected choledochal cysts, but the implication of reovirus infection in the aetiology of choledochal cyst is unclear. A single report of choledochal cysts in association with familial adenomatous polyposis raises the possibility of a genetic basis for the cysts.

Treatment is by complete cyst resection, cholecystectomy and Roux-en- Y hepaticojejunostomy as a preventive measure against the subsequent development of carcinoma. Laparoscopic repair has been successful in two recent series. Dilemmas may arise when the cysts involve the intrahepatic or intrapancreatic segments, requiring more extensive surgery, given the small risk of malignancy developing in cystic remnants at the anastomotic site or in the dilated intrahepatic bile duct of type IV or V cysts. In a report of 48 Japanese patients treated by total or subtotal excision, no malignant change occurred after a mean follow-up of 9.1 years. Similar excellent results were reported from Finland with median follow-up of 4.8 years (range, 1.3–13.2). Choledochal cysts vary greatly in size, with some larger lesions containing 5–10 litres of bile ( Figs 3.22 and 3.23 ). Histopathologically, the wall is usually thickened by inflammation and fibrosis and is bile stained. Smooth muscle fibres may be identified in the lower portion of the cyst but not in the narrow (intrapancreatic) portion. There is generally no epithelial lining, but islets of cylindrical or columnar epithelium may be preserved ( Fig. 3. 24 ). Intestinal metaplasia with mucous gland proliferation has been reported, as well as the presence of goblet and Paneth cells and neuroendocrine differentiation. According to Komi et al., the intestinal metaplasia increases with age and can be demonstrated in almost all cysts from patients over 15 years of age. Kusunoki et al. noted the absence of ganglion cells in the narrow portion of a choledochal cyst and suggested that the cyst could be the result of postganglionic neural dysfunction.

Figure 3.22

Choledochal cyst measuring about 10 cm in diameter. Note numerous vessels over the surface.

Figure 3.23

Same cyst as illustrated in Fig. 3.22 . Opened, collapsed cyst has a smooth inner lining. It did not contain bile.

Figure 3.24

Segment of wall of choledochal cyst shows inflammation and focal epithelial ulceration (H&E stain).

The majority of tumours arising in congenital cystic dilations of the bile ducts are adenocarcinomas, but some anaplastic and several squamous carcinomas have been reported, and one report mentioned sarcomatous changes. The overall incidence of carcinoma arising in all cystic dilations of the bile ducts is about 3%. The risk is age related, increasing from 0.7% in the first decade to 6.8% in the second decade to 14.3% in later decades. This finding has been confirmed in a recent series of 36 choledochal cysts describing a high rate of biliary intraepithelial neoplasia in approximately a third of cases and adenocarcinoma in about 15%. The complication is thus usually seen in adults; only three patients reviewed by Iwai et al. were under 18 years of age. An 11-year-old boy is the youngest reported to date. Reveille et al. found that stasis of bile in the choledochal cyst contributes to bacterial overgrowth and the generation of unconjugated secondary bile acids, a possible cause of biliary metaplasia and carcinoma. Interestingly, bile from congenital choledochal cyst patients is shown to promote the proliferation of human cholangiocarcinoma QBC939 cells. Certain bile acid fractions together with reflux of pancreatic enzymes may play a primary role, because pancreaticobiliary maljunction is associated with an increased risk of biliary tree malignancy irrespective of the presence of cysts.

Hereditary fibropolycystic disease (ductal plate malformation)

The term fibropolycystic diseases of the liver—not to be confused with cystic fibrosis—is used to describe a heterogeneous group of genetic disorders in which segmental dilations of the intrahepatic bile ducts and associated fibrosis can be interpreted as sequelae of persistence and aberrant remodelling of the embryonal ductal plate (see Chapter 1 ). They represent a spectrum of microscopic and macroscopic cystic lesions often associated with fibrocystic anomalies in the kidneys. The severity of the renal lesions may overshadow the liver disease, as in the early presentation of autosomal recessive polycystic kidney disease. Conversely, portal hypertension with a preserved liver function may dominate the picture later in life, as exemplified by congenital hepatic fibrosis. Cholangitis may develop, especially when the cysts communicate with the biliary system. These abnormalities are classified as ductal plate malformation , a term that refers to the histological changes of circumferentially disposed and variably ectatic bile ducts and ductules, often directly abutting the hepatocytic plates, which resemble an exuberant embryonal ductal plate. The main disorders, in particular autosomal recessive polycystic kidney disease (ARPKD), the closely associated congenital hepatic fibrosis and Caroli disease, and autosomal dominant polycystic kidney disease (ADPKD), are discussed in detail, whereas the rarer associated syndromes, which have been comprehensively reviewed by Knisely, are briefly mentioned. Over the past decade, genes and encoded proteins for several of these disorders have been identified ( Table 3.2 ). The recognition that the ‘cystoproteins’, the mutation of which causes polycystic disease, have been localized in the primary cilia or basal bodies of tubular epithelial cells has led to a renewed interest in these forgotten structures. The clinicopathological discussion of specific disorders is therefore preceded by a short account on cilia and cystogenesis; more details and comprehensive references can be found in reviews.

Table 3.2

Genes and encoded proteins in polycystic diseases of kidney and liver

Disorder Gene Product Localized in cilium
ARPKD PKHD Fibrocystin/polyductin Yes
ADPKD PKD1 Polycystin 1 Yes
PKD2 Polycystin 2 Yes
PCLD PRKCSH β-subunit of glucosidase II No
SEC63 Component of translocon in endoplasmic reticulum No

ARPKD, Autosomal recessive polycystic kidney disease; ADPKD, autosomal dominant polycystic kidney disease; PCLD, polycystic liver disease without kidney.

Primary disorders of cilia development or cystogenesis

Most studies on mechanisms of cyst development in hereditary polycystic disease have been conducted in the kidney, but there are strong suggestions that a number of pathways are common to the conversion of tubes into cysts in general, especially in the kidney tubules and hepatic bile ducts. Primary (or solitary) cilia arise from centrioles and form a finger-like extension of the cytoplasm covered with the cell membrane. In their axis, primary cilia contain a system of nine pairs of longitudinal microtubules arranged in a circle (the axoneme); in contrast, motile cilia (e.g. those in ciliated respiratory epithelium) contain a set of two centrally located microtubules within the circle of the nine pairs of longitudinal microtubules (‘9+2 pattern’). The pattern typical of, and defining, primary ciliary is the ‘9+0 pattern’. The microtubules secure the circulation of non-membrane bound macromolecules, intraflagellar transport, which is essential for the assembly and maintenance of cilia and also acts as a sensory process, in that the particles and associated peptides are changed in the cilium and carry a message back to the cell body. The solitary cilium is at present considered to represent a sensory antenna, functioning through the polycystin complex as a mechanotransducer. Loss of function of the complex results in perturbation of normal intracellular calcium ion (Ca 2+ ) concentration which underlies a multitude of pathological reactions. Severe alterations in structure and function of tubular or ductal cells will follow involving cell–cell contact, actin cytoskeleton organization, cell–extracellular matrix interactions, cell proliferation and apoptosis.

An enticing finding of recent years has been the demonstration that most proteins mutated in experimental polycystic liver disease (PCLD) have been localized in the primary cilia or basal bodies of tubular epithelial cells. They include those responsible for human disease, in particular polycystin 1 and polycystin 2 and fibrocystin/polyductin, but not the cystoproteins of isolated PCLD: hepatocystin and SEC63. Mutational analysis best studied in ADPKD suggests that both germline and somatic mutations may be involved in a two-hit model, as either a prerequisite initiating event or a later somatic event needed for cyst expansion and progression. Although the ciliary model is most attractive, it may represent only one of several parallel pathways that control essential cellular functions, such as proliferation, apoptosis, adhesion and differentiation.

Cyst epithelial lining cells show profound pathological alterations. An increased proliferation has been observed in ADPKD, comprising formation of papillary projections and overexpression of Ki-67 and PCNA (proliferating cell nuclear antigen), a loss of their ‘planar or tissue polarity’ (the ability to sense their position and orientation relative to the overall orientation of the epithelial sheet) and a switch from an absorptive to a secretory cell type, resulting in fluid secretion that is responsible (together with epithelial proliferation) for further cyst expansion. Although studies of cyst development have been mainly performed in kidney, primary cilia have been explored in cholangiocytes, and evidence showed that, at least to some extent, cystogenesis in bile ducts and ductules may be similar to that in the kidney. In ADPKD, PKD1 and PKD2 are co-localized in bile duct epithelial cells and the two-hit model of germ-line plus somatic mutations of PKD1 in focal liver cyst development appears applicable, as in the kidney cysts. As with kidney cyst, biliary cyst expansion occurs by secretion (as well as proliferation) of the cyst lining epithelial cells. Autocrine and paracrine factors, including IL-8, epithelial neutrophil attractant 78, IL-6 and vascular endothelial growth factor (VEGF), secreted into the cystic lumen modulate the rate of errant hepatic cyst growth. In ARPKD, findings suggestive of similarity with renal cystogenesis include the localization of PKHD1 in the cilia of cholangiocytes in the PCK rat, which is a model for Caroli syndrome, and that hepatocyte nuclear factor 1β (HNF1β) localized in bile duct epithelium regulates expression of PKHD1, together with other ‘cystoproteins’ that co-localize into the cilium.

Differences in cystogenetic pathways between liver and kidney in ARPKD are indicated in animal models of the disease. The mouse cpk mutation is the most extensively characterized murine model, closely resembling human ARPKD, with the exception that the B6- cpk/cpk homozygotes do not express the lesion of ductal plate malformation (DPM). However, homozygous mutants from outcrosses to some other strains express the DPM. Genetic analysis supports a loss-of-function model (two-hit model) for biliary cysts developing in an age-dependent fashion. There is no correlation between the severity of the DPM and the renal cystic disease. Expression of the biliary lesion is modulated by genetic background, and the specific biliary phenotype (DPM predominant or cyst predominant) is determined by whether loss of function of the cpk gene occurs as a germline or a somatic event.

In the mouse model generated by targeted mutation of Pkdh1 , the animals develop severe malformation of their intrahepatic bile ducts, but develop normal kidneys. The cholangiocytes maintain a proliferative state secreting transforming growth factor β1 (TGFβ1) that continuously stimulates mesenchymal cells to synthesize and put down extracellular matrix, resulting in fibrosis. The findings suggest that fibrocystin/polyductin, already expressed in the embryonic ductal plate stage, acts as a matrix sensor and signal receptor during intrahepatic bile duct development, and that its mutation results in a congenital hepatic fibrosis-like picture. Its role in liver and kidney appears to be functionally divergent, because protein domains essential for bile duct development do not affect nephrogenesis in this model.

Hepatic features of autosomal recessive polycystic kidney disease

Infantile presentation

This PKD is inherited in an autosomal recessive manner. The prevalence is estimated to be between 1:10,000 and 1:60,000 live births. The gene for this disorder, PKHD1 (polycystic kidney and hepatic disease 1), was mapped to chromosome 6p 21.2–p12 ; both the severe and the mild form of ARPKD mapped to the same locus. The gene encodes a 4074-amino acid protein, called fibrocystin or polyductin ; this large transmembrane polypeptide, also known to localize to the cilia, may be a receptor that acts in the differentiation of renal collecting duct and bile duct. An animal model is widely used. In humans, there is an equal sex incidence. Depending on the age at presentation and the degree of renal involvement, Blyth and Ockenden divided ARPKD into four types: perinatal, neonatal, infantile and juvenile. They proposed that four different mutant alleles are responsible, and that there may be a fifth group in which the onset of symptoms is later than juvenile. ARPKD has been reviewed by a number of authors.

The perinatal type is the most severe form; 50% of patients are diagnosed perinatally and die shortly after birth. In the series of Blyth and Ockenden, no infant survived beyond 6 weeks of age. The majority of patients were admitted with signs of respiratory distress and had marked abdominal distension due to huge symmetrical renal masses. Liver function test (LFT) abnormalities were uncommon. Surviving patients with the neonatal type of the disease develop gradually increasing renal insufficiency and systemic hypertension. Pyelonephritis is common. Portal fibrosis and cystic dilation of bile ducts are severe, and cholangitis is a frequent complication. In the infantile group the clinical picture is either of chronic renal failure or of increasing portal hypertension. Portal fibrosis is moderate. The juvenile group of Blyth and Ockenden typically includes children (1–5 years old) who present with portal hypertension. Liver histopathological changes are marked. It is likely that this group represents cases of congenital hepatic fibrosis, as suggested by Landing et al. Gang and Herrin have found that ARPKD has a spectrum of phenotype expression with prognostic implications, but suggest that not all cases fit into the sharply defined subgroups of Blyth and Ockenden. In their study of 11 patients, four had 90% or more renal cystic change; these patients did not survive beyond 20 days of birth. In contrast, five of the seven less severely affected patients with a 20–75% range of cystic changes in the kidneys were all alive at 6–21 years of age.

The liver in ARPKD does not appear abnormal macroscopically, although it may be enlarged and firm. About 70% of patients have enlargement of the left lobe on imaging.

Histologically, the changes range from a persistent, circular ductal plate ( Fig. 3.25 ) to a striking increase in the number of biliary channels which arise in portal areas and extend irregularly and deeply into the parenchyma ( Fig. 3.26 A ). They appear to branch or ‘anastomose’ and often show polypoid projections ( Fig. 3.26 B ). Normal interlobular ducts with corresponding arteries are not seen. According to Witzleben, the biliary channels are in continuity with the rest of the biliary system, similar to the cystic spaces of Caroli disease (‘communicating’ cystic disease), in contrast to the noncommunicating ADPKD. The supporting connective tissue is very scanty and, in the intralobular extensions, the basement membrane of the epithelium appears to be in direct contact with the liver cell plates. The epithelial lining consists of a single layer of low columnar to cuboidal cells. Cyst formation is uncommon. The dilated channels may contain a small quantity of a pink or orange-coloured material or rarely pus. Reconstruction studies by Adams et al. have shown irregularly dilated ducts running longitudinally at the periphery of the portal tract and anastomosing so extensively that they formed a single annular channel; no main interlobular duct could be identified in that portal tract or in several others from the same liver. A stereological study of 10 cases by Jörgensen showed ductal structures that could be divided into two groups: one consisted of irregular tubular structures shaped like circular cylinders, and the other of elliptical cylinders (the ‘ductal plates’). These were dilated but cysts were rare. In patients who survive for months or years, there is marked fibrosis in the liver as well as kidney lesions which appear to have been progressive lesions.

Figure 3.25

Autosomal recessive polycystic kidney disease. Unusually prominent ductal plate in the liver of a stillborn fetus with huge polycystic kidneys. (H&E stain.)

Figure 3.26

Autosomal recessive polycystic kidney disease. A, The bile ducts form an interrupted ring at the periphery of what should eventually become a portal area. There is no interlobular bile duct. Note branch of portal vein, as well as smaller vessels. B, The same case, with higher magnification of part of the ductal plate showing irregularity in outline of the ducts with polypoid projections into a dilated lumen. The lining epithelium is low cuboidal. ( A and B, H&E stain.)

Juvenile and adult presentation: congenital hepatic fibrosis

Congenital hepatic fibrosis (CHF) is considered a variant of ARPKD affecting predominantly children and adolescents, some cases being identical to the ‘juvenile’ form. The inheritance pattern is not a simple autosomal recessive one. PKHD1 missense mutation has been identified in CHF patients with minimal kidney involvement. It may be associated with dilation of the intra- or extrahepatic bile ducts, so-called Caroli syndrome (see next section), the intrahepatic cysts being detectable by US or magnetic resonance cholangiography (MRC). Infants present with abdominal distension from enlarged organs, respiratory distress and systemic hypertension. Older patients come to medical attention because of hepatosplenomegaly or bleeding from oesophageal varices, but asymptomatic cases have been reported. Cholangitis as a manifestation of CHF has been emphasized by Fauvert and Benhamou, who recognized four clinical forms: portal hypertensive, cholangitic, mixed portal hypertensive-cholangitic and latent. The pure cholangitic form is rare. In the mixed form, patients have recurrent bouts of cholangitis, with or without jaundice, in addition to the manifestations of portal hypertension.

In one series of 42 children with ARPKD from 20 sibships, 12 patients presented in the perinatal period, nine in the neonatal period, 13 in the infantile period, and eight in the juvenile period; the presentation and course of the disease was disparate in over 50% of patients. The organ predominantly affected may vary within the same family. Routine liver enzymes are usually normal, although alkaline phosphatase (ALP) levels may be increased.

When present, the usual renal disease in CHF is medullary tubular ectasia, a fusiform or cystic dilation of tubules (particularly the collecting ducts). Occasionally, patients have cystic kidneys typical of adult-type polycystic disease (ADPKD), an autosomal dominant disease, and others may have nephronophthisis.

The prognosis in patients surviving beyond the neonatal period is generally good and depends on the extent of hepatic and renal disease. Death related to renal failure or uncontrollable variceal bleed is now the exception. The combination of a patent portal vein and well-preserved liver function makes patients with CHF ideal candidates for standard portosystemic shunt surgery. Follow-up examination of 16 patients who underwent portosystemic shunt surgery revealed no impairment of liver function or hepatic encephalopathy. Development of cholangiocarcinoma may have been a chance occurrence in an adult case not associated with Caroli disease.

Pathological descriptions of CHF have been published by many authors. Grossly, the liver is enlarged, has a firm to hard consistency, and shows a fine reticular pattern of fibrosis; no cysts are visible to the naked eye. Although the entire liver is usually involved, occasional lobar cases of CHF are described.

Microscopically, there is diffuse periportal fibrosis, the bands of fibrous tissue varying in thickness. Irregularly shaped islands of hepatic tissue, some incorporating several lobules, may be seen ( Fig. 3.27 ). When the bands of fibrous tissue are thick, hepatic venules may be encroached on and become incorporated within the fibrous tissue; thus portal hypertension in this condition may not always be presinusoidal. The fibrous bands may encircle single or groups of lobules; occasionally, a small islet of hepatic tissue becomes separated from an acinus and encircled by fibrous tissue. Numerous uniform and generally small bile ducts are scattered in the fibrous tissue ( Fig. 3.28 ). An interrupted circular arrangement of the ducts (DPM) is often recognizable ( Fig. 3.29 ). The ducts are lined by cuboidal to low columnar epithelium and may contain bile or traces of mucin. They may be slightly dilated and irregular in outline. Cholestasis is not a feature of the uncomplicated case of CHF. Reduction in the number of portal vein branches is often apparent, the likely cause of the portal hypertension. There is generally minimal inflammation in CHF except in cases associated with cholangitis, when numerous neutrophils infiltrate ducts, ductules and surrounding connective tissue ( Fig. 3.30 ); rupture of the ducts can result in microabscess formation. Histopathologically, the latter cases may be difficult to differentiate from extrahepatic biliary obstruction with ascending infection, particularly since there may be an associated tissue cholestasis. The correct diagnosis must be based on the history, clinical findings and the results of imaging studies. Cholestasis is particularly prominent in those cases associated with microscopic dilation of the intrahepatic bile ducts, too subtle to be seen on imaging (‘microscopical Caroli’). Recurrent cholangitis may lead to progressive fibrosis with functional impairment similar to cirrhosis.

Figure 3.27

Congenital hepatic fibrosis. Jigsaw pattern of hepatic parenchyma and fibrous tissue. (PAS stain.)

Figure 3.28

Congenital hepatic fibrosis. Fibrous tissue contains small bile ducts, a few of which are dilated and contain bile. Note absence of inflammation. The adjacent hepatic parenchyma shows no evidence of cholestasis, and the patient is jaundice free. (H&E stain.)

Figure 3.29

Congenital hepatic fibrosis. The same case as illustrated in Fig. 3.28 . Appearance of ducts suggests part of a ductal plate malformation. (H&E stain.)

Figure 3.30

Congenital hepatic fibrosis, ‘cholangitic type’. The ducts are involved by an acute cholangitis. (H&E stain.)

A number of malformation syndromes, characterized by hepatic morphological changes that resemble those of CHF (DPM), can be differentiated by the associated findings. Some of these are recognized as being ‘ciliopathies’ in that the defective protein is part of the ciliary apparatus. They include:

  • Medullary cystic kidney disease 1 (MCKD1), which has an autosomal dominant inheritance, has been mapped to chromosome 1q21.

  • Medullary cystic kidney disease 2, via uromodulin (UMOD2), also autosomal dominant, mapped to chromosome 16p12.

  • Nephronophthisis congenital hepatic fibrosis, a recessive disorder whose gene NPHP1 has been mapped to chromosome 16p12.

  • Meckel syndrome, with encephalocoele, polydactyly and cystic kidneys.

  • Renal-hepatic-pancreatic dysplasia, with polycystic kidney disease, DPM in liver and pancreatic dysplasia, incompatible with postnatal survival; candidate genetic locus on chromosome 3.

  • Asplenia with cystic liver, kidney and pancreas , which represents Ivemark syndrome with variants, possibly in the same spectrum as renal-hepatic-pancreatic dysplasia.

  • Ellis–van Creveld syndrome or chondroectodermal dysplasia, with polydactyly, short limbs, short ribs, postaxial polydactyly and dysplastic nails and feet; mapped to chromosome 4p16.

  • Sensenbrenner syndrome ( cranioectodermal dysplasia), with dwarfism and progressive tubulointerstitial nephritis.

  • Asphyxiating thoracic dystrophy (Jeune syndrome), with skeletal dysplasia, pulmonary hypoplasia and retinal lesions.

  • Congenital disorder of glycosylation, type Ib (phosphomannose isomerase deficiency), sometimes with protein-losing enteropathy.

  • Joubert syndrome, with cerebellar vermis hypoplasia/aplasia, colobomata and psychomotor retardation ; recently defined by the acronym COACH syndrome ( c erebellar vermis hypoplasia, o ligophrenia, a taxia, c oloboma, and h epatic fibrosis), shown to carry mutation in a Meckel syndrome gene ( MKS3 ).

  • Vaginal atresia syndrome

  • Tuberous sclerosis

Caroli disease

Caroli disease generally involves the entire liver, but it may be segmental or lobar. The inheritance is autosomal recessive. By 1982, some 99 cases had been reported; they were evaluated together with 10 personally studied cases by Mercadier et al. Since then, many other case reports, as well as small series, have been reported. Clinically, patients suffer from bouts of recurrent fever and pain. Jaundice occurs only when sludge or stones block the common bile duct. Liver enzymes are generally normal except during episodes of biliary obstruction. The diagnosis is established by a variety of imaging modalities (e.g. ERCP, US, computed tomography (CT) and MRCP).

The complications of Caroli disease resemble those of choledochal cyst and include recurrent cholangitis, abscess formation, septicaemia, intrahepatic lithiasis and amyloidosis. Spontaneous rupture of a bile duct was reported by Chalasani et al. Adenocarcinomas, including some arising in cases with a lobar distribution, have also been reported, with an incidence of 7%. Hepatocellular carcinoma occurs rarely. Medical treatment consists of symptomatic or prophylactic treatment of cholangitis and promotion of bile flow with ursodiol. Surgical treatment includes internal or external drainage procedures. Transhepatic decompression has been advocated. Segmental or lobar forms of Caroli disease can be treated by partial hepatectomy. Extracorporeal shock wave lithotripsy has been used for disintegration of intrahepatic stones. Liver or combined liver and kidney transplantation has been successfully performed in patients with mono- and multilobar disease with portal hypertension, with 96 and 8 receiving liver or combined liver and kidney grafts, respectively, from 1987 to 2006 in the United States ; the authors propose an algorithm for evaluation and treatment of Caroli disease.

Macroscopically, the intrahepatic cystic dilations are round or lanceolate, 1.0–4.5 cm in diameter, and may be separated by stretches of essentially normal duct ( Fig. 3.31 ). Transluminal fibrovascular bridges are reminiscent of the periportal location of the cysts (ductal plate) and explain in part the central dot sign observed on CT. Inspissated bile or soft and friable bilirubin calculi may be found in the lumen. Microscopically, the dilated ducts usually show severe chronic inflammation, with or without superimposed acute inflammation, and varying degrees of fibrosis ( Fig. 3.32 ). The epithelium may appear normal (cuboidal to tall columnar), partly or completely ulcerated, or focally hyperplastic; all these changes can be found in different ducts in the same liver ( Fig. 3.33 ). Mucous glands (sometimes in abundance) may be present in the fibrotic and inflamed wall. Areas of severe epithelial dysplasia are seen rarely. The lumen contains admixtures of inspissated mucin and bile, calcareous material or frank pus during bouts of acute cholangitis ( Fig. 3.34 ). Caroli disease is frequently associated with congenital hepatic fibrosis (in which case it is termed Caroli syndrome ), rarely with infantile polycystic disease (ARPKD) and even adult polycystic disease (ADPKD).

Figure 3.31

Caroli disease. Section of liver shows cystically dilated bile ducts. The cystic cavities are traversed by fibrous cords, known to contain the portal vessels. The lining of the ducts is bile stained.

Figure 3.32

Caroli disease. Dilated bile ducts have thickened walls due to marked chronic inflammation. Note lymphoid follicles in the wall of one duct (left). (H&E stain.)

Figure 3.33

Caroli disease. The thickened bile duct wall reveals marked chronic inflammation. The lining epithelium is hyperplastic, except for a small segment of the duct (right). There is an area of ulceration (bottom). (H&E stain.)

Figure 3.34

Caroli disease. Markedly dilated duct contains golden-yellow inspissated bile. It shows marked inflammation, as well as ulceration (bottom). (H&E stain.)

According to Desmet, the pathogenesis of Caroli disease seems to involve total or partial arrest of remodelling of the ductal plate of the larger intrahepatic bile ducts. In Caroli syndrome the hereditary factor causing the arrest of remodelling seems to exert its influence not only during the early period of bile duct embryogenesis, but also later on during development of the more peripheral biliary ramifications (the interlobular bile ducts).

There is a single case described of ‘Marfan syndrome with diffuse ectasia of the biliary tree’. The authors suggested that the defect of connective tissue in that disease could have led to weakness of the wall of the bile ducts with resultant ectasia.

Hepatic features of autosomal dominant polycystic kidney disease

Mutations mainly in two different genes, PKD1 and PKD2, can lead to ADPKD, one of the most common genetic disorders worldwide. The PKD1 gene lies on the short arm of chromosome 16 (16p 13.3), immediately adjacent to the TSC2 , a gene responsible for approximately 50% of tuberous sclerosis. The incidence of ADPKD due to mutations in PKD1 is 1 in 1000 live births, comprising 90% of cases. The PKD1 gene encodes a protein called ‘polycystin’. It is present in plasma membranes of renal tubular cells, bile ductules, pancreatic ducts, hepatocytes and cells of large bile ducts. In the kidney, localization at the contact points (only between neighbouring cells) indicates that its main function is cell-to-cell interaction, such that loss of function contributes to cyst formation. The PKD2 gene is located on chromosome 4 (4q 21–23) and encodes a 100-kilodalton (kD) protein, polycystin 2, with sequence homology to α-subunits of voltage-activated calcium channels. As mentioned earlier, localization of polycystin 1 and polycystin 2 to primary cilia in cultured renal epithelial cells and demonstration that the proteins function as a ciliary flow-sensitive mechanosensors implicate defects in ciliary structure and function as an important mechanism of cyst formation. There is at least one unmapped focus, which accounts for 5% of the disease population. Although PKD2 is clinically milder than PKD1, it does have a deleterious impact on overall life expectancy and cannot be regarded as a benign disorder. The mean age to death or end-stage renal disease is 53 years in PKD1-associated disease, 69 years in PKD2-associated disease and 78 years in controls. PKD2 patients are less likely to have systemic hypertension, urinary tract infections and haematuria. Cerebral aneurysms are found in 10–15% of patients with PKD1 and represent a significant risk of subarachnoidal haemorrhage causing death and disability. Familial clustering of bleeding from intracranial aneurysms is recorded. In an earlier mutational analysis of 58 ADPKD families with vascular complications, 51 were PKD1 (88%) and seven PKD2 (12%). The authors found that the position of the mutation in PKD1 is predictive for development of intracranial aneurysms (59 mutations are more commonly associated with vascular disease) and is therefore of prognostic importance.

ADPKD is a multisystem disease with cysts and connective tissue abnormalities involving multiple organs. Associated conditions include colonic diverticula (70%), cardiac valve complications (25%), ovarian cysts (40%), inguinal hernia (15%) and intracranial aneurysms (10%), suggesting a diffusely abnormal matrix. Mitral valve prolapse was found in 12% of affected children. ADPKD is the cause of end-stage renal disease in 8–10% of adults; the number of cysts is age related. The renal disease can be present at birth, but hepatic manifestations are rare before 16 years of age.

The typical hepatic disorder in ADPKD is polycystic liver disease. The average age at first admission for liver-related problems was 52.8 years, with an average duration of symptoms of 3 years. The symptoms included a gradually enlarging abdominal mass, upper abdominal pain or discomfort and rare episodes of severe pain with or without nausea, vomiting and occasionally fever. The most frequent physical finding is hepatomegaly, which can be massive. Liver tests are often normal. Jaundice is unusual. Portal hypertension is rare but may occur from hepatic outflow obstruction. There is an increased risk of gallstones in patients with hepatic cysts. Rarely is treatment needed by excision or a combination of excision and fenestration. Additionally, it has become evident that CHF may occur with ADPKD; it becomes clinically evident in childhood or adolescence and may lead to portal hypertension. Liver or combined liver and renal transplantation has been performed successfully in patients with ADPKD.

The incidence of liver involvement and its complications has been reviewed in a large series of 132 patients receiving and 120 patients not receiving haemodialysis. Liver cysts were found by noninvasive imaging procedures in 85 of 124 patients on dialysis; gender distribution was equal. In contrast, the nondialysed population demonstrated a 75% incidence of liver cysts in females and 44% incidence in males, the peak incidence occurring 10 years earlier in females. The cysts were larger and greater in number in the nondialysed females, and there was a correlation with the number of pregnancies. Nineteen autopsies were reported in which five deaths were liver related. Risk factors for the development of hepatic cysts in ADPKD were also examined in 39 patients and 189 unaffected family members by Gabow. The hepatic expression of the disease was found to be modulated by age, female gender, pregnancy and severity of the renal lesion and functional impairment. Oestrogen treatment of postmenopausal women with ADPKD is associated with selective liver enlargement and abdominal symptoms.

Although the leading complication in ADPKD is infection of the liver cysts, cholangiocarcinoma is the second most common complication. A study examining hepatic cyst infection suggested that the incidence increases from 1% to 3% during end-stage renal failure. Enterobacteriaceae were cultured from the infected cysts in 9 of 12 patients. In the case of Ikei et al., Pseudomonas aeruginosa was cultured from the infected cysts. Positron emission tomography (PET) scan will probably make the diagnosis of cyst infections easier and more accurate. This may allow early treatment with appropriately selected antibiotics, often including a fluoroquinolone; the timely drainage of large infected cysts remains an option.

Hepatic cysts are rarely detected before puberty and increase with age (ultimately to 75%) in individuals over 70. However, cysts have been found in early childhood and even in the first year of life. Before the availability of a genetic probe, the criteria for identification of this disorder in children included a positive paternal history, cysts in any portion of the renal tubule or Bowman space, macroscopic cysts in the liver and cerebral aneurysms. Molecular diagnosis is useful in individuals in whom the diagnosis of ADPKD is uncertain due to lack of family history or equivocal imaging results and in younger at-risk individuals who are being evaluated as living-related kidney donors.

Grossly, the liver in ADPKD is enlarged and diffusely cystic, the cysts varying from <1 mm to 12 cm or more in diameter ( Fig. 3.35 ). One liver reported by Kwok and Lewin weighed 7.7 kg. Occasionally, only one lobe, usually the left, is affected. Diffuse dilation of the intra- and extrahepatic bile ducts has been reported in some cases. The cysts contain a clear, colourless or yellow fluid. Analysis of cyst fluid in one case disclosed similarities to the ‘bile salt-independent’ fraction of human bile, suggesting that such cysts are lined by a functioning secretory bile duct epithelium.

Figure 3.35

Autosomal dominant polycystic kidney disease. Numerous cysts of varied size are studded throughout the liver. They contained clear fluid.

Microscopically, the cysts are lined by columnar or cuboidal epithelium, but the larger cysts have a flat epithelium ( Fig. 3.36 ). Collapsed cysts resemble corpora atretica of the ovary ( Fig. 3.37 ). The supporting connective tissue is scanty except in relation to von Meyenburg complexes (see Fig. 3.36 ), a frequently associated lesion, where it may be dense and hyalinized. A small number of inflammatory cells, usually lymphocytes, may infiltrate the supporting stroma. Infected cysts contain pus and may rupture ( Fig. 3.38 ). Calcification of the wall of hepatic (and renal) cysts in ADPKD has been reported.

Figure 3.36

Autosomal dominant polycystic kidney disease. Section of liver shows multiple cysts of varied size that are lined by a flattened epithelium adjacent to a von Meyenburg complex. (H&E stain.)

Figure 3.37

Autosomal dominant polycystic kidney disease. Collapsed cyst has a corrugated wall, and the lumen is filled in with a loose connective tissue. (Masson trichrome stain.)

Figure 3.38

Autosomal dominant polycystic kidney disease. Infected cysts are filled with pus. The cyst to the right has ruptured with formation of a small cholangitic abscess. (H&E stain.)

Von Meyenburg complexes are considered part of the spectrum of adult polycystic disease, and Melnick believed that polycystic disease of the liver develops progressively over years by gradual cystic dilation of these complexes. This view is supported by a histomorphometric and clinicopathological study of 28 cases of ADPKD reported by Ramos et al. Kida et al. have suggested that cystic dilation of peribiliary glands also may lead to formation of the cysts in ADPKD. Von Meyenburg complexes are small (<0.5 cm in diameter), greyish white or green, and are usually scattered in both lobes. They have an abnormal vascular pattern in angiographic studies and are occasionally associated with cavernous haemangiomas. Microscopically, the lesions are discrete, round to irregular in shape and typically periportal in location. The constituent ducts or ductules are embedded in a collagenous stroma, are round or irregular in shape and have a slightly dilated lumen ( Fig. 3.39 ) (see Chapter 13 ). They are lined by low columnar or cuboidal epithelium and contain pink amorphous material that may be bile stained or actual bile ( Fig. 3.40 ). Congenital hepatic fibrosis has been found in some cases. Cholangiocarcinomas have been reported in association with von Meyenburg complexes, as well as with multiple hepatic cysts considered part of the spectrum of ADPKD. Intraductal papillary-mucinous neoplasm of the pancreas has been reported.

Figure 3.39

Von Meyenburg complex. Small bile ducts are embedded in a fibrous stroma. Note irregular shape of two of the ducts, each of which contains a polypoid projection. (H&E stain.)

Figure 3.40

Von Meyenburg complex. The ducts are variably dilated and contain altered bile. (H&E stain.)

Polycystic liver disease without kidney abnormalities

Isolated polycystic liver disease (PCLD), not linked to PKD1 or PKD2 , has been reported. Germline mutations in protein kinase C substrate 80K-H gene ( PRKCSH ), a known gene encoding for a previously described human protein, ‘noncatalytic β-subunit of glucosidase II’ (GIIβ), have been associated with ADPKD. This protein, now renamed hepatocystin, seems to segregate in families with PCLD. The protein is widely distributed in tissues and predicted to be an endoplasmic reticulum luminal protein that recycles from the Golgi complex. The mutations found in PCLD are all predicted to cause premature chain termination, rendering loss-of-function changes most likely. The two-hit hypothesis for cyst formation in ADPKD appears applicable to PCLD. Glucosidase II plays a major role in regulation of proper folding and maturation of glycoproteins. Polycystin 1, polycystin 2 and fibrocystin/polyductin are all glycoproteins. Mutations could compromise the processing of the N-linked oligosaccharide chains of the newly synthesized glycoproteins. Improper association and trafficking of the polycystins due to defective glycosylation by mutant GIIβ may link PCLD to the ADPKD pathway. This would be consistent with the marked similarity in clinical liver disease in both conditions. The second gene involved in PCLD, SEC63 , encodes a protein that is part of the multicomponent translocon of the endoplasmic reticulum (Sec63p) which is required in both the post-translational and the co-translational (or signal recognition particle-dependent) targeting pathway and may explain a functional link between the two genes known to be associated with PCLD. Respective lack of expression of hepatocystin and Sep63p in PCLD cyst lining appears to correlate with mutational results. Immunohistochemical analysis of cyst lining suggests that the disease involves overexpression of growth factor receptors and loss of adhesion, but proliferation or deregulated apoptosis do not seem to be implicated in PLCD. Differential findings for PRKCSH – and SEC63 -PCLD suggest a divergent mechanism for cystogenesis in the two groups. A recent study has identified mutations in the low-density lipoprotein receptor-related protein 5 ( LRP5 ) gene in patients with polycystic liver disease.

Solitary (nonparasitic) bile duct cyst

Solitary bile duct cyst is defined as a unilocular cyst lined by a single layer of columnar or low cuboidal epithelium resting on a basement membrane and a layer of fibrous tissue. The cysts occur at all ages, although the majority present in the fourth to sixth decade. They are rare in the paediatric age group; in the Boston Children’s Hospital series, 31 solitary nonparasitic cysts (26 unilocular, 5 multilocular) were diagnosed in 63 years. The female/male ratio is 4:1. Cysts smaller than 8–10 cm rarely cause symptoms. When present, symptoms include fullness or an upper abdominal mass, nausea and occasional vomiting. Rapid enlargement has been reported in infancy. Jaundice occurs infrequently. An acute abdominal crisis may result from torsion, strangulation, haemorrhage into the cyst or rupture. Diagnosis is usually established by US, CT or other imaging modalities. Solitary bile duct cysts involve the right lobe twice as often as the left. Rarely, they can arise in the falciform ligament. They are usually round and rarely are pedunculated; the lining is typically smooth ( Fig. 3.41 ). The larger ones may contain one to several litres of fluid which is usually clear, but may be mucoid, purulent (if the cyst is infected), haemorrhagic or rarely bile stained.

Figure 3.41

Solitary bile duct cyst. The sectioned cyst has a smooth lining.

Microscopically, the cyst lining usually consists of a single layer of columnar, cuboidal or flat epithelium ( Fig. 3.42 ). The epithelium rests on a basement membrane that in turn is supported by a layer of fibrous tissue. Adenocarcinomas may arise in the cyst. Other malignancies reported to arise in solitary cysts include squamous cell carcinoma, carcinosarcoma and carcinoid tumour.

Figure 3.42

Solitary bile duct cyst. The cyst is lined by a single layer of columnar cells. (H&E stain.)

The pathogenesis of solitary bile duct cyst is unknown. A congenital origin is supported by the occurrence of the cysts in fetuses and newborns, by a case presenting as a congenital diaphragmatic hernia and the association of another case with the Peutz–Jeghers syndrome.

In the past, the treatment of choice of solitary cysts was excision. This has been supplanted by aspiration and sclerotherapy, however, or laparoscopic fenestration.

Cystic fibrosis

Cystic fibrosis (CF), the most common autosomal recessive disorder, with an estimated incidence of approximately 1 in 2500 live births among Caucasians, has a worldwide distribution. CF was first recognized as a pancreatic disease distinct from coeliac disease in 1938. Originally known as ‘cystic fibrosis of the pancreas’ (mucoviscidosis), the disease was soon labelled simply ‘cystic fibrosis’, with the recognition that not only the exocrine pancreas, but also the bronchial glands (obstructive bronchopulmonary disease), the intestinal glands (meconium ileus), the sweat glands (high sweat electrolytes) and the biliary tree (focal biliary cirrhosis) may be affected by the disease.

The gene was localized at chromosome 7q31.2 in 1989. It encodes a large protein named cystic fibrosis transmembrane regulator (CFTR), whose key role is in maintaining the fluid balance across epithelial cells. CFTR belongs to the ABC family of adenosine triphosphate (ATP)-dependent channels and transporters. It functions mainly as a cyclic adenosine monophosphate (cAMP)-dependent chloride channel in the apical membrane of secretory epithelial cells of many tissues, where it promotes transmembrane efflux of chloride ions. The protein is abundantly present in the branching ducts of the pancreas, intestinal epithelium and testes, and to a lesser degree in respiratory tissue. CFTR is expressed at the apical membranes of the epithelium of branching hepatic bile ducts and the gallbladder, but not on cells of the common hepatic bile duct or hepatocytes. The CF secretory defect results in inability to maintain the luminal hydration of ducts, leading to physicochemical abnormalities of secretions and duct obstruction. More than 1000 different disease-causing mutations have been identified. They are distinguished in terms of severity on the basis of the residual activity of the codified protein. The most common mutation is ΔF508, a three-base pair (bp) deletion in CFTR , resulting in loss of phenylalanine from the protein with subsequent retention in the endoplasmic reticulum.

Since liver disease in CF is often subclinical, its prevalence is uncertain and to some extent depends on definition, type of screening and age. As overall life expectancy has improved, liver disease has become the third most frequent cause of death in CF patients. 527,528 The availability of new therapies targeting the function of CFTR that could prevent or reverse CF liver disease constitutes an additional rationale for identifying CF patients at risk of developing liver impairment. Based on prospective studies, significant clinical liver disease is seen in 4–6% of CF patients, but biochemical evidence of liver involvement without clinical symptoms occurs in 20–50%. Autopsy series in the 1950s found an incidence of multifocal biliary fibrosis (or ‘cirrhosis’) of 22–25%. The liver disease usually becomes apparent in the first decade, with sharp decline in incidence thereafter. The mode of presentation of the liver disease is variable. There may be transient neonatal conjugated hyperbilirubinaemia. In one series the majority of such cholestatic infants had meconium ileus, 28% had mucus plugs and 11% had focal biliary fibrosis. Paucity of the intrahepatic bile ducts has been reported in one patient who presented with neonatal conjugated hyperbilirubinaemia. Obstruction by pancreatic fibrosis has been reported but is much more common beyond infancy. Bile duct obstruction due to a rubbery concretion of abnormal bile has been reported in a few infants. In late childhood, intermittent elevation of serum aminotransferases and ALP, asymptomatic hepatomegaly, hepatosplenomegaly, hypersplenism or variceal haemorrhage might be the first indications of liver involvement. The unique liver lesion, termed ‘focal biliary fibrosis’ (or ‘cirrhosis’), is found in over 70% of adults who die after 24 years of age. A prospective study of 183 CF patients followed for a mean of 10 years revealed an incidence of liver disease of 17%. Clinically relevant disease suggests an incidence of 1.4–2.7%, with a peak in adolescence. Rare and unusual manifestations of liver disease in CF include common bile duct involvement proximal to the pancreas, obstruction by pancreatic fibrosis and nodular regenerative hyperplasia of the liver in CF-associated colitis with fibrosing colonopathy. At times, ERCP and liver biopsy findings may mimic those of primary sclerosing cholangitis. Irrespective of relatively rare chronic progressive liver disease, steatosis (macrovesicular) is the most common pathological finding in the liver and is present in more than 50% of autopsy cases. The cause of the steatosis is not entirely evident, although it has been attributed to malnutrition and essential fatty acid deficiency. Death from liver disease occurs in 3.4% of patients, whereas lung disease accounts for 78% of CF-related deaths.

Factors that contribute to the variability in incidence and severity of liver disease are unknown. Some studies have found significant association with meconium ileus, male gender, meconium ileus and severe genotype, or meconium ileus and pancreatic insufficiency, but such risk factors were not identified in a Swedish cohort. There is no close correlation between particular CFTR mutations and liver disease. Kinnman et al. examined the expression of CFTR and its relationship to histopathological changes in CF. They found impaired processing of ΔF508 CFTR protein in intrahepatic biliary epithelium. However, intercellular adhesion molecule 1 (ICAM1) expression on biliary epithelial cells and inflammatory infiltrates were rare findings, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease. Generally, there is discordance for liver disease within sibships, but not always. These observations have intensified the search for modifier genes which may in part explain the lack of genotypic/phenotypic correlations. The mannose-binding lectin gene, a relation with HLA-DQ6 in males, and the PI*Z allele of α1-antitrypsin have already been proposed as potential candidates. Among these, the PI*Z allele has been shown to increase the risk of developing severe liver disease in CF patients.

The development of the sweat chloride test followed the observations of salt depletion through sweat during summer heat waves. Values >60 mmol/L are diagnostic of CF. There are unusual CF patients with sweat chloride values between 40 and 60 mmol/L or even in the normal range. In such patients, abnormal bio­electric potential or immunoreactive trypsinogen screen can confirm the diagnosis. Extensive CFTR gene sequencing has been proposed to detect rare mutations and to establish a definite diagnosis in symptomatic patients with previously negative results. An elevated serum collagen type VI has been suggested as a screening test for detection of liver disease. Newborn screening using elevated serum trypsin (trypsinogen) by radioimmunoassay at 3–5 days of age or mutational analysis on Guthrie spots is followed by substantial and prolonged health gain when compared with children clinically diagnosed at a later stage.

Most investigations have shown improved liver enzymes, lipid profiles and vitamin A levels with ursodeoxycholic acid therapy. One report documents normalization of liver function tests (LFTs) and improvement in the portal tract inflammation on pathological examination. The same authors also noted improvement in ultrastructural abnormalities of bile ducts. Importantly, in CF patients, ursodeoxycholic acid is not effective for gallstone disease, the incidence of which may be 12% in older patients. Although the stones are often radiolucent, they contain calcium bilirubinate instead of increased cholesterol.

A transjugular intrahepatic portosystemic shunt (TIPS) procedure is only rarely used in patients with CF liver disease because sclerotherapy or, preferably, band ligation therapy has been reasonably successful for treating bleeding esophageal varices. Because of rebleeds, some surgeons still advocate portosystemic shunts for patients with a good synthetic function. Liver transplantation has been successful, with long-term survival comparable with that of LT for other causes. Postoperative death has resulted mainly from lung complications. A few combined liver-heart-lung, liver-pancreas and liver-intestine transplants have been reported.

Pathological studies of the liver have been described in a number of reports. The changes include steatosis, periportal fibrosis with ductular reaction and dilation (with or without inspissation of secretions) ( Fig. 3.43 ), focal biliary fibrosis (‘cirrhosis’) and multinodular biliary fibrosis (‘cirrhosis’). Steatosis is probably the most frequent lesion, present in 121 of 198 cases (61%) reported by Craig et al. ( Fig. 3.43 ). The fat was located most frequently in periportal hepatocytes but was unevenly distributed throughout when severe. No positive correlations could be made between the presence of fat in the liver and the patient’s general nutritional status. An incidental finding in infant livers is the deposition of haemosiderin in periportal hepatocytes. This finding is presumably related to the increased iron transport from the gut in pancreatic insufficiency.

Figure 3.43

Cystic fibrosis. Periportal bridging fibrosis and ductular reaction. Note marked steatosis. (Masson trichrome stain.)

The characteristic, if not pathognomonic, lesion is focal biliary fibrosis, found in 22–33% of patients in three early series. An increasing frequency with duration of survival was noted. Thus it was found at postmortem examination in 10.6% of infants younger than 3 months, in 15.6% of infants from 3 to 12 months and in 26.9% of children older than 1 year.

Macroscopically, the liver with focal biliary fibrosis shows multiple, depressed, greyish white scars that are triangular or stellate in shape. As already noted, there is variable ductular reaction and periportal fibrosis. The ductules are generally dilated and show varying degrees of atrophy of the lining cells. The lumen contains the pathognomonic pink to light-orange, rounded masses (concretions) or amorphous material ( Figs 3.44 and 3.45 ), first described by Farber in 1944. The inspissated secretions in the cholangioles stain intensely with PAS and resist diastase digestion; they do not stain positively with mucicarmine or Alcian blue, but there may be some mucin in the interlobular bile ducts. Some greatly dilated ductules eventually rupture with extrusion of their contents and induction of an acute inflammatory response. The cholangitis is thus ‘chemical’ but may be complicated by bacterial infection. Chronic inflammatory cells may also be present in the fibrous tissue. It is important to emphasize that periportal fibrosis may occur in the absence of ductular reaction and inspissation of secretion ; this nonspecific change has been found only in infants less than 3 months of age.

Figure 3.44

Cystic fibrosis. Expanded and moderately inflamed portal areas contain dilated bile ductules that contain greatly dilated ductules. Ductular lumens are filled with pink, inspissated secretion. (H&E stain.)

Figure 3.45

Cystic fibrosis. Dilated ductules (cholangioles) contain pink, amorphous secretion. Note absence of lining cells. (H&E stain.)

Over time the focal biliary lesions may coalesce, with extension of fibrosis, atrophy of the intervening parenchyma and entrapment and encirclement of groups of hepatic lobules. This type of lesion, referred to as ‘multilobular biliary cirrhosis with concretions’ or ‘multilobular cirrhosis’, occurs in 6% of patients older than 1 year. Large irregular nodules are produced, and the deep clefts between the nodules may resemble hepar lobatum. A recent study using dual-pass needle biopsy emphasizes the role of liver biopsy in predicting an individual’s risk of liver fibrosis or development of portal hypertension. A significant number of noncirrhotic portal hypertension in CF children is confirmed by others.

In addition to the presence of excessive mucus in extrahepatic bile ducts in occasional patients, there may be excessive mucus accumulation in intrahepatic bile ducts, particularly those adjacent to the porta hepatis. This change occurs in 23.4% of infants under 3 months of age and in 12.5% of patients from 3 to 12 months. In two-thirds of the patients less than 3 months of age with mucus in their ducts, histological cholestasis is seen, and a history of jaundice is noted. The gallbladder in up to 30% of patients is hypoplastic and contains mucoid material and a small amount of viscid bile. Mucous cysts are seen in the wall, and stones may be present in the lumen.

Liver biopsy material from 11 patients with CF was examined ultrastructurally by Arends et al. A finding unique to this disease was the presence of filamentous material, with an average diameter of 15 nm, in the lumen of bile ductules and ducts. Bile granules were scattered between the filaments. A reaction product of carbohydrates was found in the filaments by histochemical techniques. It was suggested that the typical mucus deposits in bile ducts are built up from these filaments. Similar ultrastructural findings were noted by others. Bradford et al. studied the ultrastructure of liver cells in detail and described the presence of membrane-bound deposits of electron-lucent material containing electron-dense cores resembling mucus. Bile duct cells with irregular shapes protruding into the lumen and the presence of necrotic cells have been observed. An increase in the number of hepatic stellate cells (HSCs) around portal areas, with deposition of collagen around bile ducts and ductules, has been noted, and bile duct destruction, rather than cholestasis, is suggested as the inducer of collagen deposition in CF-associated fibrosis and cirrhosis. A definitive role for hepatic stellate cells has been shown and TGFβ1, produced by bile duct epithelial cells, demonstrated as an inducer of stellate cell collagen gene expression.

Hereditary disorders of bile acid synthesis and bilirubin metabolism

Primary disorders of bile acid synthesis

Patients with bile acid synthetic defects present clinically with conjugated hyperbilirubinaemia within the spectrum of the neonatal hepatitis syndrome. There are no distinguishing features among routine liver tests other than age-related normal levels of γ-glutamyltransferase (GGT). In contrast to infants with other low-GGT cholestatic disorders, those with congenital bile acid synthetic defects typically are not pruritic. Patients presenting with infantile conjugated hyperbilirubinaemia and normal GGT for age can be screened for elevated cholenoic bile acids in the urine by a colour reaction with the Lifshütz reagent. (This reagent is prepared by mixing 10 mL glacial acetic acid and 1 mL concentrated H 2 SO 4 ; when 200 µL of reagent are added to 25 mL of dried urine extract, a purple colour is produced when cholenoic acids are present.) Total serum bile acids can also be measured by a 3β-hydroxysteroid dehydrogenase assay: these are absent or else found at very low concentrations, clearly disproportionately low to the degree of jaundice. (An exception is when there is amidation defect present. ) Definitive diagnosis is by determining which bile acids are present in urine or bile using electrospray-ionization tandem mass spectrometry (ESI-MS/MS). Analysis can also be performed by fast-atom-bombardment mass spectrometry (FAB-MS) or gas-liquid chromatography (GLC). Determination of urinary cholanoids by ESI-MS/MS-MS is probably most practical, and accumulating experience argues for this investigation in any infant with neonatal hepatitis syndrome.

Most of the entities in this rapidly developing sector of genetic liver disease are rare, having been identified in only a few patients, but they may be more common than this. Affected enzymes may be microsomal, cytoplasmic or peroxisomal. Importantly, infants with severe liver disease may acquire defects in bile acid synthesis, usually Δ 4 -3-oxosteroid-5β-reductase, and they must be distinguished from those with an inherited disorder of bile acid synthesis, since treatment may be different. Gene sequencing may be required to confirm or disconfirm the diagnosis in some cases. Clinical experience shows that patients with genetic disorders of bile acid synthesis often respond well to oral bile acid supplementation. Table 3.3 shows currently identified primary disorders of bile acid synthesis. The discussion here focuses on defects in (1) 3β-hydroxy-Δ 5 -C 27 -steroid dehydrogenase/isomerase (3βHSD), (2) Δ 4 –3-oxosteroid-5β-reductase, (3) oxysterol 7α-hydroxylase and (4) sterol 27-hydroxylase (CTX). Some rare defects causing infantile conjugated hyperbilirubinaemia are also discussed, as well as amidation disorders that may present with transient, apparently idiopathic, neonatal hepatitis syndrome or later in childhood with growth failure and fat-soluble vitamin deficiency. Bile acid physiology, synthesis, conjugation and transport are discussed in Chapter 9 .

Table 3.3

Primary disorders of bile acid synthesis

Deficiency Associated disorders
3β-Hydroxy-Δ 5 -C 27 -steroid dehydrogenase/isomerase deficiency Neonatal hepatitis syndrome
Δ 4 –3-Oxosteroid-5β-reductase (5β-reductase) deficiency Neonatal hepatitis syndrome
Oxysterol 7α-hydroxylase deficiency Neonatal hepatitis syndrome
Sterol 27-hydroxylase deficiency (cerebrotendinous xanthomatosis, CTX) Neonatal hepatitis syndrome, chronic diarrhoea in infancy, neurological symptoms, juvenile cataract, tuberous xanthomas (e.g. Achilles tendon)
α-Methylacyl-CoA racemase (AMACR) deficiency Neonatal hepatitis syndrome, fat-soluble vitamin deficiency, adult-onset neuropathy
Bile acid conjugation defects (BAAT) Neonatal hepatitis syndrome, fat-soluble vitamin deficiency
Zellweger syndrome Peroxisomal biogenesis disorder (see page 194)
Bile acid synthesis is completed in peroxisomes.

3β-Hydroxy-Δ 5 -C 27 -steroid dehydrogenase/isomerase deficiency

This enzyme, 3βHSD, is the second step in the pathway for synthesizing bile acids. It is involved in the conversion of 7α-hydroxy-cholesterol to 7α-hydroxy-4-cholesten-3-one and then into the pathways producing the primary bile acids. Specifically, it can proceed directly into the pathway for synthesizing chenodeoxycholic acid, or it can be 12-hydroxylated and enter the pathway for producing cholic acid. Among the primary bile acid synthetic defects, 3βHSD deficiency is most common. The first patient reported was a 3-month-old infant with cholestasis. Early biopsies of the liver in the patient and siblings demonstrated giant cell hepatitis. Primary bile acids were not detected. Concentration of the natural substrate, unesterified 7α-hydroxy-cholesterol, were elevated in the serum. Fibroblasts from this patient demonstrated complete absence of the enzyme; the parents’ fibroblasts demonstrated reduced 3βHSD activity. The patient responded to chenodeoxycholic acid, including relief from pruritus. The presentation during infancy/childhood may be quite variable. Five patients aged 4–46 months had hepatomegaly, four of whom had jaundice and two with fatty stools; none was pruritic. Microcysts were found in the kidney in two patients. All patients responded to ursodeoxycholic acid. The challenge here is to distinguish 3βHSD (and 5α-reductase) deficiency from bile canalicular transporter disorders, especially when presence or absence of pruritus does not provide a clinical distinction. In both these bile acid synthetic disorders, the abnormal bile acids that are formed inhibit the canalicular ATP-dependent bile acid transporter. In another consanguineous family, manifestations of 3βHSD deficiency were extremely variable: the proband died from end-stage liver disease at age 24 years, but one sister was asymptomatic at age 32 despite homozygosity for a null allele of the HSDRB7 gene encoding for 3βHSD.

Hepatic histopathological changes in 3βHSD deficiency depend on the age of the patient and rate of progression of the liver disease. Two of the three patients reported by Clayton et al. who were biopsied at 6 weeks and 18 months of age had giant cell hepatitis and giant cell hepatitis with bridging fibrosis, respectively. Similarly, Jacquemin et al. found giant cell hepatitis in young patients (4 and 6 months of age) and portal and perilobular fibrosis in older patients (36–46 months). Three patients studied by Bove et al. had portal and intralobular fibrosis with ‘pseudoductular metaplasia’ and cholangiolar proliferation (ductular reaction); there was no paucity of interlobular bile ducts. An adult with cirrhosis of undetermined etiology has been described as having 3βHSD deficiency.

Δ 4 –3-Oxosteroid-5β-reductase deficiency

The responsible enzyme is found only in the liver and not in fibroblasts. It is the enzymatic step in the primary pathway from cholesterol to cholic and chenodeoxycholic acid, involved in the saturation of the steroid ring. A deficiency would result in the decreased formation of these primary bile acids with an elevation of cholenoic bile acids. Δ 4 –3-Oxosteroid-5β-reductase was not detectable in the original patients presenting with neonatal hepatitis who were evaluated in follow-up after bile acid therapy. Enzyme analysis of the liver in a subsequent patient did not verify that this is a specific genetic defect, i.e. no mutation was present. Notably, this enzyme is somewhat fragile and can cease to function in the presence of severe liver disease.

A tentative diagnosis of 5α-reductase deficiency is presently based on >70% of the urinary bile acids being of the 3-oxo-Δ 4 type and significant detection of allo-(5α-hydroxy) bile acids. The gene encoding Δ 4 –3-oxosteroid-5β-reductase is AKR1D1 (also known as SRD5B1 ); mutation analysis can confirm the diagnosis. Elevation of primary bile acids should raise doubts concerning a diagnosis of a bile acid synthetic defect, and other aetiologies should be sought. Why these patients might respond to ursodeoxycholic acid is unclear other than by a decrease in the levels of the offending bile acids. If clinical response is insufficient, improvement subsequently can be observed with chenodeoxycholic acid and cholic acid therapy. More recent experience indicates that response to supplementation with chenodeoxycholic plus cholic acid is better in patients whose disease is not advanced.

Light microscopic and ultrastructural findings in 5β-reductase deficiency (and other bile acid synthetic defects) are discussed in detail by Bove et al. Light microscopic changes in most reported cases resemble nonspecific neonatal hepatitis, with prominent cholestasis, giant cell transformation and erythropoiesis. Interlobular bile ducts are consistently normal. Ultrastructurally, injury patterns common to other forms of cholestasis are seen, as well as features that may be specific for the defect. Thus there is a nonuniform mosaic of normal and abnormal canaliculi, often in adjacent clusters of hepatocytes, but there is no canalicular dilation. However, some canaliculi show diverticula, and junctional complexes are extraordinarily convoluted, often enclosing pockets of dense granular material presumed to be bile residue.

Oxysterol 7α-hydroxylase

Deficiency of oxysterol 7α-hydroxylase, the third enzyme in the ‘acidic’ pathway that can generate chenodeoxycholic acid, can produce severe infantile liver disease. The deficiency results in the accumulation of 3β-hydrocholenoic and 3β-hydroxy-5-cholestenoic acids. One infant has been described who presented with conjugated hyperbilirubinemia and low GGT and whose liver disease had already advanced to cirrhosis. Serum levels of 27-hydroxy cholesterol were greater than 4500 times normal. Histopathologically, the liver showed giant cell transformation, ductular reaction, and canalicular and bile duct plugging; no specific ultrastructural changes were noted. Both parents had an exon 5 premature terminated codon (R388X) as a heterozygous defect. There was no response to either ursodeoxycholic acid or cholic acid, either by clinical indicators or alterations in the measured bile acids. This patient underwent a liver transplant but died of complications relating to disseminated post-transplant lymphoproliferative disease. A second patient also succumbed to severe cholestatic liver disease in the first year of life. Two patients have been reported subsequently: one died at age 11 months before LT, and the other had successful living-related donor transplant from an obligate heterozygote. Both patents had cirrhosis with multinucleated giant cells on liver biopsy. A single case of successful treatment with chenodeoxycholic acids has been reported. Patients with oxysterol 7α-hydroxylase have normal serum GGT and, in general, rapidly progressive liver disease. Diagnosis based on finding elevated serum and urine 3β-monohydroxy-Δ 5 -C 24 bile acids may generate a false-positive diagnosis of oxysterol 7α-hydroxylase deficiency. Such an infant was found to have 3βHSD deficiency, not entirely surprising because 3βHSD serves as the next enzyme in that same ‘acidic’ pathway, as well as early in the ‘neutral’ pathway.

Other defects in bile acid synthesis, including amidation

Since bile acid metabolism represents a complex pathway, it seems reasonable to expect other rare defects in bile acid metabolism. Alternate pathways may be more prominent in the fetus and newborn. The effects of developmental changes in the intestinal microbiome may be relevant.

Rare defects in bile acid synthesis may account for what has been regarded as ‘idiopathic neonatal hepatitis.’ For instance, a 25-hydroxylation pathway exists which may normally contribute in a limited fashion to cholic acid production. A deficiency in this hydroxylase was reported in a family where one child died at age 13 months after lifelong jaundice, and another was stillborn. The third child developed conjugated hyperbilirubinaemia with hepatomegaly at 7 days old ; his serum GGT was normal, and his liver biopsy showed giant cell hepatitis. On treatment with chenodeoxycholic acid, he later developed pruritus, which gradually improved when cholic acid was added to the treatment regimen and chenodeoxycholic acid was stopped. Although clinically well, including improvement in pruritus, normal LFTs, and physical examination, this patient developed cirrhosis, which appeared to stabilize.

Amidation defects cause failure to conjugate a bile acid with glycine or taurine. This is the last step in bile acid synthesis. The mechanism is somewhat complicated: a coenzyme A (CoA) thioester of the bile acid is formed by the enzyme bile acid–CoA ligase (BACL, encoded by SLC27A5 ), and glycine or taurine is conjugated by bile acid–CoA:amino acid N -acyltransferase (BAAT, encode by BAAT ). Some patients with defective amidation present in early infancy with conjugated hyperbilirubinaemia, occasionally severe enough to resemble biliary atresia. Liver biopsies in those patients showed lobular cholestasis, giant cell transformation of hepatocytes and mild to severe ductular reaction with bridging fibrosis but no ductular bile plugs. Other patients are never noted to be jaundiced but have growth retardation and severe deficiency of fat-soluble vitamins resulting in rickets or coagulation abnormalities. Infants who do manifest jaundice also have prominent clinical features of fat-soluble vitamin deficiency. Based on limited observations, this seems to be a low-GGT disorder. Detailed analysis of serum bile acids shows striking lack of conjugated bile acids, but total bile acid concentrations may be elevated. Histological analysis may show a giant cell hepatitis or ductopenia. Immunostaining for BAAT is depleted or absent. Similar clinical disease appears to occur with BACL mutations. In one reported case where the amidation defect was associated with BACL deficiency, the infant was homozygous for a missense mutation in ABCB11 (thus compromising BSEP); however, other cases exclusively with BACL defects have been identified. These infants may be at greater risk for neonatal hepatitis syndrome if born premature and requiring total parenteral nutrition (TPN).

Cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive metabolic disease caused by deficiency of mitochondrial C 27 -steroid-26-hydroxylase on the inner mitochondrial membrane, which can be demonstrated in liver and cultured fibroblasts, leading to tissue accumulation of cholestanol, a minor component in the human body. The defective gene, for sterol 27-hydroxylase, localized to chromosome 2q33, has been cloned, and mutations associated with the disease have been described.

The symptoms of this disease often begin with chronic diarrhoea in infancy. Neonatal hepatitis has been recorded in a few patients. Juvenile cataracts develop during childhood, as do tuberous xanthomas (particularly over the Achilles tendons). The neurological manifestations vary from mental retardation developing during adolescence to normal intelligence throughout adult life. Spasticity and ataxia are usually detected in the second and third decades. Progression results in dementia, spinal cord paresis and peripheral neuropathy. Less frequent findings include seizures, gallstones and osteoporosis. Death in the fourth to sixth decade is usually related to neurological deterioration; however, some patients develop early heart disease and/or atheroma.

The 5α-dihydro derivative of cholesterol, cholestanol (but not cholesterol), is elevated in serum. There is excessive deposit of cholestanol and cholesterol in tissues. Bile acid concentrations are low in bile, with high bile alcohol levels in bile and urine. Heterozygotes can be detected by measurement of urinary bile alcohol levels after administration of a bile salt-binding agent, cholestyramine.

Liver specimens from two patients revealed intracellular inclusions that appeared either as amorphous pigment or in crystalloid form. The pigment is usually found in association with the smooth endoplasmic reticulum but occasionally lies free in the cytosol. An ultrastructural study of four cases revealed perisinusoidal fibrosis, bile canalicular changes (dilation, distortion and loss of microvilli, increase in microfilaments), fatty change, proliferation of smooth endoplasmic reticulum, accumulation of lipofuscin, focal cytoplasmic degeneration, proliferation of microbodies and prominent mitochondrial changes. One patient had crystalloid cores in microbodies that disappeared after therapy. The accumulated material may indicate the presence of unmetabolizable bile alcohols resulting from a defect of bile acid synthesis.

Treatment of bile acid synthesis disorders

In general, treatment is by bile acid supplementation to bypass or offset the synthetic defect. Ursodeoxycholic acid may serve as initial treatment, but this may not be curative. It is preferable to treat patients with primary bile acids: cholic acid or chenodeoxycholic acid, separately or in combination. However, these bile acids are not widely available commercially. Chronic oral cholic acid treatment for patients with 3βHSD or 5β-reductase deficiency appears to be safe and effective. With a median follow-up of 12 years, Gonzales et al. found that serum aminotransferases normalized and liver histology improved, including some with apparent regression of cirrhosis.

For CTX, a combination of chenodeoxycholic acid and pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA reductase) has reduced serum cholestanol and prevented an increase of total cholesterol. The therapy has stopped disease progression, but has not reversed clinical manifestations. Low-density lipoprotein (LDL) apheresis has been added to the regimen in a few patients.

Liver transplantation has been performed for oxysterol 7α-hydroxylase deficiency and occasionally for other of the bile acid synthesis disorders when liver disease is far advanced and unresponsive to medical treatment.

A comprehensive review of diseases of bile acid synthesis is recommended for further reading.

Disorders of bile canalicular transporters

Progressive familial intrahepatic cholestasis (PFIC) was originally described in 1965 in a Pennsylvanian Amish kindred named Byler, thus called Byler disease/syndrome. Now PFIC is recognized as occurring worldwide. Although widely referred to as PFIC types 1, 2 and 3, these terms are confusing and should be avoided. The disorders have been reclassified according to the molecular basis, with designation based on the protein that is deficient. Since genetic testing is not always immediately available, a clinical subdivision is helpful: diseases manifesting normal (or low) levels of serum GGT and those with elevated levels. Two low-GGT forms of PFIC are now termed ‘FIC1 deficiency’ and ‘BSEP deficiency’. The predominant form with a high GGT is termed ‘MDR3 deficiency’. All three are autosomal recessive disorders and involve different genes. The neonatal presentations of these three conditions are the best described. In fact, there are less severe and later-presenting forms of all three of these bile canalicular transporter defects, including but not limited to ‘benign recurrent intrahepatic cholestasis’ (BRIC). Later-onset disease may not be recognized as such, unless features indicating similarity to neonatal disease are noted. This applies particularly to the histological features.

Familial intrahepatic cholestasis type 1 deficiency

Previously known as ‘progressive’ familial intrahepatic cholestasis type 1′ (PFIC1), FIC1 deficiency is caused by mutations in ATP8B1 . Most recognized patients present in the first 6 months of life with conjugated hyperbilirubinaemia, normal serum GGT, and itching. In FIC1 deficiency, extrahepatic manifestations of disease are clinically important. Chronic diarrhoea may develop; growth failure due to maldigestion as well as fat-soluble vitamin deficiency is a major complication. Sensorineural deafness requiring hearing aids develops in 30% of patients. Recurrent respiratory infections may occur. There is a slow progression to cirrhosis.

Liver biopsy shows a bland cholestasis ( Fig. 3.46 ), without significant giant cell transformation or cellular infiltrate. Canaliculi filled with pale-appearing bile may be identified. In 1972, Linarelli et al. suggested that the ultrastructure of canalicular bile was coarsely granular in PFIC and called it ‘Byler’s bile’ ( Fig. 3.47 ). Subsequently, this type of bile was found to be characteristic of FIC1 deficiency, from which members of the Amish Byler family suffer.

Figure 3.46

Familial intrahepatic cholestasis type 1 (FIC1) deficiency. Prominent canalicular cholestasis with pseudoglandular formation. (H&E stain.)

Figure 3.47

FIC1 deficiency. Dilated canaliculus with loss of microvilli is filled with characteristically loose, coarsely granular bile.

(Courtesy of Dr. Alex Knisely.)

The function of the FIC1 protein, a P-type ATPase, has been unclear. Groen et al. have proposed a model by which the concerted action of FIC1 and MDR3, encoded by ABCB4 , is critical in maintaining the asymmetrical lipid composition of the hepatocyte plasma membrane outer leaflet (facing the canalicular lumen) and the inner leaflet (facing the cytoplasm). FIC1 is a flippase, and MDR3 is a floppase. FIC1, along with an accessory protein CDC50A, flips phosphatidylserine from the outer to inner leaflet to complement the action of MDR3, which ‘flops’ phosphatidylcholine from the inner to the outer leaflet so that it can be associated with bile salts to form micelles. This asymmetry would make the membrane outer leaflet resistant to the detergent effect of bile salts, which are at high concentration in the bile duct lumen. The precise mechanism by which a defect in this flippase function leads to cholestasis and extrahepatic manifestations is still not clear. The reduction in ectoenzymes seen in the canalicular membrane of patients probably reflects the bile acid-dependent extraction seen in mice. This finding has been proposed as an indirect feature of PFIC1, although in the absence thus far of a specific antibody available for formalin-fixed paraffin-embedded (FFPE) tissue. In PFIC1 patients, expression of GGT and neutral endopeptidase (CD10) is lost on the canalicular membrane but not on the cholangiocyte apical surface. In contrast, canalicular expression of integral membrane proteins encoded by ABCB11 and ABCB4 (BSEP and MDR3, respectively) is preserved.

Other rare cholestatic disorders have been identified as FIC1 deficiency. A form of cholestatic syndrome was reported as being specific to the inhabitants of Greenland. Jaundice, bleeding, pruritus, malnutrition, steatorrhoea, osteodystrophy and dwarfism were typical clinical features. Eight of the children died between ages 6 weeks and 3 years from bleeding or infections. Hyperbilirubinaemia, profound hypoprothrombinaemia, thrombocytosis and elevated ALP values were evident. ‘Greenland familial cholestasis’ has now been shown to be FIC1 deficiency, with all affected individuals carrying two copies of the same missense mutation. Ornvold et al. studied the liver biopsies from the 16 children and characterized the changes as early, intermediate and late. Early changes (up to 5 months of age) were restricted to the perivenular zone and consisted of cholestasis with rosette formation. Intermediate-stage changes (5–14 months) included perivenular and then periportal fibrosis in addition to persistent cholestasis. Changes in the late stage (17–60 months) included progressive cholestasis and portoportal and portocentral fibrosis in seven patients, and cirrhosis in two of the patients. Inflammation and paucity of bile ducts were not seen. Although poorly documented and now largely disappeared, it is likely that the ‘Welsh cholestasis’ was also a form of FIC1 deficiency.

Bile salt export pump deficiency

Also known as ‘progressive familial intrahepatic cholestasis type 2’ (PFIC2), BSEP deficiency is caused by mutations in ABCB11 , which encodes the human bile salt export pump. The severe form presents in infancy, with a severe hepatitis, obvious giant cells ( Figs 3.48 and 3.49 ), periportal and perivenular sinusoidal fibrosis and eventually cirrhosis. There is usually marked intracellular retention of biliary pigment and considerable hepatocyte disarray. Byler bile is not seen; instead the bile is amorphous or finely filamentous, and microvilli are lost. Mallory–Denk bodies may be present. Individual mutations do not correlate with specific histological patterns. Milder forms of BSEP deficiency do not seem to constitute as large a proportion of later-onset disease as seen with FIC1 deficiency; however, there is also clearly a spectrum of disease, which may be considerably underestimated due to difficulties in clinical diagnosis. A missense mutation in a sibling pair manifested in childhood with a BRIC phenotype but later progressed to advanced fibrosis. BSEP function was impaired, although its canalicular expression was normal, as demonstrated with immunofluorescence. Two cases of hepatocellular carcinoma in the cirrhotic liver of patients with PFIC were reported before the characterization of the various subtypes; however, BSEP deficiency appears to be the main risk factor. Mutations predicted to cause a complete loss of this protein seem to be particularly associated with malignancy. HCC complicating BSEP deficiency may reflect the effect of multiple gene mutations. Cholangiocarcinoma has been observed in two patients with BSEP deficiency.

Figure 3.48

Bile salt export pump (BSEP) deficiency. Multinucleated giant hepatocytes are variably pale or heavily loaded with bile pigment. (H&E stain.)

Figure 3.49

BSEP deficiency. The same case as illustrated in Fig. 3.48 showing early portoportal and portocentral bridging fibrosis. (Reticulin stain.)

The histological features of BSEP deficiency are not specific. In an attempt to improve this, immunohistochemistry methodology, using a number of anti-BSEP antibodies, has been developed ( Fig. 3.50 ). IHC may reveal no canalicular expression or focal, patchy and faint canalicular staining. Redistribution of BSEP expression from cytoplasmic to canalicular (retargeting) or de novo expression has been described in children treated with ursodeoxycholic acid and biliary diversion.

Figure 3.50

BSEP deficiency. The same case as shown in Figs 3.48 and 3.49 . Immunohistochemistry for BSEP shows totally absent marking at canalicular margins, whereas the control stains adequately (inset). (Peroxidase–antiperoxidase [PAP] technique.)

Genetic liver diseases in which the gene is only expressed in the liver have generally been thought to be ideal candidates for liver transplantation. BSEP deficiency has recently highlighted a problem, which may have wider significance. Patients have been noted to occasionally have ‘recurrent disease’. It is only recently that the mechanism has been determined to be caused by the presence of antibodies against the BSEP protein of the liver graft. It appears that the antibodies are capable of inhibiting the function of the protein. Various strategies have been employed to overcome the problem, but several patients have been retransplanted before the mechanism of apparently recurrent disease was understood.

Multidrug-resistance protein 3 deficiency

This entity, previously termed ‘type 3 PFIC’, has been shown to be caused by a deficiency in the multidrug-resistance protein 3 (MDR3), a floppase, caused by mutations in the encoding gene ABCB4 . This protein is essential for the entry of the main phospholipid, phosphatidylcholine, into bile. In the absence of phospholipids, bile acids cannot form mixed micelles, and the bile is extremely hydrophobic. Much or all of the phenotype is probably induced by this toxic bile, in particular the damage it causes to the biliary epithelium resulting in a cholangiopathy. In severe cases the features seen include portal inflammation, bile ductular reaction and subsequent fibrosis ( Fig. 3.51 A ). In the neonatal setting, conjugated hyperbilirubinaemia in the presence of elevated serum GGT values differs dramatically from the normal GGT levels usually observed in patients with FIC1 and BSEP disease. The histological differential diagnosis on liver biopsy specimens is with biliary atresia or other disorders manifesting in a biliary pattern. Copper and copper-binding protein accumulate considerably, and in excess of the levels usually observed with other cholangiopathies, potentially simulating Wilson disease. In the somewhat older child, the cardinal diagnostic finding is that cholangiography discloses normal biliary architecture despite the appearance of bile duct obstruction.

Figure 3.51

Multidrug-resistance protein 3 (MDR3) deficiency, liver biopsy from a 4-year-old boy. A, The portal tract is expanded due to inflammation and ductular reaction and increased similar to the changes seen with a chronic cholangiopathy (H&E stain). B, Immunohistochemistry for MDR3 shows complete absence of staining at canalicular margins. The normal control illustrates adequate labelling (inset). (PAP technique.)

The result of partial loss of function of MDR3 may have more widespread clinical importance than the neonatal disease. De Giorgio et al. have shown that a substantial proportion of adults with cholestasis liver disease including primary sclerosing cholangitis have ABCB4 mutations. Progressive cholestatic liver failure in the adult members of a Transylvanian family was characterized histologically by a small duct cholangiopathy with ductopenia and associated with ABCB4 mutation. Heterozygosity for ABCB4 mutations have been observed in children with cholestatic liver disease. Furthermore, a reduction in biliary phospholipid not only results in parenchymal damage but also reduces the amount of cholesterol that may be maintained in solution. As with BSEP, anti-MDR3 antibodies have been raised. Although this may be a useful method of establishing a complete loss of protein ( Fig. 3.51 B ), it is less clear-cut in milder cases, as also evident in cell lines. Partial loss of MDR3 function may lead to a more slowly progressive disease ( Figs 3.52–3.54 ), but may still require liver transplantation. Lipid clefts may be identified in the lumen of bile ducts.

Figure 3.52

MDR3 deficiency. Cirrhotic liver removed at transplantation shows a green discoloration and inspissated bile within dilated intrahepatic bile ducts.

Figure 3.53

MDR3 deficiency. The same case as illustrated in Fig. 3.52 . A micronodular pattern with perinodular halo is in keeping with an advanced biliary cirrhosis. (H&E stain.)

Figure 3.54

MDR3 deficiency. The same case as illustrated in Fig. 3.52 . Intraductal bile sludge showing a mixed bilirubin and cholesterol composition in keeping with phospholipid deficiency.

Cholestasis during pregnancy has also been reported in individuals heterozygous for mutations in ABCB4 (MDR3 deficiency). Histopathological findings include bile ductular reaction with inflammation, resembling the changes in the mouse model. One diagnostic test is the detection of low bile phospholipid levels. When compared to the control bile samples of cholestatic children with sclerosing cholangitis (mean phospholipid, bile acid and cholesterol concentration of 29.1 mmol/L, 116.5 mmol/L and 3.4 mmol/L, respectively), the bile of patients with a PFIC3 phenotype had much lower phospholipid concentration (mean phospholipid, bile acid and cholesterol concentration of 1.4 mmol/L, 25.7 mmol/L and 0.74 mmol/L, respectively). In patients with MDR3 deficiency, the biliary bile salt/phospholipid and cholesterol/phospholipid ratios are approximately five times higher than control bile. Biliary phospholipids in MDR3 deficiency patients are 1–15% of total biliary lipids (normal range, 19–24%). The residual percentage relates to the severity of the mutation (<2% associated with nonsense frameshift mutations). Mutations in ABCB4 may also manifest as low-phospholipid-associated cholelithiasis syndrome, characterized by biliary symptoms before 40 years of age, intrahepatic microlithiasis and recurrence of biliary symptoms after cholecystectomy.

Benign recurrent intrahepatic cholestasis

As with PFIC, benign recurrent intrahepatic cholestasis (BRIC) was also described in 1965. Although the name and the original description suggest a distinct condition, it is now understood that BRIC is really a form of PFIC. When associated with ATP8B1 mutations, it is called BRIC1, and when associated with ABCB11 mutations, BRIC2; its association with as yet undetermined transporter defects is still possible. The clinical definition of BRIC involves specific criteria: at least two episodes of jaundice separated by a symptom-free interval lasting several months to years; laboratory values consistent with intrahepatic cholestasis, notably elevated serum GGT and total serum bile acids; severe pruritus and normal intra- and extrahepatic bile ducts confirmed by cholangiography. The clinical presentation is usually jaundice with pruritus, but there is much interindividual variation in terms of severity, frequency and duration of episodes. Symptoms can be triggered by factors such as pregnancy, oral contraceptives, drugs or infection. There is an association with gallstones. Severe cholestasis in a young adult with polymorphism in ABCB11 and ATP8B1 preceded the diagnosis of Hodgkin lymphoma by 7 months. In one series, early onset of the first attack was associated with a more severe course, in terms of frequency and duration of attacks; some patients developed kidney stones, pancreatitis and diabetes. The authors proposed dropping the adjective ‘benign’ because of the profoundly detrimental effect that the disease has on the long-term quality of life of affected patients. The suggestion that BRIC is in fact not benign is supported by the finding that some cases do progress, if slowly. There is a spectrum of severity, with some degree of genotype to phenotype correlation.

Histopathological observations of biopsy specimens from 22 patients at the milder end of the BRIC spectrum were detailed by Brenard et al. During attacks of jaundice, cholestasis (hepatocellular and canalicular) occurred in all patients, and infiltration of portal areas with mononuclear cells in seven patients. Other findings included focal mononuclear cell infiltration with or without focal necrosis, infiltration of portal areas with many eosinophils, ductular reaction (one patient) and periportal fibrosis (one patient).

Treatment of bile canalicular transporter disorders

The primary medical therapies used in PFIC include ursodeoxycholic acid, phenobarbital and rifampicin (the latter two as treatment for pruritus); response to medical therapy occurs but is unusual. Some investigators have found ursodeoxycholic acid to be more beneficial in PFIC. Some patients with FIC1 deficiency respond poorly to ursodeoxycholic acid treatment. It may be of particular benefit in MDR3 deficiency, where bile salt hydrophobicity is thought to be particularly important in the disease pathogenesis; certainly, findings in the mouse model for MDR3 deficiency support this concept. Partial bile diversion by a cholecystojejunal cutaneous conduit and/or ileal diversion produces complete resolution of the liver disease, but the results are variable from one institution to another. The surgery must be performed before significant fibrosis has developed.

New treatment modalities are in development. The possibility of chemical chaperones for certain mutations is being explored. Specifically, 4-phenylbutyrate has been proposed as treatment for BSEP deficiency.

A wealth of data, mainly unpublished, regarding liver transplantation in PFIC exists. It appears to show generally good results in both BSEP deficiency and MDR3 deficiency. The results in FIC1 deficiency are less clear, in keeping with the widespread expression of the gene. In one series, hepatic transplantation was performed in 14 children with ‘Byler’s disease’. One patient died postoperatively from arterial thrombosis. In the remaining 13 patients, graft function, growth and quality of life were good after an average follow-up of 17 months, without evidence of disease recurrence. Steatosis and steatohepatitis affect the liver allograft of patients transplanted for PFIC1.

Other diseases causing intrahepatic cholestasis

North American Indian childhood cirrhosis

Weber et al. studied 14 children, all from the Cree and Ojibwa–Cree tribes, with a severe familial cholestasis. Jaundice occurred neonatally in nine children but disappeared before the end of the first year. Subsequent reports clarified that this was a high-GGT cholestatic disorder. On light microscopy, there was giant cell transformation and bilirubinostasis. Progressive liver damage was documented by persistently high levels of ALP, moderate elevation of aminotransferases severe pruritus and morphologically by fibrosis and cirrhosis. Early portal hypertension and variceal bleeding necessitated portosystemic shunts in seven children. Ultrastructural and immunohistochemical studies suggested that this group of children might represent a human model of microfilament dysfunction-induced cholestasis. The disease locus was mapped to chromosome 16q22. A single mutation was identified in all affected individuals in a gene labelled CIRH1A . The encoded protein (cirhin) has proved to be a nucleolar protein. It remains controversial. A zebrafish model suggests a role in biliary development. Cirhin also interacts with the nucleolar protein NOL11, which has a role in production of ribosomes. The common mutation found in patients with North American Indian childhood cirrhosis (NAIC) disrupts that interaction.

Familial hypercholanaemia

In familial hypercholanaemia, clinical jaundice is classically uncommon in affected infants, who usually present clinically with nutritional sequelae of chronic cholestasis, such as failure to thrive, fat-soluble vitamin deficiency, rickets and bleeding tendency. Pruritus may be severe. Serum GGT is normal or low, but serum bile acids are elevated.

The genetic defect in familial hypercholanaemia is complex. Most cases exhibit oligogenic inheritance. The genes involved are the tight junction protein 2 gene ( TJP2 , previously known as ZO2 ) and BAAT. Some patients are homozygous for a disease-causing mutation in TJP2 and have normal BAAT ; others have mutated TJP2 plus they are heterozygous for a mutation in BAAT ; still others are homozygous for a mutation in BAAT and have normal TJP2 . Some children are homozygous for the TJP2 mutation, with normal BAAT , and they are entirely asymptomatic. TJP2 encodes a tight junction protein (TJP2, formerly ZO-2) and BAAT encodes the protein bile acid CoA:amino acid N -acyltransferase, which plays an important role in amidation, the process by which bile acids are conjugated to taurine or glycine (see earlier). The postulated mechanism is that bile acids do not enter the bile because of BAAT mutation or leak back into plasma because of faulty tight junctions caused by TJP2 mutation. Other genes have not been excluded. Mutations in the microsomal epoxide hydrolase gene ( EPHX1 ) have also been reported.

Recent observations indicate that truncating mutations in TJP2 can produce a much more severe clinical phenotype. Affected children presenting in the first 3 months of life with severe cholestatic liver disease and low serum GGT (without mutations in ATP8B1 and ABCB11 ) were found to have homozygous mutations TJP2 in the absence of BAAT mutations. In these patients, claudin 1 is normally expressed in the cytoplasm but cannot co-localize at tight junctions without TJP2. These children have unfavourable outcomes, and the majority have required liver transplantation. Likewise, BAAT mutations may be associated with more severe liver disease, classified as an amidation disorder of bile acid synthesis.

Citrullinaemia type II

This disorder, which is caused by an abnormal mitochondrial protein ( citrin, a mitochondrial aspartate-glutamate carrier; see urea cycle disorders later), encoded by the gene SLC25A13 on chromosome 7q21, has different clinical presentations dependent on age. Infants with citrullinaemia type II typically present with infantile conjugated hyperbilirubinaemia. The features are distinctive enough to be called ‘neonatal intrahepatic cholestasis with citrin deficiency’ (NICCD). This name is misleading, however, in that the degree of hepatic inflammation can be very severe. Moreover, the infant can have various metabolic defects affecting glucose metabolism (resulting in hypoglycaemia), urea synthesis and fatty acid synthesis. The infant may appear to have galactosaemia due to decreased UDP-galactose epimerase activity, but in citrullinaemia type II, measurement of serum amino acids shows elevated plasma levels of citrulline and methionine. Liver biopsy may display steatosis in addition to inflammatory changes.

Neonatal sclerosing cholangitis

Although primary sclerosing cholangitis can present clinically in infancy, it is extremely rare for it to present in the first 6 months of life. By contrast, some infants with neonatal conjugated hyperbilirubinaemia clear the jaundice and experience a period of apparent recovery. Later, cholestasis recurs, and imaging of the biliary tree reveals an appearance similar to that of primary sclerosing cholangitis. These infants are said to have ‘neonatal sclerosing cholangitis’ (NSC). It was first described in 1987, with subsequent reports. Serum GGT is elevated. The disease mechanism is likely multiple, and it has not been determined for every case. Affected infants should not undergo a Kasai portoenterostomy; medical management is that of childhood chronic cholestatic liver disease; with the development of end-stage biliary cirrhosis, liver transplantation is the required intervention.

Neonatal ichthyosis-schlerosing cholangitis (NISCH) is one form of NSC where the genetic basis is known. Affected infants have prominent cutaneous features (scanty scalp hair, scarring alopecia, ichthyosis) as well as hepatic findings of NSC. It involves mutations in the gene encoding claudin-1, a tight junction protein, CLDN1 (found on chromosome 3q27-q28). Biliary damage may be caused by disturbed regulation of hepatic paracellular permeability. The severity of the neonatal liver disorder may resemble biliary atresia. Serum GGT level is increased. Liver biopsy shows features of duct obstruction.

Arthrogryposis, renal dysfunction and cholestasis syndrome

The full-blown arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome involves skeletal changes, renal tubular dysfunction and cholestasis, and some infants also have severe ichthyosis, central nervous system (CNS) deformity (lissencephaly) and prominent growth failure. Approximately 10% have cardiac defects. However, ARC syndrome is frequently incomplete, lacking the skeletal changes. Infants with incomplete ARC may present with conjugated hyperbilirubinaemia plus renal tubular dysfunction. This appears to be a disorder characterized by normal serum GGT. The genetic abnormality is twofold. Some patients have germline mutations in the gene VPS33B on chromosome 15q26, encoding a protein that regulates vesicular membrane fusion by interacting with SNARE proteins. Others have mutations in VIPAS39, which encodes the VIPAR protein Mutations in VPS33B are approximately twice as frequent as those in VIPAS39 .

The histological pattern in the liver includes giant cell transformation of hepatocytes, lobular cholestasis and paucity of bile ducts. However, liver biopsy can be hazardous in ARC syndrome because of intrinsic platelet dysfunction severe enough to lead to haemorrhage after liver biopsy ; consequently, mutation analysis is the diagnostic test of choice. Prognosis for patients with ARC syndrome is generally poor, with limited survival beyond infancy. Cirrhosis may develop.

Familial benign chronic intrahepatic cholestasis

Three of four adult siblings in a family studied for three generations had clinical and/or laboratory evidence of slowly progressive intrahepatic cholestasis. Slight hyperpigmentation, facial hypertrichosis and hypothyroidism were seen in affected individuals, who also had prolonged increases in serum aminotransferases GGT and ALP levels. A biopsy of one patient who was jaundiced showed cholestasis and other changes indistinguishable from those of extrahepatic obstruction. Asymptomatic intervals were characterized by abnormal BSP retention, reduced N -demethylation capacity, elevated fasting total bile acid levels and normal light microscopic findings. A high serum α-lipoprotein level was found in affected individuals. The inheritance is believed to be autosomal recessive. A defect in prekeratin–keratin metabolism is postulated to account for the lesions in liver and other tissues.

Progressive cholestasis in McCune–Albright syndrome

This syndrome is characterized by café au lait spots, polyostotic fibrous dysplasia and sexual precocity. Silva et al. reported two patients with McCune–Albright syndrome (MAS) who presented with neonatal cholestasis. Despite the severity of presentation, which can resemble biliary atresia on a nondraining hepatobiliary scan, both patients cleared their jaundice in 6 months but continued to have mild LFT abnormalities. The patients have been shown to carry an activating mutation in the gene encoding the α-subunit of the G protein. The protein stimulates adenylcyclase in liver tissue, suggesting that this metabolic defect could be responsible for their cholestatic syndrome. Serum GGT is elevated in MAS. A variety of hepatobiliary and pancreatic neoplasms have been observed in MAS patients, in particular inflammatory hepatocellular adenomas.

Aegenaes syndrome

Also known as lymphoedema cholestasis syndrome type 1 (LCS1), Aegenaes syndrome is a rare disorder with limb oedema and cholestasis, originally reported in Norwegian kindreds. Infants present clinically with conjugated hyperbilirubinaemia and elevated serum GGT along with a lymphatic disorder, typically lower-limb oedema, although a generalized oedema or lymphangiomas may occur. The intrahepatic cholestasis may be severe, with fat-soluble vitamin deficiencies and pruritus. The neonatal hepatitis syndrome may predate the lymphatic disorder and resolve before the lymphatic component is evident. The typical course is relapsing with intermittent bouts of cholestasis. Progression to end-stage liver disease is uncommon; however, a few young children have required liver transplantation. Hepatocellular carcinoma was reported in a teenager who was thought to have LCS1. In adulthood the lymphatic disorder predominates. The disease mechanism is unknown; the gene has been localized to chromosome 15q, but there may be genetic heterogeneity.

Microvillus inclusion disease

Some patients with microvillus inclusion disease (MVID), associated with mutations in the MYO5B gene, develop intrahepatic cholestasis with jaundice, severe pruritus, normal serum GGT and increased serum bile acid concentrations. The genetic basis is not known; no mutations in ATP8B1 or ABCB11 were found. The degree of cholestasis may worsen after intestinal transplantation.

Hereditary defects of bilirubin metabolism

Patients present with asymptomatic jaundice from elevated serum unconjugated bilirubin (Crigler–Najjar syndrome I and II, Gilbert syndrome) or conjugated bilirubin (Dubin–Johnson syndrome, Rotor syndrome). Bilirubin metabolism is discussed in Chapter 9 ; see also reviews by Bosma and Erlinger et al.

Crigler–Najjar syndrome type I

Crigler–Najjar syndrome is characterized by severe nonhaemolytic unconjugated hyperbilirubinaemia beginning in the newborn period. It was named after the physicians who provided the first description in 1952. Patients with Crigler–Najjar syndrome type I can develop kernicterus at any age. The diagnosis should be entertained in the first 3 days of life. Levels of unconjugated bilirubin are usually over 20 mg/dL and may be as high as 50 mg/dL before therapy. Routine LFTs are in the normal range.

Crigler–Najjar syndrome type I is rare, with an incidence of about 1 per 1 million. Inheritance is autosomal recessive. Patients lack the enzyme uridine diphosphate (UDP)-glucuronosyltransferase completely and consequently are unable to conjugate bilirubin. Mutations in exons 2–5 of the UGT1A gene affect all isoforms influencing glucuronidation of bilirubin and other substrates (type Ia). Mutations of exon 1 affect bilirubin glucuronidation only (type Ib).

Therapy is difficult and limited. Patients do not respond to phenobarbital. Phototherapy has been used long-term with plasmapheresis for acute bilirubin elevations and, if initiated promptly, such therapy can reverse early bilirubin encephalopathy. Therapeutic effectiveness decreases during adolescence. Currently, light-generating blankets or mattresses have proved effective. Liver transplantation results in a cure. This can also be achieved by an orthotopic auxiliary transplant. Experimental hepatocyte transplantation has decreased the hours needed for phototherapy. Gene therapy is under investigation. The Gunn rat has been a valuable animal model.

The histopathology of the liver in Crigler–Najjar syndrome type I is characterized by intrahepatic cholestasis ( Fig. 3.55 ), but the liver may be normal.

Figure 3.55

Crigler–Najjar syndrome type I. Liver removed at transplantation is normal except for bile pigment seen in some canaliculi and in Kupffer cells.

Crigler–Najjar syndrome type II

Crigler–Najjar syndrome type II appears to be a milder variant of type I. It is also characterized by unconjugated hyperbilirubinaemia. The deficiency of bilirubin UDP-glucuronosyltransferase is less severe, but the mechanism of this difference is not entirely clear.

Arias et al. described Crigler–Najjar syndrome type II in 1969. Patients present with neonatal jaundice, but the jaundice is less severe than in patients with type I. Bilirubin levels fluctuate between 7 and 20 mg/dL. These patients generally do not develop the severe intellectual compromise of type I patients, although during an intercurrent illness, serum bilirubin levels may rise to 40 mg/dL and cause kernicterus.

Crigler–Najjar syndrome type II is inherited as an autosomal recessive disease, although occasional patients with autosomal dominant inheritance have been reported. Type II patients also have significantly reduced levels of bilirubin UDP-glucuronosyltransferase 1. Analysis of bile before administration of phenobarbital demonstrates the presence of significant quantities of bilirubin monoglucuronide, which are not seen in type I disease. Patients with Crigler–Najjar II and significant bilirubin elevations can be distinguished from type I patients by the response to phenobarbital: in type II patients, the drug lowers the serum bilirubin by 30%.

Gilbert syndrome

Gilbert syndrome is a mild, and usually intermittent, form of unconjugated hyperbilirubinaemia. Patients often present at puberty, when jaundice is first noticed. Bilirubin levels increase following starvation or other stress. In newborns it can exacerbate neonatal jaundice in the first 2 days and can contribute to higher bilirubin levels in patients with concomitant glucose-6-phosphate dehydrogenase (G6PD) deficiency. Mild haemolysis and hepatocyte bilirubin uptake defects have been found. Liver enzymes are in the normal range.

Gilbert syndrome is the most common genetic bilirubin defect in Caucasians. The incidence is 3–16% of the population; inheritance is autosomal recessive. Hepatic bilirubin UDP-glucuronosyltransferase-1 activity may be 25% of normal. Mutations have been found in both the promoter and coding regions of the UDP-glucuronosyltransferase-1 gene in patients with Gilbert syndrome, resulting in reduced enzyme expression.

As with Crigler–Najjar syndrome type II, analysis of bile before administration of phenobarbital demonstrates the presence of significant quantities of bilirubin monoglucuronide. Diagnosis of Gilbert syndrome is clinical. Liver biopsy, even if direct measurement of bilirubin UDP-glucuronosyltranserfase can be performed, is not justified.

No therapy is necessary for Gilbert syndrome. Prolonged fasting should be avoided. A primate animal model has been reported. Increased lipofuscin pigment accumulation in liver cells has been described in Gilbert syndrome . The pigment granules do not exhibit the coarse granularity of those of the Dubin–Johnson syndrome. Ultrastructurally, hepatocytes reveal hypertrophy of the smooth endoplasmic reticulum.

Dubin–Johnson syndrome

Dubin–Johnson syndrome was first described in 1954 by Dubin and Johnson and Sprinz and Nelson. Dubin subsequently reviewed in detail 50 cases reported up to 1958. Patients typically present with asymptomatic conjugated hyperbilirubinaemia, but they have normal liver enzymes and no other evidence of hepatic dysfunction. Some patients have hepatomegaly and abdominal pain. Cholecystitis associated with minute bilirubinate stones may develop.

Elevations of predominantly conjugated bilirubin range from 2 to 5 mg/dL. Sulphobromophthalein is retained in these patients, with levels increased at 60–90 min over those at 45 min after intravenous administration. In urine the total coproporphyrin level is normal, but isomer I is >80%, which is characteristic of the syndrome.

Age of onset may be as late as the sixth decade. Dubin–Johnson syndrome is rare in infancy, except in an inbred population of Iranian Jews, where the incidence is as high as 1 in 1300. Inheritance is autosomal recessive, with reduced penetrance in females. The underlying defect is complete absence of the canalicular multispecific organic anion transporter (cMOAT). The ABCC2 gene is localized to chromosome 10q24. It was cloned in humans in 1996. Human multidrug-resistance protein 2 (MDR2) is the same gene with 32 exons. Multiple gene mutations have been documented and can be detected in fibroblasts. The most common mutation is R1066X, which leads to a premature termination of cMOAT. On liver biopsy, IHC for cMOAT (MDR2) reveals a complete absence of staining ( Fig. 3.56 C ). Animal models include the Corriedale sheep and the transport-deficient (TR) rat. Pigment accumulation resembling that of the Dubin–Johnson syndrome has been described in the howler monkey.

Figure 3.56

Dubin–Johnson syndrome. Liver cells contain an abundance of a brown, coarsely granular pigment. ( A, H&E stain; B, Fontana stain.) C, Immunostain for canalicular multispecific organic anion transporter (cMOAT) showing no specific staining. Only the coarsely granular pigment is still seen. Normal control shows the expected pattern of staining (inset). (PAP method.)

Accumulation of a coarsely granular brown pigment, called lipomelanin, in liver cells is characteristic of the Dubin–Johnson syndrome ( Fig. 3.56 A ). Lobular distribution is diffuse, although more heavily concentrated in the perivenular zone. The accumulation of the pigment in hepatocytes gives the liver a grey to black colour grossly, although this feature may be less prominent in younger patients. Thus the gross (black) colour of the liver biopsy core may be diagnostic. The pigment shares some of its physicochemical properties with lipofuscin and melanin in that it is Oil Red O-positive (in frozen sections), stains black with the Fontana stain ( Fig. 3.56 B ), is variably PAS positive and is autofluorescent when examined by ultraviolet microscopy. Ultrastructurally, the Dubin–Johnson pigment is lysosomal and thought to differ from lipofuscin in being pleomorphic and having a more variegated appearance and less lipid. An electron spin resonance spectroscopy study suggested that the pigment is not melanin but rather is a stable free radical with unusual properties. Phillips et al. consider the lysosomes distinctive in having very dense areas as well as moderately electron-dense, finely stippled areas.

Rotor syndrome

A form of conjugated hyperbilirubinaemia, Rotor syndrome was reported in 1948. Although clinically similar to the Dubin–Johnson syndrome, there is no abnormal liver pigmentation. Total coproporphyrin excretion in the urine is increased, and isomer I is elevated but is less than 80% of the total. Inheritance is autosomal recessive, but the genetic defect is unknown. Hepatobiliary scanning reveals no minimal visualization. There is no pigment storage or other morphologic change in Rotor syndrome. A number of ultrastructural changes were described by Phillips et al., including distinctive ‘two-tone’ lysosomes.

Disorders of porphyrin metabolism

The porphyrias are a group of eight disorders of the biosynthesis of porphyrins and haem that lead to the excessive accumulation and excretion of porphyrins and porphyrin precursors. Caused by a defect in one of the eight enzymes involved in haem synthesis, porphyrias are classified into hepatic or erythropoietic depending on where overproduction of the haem precursors takes place. Porphyrias manifest with cutaneous sensitivity, acute neurovisceral symptoms or both. Acute hepatic porphyrias are associated with primary liver cancer and in particular hepatocellular carcinoma. Only two of the porphyrias importantly affect the liver: porphyria cutanea tarda and the erythrocytic protoporphyrias. Severe liver injury is increasingly recognized in association with X-linked protoporphyria, the variability of its manifestations probably depending on random X chromosome inactivation. Liver transplantation is used to treat the liver disease complicating porphyria or to correct the genetic defect causing intractable neurovisceral involvement.

Porphyria cutanea tarda

The classification, clinical aspects and therapy of porphyria cutanea tarda (PCT) and other porphyrias are covered in several comprehensive reviews. PCT is classified as sporadic or familial. Most patients with PCT have the sporadic form (type I). Decreased activity of uroporphyrinogen decarboxylase is restricted to the liver, and the family history is negative. Familial (type II) PCT, accounting for approximately 20% of cases of PCT, is inherited as an autosomal dominant trait. The gene ( UROD , encoding enzyme hepatic uroporphyrinogen decarboxylase) is located on chromosome 1p34. The enzyme defect (50% reduction of activity) is present in all tissues. Clinical penetrance is low, with less than 10% of affected persons developing symptoms. Of note, some familial PCT occurs with mutations in gene(s) other than UROD ; this is classified as PCT type III. Familial (type II) PCT has an earlier onset than sporadic (type I) PCT, with some cases presenting in childhood.

Sporadic (type I) PCT occurs typically in male patients between 40 and 50 years of age. Precipitating factors in the sporadic form include toxins (e.g. hexachlorobenzene), oestrogens, alcohol, mutations in the HFE gene and viral infection. An isolated case of PCT has followed interferon-alfa therapy of hepatic metastases. The interaction of the HFE mutation and susceptibility to sporadic PCT has been discussed in several studies. Mild to moderate iron overload is found in 60–70% of patients with PCT; 10% have increases in the range of hereditary haemochromatosis. In almost all studies, a high prevalence of HFE gene mutations has been detected, with differences between patients of northern European ancestry and those of Mediterranean origin. In a U.S. study, 19% of the PCT patients tested were homozygous for the C282Y mutation, and 7% were compound heterozygous (0.5% and 1.0%, respectively, in the general population). Viral infections may trigger PCT. Initial reports documented a high incidence (40%, 57% and 70.7% in three series) of hepatitis B serological markers in PCT and anecdotal cases of HIV infection. The strong association between hepatitis C virus (HCV) and PCT is most important. In the series reviewed by Elder, the prevalence of antibodies to HCV in PCT has ranged from 9% to 79%. This is broadly related to the level of endemicity of HCV infection in the general population in various countries. A subsequent meta-analysis allowed Gisbert et al. to conclude that the mean prevalence of HCV infection, detected by polymerase chain reaction (PCR) in PCT patients, is approximately 50%, much higher than that reported in the general population. Prevalence similarly varied depending on the country and the type of PCT (57% in sporadic, 25% in familial). It must be noted that HCV infection in itself is unlikely to derange the haem synthesis pathway; only a small proportion of HCV patients show an increase in urinary porphyrins, and prevalence of PCT among HCV patients is 1–5%. Treating PCT patients with chloroquine apparently does not affect the course of the underlying liver disease.

Histopathological findings in the liver in PCT include the accumulation of uroporphyrin in the cytoplasm of hepatocytes. Needle-shaped cytoplasmic inclusions have been identified in hepatocytes by light, fluorescent and electron microscopy in biopsy specimens; the crystals are also birefringent under polarizing light. According to Cortes et al., the inclusions are best seen by light microscopy in unstained paraffin sections. The crystals can be specifically stained in paraffin sections by the ferric ferricyanide reduction test ( Fig. 3.57 ). They are often found close to ferritin-like deposits. Morphometric studies have shown significantly greater amounts of uroporphyrin crystals in familial than sporadic PCT. The crystals have been induced experimentally in mice by iron overload. Ultrastructurally, they reveal alternating areas of differing electron density. The crystals appear to be a mixture of porphyrinogens and porphyrins surrounding uroporphyrin crystals. Other changes in liver biopsy specimens from patients with PCT have been described in several series. They include steatosis, variable haemosiderosis in periportal hepatocytes and fibrosis. Cortes et al. found periductal lymphoid aggregates in 43% of their cases. The incidence of cirrhosis in two series was 33% and 34%. It should be emphasized that tests for chronic viral hepatitis, particularly chronic HCV infection, were not performed in these series.

Figure 3.57

Porphyria cutanea tarda. Needle-shaped crystals of varied length are present in the cytoplasm of liver cells. (Ferric ferricyanide stain.)

Hepatocellular carcinoma (HCC) has been reported in association with PCT and also in patients with acute intermittent porphyria, variegate porphyria and hereditary coproporphyria. The risk in patients with acute hepatic porphyria is high particularly after 50 years of age. Factors related to an increased risk of HCC in PCT are a long symptomatic period before start of therapy, chronic hepatitis (particularly HCV), iron overload and advanced fibrosis or cirrhosis. In one series, there was a direct relationship between increasing age and extent of liver damage, with fibrosis present at a mean of 48 years, cirrhosis at 57 years and HCC at 66 years. A somatic second-hit mutation has been proposed as the critical mechanism for the development of HCC in acute porphyrias.

Erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) was first reported in 1926, with further definition of the dermatological manifestations in 1961, and liver disease reported in 1965. Clinical presentation begins with photosensitivity, usually before 6 years of age; patients have an extreme burning sensation with erythema and swelling. Onset of liver disease is usually later; most cases are diagnosed after 30 years of age, although liver disease can become evident in adolescence. Males are affected twice as often as females. Patients with EPP often present with right upper quadrant pain radiating to the back. Cirrhosis develops in only 1–10% of patients. There is no evidence of biliary obstruction. Death occurs within 3–5 months (range, 1 month to 2 years) following the development of jaundice. A few patients with EPP have exhibited haemolysis and neurological dysfunction.

Ferrochelatase, the defective enzyme, catalyses the insertion of iron into protoporphyrin as the final step in haem synthesis. Functional enzyme levels are usually less than 50%. Diagnosis of EPP is by measurement of elevated plasma, erythrocyte, and faecal protoporphyrin. Cord blood analysis is not a reliable prognostic tool. The bone marrow contributes 80% of the protoporphyrins and the liver up to 20%. Protoporphyrin is poorly water soluble and is secreted by hepatocytes only into bile. When this hepatobiliary transport is overwhelmed, insoluble aggregates of protoporphyrin form crystals in hepatocytes, canaliculi and proximal bile ducts, resulting in ‘black liver disease’. Autosomal dominant inheritance is found in most families, but autosomal recessive inheritance has been documented. The incidence of EPP is 1 in 75,000 to 1 in 200,000 live births worldwide.

Approximately 90 different mutations have been identified in the ferrochelatase gene, localized on chromosome 18, including point mutations, nucleotide deletions and a partial chromosome deletion. The ferrochelatase gene is composed of 11 exons and 10 introns with a size of approximately 4.5 kilobases (kb), and the enzyme is localized to the matrix side of the inner mitochondrial membrane. Patients with EPP who develop liver disease usually have a mutation in one ferrochelatase allele that alters enzyme function, together with a polymorphism in the nonmutant allele that causes low gene expression. This results in significant increase in the hepatobiliary excretion of protoporphyrin, which can damage the liver through both cholestatic injury and oxidative stress.

Haematin has been the most successful medical therapy. Liver transplantation is appropriate for patients with liver failure or end-stage cirrhosis. However, recurrence of protoporphyric liver disease has been reported. Bone marrow transplantation (BMT) is needed after LT to prevent disease recurrence in younger patients. A mouse model is available for future evaluation of therapy. The hepatobiliary alterations in this murine model have been reversed by BMT. It has thus been suggested that BMT may be an option for EPP patients at risk of developing hepatic complications. A recent study has confirmed that bone marrow-derived cells play a significant role in restoring and regenerating hepatic tissue in this mouse model.

Macroscopically, the liver affected by EPP is generally black. The first description of the characteristic, if not pathognomonic, changes was published by Cripps and Scheuer. Sections from percutaneous hepatic biopsy specimens from five patients showed focal accumulations of a dense, dark-brown pigment in canaliculi, interlobular bile ducts, connective tissue and Kupffer cells ( Fig. 3.58 ). The pigment had an intense red autofluorescence in frozen sections examined by fluorescence microscopy with an iodine-tungsten-quartz light source. Two of the patients had portal and periportal fibrosis, and in one case, the portal areas were infiltrated by mononuclear cells. Cholelithiasis occurs in some patients with EPP, and the calculi contain protoporphyrin. Crystals isolated from the liver in one EPP patient had the same fluorescence spectrum as protoporphyrin.

Figure 3.58

Erythropoietic protoporphyria. Brown deposits are present in canaliculi, hepatocytes and Kupffer cells. (H&E stain.)

The deposits in canaliculi, bile ducts and Kupffer cells display bright-red birefringence with a centrally located, dark Maltese cross ( Fig. 3.59 ). Some of the larger deposits and most of the smaller ones in the cytoplasm of hepatocytes and Kupffer cells appear as clusters of brilliantly illuminated granules on polarizing microscopy.

Figure 3.59

Protoporphyrin deposits have a red to yellow birefringence with a Maltese cross configuration (H&E stain).

Transmission EM studies have demonstrated that the deposits of protoporphyrin in EPP consist of numerous, slender, electron-dense crystals arranged singly in sheaves or in a ‘star-burst’ pattern ( Fig. 3.60 ). The crystals are straight or slightly curved and are 43–646 nm in length and 6.1–22.0 nm in width. The crystalline accumulations in the cytoplasm of liver cells are not surrounded by a membrane, but in Kupffer cells they are intralysosomal. Non-membrane-bound crystals have also been demonstrated in the cytoplasm of bile duct cells. The protoporphyrin casts in canaliculi are readily visualized by scanning EM ( Fig. 3.61 ).

Figure 3.60

Erythropoietic protoporphyria. Electron micrograph showing a mass of radiating, hair-like pigment crystals in a dilated canaliculus. (Magnification ×1800.)

(Courtesy of the late Dr. G Klatskin.)

Figure 3.61

Erythropoietic protoporphyria. Rounded protoporphyrin casts in canaliculi show fracture lines. Casts are solid and have a granular structure at higher magnification. Hypertrophied Kupffer cell (upper left) probably contains phagocytosed protoporphyrin. (Scanning electron microscopy [SEM] ×1250.)

As already noted, the liver disease in EPP may progress to cirrhosis, although the overall incidence of significant hepatic disease is probably small. Hepatic failure may be precipitated by viral infection (e.g. Epstein–Barr virus) or by alcohol. A direct toxic effect of protoporphyrin in the pathogenesis of liver disease is suggested by the changes observed in protoporphyrin-perfused rat livers. The occurrence of cirrhosis in two sisters with EPP also raises the possibility of a genetic predisposition for hepatic disease. A unique association of two rare diseases, Langerhans cell histiocytosis and EPP, has been reported.

A separate disorder closely resembling erythropoetic protoporphyria is X-linked dominant protoporphyria (XLP). It is caused by gain-of-function mutations in ALAS2 , the 5-aminolaevulinate synthase gene on Xp11.2. The mutation is typically a deletion of two or four bases in exon 11. The increased activity of the mutated enzyme generates high levels of protoporphyrin in erythrocytes, found either as free protoporphyrin or as zinc chelated. These patients may be at higher risk for hepatic complications. Phenotypic variation is considerable. Prevalence in a North American series was greater than that in European series, accounting for 10% of patients with a disorder appearing to be EPP.

Disorders of carbohydrate metabolism and related conditions

Glycogen storage diseases (glycogenoses)

Liver disease is significant with glycogen storage disease (GSD) types I, III, IV, VI and IX. Strictly speaking, GSD type IV is an amylopectin storage disorder. GSD type II may be associated with hepatomegaly ( Table 3.4 ).

Table 3.4

Glycogen storage diseases: enzyme defects, gene locations, diagnostic tests and major histological features

Type Enzyme Gene locus Enzymatic test Liver abnormalities
0 Glycogen synthase 12p12.2 Liver Steatosis
Ia Glucose-6-phosphatase 17q21 Liver Steatosis and GHD
Adenoma, later HCC
Ib (non-a) Glucose-6-phosphate translocase 11q23 Freshly removed liver Steatosis and GHD
Adenoma, later HCC
II Lysosomal-γ1-4 and-γ1-6-glucosidase 17q25 Leukocytes, liver, muscle, amniocytes Cytoplasmic vacuoles
Lysosomal monoparticulate glycogen in EM
IIIa/b Amylo-1-6 glycosidase (debrancher) 1p21 a. Liver, muscle, heart
b. Liver
GHD and steatosis, fibrosis, rarely cirrhosis
IV Amylo-1-4 glycan-6-glycosyltransferase 3p12 Leukocytes, liver, amniocytes Ground-glass, diastase-resistant inclusions
Non-membrane-bound fibrillar material on EM
VI Liver phosphorylase E 14q21-22 Liver GHD, steatosis, fibrosis, rarely cirrhosis
IX Liver phosphorylase kinase Xp22.1-22.2
Liver, muscle, erythrocytes, leukocytes Non-uniform GHD, steatosis
XI GLUT2 transporter 3q26.1-q26.3 Liver GHD

EM, Electron microscopy; GHD, glycogenic hepatocyte distension; GLUT2, glucose transporter 2; HCC, hepatocellular carcinoma.

Type I glycogen storage diseases

Type I GSD is caused by a deficiency of glucose-6-phosphatase, an enzyme that converts glucose-6-phosphate (G6P) to glucose, resulting in decreased hepatic production of glucose and accumulation of glycogen in the liver, kidney and intestine. Von Gierke described type Ia GSD in 1929. The incidence of the disease is 1 in 100,000. Inheritance is autosomal recessive. The G6PC gene is mutated in type Ia GSD and is located on chromosome 17q21, while the SLC37A4 gene is mutated in type Ib and is located on chromosome 11q23. At least 84 mutations have been reported in type Ia and 80 mutations in type Ib GSD. Overall, type Ia is more common, accounting for approximately 80% of GSD type I cases. Of note, previous classification of ‘types Ic and Id’ GSD was based on the belief that additional genes were involved, but subsequent studies have shown that these cases have type Ib SLC37A4 mutations.

In 1952, Cori and Cori reported that G6P activity was virtually absent in the livers of individuals with type I GSD. This enzyme is necessary for gluconeogenesis and glycogenolysis, providing 80% of the normal hepatic glucose production. In addition to the liver, glucose-6-phosphatase also is present in the kidney, pancreas and intestinal epithelium, but not in white blood cells (WBCs). In type Ia GSD, glucose-6-phosphatase protein expression is absent due to G6PC gene mutations, while in type Ib GSD, a transmembrane transport protein is defective (glucose-6-phosphatase translocase, encoded by SLC37A4 ). This transporter is present in many organs, as well as being active in neutrophils and monocytes. In fact, the loss of glucose-6-phosphatase translocase activity in WBCs leads to neutrophil dysfunction through enhanced endoplasmic reticulum stress, oxidative stress and apoptosis, all leading to neutropenia.

In the first year of life, type I GSD usually presents with hypoglycaemia in the presence of a greatly enlarged liver. Additional features associated with type Ib GSD include neutropenia, recurrent infections and inflammatory bowel disease, which may resemble Crohn’s disease. Affected individuals also can have oral, perioral and perianal ulcers, infections, abscesses and fistulas.

Effective medical management permits survival into adulthood, if death does not occur in the first year of life. However, in adulthood, type I GSD is a multisystemic disorder. All adults with GSD type Ia have hepatomegaly. Other common findings include short stature in 90%; hepatocellular adenomas in 75%, usually multiple; iron deficiency in 81%; proteinuria from focal glomerulosclerosis in 67%; renal calcification in 65%; and osteopenia and fractures in 27% of patients. Hypertriglyceridaemia occurs in all patients, hypercholesterolaemia in 76%, and hyperuricaemia in 89%. Less common complications include pancreatitis (thought to be secondary to high lipid levels) and myocardial infarction (from coronary artery disease). Pericardial effusion with valvular disease has been reported. In children, epistaxis and bleeding during invasive procedures is associated with long bleeding times. Studies indicate a decrease in platelet adhesion, aggregation and ATP release associated with hypoglycaemia, which can improve after normalization of blood glucose levels. Von Willebrand factor may be deficient. Correction has been documented with intravenous 1-deamino-8- d -arginine vasopressin (DDAVP).

Two serious complications of this GSD type I may develop by adulthood. Chronic renal disease is caused by focal segmental glomerulosclerosis without immune deposits and uric acid nephropathy. Hyperfiltration occurs throughout childhood. The second major complication is the hepatocellular adenomas. The male/female ratio is at least 2:1 for individuals who develop adenomas. Adenomas are seen as early as 3 years of age and as late as 40 years, the vast majority developing in the second decade. The development of adenomas appears to be independent of dietetic treatment, compliance to treatment or biochemical parameters related to metabolic balance. On IHC subclassification, most of the hepatic adenomas are inflammatory adenomas or unclassified adenomas. For example, one large study found 26 of 33 hepatic adenomas were inflammatory adenomas, while the remaining five were unclassified. There is a well-documented risk of malignant transformation in the hepatic adenomas, although it is not clear if the risk is the same for inflammatory adenomas versus unclassified adenomas. In one study, mean patient age was 23 years, and adenomas were followed between 2 and 7 years before malignant transformation. Of note, serum alpha-fetoprotein (AFP) levels are often not elevated; thus the diagnosis of malignant transformation of a hepatic adenoma depends on the presence of careful liver imaging and/or liver biopsy. HCCs can develop even if successful dietary management leads to adenoma regression.

The goal of therapy for GSD type I in infancy and childhood is to keep the glucose level at 70 mg/dL. This can be achieved by nocturnal gastric infusions of glucose-containing solutions, or by the administration of uncooked cornstarch around the clock, or by a combination of both.

Infections and inflammatory bowel disease in type GSD type Ib can be improved by granulocyte colony-stimulating factor (G-CSF), which seems to ameliorate neutrophil membrane function, but does not reduce the number of apoptotic neutrophils in the circulation. In one study of 17 patients with long-term follow-up, near-normal growth was achieved; however, puberty was delayed in five patients. Five patients also received allopurinol for hyperuricaemia, while two were treated for hypertriglyceridaemia. Some 35% of the patients were anaemic. Hepatocellular adenomas developed in 29%. All but one patient had glomerular hyperfiltration, with protein loss in two patients.

Liver transplantation has been successful in curing GSD type Ia but does not alter the course of the renal disease. Combined liver-kidney transplantation has been successful. Management of hepatocellular adenomas includes resection or, preferably for multiple adenomas, hepatic transplantation, because the potential trophic effect on residual adenomas of resecting an adenoma is not known. Despite the persistence of neutropenia after LT, dramatic catch-up growth and reduced infections have been observed. LT in four patients with GSD type Ib was followed by stabilization of glucose intolerance, decreased hospital admission and normalized neutrophil counts, but long-term observation is needed to confirm a sustained benefit.

Type II glycogen storage disease (acid maltase deficiency, Pompe disease)

Type II GSD, first described by Pompe, is caused by a deficiency of the lysosomal enzyme acid α-glucosidase (also called acid maltase), encoded by GAA . The prevalence of type II GSD is 1 in 100,000 live births (infantile form), 1 in 720,000 (juvenile form) and 1 in 53,000 (adult form). The defect in acid maltase can be demonstrated in all cells, including fibroblasts, lymphocytes, amniotic cells and chorionic villi. The milder forms have more enzyme activity.

The gene is localized to chromosome 17q25. There are more than 40 mutations, which influence the clinical phenotypes. However, there is tremendous phenotypic variation, even for individuals with the same mutation. Type II GSD can present as a generalized infantile type, as a milder form that presents during childhood (juvenile) or in adulthood. In one study, the median age at presentation was 33 years.

The fatal infantile disease presents as a ‘floppy baby’ with massive cardiomegaly, macroglossia, and progressive muscle weakness leading to death at a median age of 6–8 months. Hepatomegaly is mild, there is no liver synthetic dysfunction, and hypoglycaemia is absent.

The milder forms have a later onset and present with exercise intolerance, myalgia, weakness with or without rhabdomyolysis and myoglobinuria; contractures develop eventually. The juvenile form also involves respiratory muscles, and death may result from respiratory failure. The adult form presents from the second to sixth decade without cardiac involvement. Creatinine phosphokinase is usually elevated. There is no abnormality in glucose metabolism. Vacuolated lymphocytes are found in the peripheral blood and bone marrow; the percentage of PAS-stained lymphocytes on blood smears is of diagnostic value. Many organs show vacuolated cells due to enlarged lysosomes containing glycogen. For example, skeletal, cardiac and smooth muscle fibres contain glycogen-filled vacuoles. Vacuolated cells are particularly prominent in autopsy material as a result of the disappearance of extralysosomal glycogen. In the kidneys, glycogen accumulation is primarily in the loops of Henle and collecting tubules, a location that distinguishes this variety from GSD type I.

Type III glycogen storage diseases (Forbes disease, limit dextrinosis)

The autosomal recessive GSD type III is caused by a deficiency in amylo-1,6-glucosidase, 4α-glucanotransferase ( AGL or glycogen debranching enzyme), whose gene is located on chromosome 1p21. Multiple mutations have been identified, which mirror the biochemical and clinical heterogeneity of the disease. Clinical presentation may be similar to type I GSD, with hypoglycaemia, hepatomegaly and hyperlipidaemia. Growth failure is less severe, with better resolution at puberty. Hepatocellular adenomas occur in up to 25% of patients. Type IIIb is characterized by liver involvement alone, whereas type IIIa involves muscle as well as liver; type IIIb is specifically associated with mutations in AGL exon 3. Adults may develop adenomas, cirrhosis and, rarely, HCC. Portal hypertension can be present despite normal liver enzymes. Some children have mild splenomegaly at initial clinical presentation. Muscle involvement, including cardiac, can become an increasing problem in adulthood. Treatment consists of a high-protein diet and uncooked cornstarch. Prenatal diagnosis is available. Liver transplantation is sometimes required.

Type IV glycogen storage disease (branching enzyme deficiency; amylopectinosis; Andersen disease)

Type IV GSD is a rare autosomal recessive disorder caused by a deficiency of glycogen branching enzyme, leading to the accumulation of amylopectin-like polysaccharides in affected tissues. The deficient enzyme can be measured in the liver, WBCs or cultured fibroblasts, and prenatal diagnosis is available. The GBE1 gene is located on chromosome 3p12; multiple defects have been described, which may correlate with clinical heterogeneity.

The prenatal course may be complicated by polyhydramnios, decreased fetal movement and dilated cardiomyopathy. A rare infantile variant exists with early perinatal death. Infantile presentation includes hepatosplenomegaly and failure to thrive. Some patients may have arthrogryposis of the lower limbs. Hypoglycaemia is rare except as a feature of liver failure. Occasionally, patients may not have progression of the liver disease. Later presentation includes congestive cardiac failure, skeletal muscle weakness and nerve involvement. In adults, progressive involvement of the upper and lower motor neurons, sensory loss, early neurogenic bladder and, in some patients, late dementia may be presenting features. Adult cases are sometimes referred to as ‘adult polyglucosan body disease’.

Liver transplantation is an effective treatment for patients who develop liver failure. In a study of 14 patients, death from cardiac failure occurred in a single case, while nine of the survivors did not have neurological, muscular or cardiac complications up to 13.5 years after LT. A reduction in myocardial amylopectin storage was reported by one group but was not found by others.

Types VI and IX glycogen storage disease and related subtypes (phosphorylase system deficiency)

Defects in the phosphorylase system are caused by either deficient phosphorylase enzymes or deficiencies in the phosphorylase kinases, which activate the phosphorylase enzymes. The phosphorylase systems are enzymatically distinct in the liver and skeletal muscle. Muscle phosphorylase deficiency (GSD V) does not involve the liver and is not discussed further. Of the liver phosphorylase system defects, phosphorylase kinase defects (GSD IX) are much more common than liver phosphorylase deficiency (GSD VI). Enzyme activity is measured most accurately in liver tissue. Blood cells may or may not demonstrate these deficiencies.

Mutations in the PYGL gene lead to deficient activity of liver phosphorylase in type VI GSD (Hers disease), leading to glycogen accumulation because of a decreased ability to degrade glycogen by cleavage of the α1, 4-glycosidic bonds. The disease is inherited in an autosomal recessive manner and appears to be more common in the Mennonite population. Signs and symptoms can be very mild in children, whereas other children can present with hepatomegaly and growth retardation. Hypoglycaemia can also be induced by limited dietary intake, e.g. during an illness. However, almost all patients with type VI GSD improve with age to the point that as adults, they are generally asymptomatic.

In contrast to phosphorylase deficiency seen in GSD type VI, phosphorylase kinase deficiency, GSD IX, is clinically and genetically more complex, since the enzyme consists of four tissue-specific subunits encoded on different genes (including chromosome X) and differentially expressed in different tissues. The phosphorylase kinase liver α-subunit genes PHKA1 and PHKA2 are on chromosome X, the β-subunit gene PHKB is on 16q12-13, and the γ-subunit PHKG2 is on 16p11.2.

Phosphorylase kinase activates phosphorylase and is itself activated by a cAMP-dependent protein that can be stimulated by glucagon or adrenaline. The α- and β-subunits modulate the γ-subunit, which contains the catalytic portion of the enzymes.

The most common phosphorylase kinase deficiency is GSD IXa (~75% of cases), which manifests between ages 1 and 5 years with hepatomegaly (92%), growth retardation (68%), hypercholesterolemia (76%), hypertriglyceridemia (70%) and mild elevation in serum aminotransferases (56%), while hypoglycemia and metabolic acidosis are rare. Hepatomegaly and growth retardation usually resolve at puberty. Autosomal recessive forms of phosphorylase kinase deficiency (GSD IXb, c) present with more severe liver disease, which may progress to cirrhosis. Renal tubular acidosis and neurological complications are seen, including peripheral sensory neuropathy.

Therapy is usually not necessary in types VI and IX GSD; in some patients, uncooked cornstarch may be helpful.

Fanconi–Bickel syndrome (glycogen storage disease type XI)

This rare form of GSD is characterized by hepatorenal glycogen accumulation, fasting hypoglycaemia, hypergalactosaemia and renal tubular acidosis. GSD type XI results from defective function of GLUT2, which is the most important glucose transporter in hepatocytes, pancreatic β-cells, enterocytes and renal tubular cells. The SLC2A2 gene encoding GLUT2 is on chromosome 3q26.1-q26.3, and 34 different mutations have been reported. Deficiency results in impaired import and export of glucose and galactose in affected tissues. Renal glycogen accumulation leads to impaired tubular function, Fanconi nephropathy and rickets. Patients may be picked up by the galactosaemia screen test because galactose, as well as glucose, is transported by GLUT2. Untreated, the disease can be compatible with survival into adulthood, but patients continue to be symptomatic even after hepatomegaly recedes.

Glycogen synthase deficiency (glycogen storage disease type 0)

Glycogen synthase deficiency is a rare inborn error of metabolism leading to fasting hypoglycaemia in infancy or early childhood that is accompanied by high blood ketone levels and low alanine and lactate concentrations. The liver glycogen synthase gene ( GYS2 ) is on chromosome 12p12.2. Glycogen synthase deficiency can present with nonspecific symptoms after overnight fasting, as an incidental finding or because of a positive family history. The diagnosis is based on the characteristic biochemical profile and the demonstration of absent glycogen synthase activity and slightly reduced glycogen content in liver tissue, which is frequently steatotic.

Gross, light microscopic and ultrastructural features of glycogenoses

Grossly, the liver in the glycogenoses tends to be enlarged, smooth and paler than normal. GSD type IV livers have a tan and waxy appearance, with myriad tiny nodules that occasionally aggregate into larger nodules. A reticular pattern of fibrosis or rarely a micronodular or mixed type of cirrhosis may be seen in some types of GSD, such as types III and IV. Tumour nodules of varied size (representing either adenomas or carcinoma) may be visible macroscopically.

The diagnosis of a specific type of GSD requires biochemical determination of the enzyme defect or gene sequencing. Although subtle histological differences have been described, the pathological features are distinctive in only a few of the glycogenoses, such as the light microscopic appearance of the liver in type IV and the ultrastructural features of types II and IV.

In the majority of the glycogenoses, hepatocytes are swollen and rarefied, with wisps of pinkish material in an otherwise empty cytoplasm ( Fig. 3.62 ). Cell membranes appear thickened due to peripheral displacement of organelles by the stored glycogen. The overall appearance of hepatocytes has been likened to that of plant cells. Nuclei of liver cells also may be glycogenated. The excess cytoplasmic glycogen can be stained with periodic acid-Schiff (PAS) and readily digested by diastase ( Fig. 3.63 ). However, the haematoxlyin and eosin (H&E) findings are a better clue to the diagnosis than PAS staining, because even the normal liver can have abundant glycogen. Thus strong PAS staining plus diastase digestion is not strongly suggestive of anything per se, in the absence of typical H&E findings. The presence of sharply defined vacuoles in the cytoplasm, particularly in GSD types I and III, indicates the simultaneous accumulation of fat. Perivenular Mallory–Denk bodies have been reported in glycogenosis type Ia.

Figure 3.62

Glycogenosis type III. Liver cells are swollen and have pyknotic, centrally or eccentrically located nuclei. The cytoplasm is rarefied. (H&E stain.)

Figure 3.63

Glycogenosis type III. A large quantity of glycogen in liver cells (left) has been digested by diastase (right). (PAS stain before and after diastase.)

The histopathology of the liver in glycogenosis type IV is extremely different from that of the other glycogenoses. The changes closely resemble those of Lafora disease, but in Lafora disease, progression to cirrhosis does not occur. Typically, hepatocytes in type IV GSD are enlarged and contain colourless or lightly eosinophilic ground-glass inclusions that are round, oval or bean shaped ( Fig. 3.64 ). An artefactual space may surround the inclusions. They are most heavily concentrated in the periportal zone but can be found in other zones. Both the inclusions and the rest of the hepatocyte cytoplasm in type IV glycogenosis are stained deeply with PAS ( Fig. 3.65 ); diastase treatment removes the normal glycogen but not the abnormal amylopectin-like material in the inclusions. The latter can, however, be digested by pectinase ( Fig. 3.65 ), and α- or β-amylase. The inclusions can be nonspecifically stained with colloidal iron (green), Best carmine (red) and Lugol iodine (mahogany brown). Globular, PAS-positive inclusions with a Maltese cross birefringence have been reported in the liver, skeletal muscle and CNS in several congenital cases.

Figure 3.64

Glycogenosis type IV. Lightly eosinophilic (ground glass-like) inclusions are present in many liver cells. (H&E stain.)

Figure 3.65

Glycogenosis type IV. Cytoplasmic inclusions, intensely stained with PAS (left), have been digested by pectinase (right). (PAS before and after pectinase.)

Fibrosis, which can progress to cirrhosis, is a frequent finding in glycogenosis type IV but can also occur in types III and IXb, c ( Fig. 3.66 ). As already noted, patients with glycogenosis type I may develop hepatocellular adenomas or HCC. The hepatocellular adenomas are often multiple, and most are subtyped either as inflammatory adenomas or unclassified adenomas. Unusual histological features in some adenomas have been described, including a steatohepatitic morphology with marked steatosis, Mallory–Denk bodies and fibrosis. Amyloid deposition has also been described in a small subset of GSD-related adenomas. Focal nodular hyperplasia can also develop in livers with GSD, including types I and VI.

Figure 3.66

Glycogenosis type III. Micronodular cirrhosis. Note absence of inflammation and interface hepatitis. (H&E stain.)

The ultrastructural features of the glycogenoses are quite comparable, with the exception of types II and IV. In the cytoplasm of the hepatocytes, large pools of glycogen rosettes displace organelles, such as mitochondria and the endoplasmic reticulum (ER), to the periphery of the cell. The glycogen may be associated with vesicles of smooth ER, or it may assume a starry-sky pattern, as observed in glycogenosis types III, VI and IX. Morphometric analysis in glycogenosis type I has shown an increased volume of glycogen per unit volume of the hepatocyte cytoplasm. Additionally, there is marked reduction (by 50%) of the rough ER per unit volume of liver tissue. Double-contoured vesicles in the ER are characteristic of type I GSD. Lipid droplets of varied size are seen in most of the glycogenoses, but appear to be more prominent in types I, II and VI. Nuclear glycogenation is minimal or absent in type Ib GSD, in comparison to type Ia. Increased collagen deposition in the space of Disse is observed in the GSD types I, III, IV, VI and XI.

Glycogenosis type II differs from the other types in the accumulation of monoparticulate glycogen (versus rosettes) in lysosomes that vary from 1 to 8 µm in diameter ( Fig. 3.67 ). However, increased accumulation of glycogen rosettes may also be noted in the cytoplasm. The ultrastructural features of glycogenosis type IV are pathognomonic. The inclusions noted by light microscopy consist of undulating, randomly oriented, delicate fibrils that measure up to 5 nm in diameter; these accumulations are not membrane bound ( Fig. 3.68 ).

Figure 3.67

Glycogenosis type II. Electron micrograph reveals accumulation of monoparticulate glycogen in enlarged lysosomes. Note glycogen rosettes between the lysosomes. (×15,000.)

Figure 3.68

Glycogenosis type IV. Electron micrograph shows segment of inclusion (left) consisting of non-membrane-bound fibrillar material; some glycogen rosettes are present between the inclusion and the hepatocyte cytoplasm (right) (×21,130).

Differential diagnosis of histological findings in glycogenoses

The differential diagnosis for the light microscopic findings in the glycogenoses includes primarily glycogenic hepatopathy and mutations leading to urea cycle defects. Individuals with glycogenic hepatopathy present with enlarged livers and elevated liver enzymes. The hepatocytes in glycogenic hepatopathy are diffusely swollen with glycogen, and the H&E findings can be essentially indistinguishable from a GSD. However, the clinical settings are distinct: glycogenic hepatopathy occurs in the setting of elevated and poorly controlled blood sugar levels (typically type 1 diabetes mellitus), often in children and teenagers, and responds dramatically to diabetic control, whereas GSDs, despite their variable findings at presentation, present with low or normal blood glucose levels.

Urea cycle defects also can show variable and sometimes marked glycogen accumulation in hepatocytes. As with glycogenic hepatopathy, the clinical setting is distinct because urea cycle defects present most often in the neonatal period with hyperammonaemia, lethargy, hypotonia and seizures. The elevated ammonia levels are a critical clue to the diagnosis, which can be further supported by quantitative amino acid level analysis of the serum. Children with milder forms of urea cycle defects who present later in life can have a history of avoiding meat and other high-protein foods. The H&E findings can closely mimic a GSD, and correlation with clinical and laboratory findings is typically needed to secure the proper diagnosis. Ultrastructural differences can exist between urea cycle defects and glycogenoses. One study found that the intracytoplasmic organelles in urea cycle defects are not displaced to the cell periphery by the abundant glycogen, in contrast to glycogen storage diseases.

Myoclonus epilepsy, Lafora type (Lafora disease)

Lafora disease is an autosomal recessive and fatal form of epilepsy with onset in late childhood or adolescence. One of the characteristic features of the disease is the presence of PAS-positive inclusion bodies in hepatocytes that are morphologically and immunohistochemically similar to those of glycogenosis type IV. Two genes can be mutated in Lafora disease: EPM2A on chromosome 6q24 and NHLRC1 ( EPM2B ) on chromosome 6p22.3. The EPM2A gene produces a protein, tyrosine phosphatase, called laforin, whereas NHLRC1 encodes a protein named malin. At least 43 different variations in EPM2A and 23 in NHLRC1 are known. Laforin, through its phosphorylase activity, and malin, possibly through inactivation of glycogen synthase, seem to have an inhibitory activity to polyglucosan accumulation. Their deficiency leads to an accumulation of insufficiently branched, and hence insoluble, glycogen molecules, which are deposited in the form of polyglucosan or Lafora bodies.

The disease begins in adolescence with epileptic seizures (grand mal attacks being the most common) followed by myoclonus (beginning in the face and extremities, but progressively involving other muscles) and dementia. Atypical cases without myoclonus or epilepsy have been reported. Axillary skin biopsy is the simplest, least invasive diagnostic procedure, with characteristic inclusion bodies reliably found in duct cells of eccrine sweat glands. Most patients die between ages 16 and 24. However, a late-onset slowly progressive form, possibly related to a distinctive mutation, has been reported.

The histological hallmark of Lafora disease is the presence of distinctive intraneuronal inclusions (Lafora bodies) most frequently found in the substantia nigra, globus pallidum, dentate nucleus, parts of the reticular system and cerebral cortex. The Lafora bodies vary in size from dot-like to as large as 35 µm. They stain positively with PAS, Best carmine, Lugol iodine (dark brown), colloidal iron and methenamine silver nitrate. Three types have been described. With the PAS stain, type I bodies (the most common) are granular and red, type II bodies have a densely stained peripheral zone, and type III bodies (the least common) are homogeneously bright red. The bodies are digested by α-amylase, amylopectidase and pectinase. On the basis of histochemical studies, Yokoi et al. concluded that Lafora bodies are composed predominantly of an unusual branched polyglucosan. Ultrastructurally, they are not membrane bound and are composed of varying proportions of fibrillar and granular material. The core is usually granular while the fibrils, which measure from 6 to 13 nm, radiate outward and branch repeatedly.

Extraneural deposits of abnormal material are frequently present. The histochemical and ultrastructural characteristics of these deposits in skeletal and smooth muscle, myocardium, liver and other organs resemble those of Lafora bodies in the brain, with minor differences. Hepatic involvement has been reported by a number of investigators. In H&E preparations, liver cells contain round, oval or kidney-shaped inclusions that are sharply circumscribed, homogeneous or finely granular and lightly eosinophilic; they may be surrounded by a ‘halo’ that is probably artifactual ( Fig. 3.69 ). The nuclei of hepatocytes are frequently displaced to the periphery.

Figure 3.69

Lafora disease. More or less rounded pink inclusions in the cytoplasm of periportal hepatocytes; note artefactual ‘halo’ around the inclusions. (H&E stain.)

Affected hepatocytes in Lafora disease are predominantly periportal in location. They resemble those seen in type IV glycogenosis and show identical staining reactions, with the exception of the colloidal iron stain; the cytoplasm in Lafora disease is homogeneous ( Fig. 3.70 ), whereas the stored material in type IV glycogenosis is coarse and clumped. Hepatocytes not harbouring Lafora bodies contain a large quantity of glycogen. The inclusions also bear a resemblance to ground-glass hepatocytes of hepatitis B antigen carriers (but do not stain HBsAg), as well as to liver cells injured by cyanamide, a drug used in alcohol aversion therapy. Lafora body-like inclusions have also been reported in patients who did not have myoclonus or epilepsy. These inclusions have been observed after chemotherapy, particularly in the liver of leukaemic children treated with 6-thioguanine and other drugs, especially cyanamide (see Chapter 12 ). Cirrhosis has not been reported in myoclonus epilepsy, but there may be slight portal fibrosis.

Figure 3.70

Lafora bodies are positively stained with colloidal iron. Note varied shapes and location in periportal hepatocytes. (Hale colloidal iron method.)


Galactosaemia is an autosomal recessive disorder caused by a deficiency of galactose 1-phosphate uridyltransferase and first described by von Ruess in 1908. The deficient enzyme was identified by Isselbacher et al. in 1956. The incidence is 1 in 45,000 births. The GALT gene is located on chromosome 9q13, and several allelic variants have been reported, resulting in varying degrees of residual enzyme activity. Missense mutations are associated with low to undetectable enzyme activity, resulting in the most profound symptoms. The ‘Duarte’ variant produces some active enzyme, and patients with this allele have a better prognosis. Compound heterozygotes (e.g. a classic allele with a Duarte allele) can be affected, although less severely than homozygotes. Much more rarely, a defect in the epimerase enzyme (encoded by GALE ) produces the same neonatal symptoms.

Common symptoms in early infancy are conjugated hyperbilirubinaemia, failure to thrive, vomiting and diarrhoea after galactose ingestion. Oil-drop cataracts may be noted. Newborns are predisposed to overwhelming sepsis, most often from Escherichia coli . Haemolytic anaemia may occur early. Increased intracranial pressure and cerebral oedema may be presenting features. Neonatal liver failure with acute or chronic pattern may occur, without predisposing sepsis; clinical features include lethargy, with severe cases manifesting oedema, ascites and bleeding. Long-term complications such as mental disability, speech defects, ataxia and ovarian failure may occur despite dietary restriction.

The diagnosis is classically suggested by the detection of urinary reducing substances (nonglucose), which is neither sensitive nor specific, and may be positive with any liver failure. Erythrocyte galactose 1-phosphate uridyltransferase activity can be measured, but the test must be done before the infant receives a blood transfusion. Erythrocyte galactose 1-phosphate levels can be assayed in Guthrie spots. Testing for the main genetic mutation or measurement of parental-erythrocyte galactose 1-phosphate uridyltransferase may be confirmatory. Since routine clinical management of an infant with conjugated hyperbilirubinaemia involves withholding lactose-containing feed until galactosaemia has been excluded as the diagnosis, speedy diagnosis is important. Prenatal diagnosis is performed by assaying chorionic villus biopsies or amniotic cells for the enzyme deficiency.

Complete dietary restriction of galactose is the only available therapy. The liver disease undergoes remarkable recovery, but neurodevelopmental complications are not necessarily avoided by dietary therapy. Dietary therapy is difficult, since fruits and vegetables contain significant amounts of soluble galactose. In addition, there is an endogenous synthesis of galactose. Long-term follow-up should be considered in peripheral epimerase deficiency. Dietary restriction is recommended during an affected pregnancy; but it is unknown whether this will prevent or reduce neurological damage.

Of the histopathological changes, those in the liver are the most distinctive, although they are not pathognomonic. The earliest change, which may appear within 10 or 11 days of birth, is marked steatosis and a bile ductular reaction; the cholangioles usually contain bile plugs and may be surrounded and infiltrated by neutrophils. Fibrosis can already be present at this early stage ( Fig. 3.71 A and B ). The next change, which begins as early as 2 weeks and is fully developed by 4–6 weeks, is a striking pseudoglandular (pseudoacinar) transformation of the hepatic plates. The hepatocytes surround dilated canaliculi that are either empty or contain bile or some pink or orange-coloured material. During this and the preceding phase, extramedullary haemopoiesis and haemosiderosis may at times be prominent ( Fig. 3.71 C ). Fibrosis can be significant by age 6 weeks and culminate in cirrhosis at 3–6 months, typically with a micronodular pattern. A galactose-free diet can lead to regression of liver damage. One patient who survived to age 52 years with established cirrhosis has been reported. Occasional findings in the liver in galactosaemia include giant cell transformation. Hepatic adenomas have also been reported, but in current terminology, the ‘adenomas’ would be called macrogenerative nodules (or dysplastic nodules), since they occurred in a cirrhotic liver.

Figure 3.71

Galactosaemia. Steatosis, cholestasis and fibrous expansion of the portal tract are associated with mild ductular reaction and conspicuous periportal haemosiderosis in this liver biopsy specimen taken from a 3-week-old baby. ( A, H&E stain; B, reticulin; C, Perls method for iron.)

Hereditary fructose intolerance

Hereditary fructose intolerance is caused by a deficiency of aldolase B, resulting in the inability to convert fructose 1-phosphate into dihydroxyacetone and glyceraldehyde. This may result in acute liver failure or in cirrhosis. The disorder was first described by Hers and Joassin in 1961. The clinical presentation is quite variable. Typically, liver disease appears in infants on introduction of fructose into the diet at weaning. The predominant symptoms are poor feeding, vomiting and failure to thrive. Other findings may include hepatomegaly, pallor, haemorrhage, trembling and jerkiness, shock, jaundice, oedema, tachypnoea, ascites, splenomegaly, fever and rickets. Patients who survive beyond infancy develop an aversion to sweet foods and characteristically have caries-free teeth. Some present later with cirrhosis.

Laboratory tests may reveal fructosaemia and fructosuria after recent fructose ingestion, together with hypophosphataemia, metabolic acidosis and abnormal LFTs. Uric acid levels may be elevated. A generalized amino aciduria with excretion of organic acids, as well as elevated serum levels of tyrosine and methionine, can simulate the findings in tyrosinaemia. None of these findings is diagnostic.

An unequivocal diagnosis is made by mutational analysis of the aldolase B gene, located on chromosome 9q22. More than 30 mutations have been identified. The A150P (65%), A175D (11%) and N335K (8%) were the most common mutated alleles in a large series from Europe, where the disease occurs in an estimated 1 in 26,000 births. Analysis of liver or intestinal tissue for aldolase B enzyme activity can also confirm the diagnosis. Administration of fructose as a diagnostic test (so-called fructose tolerance test) is potentially dangerous; this obsolete test should be avoided.

Dietary treatment with restriction of fructose, sorbitol and sucrose may lead to full restoration of normal health, growth and development, provided liver and renal diseases are not advanced at initiation of treatment. Patients need to be aware of high-fructose corn syrup and honey in the diet. Life-threatening acute liver failure may develop on the reintroduction of fructose, sucrose or sorbitol. Since there is endogenous production of fructose, some worsening of liver disease may occur despite meticulous avoidance of fructose.

Histopathological findings in hereditary fructose intolerance include neonatal hepatitis with giant cell transformation, steatosis, fibrosis and cirrhosis. The resemblance of the lesions to those of galactosaemia has been noted. As noted by Hardwick and Dimmick, most descriptions of the histopathology of the liver in hereditary fructose intolerance refer to changes consistent with the early phases of cirrhosis rather than with an established cirrhosis. Hepatocellular carcinoma has been reported in a 49-year-old man suspected of having hereditary fructose intolerance.

Ultrastructural changes include concentric and irregularly disposed membranous arrays that are present in the glycogen areas of most hepatocytes and are associated with marked rarefaction of the cytoplasm (‘fructose holes’) ( Fig. 3.72 ). Many of the membranous formations resemble cytolysosomes. It has been suggested that these changes are related to the intracellular accumulation of substrates. Similar ultrastructural changes have been reproduced in rats whose livers were infused with fructose through the portal vein. Survival of injured cells is thought to depend on the sequestration of damaged areas (cytolysosome formation), as might occur with minimal or mild exposure to fructose. Larger quantities of fructose, particularly if repeated, may lead to more severe liver injury with necrosis and ultimately to cirrhosis.

Figure 3.72

Hereditary fructose intolerance. Electron micrograph of liver from a patient before instituting a fructose-free diet. Note prominence of the smooth endoplasmic reticulum (ser) and formation of membranous arrays (ma) . Glycogen particles are aligned between layers of the smooth membranes, and enclosed cytoplasm is rarefied and watery. A cytolysosome (arrow) is also illustrated. (×28,200.)

(Courtesy of Dr. M.J. Phillips.)

Disorders of glycoprotein and glycolipid metabolism

These diseases involve systemic disorders resulting from deficient activity of enzymes mediating the catabolism of glycosamino­glycans (mucopolysaccharidoses) or the synthesis or degradation of glycoproteins and glycolipids (aspartylglycosaminuria, α-mannosidosis, fucosidosis, mucolipidoses and congenital disorders of glycosylation).


In the mucopolysaccharidoses, excessive amounts of mucopolysaccharides accumulate in somatic and visceral tissues, and their partial degradation products are excreted in the urine; in addition, there may be accumulation of gangliosides. These extremely rare disorders are distinguished by their clinical manifestations and by two-dimensional (2D) electrophoresis of glycosaminoglycans (GAGs) and specific enzyme assay. Enzyme analysis is performed in WBCs and fibroblasts, and the use of dried blood spots appears promising. The selective measurement of GAG-derived oligosaccharides in urine provides a sensitive method for the early identification of individuals with mucopolysaccharidosis (MPS) and allows the determination of oligosaccharide profiles that not only characterize subtype but also can be used for the biochemical monitoring of therapies. The role of genetic testing has been reviewed recently, particularly in terms of identifying carriers or undiagnosed family members. Except for type II, which is X-linked, all mucopolysaccharidoses are inherited as autosomal recessive traits. Six mucopolysaccharidoses (Hurler syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio syndrome, Maroteaux–Lamy syndrome and type VII β-glucuronidase deficiency) are associated with variable degree of enlargement of the liver and spleen.

Mucopolysaccharidosis type I: Hurler, Scheie and Hurler–Scheie subtypes

MPS type I is caused by a deficiency of the degradative lysosomal enzyme α- l -iduronidase. It results in clinical symptoms ranging from mild somatic complications and a normal lifespan to severe CNS involvement and a significantly shortened lifespan. A particularly high incidence of 1 in 26,206 births, with a carrier frequency of 1 in 81 (1 in 10 in ‘travellers’), has been reported from the Irish Republic. The leucocytes of obligate heterozygotes have about half the mean specific enzyme activity of normal controls. The gene is located on chromosome 4q16 ; multiple mutations have been characterized, with disease severity linked to the specific mutation. Patients with the most severe phenotype (Hurler [MPS IH] subtype) manifest developmental delay by 12–14 months of age. Features include corneal clouding, dysostosis multiplex, organomegaly, macroglossia, a prominent forehead and stiff joints. Death occurs by 10 years of age, usually from obstructive airway disease, respiratory infections or cardiac complications. Scheie (MPS IS; formerly MPS type V) and Hurler–Scheie (MPS IH/S) subtypes are associated with less severe clinical disease.

Prenatal diagnosis by amniocentesis is now routine, and in utero therapy has been attempted. BMT is very effective if performed before significant loss of cognitive function, usually at under 2 years of age. Cord blood or haematopoietic stem cell transplantation (HSCT) can achieve a favourable outcome in Hurler syndrome, with improved cognitive function, but with a limited effect on corneas and skeleton. Enzyme replacement with intravenous laronidase therapy is well tolerated and effective for patients who do not have neuronal pathology. It may safely be combined with subsequent HSCT.

Mucopolysaccharidosis type II: Hunter syndrome

MPS type II is caused by a deficiency of iduronate-2-sulphatase and is the only X-linked MPS. The gene has been localized to Xq27.3-28. Affected children have coarse facies, deafness, cognitive deterioration, growth failure, joint contractures, severe hepatosplenomegaly and chronic diarrhoea. Death usually occurs before age 15 years, with mean age at death before and after 1985 being 11.3 and 14.1 years, respectively, which may reflect improvements in patient identification, care and management. A milder form of this disease exists with complications that include hearing impairment, and papilloedema resulting from local infiltration in the eye. Death is usually from airway obstruction or cardiac failure. BMT does not prevent the severe cognitive deterioration and is not currently recommended. Enzyme replacement therapy with idursulfase has the potential to benefit many patients with MPS II, especially if started early in the disease course.

Mucopolysaccharidosis type III: Sanfilippo disease

MPS type III comprises four phenotypically identical subtypes with different enzyme defects. All forms are characterized by coarse features, hepatomegaly, variable splenomegaly and mental retardation, but there is no corneal clouding. Type A results from deficiency of heparan- N -sulphatase; type B from deficiency of N -acetyl-α- d -glucosidase; type C from deficiency of acetyl CoA:α-glucosaminide acetyltransferase; type D from deficiency of N -acetylglucosamine 6-sulphatase. The gene for type A has been cloned, and several mutations associated with Sanfilippo disease have been described. A locus for type C has been mapped to the pericentromeric region of chromosome 8. The gene for type D has been cloned and localized to chromosome 16q24. Prenatal diagnosis is available. There is no known therapy for MPS III.

Mucopolysaccharidosis type IV: Morquio syndrome

MPS type IV is caused by deficiency in N -acetylglucosamine 6-sulphate sulphatase (type A) or β-galactosidase (type B). Both types are phenotypically similar, with severe skeletal deformities and growth retardation, cervical myelopathy, a potentially fatal hazard, mild corneal clouding and hepatomegaly, but no mental retardation. Late onset has characterized type B MPS IV.

Mucopolysaccharidosis type VI: Maroteaux–Lamy syndrome

Deficiency of N -acetylgalactosamine 4-sulphatase (arylsulphatase B) is the cause of MPS type VI, which has three clinical subtypes—infantile, intermediate and adult—that vary in severity. The infantile form is characterized by growth retardation, dysostosis multiplex, coarse facial features, restricted movement, hepatosplenomegaly and corneal clouding. Clinical presentation with neonatal conjugated hyperbilirubinaemia has been reported. Death in the second and third decades is the result of cardiac failure. Intellect is not compromised in any form of the disease. The gene has been localized to chromosome 5q13-14; several mutations in the arylsulphatase gene have been found, but clear mutational-phenotype correlations are not yet possible. Sensitive immune assays to detect 4-sulfatase activity may allow an early detection of MPS VI on blood spots. BMT has been helpful in this disorder. Recent studies have concentrated on recombinant enzyme therapy, which is well tolerated and reduces lysosomal storage, as evidenced by reduction in urinary GAG, with clinical responses in all patients; the largest gains occur in patients with advanced disease receiving high-dose recombinant enzyme. Long-term enzyme therapy resulted in significant improvement in pulmonary function.

Mucopolysaccharidosis type VII: Sly syndrome

MPS type VII is caused by a deficiency of β-glucuronidase. This rare disorder is characterized by dysostosis multiplex, hepatosplenomegaly, mental retardation and frequent pulmonary infections. MPS VII is one of the storage disorders that can present with hydrops fetalis. One patient had isolated neonatal ascites as the first manifestation. Another infant presented with conjugated hyperbilirubinaemia with hepatosplenomegaly. The gene has been localized to chromosome 7q21.1-22; it appears that more than one mutation can result in MSP VII. Prenatal diagnosis is possible using amniotic fluid. In one case, BMT resulted in improved motor function and fewer infections.

Pathological changes in mucopolysaccharidoses

Macroscopically, the liver in the mucopolysaccharidoses is enlarged, firm or hard and has a pale, slightly yellowish or greyish colour. There may be extensive fibrosis or cirrhosis. Microscopically, both hepatocytes and Kupffer cells are swollen and have an empty or faintly vacuolated cytoplasm ( Fig. 3.73 ). Since much of the stored acid mucopolysaccharide is leached out by aqueous fixatives, other methods of fixation, such as Lindsay dioxane picrate solution, are preferable. Alternatively, addition of a 10% solution of acetyl trimethylammonium bromide to the formalin fixative is said to preserve acid mucopolysaccharide in the cells.

Figure 3.73

Hurler disease. Liver cells are large and have a rarefied cytoplasm. (H&E stain.)

The stored acid mucopolysaccharide is best demonstrated by a colloidal iron stain, and most of it can be digested with hyaluronidase ( Fig. 3.74 ). It is weakly positive with PAS stain but cannot be digested by diastase. Acid mucopolysaccharide is metachromatic when stained with toluidine blue. Little or no neutral lipid can be demonstrated in frozen sections of the liver stained with the sudanophilic dyes.

Figure 3.74

Hurler disease. Greenish blue, punctate mucopolysaccharide is present in the cytoplasm of hepatocytes and Kupffer cells. (Hale colloidal iron method.)

When fibrosis is present in the mucopolysaccharidoses, it is diffuse, with heavy deposition of collagen bundles in the space of Disse and gradual microdissection of the parenchyma into nodules ( Fig. 3.75 ). Periportal bridging fibrosis has been emphasized by Parfrey and Hutchins. The type of cirrhosis associated with the mucopolysaccharidoses may be macronodular or micronodular. When cirrhosis supervenes, it generally does so in older children and adults.

Figure 3.75

Hurler disease. Perisinusoidal fibrosis in the hepatic parenchyma. Note also the swollen, vacuolated appearance of the hepatocytes. (Masson trichrome stain.)

Ultrastructural studies of the liver in MPS I, MPS II and MPS III have demonstrated the presence of vacuoles of varied size in both hepatocytes and Kupffer cells; these are bounded by a single membrane and contain some electron-dense, poorly structured material. 5,1117,1124,1125 It is now generally accepted that the clear vacuoles represent lysosomes filled with acid mucopolysaccharide. Cytoplasmic vacuoles may form by mitochondrial ‘budding’ in the hepatocytes of patients with MPS I and MPS III. A peculiar crystalloid structure has been described in mitochondria of hepatocytes in MPS III. In MPS IV, Kupffer cells but not hepatocytes contain clear vacuoles. The ultrastructural changes in MPS IS (Scheie syndrome; formerly MPS V) are similar to those of MPS I and MPS II. In MPS VI there is moderate storage of electron-lucent material in lysosomes of hepatocytes, but Kupffer cells and hepatic fibroblasts contain as much acid mucopolysaccharide as in MPS I.

Noncirrhotic portal hypertension and nodular regenerative hyperplasia of the liver have been reported in dogs with MPS I. The nodular regenerative hyperplasia is associated with a venopathy of small portal veins, but the pathogenesis of these lesions is undetermined.


The deficient enzyme, aspartylglycosaminidase, is normally present in the liver and brain as well as other tissues. Several different mutations in the gene, located on chromosome 4q32-33, can result in clinical disease. Peripheral blood and bone marrow examinations show vacuolated lymphocytes that do not stain with Best carmine or PAS. The urine contains increased quantities of 2-acetamido-1 (B- l -aspartamido)-1,2-dideoxy B- d -glucose.

The diagnosis of aspartylglucosaminuria should be considered in children who present with a mucopolysaccharide-like disorder but with negative urinary screens. Clinical signs include coarse facial features (which increase with age), a protuberant abdomen with an umbilical defect, hepatosplenomegaly (which regresses with age) and the heart murmur of mitral insufficiency. Other features may include acne, macroglossia causing tooth malocclusion, hoarseness and short stature. Oral complications include dental cavities, gingivitis, candidiasis, extensive gingival overgrowth, benign odontogenic tumours or tumour-like lesions and reduced maxillary sinuses.

Appearing normal at birth, patients have accelerated growth through early puberty but end up short in stature. Mental development may be normal except for delayed speech until 5 years of age. Deterioration is noted between 6 and 15 years of age with development of clumsiness and hyperkinesia. Rapid mental and somatic deterioration occurs in adulthood; adults usually have an IQ under 40, along with uncontrollable behaviour and seizures. Patients seldom reach age 45. The neurological abnormalities are related to significant changes in the grey matter; the white matter is characterized by delayed myelination.

The incidence of aspartylglucosaminuria is highest in Finland, where the frequency of carriers is 1 in 36, and DNA testing is available. Prenatal diagnosis is achieved by enzyme assay in amniotic cells.

BMT may be beneficial. No loss of capabilities and an improvement in biochemical markers and white-matter signals on magnetic resonance imaging (MRI) were observed in two siblings followed for 5 years after BMT.

Histopathologically, liver cells contain large and small vacuoles that stain variably with PAS. Kupffer cell vacuoles do not react with PAS. There is no increase in fibrous tissue. Ultrastructurally, enlarged lysosomes are present in hepatocytes and Kupffer cells. The fine matrix background is similar to that of the mucopolysaccharidoses. Membranous structures are found in these large lysosomes, as well as electron-lucent lipid droplets. Round, electron-dense structures and, less frequently, single membranous structures of varied sizes are also present.


α-Mannosidosis is a rare autosomal recessive disease caused by a deficiency in acidic α-mannosidase A and B in various tissues and in leukocytes, which leads to the tissue accumulation and abnormally high urinary excretion of mannose-rich oligosaccharides. Diagnosis is established by analysing urinary oligosaccharides by thin-layer chromatography (TLC) to demonstrate increased mannose enrichment or by assaying α-mannosidase activity level in leukocytes. Prenatal diagnosis is made by measurement of α-mannosidase in chorionic villi or trophoblast biopsy. The gene for acidic α-mannosidase has been localized to chromosome 19p13.2-q12, and several mutations have been identified.

Patients present with psychomotor retardation. They have a distinctive coarse facies, corneal and lenticular opacities, hearing loss, hepatosplenomegaly (usually early in the disease course) and symptoms and signs of recurrent infection. Dysostosis multiplex is demonstrated by skeletal radiology in the majority of patients. Vacuolated lymphocytes are seen in the peripheral blood. About half the patients have decreased levels of serum immunoglobulin G (IgG). The susceptibility to infection may be a direct consequence of impaired leukocyte membrane recognition processes, which result from defective catabolism of substrates with α- d -mannose residues. The disorder may contribute to the onset of systemic lupus erythematosus (SLE) in predisposed patients.

Two types of α-mannosidosis are recognized. Type I is the more severe infantile type, with death between 3 and 10 years of age. Type II is a milder disease with a juvenile or adult onset. There is no known treatment, but despite recurrent infections, some patients may survive into adulthood. Allogeneic haematopoietic stem cell or T-cell-depleted peripheral blood stem cell transplantation may halt the progressive cognitive loss and reverse the pattern of urinary oligosaccharides.

Hepatocytes in mannosidosis contain PAS-negative vacuoles in the cytoplasm. Ultrastructurally, the vacuoles are bounded by a single membrane and contain amorphous and occasionally membranous and filamentous material. Vacuoles may also be present in sinusoidal lining cells. Light microscopic changes, which are nonspecific, include vacuolization of liver cells, steatosis and perisinusoidal fibrosis.


Fucosidosis is caused by a profound deficiency of tissue α1-fucosidases resulting in storage of fucose-containing glycolipids, glycoproteins and polysaccharides or oligosaccharides. There seems to be no biochemical difference in α1-fucosidase between the phenotypically different type I and II disease. The gene has been localized to chromosome 1q34. The disease results from several different mutations in one subunit of the α-fucosidase gene.

Type I fucosidosis is rapidly progressive and fatal between 4 and 6 years of age. Type II pursues a much slower course, with survival into adolescence and beyond. Common to both variants are psychomotor retardation, neurological deficits and skeletal abnormalities, Hurler syndrome-like features, hepatosplenomegaly, thickness of the skin, tendency to hernias and cardiomegaly. Almost all patients experience repeated bouts of respiratory infection. The patients with the slower evolution are characterized by angiokeratoma corporis diffusum. Diagnosis is made by TLC of urine. Prenatal diagnosis has been made by both DNA analysis and enzyme analysis but requires an experienced laboratory. BMT in one patient has been followed by progressive rise of enzymatic levels and some improvement in psychomotor development.

The gross and light microscopic changes are similar to those of Hurler disease. Both hepatocytes and Kupffer cells are vacuolated. Ultrastructurally, hepatocytes show membrane-bound vacuoles representing lysosomes that contain granular or reticulogranular material suggesting polysaccharide, as well as lamellar bodies which indicate the accumulation of complex lipids. Kupffer cells contain similar material but the vacuoles are smaller and are poor in lipid content. Biliary epithelial cells are markedly vacuolated on light microscopy and contain numerous clear vacuoles on EM.


The mucolipidoses are a group of diseases with features overlap­ping those of the mucopolysaccharidoses and sphingolipidoses. Mucolipidoses are lysosomal storage diseases with evidence of multiple primary defects of mucopolysaccharide, lipid and glycoprotein metabolism in various combinations.

Mucolipidosis I (sialidosis)

Mucolipidosis I is a rare syndrome caused by a deficiency in lysosomal sialidase (neuraminidase) resulting in defective intralysosomal catabolism of sialylated glycoconjugates and sialic acid transport out of the lysosomes. Mutations in the sialidase gene NEU1 , located on chromosome 6p21.3, result in the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides.

Two major forms are recognized. Sialidosis type I (Salla disease) is a milder, late-onset, nondysmorphic form of the disorder; patients develop visual defects, myoclonus syndrome, cherry-red macular spots, ataxia, hyperreflexia and seizures. Type II, the severe infantile form, is characterized by gargoyle-like facial dysmorphism, and dysostosis is apparent by 4 or 5 years of age. Peripheral neuropathy develops, with muscular weakness and difficulties in coordination. Findings include corneal opacities, impaired hearing and hepatosplenomegaly. Vacuolated lymphocytes are found in all cases. An increased amount of bound sialic acid is excreted in the urine, a finding that can be used as a simple screening test.

Definitive diagnosis is established by demonstration of a deficiency of sialidase in peripheral leukocytes or cultured fibroblasts of both type I and type II patients. Some patients with similar features appear deficient in both sialidase and β-galactosidase, a disease termed galactosialidosis (gene map locus 20q13). Sialidase can also be measured in cells from amniotic fluid, but special fresh preparations are necessary. A mouse model of sialidosis is currently used to evaluate enzyme replacement therapy.

Light microscopy of the liver reveals marked enlargement of portal macrophages and Kupffer cells, both of which have a foamy cytoplasm. Ultrastructurally, hepatocytes contain membrane-bound vacuoles that are electron-lucent. The vacuoles frequently contain numerous osmiophilic droplets as well as a reticulogranular or flocculent material. Riches and Smuckler also described multilamellar bodies and fragments of membrane-like material in the vacuoles. Similar vacuoles are found in Kupffer cells and to a lesser extent in endothelial cells, stellate cells and biliary epithelial cells.

Mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy)

Mucolipidosis II and III (MLII and MLIII) are biochemically related diseases with different clinical manifestations. These disorders of lysosomal enzyme targeting are caused by a defective N -acetylglucosamine 1-phosphotransferase (termed phosphotransferase) activity in Golgi compartments of the cells and lead to the impaired formation of mannose 6-phosphate recognition markers in soluble lysosomal enzymes, followed by their defective transport to lysosomes and increased excretion into the serum. The diseases are autosomal recessive and linked to chromosome 4q21-23.

MLII presents in the first year of life, with severe psychomotor retardation, early growth retardation, facial dysmorphism with characteristic gingival hyperplasia, equivocal or absent corneal clouding, hepatomegaly, skeletal dysplasia and severe dysostosis multiplex, which may resemble Pacman dysplasia. MLIII has similar symptoms, but these are milder and present later in life. Patients may survive into adulthood with the milder form of the disease.

Fibroblasts cultured from patients with MLII contain numerous dense inclusions (thus the term ‘I cell’) which are best seen on phase-contrast microscopy. Vacuolated lymphocytes of B-cell lineage in lymph nodes, spleen and kidney have been found to contain large amounts of hexosamine. Cytoplasmic vacuoles were also noted in Kupffer cells, fibroblasts in the myocardium, renal podocytes and acinar cells of the pancreas. The vacuolated cells stain positively with Hale colloidal iron method.

Diagnosis is made by measurement of the activity of the phosphotransferase in patient’s fibroblasts or by demonstration of elevated levels of serum lysosomal enzymes (as a result of hypersecretion rather than targeting into the lysosomes). Prenatal diagnosis is possible by measurement of lysosomal enzymes or phosphotransferase activity on cultured amnion cells. There is no definitive treatment, although BMT in one MLII patient has been followed by neurodevelopmental gains and prevention of cardiopulmonary complications.

Light microscopy may show no changes in hepatocytes, but there is enlargement of Kupffer cells and portal macrophages that have a foamy cytoplasm, and granulomas composed of finely vacuolated epithelioid cells may be seen in portal areas. The vacuoles are limited by single membranes and contain fibrillogranular material, membranous lamellae or lipid globules. Hepatocytes are generally only slightly affected but may contain different types of dense polymorphic inclusions. Vacuoles in hepatocytes correspond to triglyceride droplets and membranous inclusions, as well as enlarged lysosomes containing granular material on EM. Kupffer cells contain electron-dense membranous lamellae.

Vacuolated cells (leukocytes and, in MLIII, bone marrow cells) similar to those of mucolipidosis I are present. Cultured fibroblasts show inclusions resembling those of the I cells of MLII. Ultrastructural findings in the liver include slightly enlarged secondary lysosomes without storage of any abnormal material, while Kupffer cells are relatively rich in unstructured osmiophilic material.

Mucolipidosis IV (sialolipidosis)

The rare disease of sialolipidosis is postulated to result from a defect in intracellular packaging or transport of lysosomal enzymes. Chromosome assignment is to 19p13.2-13.3. Most often seen among Ashkenazi Jews, mucolipidosis IV is characterized by an early onset of corneal opacification and psychomotor retardation; a small head circumference is common. Gargoyle-like facies, skeletal deformities and organomegaly are absent.

Diagnosis is made by demonstration of characteristic inclusions in epithelial cells and conjunctival biopsies. Biochemically, mucolipidosis IV is characterized by the accumulation of gangliosides, phospholipids and acidic mucopolysaccharides in skin fibroblasts. The heterogeneity of the stored material accounts for cell vacuoles being variably stained with PAS, Sudan Black and Luxol fast blue. Prenatal diagnosis of mucolipidosis IV is performed by TEM to demonstrate lamellar bodies in endothelial cells of chorionic villi.

Ultrastructural studies of conjunctival biopsies from four patients have shown two types of abnormal inclusion bodies, both in stromal fibroblasts and in epithelial cells: (1) single-membrane-limited cytoplasmic vacuoles containing both fibrillogranular material and membranous lamellae and (2) lamellar and concentric bodies similar to those found in Tay–Sachs disease. In liver biopsy material, Berman et al. have shown that hepatocytes contain inclusions composed predominantly of concentric lamellae, whereas vacuoles in Kupffer cells have clear contents. The prenatal diagnosis of mucolipidosis IV was reported by Kohn et al. A partial ganglioside sialidase deficiency has been found in cultured fibroblasts, but it is doubtful that this is the primary enzyme defect.

Congenital disorders of glycosylation (carbohydrate-deficient glycoprotein syndrome)

A congenital disorder of glycosylation (CDG; previously called ‘carbohydrate-deficient glycoprotein syndrome’) refers to any of a heterogeneous group of metabolic defects in glycosylation pathways. Most recently discovered CDGs are caused by defects in three of the currently eight recognized major glycosylation pathways ( N -glycosylation, GPI anchor synthesis and O -mannose-based glycosylation). The list is rapidly expanding, with eight new disorders described in 2013 alone. They are divided into type 1 (defective assembly of dolichol lipid-linked oligosaccharide [LLO] chain) and type 2 (defective trimming and processing of the protein-bound glycan). In the common form of this disease (CDG type Ia), patients present with neurodevelopmental delay, hypotonia, diarrhoea leading to poor weight gain and dysmorphism (esotropia, inverted nipples, peculiar distribution of fat). Cerebellar hypoplasia is seen on computed tomography (CT). Patients have deficiencies in many serum proteins and elevated hepatocellular enzymes. The illness may progress to death in infancy, usually from infections, stroke and/or inanition.

If patients survive early childhood without diagnosis, many of the characteristic dysmorphisms become less apparent, and it becomes more difficult to recognize older patients with this disease. Older patients are characterized by nonprogressive neurodevelopmental delay with or without seizures, a stooped posture and a high risk of thrombosis. Ataxia is present in most patients. Some patients have progressive retinitis pigmentosa. Puberty is delayed in females, but not in males. Impaired linear growth is common.

The characteristic serological findings are the result of deficiencies in a wide spectrum of serum proteins, all of which are normally glycosylated. Glycosylated residues on secretory proteins perform several essential functions: they assist in achieving proper three-dimensional (3D) conformation, facilitate secretion of mature protein, protect the polypeptide from degradation and serve as recognition sites for some receptors. In all congenital disorders of glycosylation, the failure of normal glycosylation leads to deficiencies in the glycoproteins essential for coagulation and inhibition of coagulation (e.g. factor XI, antithrombin III, proteins C and S, heparin cofactor II). These defects contribute to the significant potential for strokes and transient stroke-like episodes. Defective synthesis of hormonal binding proteins or receptors cause the abnormal endocrine findings.

There is no specific screening test for CDGs. The disease should be suspected in a patient with the characteristic clinical findings or an unexplained deficiency of more than one glycosylated serum protein, particularly albumin, transferrin, antithrombin III and α1-antitrypsin. Studies for several different diseases which depend on the absence of a glycosylated protein may be abnormal, suggesting several different unrelated diseases.

Diagnosis is made by isoelectric focusing of serum transferrin, with quantification of the glycosylation variants. The absence of sialic acid residues in transferrin from patients with CDGs results in a more cathodal migration of serum transferrin. The test is simple, easily available and relatively inexpensive. Patterns of transferrin isoelectric focusing are used to determine the type of CDG, in combination with the clinical presentation. False-positive results in the transferrin assay are not common: hypoglycosylation of serum proteins can occur in chronic alcoholism, in classic galactosaemia or in hereditary fructose intolerance. In these cases, treatment of the primary disease and clearance of the abnormal metabolic by-products results in resolution of the glycosylation abnormalities. All patients with positive transferrin assays should have galactose 1-phosphate uridyltransferase testing and diagnostic testing for hereditary fructose intolerance.

In a patient with abnormal glycosylation of transferrin, the exact enzyme defect must be determined, if possible. The specific CDG syndrome is typed by a combination of the clinical presentation and the pattern of glycosylation present in isoelectric focused transferrin. CDG IIx, as well as phosphoglucomutase 1 deficiency, can resemble Wilson disease clinically; serum ceruloplasmin is low, hepatic copper concentration moderately elevated, but basal urinary copper excretion is normal.

The incidence of CDGs has been difficult to determine; estimates have ranged from 1 in 50,000 to 1 in 80,000 live births, although these may be low. Cases of CDG have been described worldwide.

Gene localization for the CDGs varies according to types. Phosphomannomutase (type Ia, PMM2 gene) has been localized to chromosome 16p13, phosphomannose isomerase (type Ib) to 15q22-qter, and N -acetyl glucosaminyltransferase II (type II) to chromosome 14q21. Further gene loci are expected as identification of mutant genes continues.

There is no reliable method for prenatal diagnosis in CDGs; a combination of enzymology and genetic linkage analysis may allow prenatal diagnosis. Therapy in CDGs is symptomatic. Oral mannose supplementation has been successful in treating several cases of CDG Ib.

Histopathological features in the liver in CDGS appear to be nonspecific. Conradi et al. studied liver biopsies from seven children and found slight to moderate fibrosis and steatosis. Similar findings have been reported recently by Socha et al. In the case report by Jaeken et al., a biopsy at the age of 5.5 months showed microvesicular steatosis and periportal fibrosis. A second biopsy at age 1 year revealed more pronounced fibrosis and ductular reaction, a picture resembling congenital hepatic fibrosis. Postmortem findings in twins with the deficiency included diffuse steatosis (particularly in periportal areas) and fibrosis in one twin and cirrhosis in the other. Ultrastructural findings in the cases of Conradi et al. included lysosomal vacuoles with concentric electron-dense membranes, and variable electron-lucent and electron-dense material in liver cells.

Endoplasmic reticulum storage diseases

The term ‘endoplasmic reticulum storage disease’ was first used by Callea et al. to emphasize that a group of inborn errors of metabolism have as a common denominator the ER as the site of protein retention. The disorders affect secretory proteins: many of them, in particular α1-antitrypsin, α1-antichymotrypsin and C1 inhibitor, belong to the serine proteinase inhibitors (serpins), a superfamily of proteins whose gene cluster is located on chromosome 14q32.1. These storage diseases result from molecular abnormalities leading to protein misfolding and polymer formation, which prevents protein secretion. The resulting protein aggregates accumulate in the hepatocyte ER, with consequent low levels of the corresponding protein in the plasma.

α1-Antitrypsin deficiency

α1-Antitrypsin (α1-AT) is a plasma glycoprotein of approximately 52 kD which is synthesized almost exclusively by hepatocytes. It is encoded by SERPINA1 on chromosome 14. α1-AT is a competitive inhibitor of leukocyte elastase with reaction kinetics favouring elastase complexing with α1-AT rather than with its substrate elastin. The major factors controlling serum levels of α1-AT are inflammatory stimuli, although some liver diseases, oestrogenic hormones and androgenic hormones can also alter serum levels.

The association of panlobular emphysema with α1-AT deficiency was recognized by Laurell and Eriksson in 1963. Subsequently, Sharp et al. reported cirrhosis in 10 children from six different kindreds with α1-AT deficiency. Liver disease was subsequently shown to be most frequently associated with the α1-AT variant Z. α1-AT deficiency was one of the first specific disorders to be dissected from the ‘idiopathic neonatal hepatitis’ category.

The diagnosis of α1-AT deficiency should be considered in all patients with undefined liver disease by measuring the serum level of α1-AT. In homozygotes, low levels of serum α1-AT (normal, >1.0 g/L) suggest the diagnosis of deficiency. However, α1-AT is an acute-phase reactant, and serum levels may increase as a result of hepatic inflammation, leading to normal values in heterozygotes. The diagnosis can be confirmed by protease inhibitor (PI) typing of the patient. PI typing is based on amino acid variations, which produce variably charged proteins that can be detected by isoelectric focusing in polyacrylamide gels. At least 90 allelic variants of α1-AT have been described by isoelectric focusing or DNA sequencing. By PI typing, the three most common alleles are M (normal) and the deficiency alleles S (plasma levels about 60% of normal) and Z (plasma levels about 15% of normal). Liver disease is predominantly seen in individuals carrying the Z allele, of whom only a relatively small proportion ever develops liver disease.

The PI*Z allele is found in approximately 1–2% of Caucasians of northern European ancestry, being highest in Scandinavian populations. It is virtually absent in black and Asian populations, but a review of epidemiologic surveys shows that it can affect individuals in all racial subgroups worldwide. In the West, α1-AT deficiency is estimated to affect about 1 in 2000–5000 individuals.

The phenotypic expression of α1-AT deficiency depends on the protein production of both alleles: inheritance is codominant. Most allelic variants are the result of an amino acid substitution in the polypeptide chain. In the Z allele, the substitution of Lys342 for Glu342 directly inhibits secretion of α1-AT, which then accumulates in the ER. The marked variation in phenotypic expression of the liver disease results from genetic modifiers and environmental factors that influence the intracellular disposal of the mutant glycoprotein.

Neonates and children

Clinically, neonates who develop liver disease present with conjugated hyperbilirubinaemia. Severe cholestasis may be associated with acholic stools and must be differentiated from extrahepatic biliary atresia. Cholestasis usually resolves by 6 months of age without therapy, but a proportion of infants develop chronic liver disease. Up to 10% of neonates may have paucity of the intrahepatic bile ducts, with persistence of cholestasis and development of pruritus. A very small proportion of children with α1-AT deficiency never develop neonatal hepatitis syndrome but present clinically in the preschool age bracket, usually as toddlers, with hepatosplenomegaly caused by cirrhosis.

In prospective studies on the incidence of liver disease in PI*Z individuals, 127 homozygous PI*Z children and 54 PI*SZ children were followed into adolescence by physical examinations and routine LFTs. Initially, symptomatic liver disease was found in only 11% of infants. In addition, 75% of all the infants with a PI*Z phenotype had elevated serum alanine aminotransferase (ALT) levels. By age 12 years, only 33% of the infants who presented with liver disease had ALT elevation, although 14% of patients who had had no liver disease in infancy now had an elevated ALT. Thus overall, evidence of hepatocyte injury decreased as the children approached the teenage years. Chronic liver disease led to death in only 2.5% of PI*Z individuals by age 12 years. In PI*SZ children, elevated ALT levels were not seen when serum α1-AT levels were at least 40% of normal. In another study of 85 children with neonatal hepatitis and α1-AT deficiency, poor outcomes were associated with ALT >260 U/L, prothrombin time >16 s and α1-AT concentration <0.25 g/L. On follow-up, persisting total serum bilirubin elevations and abnormal coagulation tests associated with abnormal factor V levels predicted death within 1 year and served as criteria for liver transplant evaluation.

In a survey of children with α1-AT liver disease in the United Kingdom, clinical outcomes fell into four categories. Approximately 25% of the individuals gradually improved and appeared normal at ages ranging from 3–10 years. In contrast, 25% had persistently abnormal serum aminotransferases levels but were otherwise well; 25% had raised aminotransferase levels with enlarged liver and spleens; and 25% died of cirrhosis or required transplantation at ages ranging from 6 months to 17 years.

PI*MZ and PI*SZ are not associated with significant chronic liver disease in children; however, some may have elevated serum aminotransferases, which typically normalize by age 10 years.



α1-AT-deficient individuals who escape severe liver disease in childhood generally are free of clinical liver disease until late in life. From ages 20–40 years, the incidence of liver disease in the α1-AT-deficient population is approximately 2%. From 41–50 years, the incidence increases to approximately 5%, with a 2:1 male predominance. Between 51 and 60 years of age, the reported incidence is 15% in males and 0% in females. In an autopsy series of 246 Swedish PI*Z patients, the incidence of cirrhosis was 12%, but this increased to 19% in individuals over age 50. Registry data show that emphysema and cirrhosis are the most common underlying causes of death (72% and 10%, respectively), with malignancy and diverticulitis accounting for 3% of deaths each. Death usually occurs within 2 years of the clinical diagnosis of cirrhosis.

Swedish investigators have documented an increased incidence of malignancy, with or without cirrhosis, in adult homozygous PI*ZZ patients. In this study, the odds ratio in α1-AT deficiency for cirrhosis was 7.8 and for hepatocellular carcinoma (HCC) was 20. Although the mechanisms of carcinogenesis are not clear, recent studies suggest that hepatocytes with marked accumulation of α1-AT globules engender a cancer-prone state, by surviving with intrinsic damage and by chronically stimulating in ‘trans’ adjacent, relatively undamaged hepatocytes that have a selective proliferative advantage.


Prospective, non-disease-oriented population surveys do not demonstrate a risk in heterozygotes (PI*MZ) for either liver or lung disease. However, liver disease population studies in England, Sweden and Norway have shown an increased frequency of the PI*MZ phenotype in patients with cryptogenic cirrhosis. It is not entirely clear to what extent the heterozygous state of α1-AT deficiency contributes to liver disease in the setting of another chronic liver disease, but in one large study, α1-AT heterozygosity was associated with an increased risk of liver decompensation in individuals with hepatitis C or fatty liver disease.

Overall, PI heterozygosity does not appear to be a major independent risk factor for HCC. In contrast, several studies found an association between PI*Z heterozygosity and cholangiocarcinoma.

As one example, in a series of 317 consecutive cancers, cholangiocarcinomas or combined HCC and cholangiocarcinoma were seen significantly more frequently in PI*Z-associated carcinomas (57.9%) than in non-PI*Z-associated carcinoma (27.2%). Interestingly, cirrhosis was found in only 3 of the 19 PI*Z-associated carcinomas. Other studies also report that cholangiocarcinomas in noncirrhotic livers can be associated with α1-AT deficiency.

Rare variants

Overall, rare variants causing α1-AT deficiency are seen in about 1% of cases. Interestingly, many of these rare variants are initially misclassified on PI* typing. In some cases that were initially misclassified, unexpectedly low serum α1-AT levels for the initial PI* type suggested the need for further study. Overall, the most common of the rare variants is PI*M malton . PI*M malton is most likely to cause clinical lung and/or liver disease when individuals are PI*M malton homozygous or have a PI*M malton /Z phenotype. However, rare cases of severe liver disease have been reported in individuals heterozygous for Pi*M malton . PI*S iiyama is molecularly similar to Pi*M malton and is also capable of retention in hepatocytes.

General microscopic findings

Morphologically, the hallmark of Z-type α1-AT is PAS-positive, diastase-resistant globules in periportal hepatocytes ( Figs 3.76 and 3.77 ). Overall, the morphology of the globules is essentially identical for Z and other α1-AT deficiency alleles. The globules represent the retention of polymers of the abnormal protein within the rough ER ( Figs 3.78 and 3.79 ). The globules are located in zone 1 hepatocytes in the noncirrhotic liver but may show a more diffuse distribution when a liver becomes cirrhotic. The globules may show a patchy distribution in both noncirrhotic and cirrhotic livers, so the entire biopsy should be examined on D-PAS stain. Histological data are sparse, but rare phenotypes such as PI*M malton and PI*M duarte and S iiyama also produce globules similar to those with Z alleles. Likewise, histological descriptions are sparse for individuals with the PI*QO (null) phenotype. Individuals producing no α1-AT would have no globules, whereas those producing truncated but unsecreted forms of α1-AT (e.g. PI*QO hongkong ) might have globules if the rate of protein degradation in the ER were sufficiently slow. The S-deficiency allele polymerizes at a rate much lower than that of Z or M malton , and therefore it does not ordinarily accumulate in the liver. However, typical globules were reported in a PI*SS individual who also had alcoholic liver disease. Several early studies reported the accumulation of α1-AT globules in hepatocytes, despite PI*M phenotypes, in individuals with chronic inflammatory conditions. However, these reports are rather exceptional, and most individuals with significant liver inflammation and systemic illnesses do not develop α1-AT globules in the absence of an abnormal α1-AT genotype. Zone 3 eosinophilic globules have also been reported in congestive hepatopathy. In some cases the globules were either weakly positive on immunostaining for α1-AT or were positive for both α1-AT and fibrinogen, suggesting the globules represent a more generalized protein metabolism problem. Also of note, in congestive hepatopathy, the globules are located in zone 3 and not zone 1.

Figure 3.76

Homozygous α1-antitrypsin (Pi*ZZ) deficiency. Eosinophilic globules of varied size are present in the cytoplasm of periportal liver cells. (H&E stain.)

Figure 3.77

α1-Antitrypsin (α1-AT) deficiency. The globules are intensely PAS positive. (PAS stain after diastase digestion.)

Figure 3.78

Presence of α1-AT in the globules is confirmed by immunohistochemistry. Note that the periphery of the larger globules is more intensely stained than the centre. (PAP technique.)

Figure 3.79

α1-AT deficiency. Electron micrograph showing moderately electron-dense, amorphous material in the cisterns of the dilated endoplasmic reticulum of a hepatocyte. The larger deposits have a serrated edge. Note the ‘halo’ surrounding the deposits. (×6500.)

Histological findings


The eosinophilic globules of α1-AT can be subtle on H&E staining. In particular, they can be absent or very difficult to detect in infants less than 12 weeks of age, and the diagnosis in the first few months of life rests on PI phenotyping. Immunoperoxidase staining for α1-AT can occasionally help in ambiguous cases, but the stains can be challenging to interpret, because there can be background staining in inflamed, reactive hepatocytes without α1-AT deficiency. Inclusions can be found in bile duct epithelium.

The histological features of α1-AT deficiency in cholestatic infants can mimic neonatal hepatitis or extrahepatic biliary atresia, since the most common findings are lobular cholestasis, bile ductular proliferation and varying degrees of fibrosis ( Figs 3.80 and 3.81 ). This emphasizes the need for screening for α1-AT deficiency by PI typing before a patient is treated by a Kasai portoenterostomy. The distinction between these two entities is made more difficult by the often small size of the extrahepatic biliary tree in patients with α1-AT deficiency, likely because of disuse atrophy secondary to cholestasis. Inexperienced surgeons may misinterpret operative cholangiograms from these patients as showing biliary atresia and perform an unnecessary Kasai portoenterostomy.

Figure 3.80

Neonatal hepatitis syndrome in homozygous an α1-AT-deficient infant, 3 weeks old. Canalicular cholestasis is present, with minimal lobular and portal inflammation. Note presence of haemopoietic foci and apparent absence of bile duct. (H&E stain.)

Figure 3.81

Neonatal hepatitis syndrome in a homozygous α1-AT-deficient infant, 4 weeks old. Portal tract oedema, ductular reaction and inflammation with cholestasis in the adjacent parenchyma suggest the differential diagnosis of a distal obstructive cholangiopathy. (H&E stain.)

In α1-AT deficiency, inflammation is rarely a prominent feature. However, residual extramedullary haemotopoiesis may be present, depending on the age of the neonate, and can be confused with inflammation. Hepatocyte necrosis, if present, is minimal. Interestingly, mild zone 1 steatosis is also a common feature ( Fig. 3.82 ).

Figure 3.82

α1-AT deficiency. Liver biopsy specimen taken from an 8-week-old infant. There is portoseptal fibrous expansion with mild ductular reaction and inflammation. Note the minimal erosion of the parenchymal limiting plate, but significant periportal/periseptal steatosis. (H&E stain.)

Cholestasis can be seen in hepatocytes, canaliculi and even bile ductules. Hepatocytes vary in size, with some tendency for pseudoacinar formation. Giant cell transformation of hepatocytes can be present as in idiopathic neonatal giant cell hepatitis. Also of note, up to 10% of biopsies in the setting of α1-AT deficiency may have paucity of the intrahepatic bile ducts (see Fig. 3.80 ) and chronic cholestasis ( Fig. 3.83 ), mimicking ‘paucity of intrahepatic bile duct’ disorders. In fact, a patent but narrow extrahepatic biliary tree is often found at liver transplantation for α1-AT deficiency, and a Roux-en- Y procedure may be necessary rather than a duct-to-duct anastomosis. Risk factors for rapidly progressive liver disease include a prominent ductular reaction, bridging fibrosis or cirrhosis on the initial biopsy specimen. The cirrhosis is typically mixed macro- and micronodular in type ( Figs 3.84–3.86 ).

Figure 3.83

Abundant copper accumulation ( red granules) in periseptal liver cells (rhodanine stain).

Figure 3.84

α1-AT deficiency. Cirrhotic liver removed at transplantation of a 6-year-old child. A, The liver surface is greatly deformed by large, protruding nodules separated by deep grooves. The tip of the gallbladder is seen ( lower part of the field). B, Cut section of the same liver shows large areas of re-expanded parenchyma separated by broad scars, a pattern reminiscent of focal nodular hyperplasia.

Figure 3.85

α1-AT deficiency. Cirrhotic nodules are surrounded by a ring of dense collagen and separated by loose fibrous tissue. Note lack of interface hepatitis and of significant inflammation. (H&E stain.)

Figure 3.86

Many α1-AT globules are present in hepatocytes throughout the cirrhotic nodules in the case illustrated in Fig. 3.85 (PAP technique).


Other than the characteristic globules, the histopathological findings are nonspecific in adults with both homozygous and heterozygous α1-AT deficiency. There can be minimal to mild portal inflammation with a predominance of lymphocytes. Fibrosis varies from none to cirrhosis. A ductular reaction is only obvious when cirrhosis is present. Mild fat accumulation is often seen in the periportal hepatocytes, but ballooning, cholestasis and iron accumulation are all uncommon. However, cirrhotic livers with α1-AT deficiency and moderate to severe iron accumulation (3–4+ on a scale of 4) can be associated with HFE mutations. The α1-AT globules are generally less prominent in heterozygous livers. In many cases, additional independent disease processes may be present, such as fatty liver disease or chronic viral hepatitis, and the histological findings of these diseases can dominate the histological changes.

Occasionally, individuals undergoing cadaveric LT can receive grafts heterozygous for the Z α1-AT deficiency allele. One study identified a frequency of 0.8% for the Z α1-AT allele in presumed healthy donor cadaveric livers. However, the globules may not be evident for up to several years after transplant, with no globules in earlier biopsies. Data are insufficient at this point to indicate clearly whether there is significant impact on the post-transplant course.

Medical therapy

Medical therapy is mainly supportive, with attention to malabsorption associated with severe cholestasis. Ursodeoxycholic acid (UDCA) may significantly improve clinical status and liver test results in some children, but no beneficial effect was shown in children with the most severe liver disease. The drug 4-phenylbutyrate has been proposed as a pharmacologic protein chaperone. More recently, drugs which specifically enhance autophagy have been shown to improve liver disease and are currently in clinical trial. These drugs include rifamycin and carbamazepine. Infusions of recombinant α1-AT are neither indicated for nor effective in treating the liver disease.

Liver transplantation corrects the genetic defect and cures the patient. α1-AT deficiency is the second most frequent indication for LT in children and the most common inherited disease for which LT is performed. Although α1-AT deficiency does not recur in the allograft, globules can develop over time in allograft livers obtained from living-related donors or deceased donors with deficiency alleles, even if no globules are evident on liver graft biopsies at LT.

α1-Antichymotrypsin deficiency

α1-Antichymotrypsin (α1-ACT) deficiency is familial, with a gene frequency of up to 0.003. Deficient individuals described thus far are heterozygotes; homozygosity may be incompatible with life. Diagnosis is by quantification of serum α1-ACT levels, with demonstration of a level of approximately 64% of normal. The inheritance is autosomal codominant. The α1-ACT gene maps to chromosome 14q31-32, which is the same site as the α1-antitrypsin locus. Treatment at present is supportive.

In adults, low levels of α1-ACT can be associated with cryptogenic chronic hepatitis. In addition, there is an increased incidence of cryptogenic liver and lung disease among relatives with the α1-ACT deficiency. In a U.S. study, a strong association was found between α1-ACT deficiency and cirrhosis, particularly HCV-related cirrhosis. Thus 34% of patients with HCV-related cirrhosis were α1-ACT deficient, compared with 11% of patients with cirrhosis of other aetiologies. Combined heterozygosity for both PI*SZ α1-AT and α1-ACT deficiencies may enhance the risk of developing liver disease.

On liver biopsy, the zone 1 hepatocytes can have globular inclusions. In most cases the inclusions are smaller than those of α1-AT deficiency, and they are usually not visible on H&E stains. On D-PAS stain the globules are weakly positive. Immunohistochemical staining can highlight granular inclusions in liver cells, particularly those adjacent to portal areas and fibrous bands. A fluffy material has been noted in dilated cisterns of the ER ultrastructurally.

Afibrinogenaemia and hypofibrinogenaemia

Afibrinogenaemia and hypofibrinogenaemia are very rare diseases affecting approximately 1–2 individuals per 1 million population; consanguinity is common. Plasma fibrinogen levels are low or absent and can also be functionally impaired. The inheritance of afibrinogenaemia is likely to be autosomal recessive, but hypofibrinogenaemia may be either autosomal recessive or autosomal dominant. The fibrinogen gene cluster is located on chromosome 4q28-31 and consists of three related fibrinogen genes: α ( FGA ); β ( FGB ) or γ ( FGG ). Mutations in the α-fibrinogen gene ( FGA ) account for the majority of cases of congenital afibrinogenaemia, although mutations in all three fibrinogen genes, FGG , FGA and FGB , can cause congenital afibrinogenaemia.

In afibrinogenaemia, where there is complete absence of fibrinogen, individuals often present with umbilical bleeding. Other complications can include intracranial haemorrhage following mild trauma, severe epistaxis, gingival and gastrointestinal bleeding, ecchymoses and spontaneous splenic rupture. Affected females may experience menorrhagia, recurrent abortions and postpartum haemorrhage. Other findings include greatly prolonged prothrombin time (PT), partial thromboplastin time (PTT) and thrombin time (TT). The erythrocyte sedimentation rate (ESR) is also very low, since fibrinogen is the primary factor affecting this test. Mild thrombocytopenia is present in about 25% of patients. The differentiation of primary from secondary hypofibrinogenaemia (e.g. drug induced) is important.

In hypofibrinogenaemia, fibrinogen levels are low, yet variable, which accounts for the differences in symptomatology. The majority of patients are asymptomatic, and elevated serum aminotransferase levels may be the only manifestation of liver disease. Perhaps counterintuitively, some individuals have an increased risk for thrombosis due to additional mutations, such as factor V Leiden mutations, or because the fibrinogen mutations lead to fibrinolysis-resistant fibrin polymers.

Although histological data are sparse, the inclusions in hypo­fibrinogenaemia can vary in size, shape and color, presumably reflecting differences in underlying mutations. The inclusions can be eosinophilic, small and round, or can manifest as somewhat larger eosinophilic globules similar in size to those of α1-AT deficiency ( Figs 3.87–3.89 ). In other cases, the globules are large and pale and resemble that of ground-glass inclusions seen in chronic HBV infection, or the pseudo-ground-glass change seen in drug reactions. The globules are negative or weakly positive on D-PAS. They are also positive with the phosphotungstic acid-haematoxylin stain. Immunoreactivity to fibrinogen antibody can be demonstrated (see Fig. 3.89 ). Ultrastructurally, the cisterns of the rough endoplasmic reticulum (RER) are filled with densely packed, curved tubular structures arranged in a fingerprint-like pattern ( Fig. 3.90 ). Rarely, inclusions of hypofibrinogenaemia may be found incidentally in patients without clinical evidence of hypofibrinogenaemia and in whom mutational analysis has not been found or investigated. Conversely, an afibrinogenic patient with proven mutation has shown no evidence of storage in liver.

Figure 3.87

Fibrinogen storage disease. Hepatocytes contain eosinophilic globules of varied size. (H&E stain.)

Figure 3.88

The same case as illustrated in Fig. 3.87 . Some globules are vacuolated, and others have a dark core. (PAS stain after diastase digestion.)

Figure 3.89

The same case as illustrated in Figs 3.87 and 3.88 . Globules are immunoreactive to fibrinogen antibodies. (PAP technique.)

Figure 3.90

Electron micrograph of the same case as illustrated in Figs 3.87–3.89 . The fibrinogen inclusion in the liver cell has a fingerprint-like pattern. (×31,700.)

The inclusions are the main finding in hypofibrinogenaemia and afibrinogenaemia, and the biopsies typically do show significant inflammation or other findings, unless there is an additional superimposed disease process. Some patients progress to cirrhosis, although in afibrinogenaemia the bleeding diathesis is the more common cause of death.

Treatment consists of administration of plasma-derived fibrinogen concentrate, cryoprecipitate or fresh-frozen plasma.

Antithrombin III deficiency

This uncommon disorder has an autosomal dominant inheritance with complete penetrance. The gene for antithrombin III is located at chromosome 1q 23-25. There is a propensity for venous thromboembolism, ranging from superficial thrombophlebitis to pulmonary embolism. The deficiency may be complicated by the Budd–Chiari syndrome. The deficiency does not lead to excess mortality, according to Rosendaal et al., who do not recommend prophylactic anticoagulation. Antithrombin concentrates are used in high-risk clinical settings. An 8-month-old infant with antithrombin III deficiency developed multiple large venous and arterial thromboses and E. coli sepsis. He had a micronodular cirrhosis, and liver cells contained multiple, eosinophilic, PAS-positive globules resembling those of α1-antitrypsin deficiency.

Disorders of amino acid metabolism

Hereditary tyrosinaemia type 1

Hereditary tyrosinaemia type 1 (HT1) is caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine degradation pathway ( Fig. 3.91 ). This leads to accumulation of highly reactive intermediate metabolites such as maleyl- and fumarylacetoacetate, which are toxic and mutagenic within the liver. The secondary metabolite succinylacetone is excreted in the urine; a high urinary level is a diagnostic feature. Succinylacetone also has an inhibitory effect on porphobilinogen synthase, accounting for episodes similar to acute hepatic porphyria, with neurological crises and excretion of 5-aminolaevulinic acid in the urine ( Fig. 3.91 ).

Figure 3.91

Tyrosine metabolic pathway. In tyrosinaemia type 1, deficient fumarylacetoacetate hydrolase (FAH) leads to accumulation of highly reactive intermediate metabolites (**) and production of succinylacetone, which is both excreted in urine (diagnostic test) and having an inhibitory effect on porphobilinogen (PBG) synthase (acute porphyria crisis and urinary excretion of 5-aminolaevulinic acid). NTBC (nitisinone) therapy blocks the degradation pathway, preventing reactive metabolite formation.

Clinical presentation of HT1 is heterogeneous, even within the same family. Progressive liver damage is often not reflected in routine liver tests. The earlier the presentation, the worse is the prognosis. Patients present with vomiting, diarrhoea, failure to thrive, abdominal distension, anaemia, bleeding and rickets. Findings consistent with advanced liver disease include ascites, hepatosplenomegaly, peripheral oedema, hypoglycaemia, hypoproteinaemia and severe coagulopathy, as well as urinary findings compatible with the renal tubular changes of Fanconi syndrome.

Liver crisis, often precipitated by an infection, typically presents in the first 2 years of life and is characterized by increased severity of hepatic dysfunction. Coagulation tests (PT, PTT) worsen and are not responsive to vitamin K; jaundice heralds the terminal event.

Neurological crisis may also be precipitated by infection. Children become irritable and less active and develop severe pain, often localized to the legs. Paraesthesias, hypertension, tachycardia and paralysis may develop. The paralysis may progress to complete flaccid quadriplegia, including paralysis of the diaphragm, and may require mechanical ventilation. Cerebrospinal fluid analysis is unremarkable. This complication may account for 10% of deaths, but the incidence varies greatly. Nerve toxicity due to δ-aminolaevulinic acid retention is thought to be responsible ( Fig. 3.91 ).

More than 80% of HT1 patients have renal tubular defects. Histologically, 25% have some degree of glomerulosclerosis, and 50% have mild to moderate interstitial nephritis and fibrosis. Nephrocalcinosis can be detected by ultrasound in one-third of patients; 50% may have an abnormal glomerular filtration rate. Hypertrophic cardiomyopathy has been documented.

Hepatocellular carcinoma occurs in 10–37% or more of HT1 patients, based on observations obtained before the routine use of the drug nitisinone, and may rarely be the presenting feature. HCC is associated with cirrhosis and may occur as early as the first year of life or in adulthood. Serum alpha-fetoprotein (AFP) screening may be helpful, but the complication has been seen with normal AFP levels.

HT1 occurs worldwide, although it is particularly common in individuals of French-Canadian descent and is most common in the Lac St.Jean–Chicoutimi region of the Canadian province of Quebec. A variety of mutations in the FAH gene have been described; 100% of patients in the province of Quebec and 28% elsewhere carry a splice mutation in intron 12 of the FAH gene. The gene is found on chromosome 15q 23-25. Prenatal diagnosis is available. Early diagnosis by screening of Guthrie spots is available ; it permits immediate introduction of treatment so that most of the severe liver disease can be abrogated.

Interestingly, many patients with HT1 have a mosaic pattern of FAH expression in liver tissue. This phenomenon has been explained by a spontaneous reversion of the mutation in one allele to a normal genotype. There seems to be no evidence that it could be of maternal origin due to transplacental cell trafficking and subsequent fusion. In one study, the extent of mutation reversion of the FAH gene in the liver correlated inversely with the clinical severity of the disease, suggesting that the corrected hepatocytes play a substantial protective role in liver function.

NTBC (2-(2-nitro-4-trifluoromethyl-benzoyl)-1, 3-cyclohexanedione), or nitisinone, inhibits 4-hydroxyphenylpyruvate, the enzyme deficient in tyrosinaemia type 2, and prevents the accumulation of toxic metabolites (see Fig. 3.91 ). Since the first clinical trial of NTBC therapy in 1991, more than 220 HT1 patients have been treated with the drug and shown initial good response, including normalization of liver function and metabolic parameters in most patients. Recent experience in Quebec indicates that commencing nitisinone treatment before the patient is 1 month old eliminates acute complications of the disorder. Long-term studies are still necessary to determine whether NTBC may prevent or only delay long-term complications, and to examine the possible risks of the drug. Of the patients who started receiving NTBC early in life, two (1%) developed HCC during the first year of treatment, but no further cases occurred among these patients followed for up to 9 years. In one patient, liver tumour with lung metastases developing at age 15 months was reclassified as hepatoblastoma after a good response to chemotherapy and partial hepatectomy, which resulted in a 12-year disease-free period. However, with late onset of treatment, the risk of malignancy remains despite NTBC therapy, and lifelong surveillance is required. Besides an increase in AFP, a slow AFP decrease and never-normalizing levels of AFP are important predictors of liver cancer development in later life.

Dietary therapy must be maintained despite NTBC therapy. Corneal lesions reported in 13% of treated patients can be controlled by using a low-tyrosine, low-phenylalanine diet. The unique renal tubular dysfunction of HT1 resulting in hypophosphataemic rickets is usually responsive to dietary therapy, but it is completely reversed by NTBC therapy. However, the marked coagulopathy, neurological complications simulating porphyria and development of HCC are not responsive to dietary therapy alone. Liver transplantation remains the treatment of choice for medical treatment failures. Renal disease does not progress after LT ; however, renal impairment associated with HT1 may be greater than appreciated.

Pathological aspects of HT1 have been described in several reports. The liver, at autopsy or removed at transplantation, is slightly to moderately enlarged, yellow, firm and nodular ( Fig. 3.92 ). The microscopic features include fatty change, cholestasis, pseudoacinar transformation of hepatic plates, pericellular and periportal fibrosis, variable haemosiderosis, extramedullary haematopoiesis and varying-sized foci of nodular regeneration, some qualifying as macroregenerative nodules ( Figs 3.93–3.95 ). The regenerating nodules, which appear as high-attenuation foci on CT scans, are difficult to differentiate from multifocal HCC. The nodules often show more fat accumulation than the adjacent liver ( Fig. 3.93 ), and some may exhibit dysplastic changes ( Fig. 3.94 ). Periportal ductular reaction, although present, is usually not striking. Cirrhosis may be micronodular, macronodular or mixed. Jaffe has noted the prominence and variegated colours of the macronodules (yellow, tan, green) (see Fig. 3.92 ). Transition from a micronodular to a macronodular cirrhosis has been documented. Liver cell dysplasia, both the large and small cell varieties, is frequently observed, and the distinction between dysplastic nodules and HCC may be difficult, if not impossible.

Figure 3.92

Tyrosinaemia. Section of explanted liver showing cirrhotic nodules of varied sizes and colour.

Figure 3.93

Tyrosinaemia. The same case as illustrated in Fig. 3.92 . A segment of a macroregenerative nodule shows marked steatosis. (H&E stain.)

Figure 3.94

Tyrosinaemia. Cellular and canalicular cholestasis with neocholangioles. (H&E stain.)

Figure 3.95

Tyrosinaemia. The same case as illustrated in Fig. 3.92 . Another macroregenerative nodule shows dysplastic changes with increased cell basophilia and nucleocytoplasmic ratio.

Cytogenetic studies on skin fibroblasts from a patient with HT1 and HCC have demonstrated chromosome breakages in 71% of cells. DNA ploidy abnormalities detected in three patients may be a useful marker for early malignant transformation. Ultrastructural studies have confirmed the presence of fat in hepatocytes and the cholestasis, as well as a number of nonspecific changes. In the case studied by Jevtic et al., hepatocytes surrounded a central canaliculus forming tubules; the cells were joined by many desmosomes. The basal portions of the cells were not covered by microvilli or a space of Disse, and basement membrane-like material was present. Hepatocellular mitochondria can be greatly increased in number in HT1 and may show mild to moderate pleomorphism with randomly oriented cristae. Peroxisomes are frequently enlarged and may contain nucleoids or small lipid droplets.

In the pancreas, about 50% of HT1 patients have hyperplasia of the islets of Langerhans. Renal changes include interstitial oedema, tubular dilation with vacuolar and granular degeneration, loss of glomeruli and hypertrophy and hyperplasia of the juxtaglomerular complex.

Mouse models of HT1 are used to study mechanism of injury and potential therapy.

Congenital hyperammonaemia syndromes and urea cycle disorders

Defects in any of the enzymes of the urea cycle ( Fig. 3.96 ) may lead to hyperammonaemia and encephalopathy at any age, although presentation is typically in the neonatal period. Such patients are normal at birth but by 24 hours of age develop irritability, poor feeding, vomiting, lethargy and respiratory distress. This is quickly followed by hypotonia, seizures, coma and respiratory arrest. If treatment is not instituted early, neurological function may be irreversibly compromised. Patients may be misdiagnosed as having respiratory distress syndrome, sepsis or intraventricular haemorrhage in the newborn period, or Reye-like syndrome when presentation is delayed. Thus the evaluation of an acutely ill newborn should include the possibility of a hyperammonaemia syndrome, more so in families who have already lost children to one of the more common urea cycle disorders. These disorders are autosomal recessive except for ornithine transcarbamylase (OTC) deficiency, which is X-linked.

Figure 3.96

Urea cycle metabolic pathway. Shaded oval indicates parts of the reaction inside mitochondrion.

The urea cycle disorders have been comprehensively reviewed. Among the classic urea cycle disorders, the only one with significant liver disease is argininosuccinic aciduria (ASA). The urea cycle enzyme that is deficient in this disorder, argininosuccinic lyase, is located on chromosome 7. Several mutations within this gene may result in argininosuccinate lyase deficiency. The enzyme protein may be absent in the liver ; it can also be measured in red blood cells, fibroblasts and amnion cells, with substrate accumulation occurring in the fluid of cultured cells. Liver disease is manifested clinically by hepatomegaly and by serum ALT elevation and severe fibrosis on liver biopsy. Marked macrovesicular steatosis or clarification of hepatocyte cytoplasm has been noted. Ultrastructural changes include dilation of the RER or smooth endoplasmic reticulum (SER) and the presence of mega­mitochondria in zones affected by steatosis.

Despite the structural changes, survival in ASA patients and in those with argininosuccinate synthetase deficiency (ASS, also known as ‘citrullinaemia type 1’) is much better than in those with OTC and carbamyl phosphate synthetase (CPS) deficiencies. Nonspecific changes, including steatosis and cholestasis, have been observed in patients with CPS1 and ASS deficiency, respectively.

Pathological studies of the liver in males with OTC deficiency have generally been normal. Occasional case reports suggest some mitochondrial and peroxisomal changes. Acquired abnormalities, more likely to be seen over time, are documented in females who have varying degrees of deficiency. At the light microscopic level, the mild changes include fat accumulation, inflammation, interface hepatitis and mild periportal fibrosis. Organelles in the hepatocytes usually are normal. Intracytoplasmic D-PAS+ inclusions were identified at autopsy of a neonate affected by a chromosomal microdeletion including the OTC gene. Of note, OTC deficiency manifested clinically in both recipient and donor after living-related transplantation.

In the neonatal period, management of hyperammonaemia in the intensive care setting is often necessary. Long-term treatment consists of a combination of dietary restriction of protein, bypassing the defect by administering arginine and, on occasion, the use of ammonia scavengers.

Liver transplantation is the definitive treatment for patients with severe variants, progressive liver disease, and for some patients whose disease manifestations have proved very difficult to control with conventional therapy.

Citrullinaemia type 2 is caused by deficiency of citrin, a transporter involved in the urea cycle ( Fig. 3.96 ). Clinical features depend on age. In the infant it may present clinically as a conjugated hyperbilirubinaemia disorder, also called ‘neonatal intrahepatic cholestasis with citrin deficiency’ (NICCD). In older children it may present as failure to thrive and dyslipidaemia (FTTDCD). In adults it causes hyperammonemia and neuropsychiatric symptoms.


Cystinosis is a rare autosomal recessive disease (1 in 100,000–200,000 live births). It is characterized by the accumulation of l -cystine crystals in lysosomes from a defective carrier-mediated transport system for cystine. The cystine accumulates in the eye, reticuloendothelial system, kidney and other internal organs. The gene responsible for cystinosis, located on chromosome 17p13, is called CTNS ; it encodes a lysosomal membrane protein named cystinosin, whose cysteine transport activity is hydrogen ion (H + ) driven. Several mutations of CTNS are associated with cystinosis. Massive hepatomegaly has been reported in this disorder, with the incidence of detectable hepatomegaly as high as 42% in patients over 10 years of age. Most patients do not have significant liver dysfunction.

The most severe clinical form is nephrogenic (infantile) cystinosis. Patients are normal at birth, but symptoms of renal tubular dysfunction (Fanconi syndrome) develop by 6–12 months and progress to renal failure by 10 years of age. This tubular defect accounts for the presenting symptoms of polyuria, polydipsia and failure to thrive. Affected children are often hospitalized because of dehydration, acidosis resulting from potassium and bicarbonate urinary loss, vomiting and electrolyte imbalance. Renal phosphate loss causes the subsequent development of vitamin D-resistant rickets. Mental development is normal. Patients develop photophobia from accumulation of cystine crystals in the cornea and conjunctiva. The crystals can be detected by slit-lamp examination and are diagnostic of the disease. Hot weather accentuates symptoms because of a decreased ability to sweat. Crystal accumulation in the thyroid may lead to hypothyroidism.

Dialysis or renal transplantation is usually undertaken between 6 and 12 years of age. Although cystinosis does not recur in the donor kidney, storage continues in other organs, resulting in blindness, corneal erosions, diabetes and neurological deterioration between 13 and 30 years of age. Medical therapy consists of fluid and electrolyte replacement for the renal tubular defects and carnitine to replace urinary losses. Cysteamine has been given both systemically and topically (in the eyes). Although significant toxicity is involved in using this drug, large-scale trials have shown that it delays loss of renal function, particularly in patients treated very early in their disease course. Drug-induced lupus and antiphospholipid syndromes have been ascribed to cysteamine therapy in a paediatric patient.

There are adolescents and adults with more benign forms of cystinosis. A late form, not associated with symptoms before the fifth year of life, is compatible with survival well into the second decade. Clinical manifestations include retinal depigmentation, rickets, mild renal failure and accumulation of cystine crystals in the conjunctiva and bone marrow. In benign cystinosis, patients are asymptomatic and have a normal life expectancy, presumably because substantially less cystine accumulates within cells than in the nephropathic or intermediate forms. This form of the disease is diagnosed only by slit-lamp examination of the eyes.

Diagnosis in all forms is made by identification of crystals in polymorphonuclear leukocytes or by slit-lamp examination to identify corneal crystals. The latter procedure may give false-negative results in infants. Prenatal diagnosis is available by measurement of cystine in amniotic cells or chorionic villi. The diagnosis of cystinosis is established by visualization of the rectangular and hexagonal crystals in bone marrow aspirates, in conjunctival, rectal or renal biopsy tissue, or by ophthalmological examination. Phase-contrast and polarizing microscopy are especially useful in searching for the crystals in biopsy material. Because of the solubility of the crystals in water, all tissues should be fixed in alcohol, and aqueous stains should be avoided. The crystals are therefore best seen in unstained frozen sections or in sections made from alcohol-fixed tissue, and examined by phase or polarizing microscopy. Electron microscopy is a useful method of diagnosis if light microscopic examination of biopsy tissue fails to reveal the crystals. The diagnosis has been made before birth by light and electron microscopic examination of fetal tissues. Other methods of diagnosis include the determination of the amount of cystine in white blood cells or in cultured skin fibroblasts. A noninvasive method of diagnosis is based on infrared spectroscopy of hair. Fluorescence in situ hybridization (FISH) may provide a rapid detection of the 57-kb deletion in CTNS .

The cystine crystals accumulate within lysosomes. The most severely affected organ is the kidney. Most of the cells containing the crystals are of reticuloendothelial origin. Crystals in many organs, such as the cornea, conjunctiva, bone marrow, spleen and liver, excite little or no reaction. In the liver, markedly hypertrophied Kupffer cells packed with cystine are located mainly in perivenular zones ( Fig. 3.97 ). They have a brilliant silvery birefringence when viewed in polarized light ( Fig. 3.98 ). The spaces made by the crystals in Kupffer cells can be seen on EM ( Fig. 3.99 ), and the crystals have a characteristic appearance on SEM ( Fig. 3.100 ).

Figure 3.97

Cystinosis. Cystine crystals are packed in the cytoplasm of hypertrophied Kupffer cells. (H&E stain.)

Figure 3.98

The same case as illustrated in Fig. 3.97 . Cystine crystals display brilliant silvery birefringence. (H&E stain.)

Figure 3.99

Electron micrograph shows spaces created by cystine crystals that were dissolved during processing. Several have rectangular shapes. (×32,000.)

Figure 3.100

Three-dimensional appearance of cystine crystals; some have a hexagonal shape (SEM, ×5000).

Hepatic fibrosis in cystinosis was reported in one case. The patient, who died at the age of 24 years after multiple renal transplants, had presented with portal hypertension. In addition to massive crystal accumulation, the liver showed extensive fibrosis without cirrhosis, and numerous hepatic stellate cells were seen in association with the fibrous tissue. The authors suggested that the stellate cells were activated by injured cystine-laden Kupffer cells but could not dismiss a possible role of the multiple renal transplants in the fibrosis. A patient with cystinosis treated with oral cysteamine developed veno-occlusive disease that eventually required LT. The hepatectomy specimen disclosed many cystine crystals in the fibrous scars around terminal hepatic venules. Ten years later, after a kidney transplant that failed because of disease recurrence, the patient presented with hepatic dysfunction, once again with veno-occlusive disease and crystal reaccumulation. Additional cases of noncirrhotic portal hypertension have been ascribed to cystine accumulation in Kupffer cells. In two patients, long-term infantile nephropathic cystinosis was associated with a form of sclerosing cholangitis, which responded to therapy with UDCA. Liver biopsy specimens showed severe accumulation of cystine, predominantly localized in Kupffer cells, together with morphological features of sclerosing cholangitis. One patient showed changes compatible with sclerosing cholangitis on MRI.

Homocystinuria (cystathionine β-synthase deficiency)

Patients present with ectopia lentis, osteoporosis, genu valgum, pes cavus, kyphoscoliosis and progressive mental retardation. In addition, arterial thrombosis is common; however, most patients with homocystinuria and strokes have a concurrent factor V Leiden mutation. Hepatomegaly with normal liver enzymes is common. Screening is by detection of high levels of methionine and homocystine in urine. Diagnosis includes evaluation for other causes of homocystinuria, including vitamin B 12 or an abnormality in its processing. Cystathionine β-synthase (CBS) can be measured in cultured fibroblasts.

Homocystinuria has an estimated worldwide frequency of 1 in 344,000, but the frequency is higher in Ireland (1 in 65,000). The cystathionase gene is found on chromosome 21q-22.3 ; it has been cloned. Several different defects have been found to be associated with homocystinuria. Prenatal diagnosis can be performed. Treatment is with supplementation with folic acid and betaine.

Light microscopic studies of the liver in homocystinuria have shown steatosis, more prominent in the perivenular regions. Mild to moderate portal fibrosis and thickened arterioles with intimal hyperplasia or fibrosis have been observed in some patients. Ultrastructural studies have demonstrated mitochondria with unusual shapes, increased SER and numerous pericanalicular lysosomes. Hyperhomocystinaemia in liver of CBS-deficient mice has been shown to promote oxidative stress, leading to fibrosis and steatosis.

Disorders of lipoprotein and lipid metabolism


The disease was first described by Bassen and Kornzweig. Patients with abetalipoproteinaemia have malabsorption of fat, acanthocytosis, retinitis pigmentosa and ataxic neuropathic disease. Symptoms usually begin in infancy with steatorrhoea and failure to thrive. Acanthocytes are evident by 12 months of age. Neurological abnormalities causing an unsteady gait develop between 2 and 17 years. Degenerative changes affect the posterior and lateral columns of the spinal cord, spinocerebellar pathways and peripheral nerves. The diagnosis is suspected on the basis of the clinical features, a serum cholesterol level <1.3 mmol/L (50 mg/dL) and an extremely low serum triglyceride level. Patients tend to be lean. The diagnosis is confirmed by determination of the β-lipoprotein concentration using lipoprotein electrophoresis, ultracentrifugation or immunochemical methods. Small bowel biopsies reveal fat droplets in the epithelium ( Fig. 3.101 ).

Figure 3.101

Abetalipoproteinaemia. Intestinal microvillus reveals marked vacuolization of epithelial cells. (H&E stain.)

The inheritance is autosomal recessive. The defective gene ( MTTP ) codes for the microsomal triglyceride transfer protein. This protein is essential for the transport of triglyceride, cholesteryl ester and phospholipid from phospholipid surfaces. It is synthesized as a heterodimer; in abetalipoproteinaemia the large subunit of the protein is absent. Mutations in MTTP were demonstrated in two patients with abetalipoproteinaemia.

Treatment consists of a low-fat diet and replacement of vitamins A and E. Late manifestations include essential fatty acid deficiency and peripheral vascular disease. Supplementation with medium-chain triglycerides for calories was a common recommendation in the past; this therapy has been implicated as the cause of cirrhosis in some patients and is no longer recommended. Liver transplantation cures the liver disease and corrects the serum lipid profile, but it does not cure the steatorrhoea, as the mutated MTTP gene is also expressed in the intestine. LT in a 20-year-old woman with untreated abetalipoproteinaemia who had developed cirrhosis was followed by a dramatic rise in serum cholesterol and triglycerides, as well as an increase in apolipoprotein B from barely measurable (<1 mg/dL to 76 mg/dL). Study of apo B and apo B mRNA in cultured liver and intestinal cells suggested that the defect in abetalipoproteinemia did not involve the apolipoprotein B gene or the synthesis or glycosylation of the apolipoprotein, but rather some aspect of lipoprotein assembly or secretion.

Histological studies of the liver in several cases of abetalipoproteinaemia have shown variable steatosis. The patient studied by Partin et al. underwent liver biopsy before and after 2, 14 and 20 months of therapy with medium-chain triglycerides. Before treatment, hepatocytes contained large fat droplets that had ruptured to form perivenular ‘fatty lakes’. Ultrastructurally, the Golgi apparatus was almost completely deficient in trans-Golgi vacuole formation. Endogenous triglyceride particles could not be found, and the circum-Golgi SER was absent. During the 14 months of dietary treatment, the fat droplets in hepatocytes became smaller and less numerous, but there was progression of initially mild hepatic fibrosis to a micronodular cirrhosis. Mallory–Denk bodies were identified in hepatocytes of the cirrhotic liver by EM. Clinically, the patient had substantial hepatomegaly and a persistent increase of serum aminotransferase activity. Whether the hepatic lesions were part of the natural course of the disease or related to therapy remains undetermined. The ultrastructural findings of one reported case included fatty change, a normal Golgi apparatus and ER, acanthocytes in sinusoids and phagocytosis of deformed erythrocytes by Kupffer cells ( Fig. 3.102 ). An ultrastructural study of another case by Collins et al. demonstrated striking changes in hepatocellular peroxisomes. They included pleomorphism and a broadened range of size, often larger than normal with marginal bars in some. Whether the changes were those of a peroxisomal disorder or a reflection of the disturbed lipid metabolism remains uncertain.

Figure 3.102

Abetalipoproteinaemia. Kupffer cell with phagocytosed acanthocyte (transmission electron microscopy [TEM] ×40,000). Inset, Sinusoidal acanthocyte (SEM ×5000).

Familial hypobetalipoproteinaemia

Familial hypobetalipoproteinaemia (FHBL) is characterized by reduced serum levels of β-lipoprotein and apo B-containing lipoprotein. Taruji et al. reported a mutation of the apo B gene leading to synthesis of a truncated form of apo B (apo B-38.95). Many other truncations of apo B have been identified and are reviewed by Kane and Havel. Sporadic cases of FHBL with an apparently recessive transmission may be caused by de novo mutations of apo B gene. Mutations in the gene encoding for the angiopoietin-like protein 3 ( ANGPTL3 ) are also a cause of some forms of FHBL. Welty recently reviewed the different forms of FHBL.

In homozygous hypobetalipoproteinaemia, apo B and low-density lipoprotein (LDL) cholesterol levels are very low or undetectable. When β-lipoproteins are absent, the clinical phenotype is indistinguishable from that of abetalipoproteinaemia with fat malabsorption, acanthocytosis, retinitis pigmentosa and neuromuscular degeneration. Liver biopsies also reveal marked steatosis. Perisinusoidal fibrosis in one case was attributed to hypervitaminosis A. A mouse model should provide new insights into apo B metabolism. Treatment is similar to that of abetalipoproteinaemia: restriction of dietary fat and intensive vitamin E supplementation.

Patients with heterozygous hypobetalipoproteinaemia are usually asymptomatic and have low plasma levels of total cholesterol (45–150 mg/dL) and low to normal levels of triglycerides (11–140 mg/dL). Mild to moderate increases in the levels of the serum aminotransferases have been reported. Ultrasound examination discloses a hyperechoic liver. Liver biopsies have revealed mild to moderate steatosis without necroinflammatory changes or fibrosis. The long-term prognosis is excellent; patients may actually be protected from developing coronary atherosclerotic disease.

Familial high-density lipoprotein deficiency (Tangier disease)

This rare autosomal recessive disease is caused by a mutation in the ATP-binding cassette transporter 1 ( ABCA1 ) gene on chromosome 9q31. The ATP-binding cassette transporter 1 protein is also called the ‘cholesterol efflux regulatory protein’. In the absence of this regulatory protein, apolipoprotein-mediated cholesterol removal from cells is blocked, leading to the characteristic accumulation of cholesterol esters in reticuloendothelial cells and low or absent plasma high-density lipoprotein (HDL) and low plasma cholesterol levels.

Patients with classic familial HDL deficiency present with a striking tonsillar enlargement with orange discolouration, lymphadenopathy, hepatosplenomegaly and peripheral neuropathy. Thrombocytopenia, corneal opacities and xanthomas are less common. Some patients have hyperbilirubinaemia. Others present an atypical clinical picture with hepatosplenomegaly, low serum cholesterol and HDL cholesterol concentrations, early-onset coronary artery disease and corneal opacities, but no orange tonsils; the hepatosplenomegaly and corneal opacities may also be absent.

The pathological aspects of Tangier disease have been comprehensively discussed. Deposition of cholesterol esters is widespread, with involvement of tonsillar and adenoidal tissue, liver, spleen, lymph nodes, bone marrow, thymus, intestinal mucosa, skin and cornea. The foam cells contain birefringent, needle-shaped cholesterol crystals, are sudanophilic and PAS negative, and stain positively with the Schultz modification of the Lieberman–Burchard reaction. Involvement of the liver by clusters of cholesterol-containing cells has been noted by several investigators. The differential diagnosis from other cholesterol storage diseases is discussed by Bale et al.

Familial hypercholesterolaemia

Patients with familial hypercholesterolaemia have elevated serum cholesterol, which is deposited in tendons and skin (xanthoma), cornea (arcus senilis) and arteries (atheroma). The severity of disease is related to the gene dosage; this is an autosomal dominant trait with homozygotes affected more severely than heterozygotes. Heterozygotes have a twofold elevation of serum cholesterol from birth, with onset of tendon xanthomas and coronary atherosclerosis after age 20 years. Homozygotes have severe hypercholesterolaemia with complications developing in the first decade of life; xanthomas may be present at birth. Coronary heart disease begins in childhood and frequently leads to death before age 20 years. Most patients have mutations in the gene for low-density lipoprotein receptor (LDLR), although two other sets of mutations contribute to the phenotype. However, similar phenotypes are described in association with mutations of APOB and PCSK9.

Patients have elevated concentrations of low-density lipoprotein, the major cholesterol transport lipoprotein in plasma. The incidence of heterozygotes is about 1 in 500 and of homozygotes about 1 per 1 million live births. The defect is an abnormal LDLR gene. Several different mutations have been described. The gene is on chromosome 19p. Therapy is directed at lowering plasma cholesterol to control complications. Homozygotes have been managed by combined liver–heart transplantation.

Reported pathological findings in homozygous familial hypercholesterolaemia have been reviewed by Buja et al. In addition to atherosclerosis of the aorta and coronary vessels, there is neutral lipid accumulation in extravascular sites that include the skin, tendons, spleen, thymus and other organs. In the liver, there is accumulation of lipid in hepatocytes and Kupffer cells. Ultrastructurally, accumulation of both neutral lipid and cholesterol has been observed in hepatocytes.

Wolman disease and cholesteryl ester storage disease

Wolman disease and cholesteryl ester storage disease (also called ‘cholesterol’ ester storage disease in older literature) are two disorders caused, respectively, by absent or by reduced (3–8%) activity of the enzyme lysosomal acid lipase. The enzyme is essential for the intralysosomal metabolism of cholesterol esters and triglycerides, namely, their uptake by receptor-mediated endocytosis into lipoprotein particles. The enzyme is trafficked to the lysosome by the mannose-6-phosphate receptor systems. The gene encoding the lipase is LIPA on chromosome 10q23.31 and contains 10 exons spread over 36 kb.

Wolman disease was first described in 1956. Patients may present with hydrops fetalis or congenital ascites ; clinical presentation with conjugated hyperbilirubinaemia is uncommon. The usual presentation is failure to thrive with vomiting, frequently with severe diarrhoea accompanied by steatorrhoea. Physical findings include severe malnutrition, hepatosplenomegaly, ascites, pallor and mild lymphadenopathy. Neurological development is abnormal. Radiological studies reveal large adrenals with calcification, a critical finding for clinical diagnosis. Examination of the peripheral blood smear reveals vacuolated lymphocytes, while bone marrow examination discloses foam cells which stain positively for cholesterol and neutral fat or triglyceride. Serum lipid levels are usually low or normal, but in rare instances they are elevated. Adrenal responses become depressed with time. Death usually occurs in the first year of life despite aggressive nutritional support.

The relatively milder form of the disease, cholesteryl ester storage disease (CESD), presents at any age with hepatomegaly, usually caused by lipid retention in hepatocytes and Kupffer cells, and variable splenomegaly caused by portal hypertension. In children, diarrhoea may occur because of lipid retention in enterocytes; resulting malnutrition may lead to short stature. Recurrent abdominal pain may be a feature. The typical natural history of CESD includes premature atherosclerosis and progressive liver disease resulting in cirrhosis. There have been reports of pulmonary vascular obstruction and mesenteric lipodystrophy. An asymptomatic 51-year-old man with CESD and accelerated atherosclerosis developed cholangiocarcinoma and died of liver failure as a result of extensive tumour infiltration. Cholangiocarcinoma had similarly developed in a 51-year-old woman with CESD (unpublished observation by authors). Laboratory abnormalities in CESD include elevated serum levels of aminotransferases cholesterol and its esters, LDL, triglycerides and bile acids. Sea-blue histiocytes have been noted in the liver, small bowel and bone marrow. Jaundice suggests the development of cirrhosis, which may be followed by liver failure. Prenatal diagnosis is available.

Wolman disease can result from a number of mutations in the lysosomal acid lipase gene LIPA , all resulting in absence of enzyme activity. CESD appears to have a somewhat different array of mutations associated with it. CESD seems distinct from Wolman in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype. The most common mutation in CESD is caused by aberrant splicing of an allele due to a point mutation, resulting in the production of a shortened lysosomal acid lipase mRNA lacking 72 nucleotides, and in the synthesis of an enzyme missing 24 amino acids. Compound heterozygosity for a mutation causing Wolman disease is common among CESD patients. CESD is probably underdiagnosed, and the incidence, estimated at 1 in 40,000, is not really known. Since these patients typically present with a clinical picture of liver enlargement and dyslipidaemia, misdiagnosis as nonalcoholic fatty liver disease (NAFLD), including normal-body-weight NAFLD, is possible. Liver biopsy showing typical findings of CESD may contribute to sorting out these two divergent diagnoses.

Therapy in Wolman disease, nutritional or otherwise, is difficult. Wolman suggests avoidance of lipid esters despite the presence of an essential fatty acid deficiency. The role of antioxidant therapy remains unclear, although vitamin E has been routinely used. Bone marrow transplantation corrects the enzyme defect. BMT performed early in the disease course preserved the life of one patient with Wolman disease; growth and mental development improved over the years. In another patient, successful long-term engraftment was followed by continued normalization of acid lipase enzyme activity in peripheral leukocytes, weight gain, and normal cholesterol, triglyceride and liver enzyme levels at 4 years of age. Previous attempts had been unsuccessful, presumably because of advanced liver or systemic disease at BMT.

In CESD, a favourable clinical response with a stage I American Heart Association diet and large doses of cholestyramine has been reported from the University of Minnesota. Medical therapy with an HMG-CoA-reductase inhibitor has had reasonable success, although its effect on lipid levels and liver tests has been variable. One patient had a favourable response in terms of cholesterolaemia to lovastatin plus ezetimibe, an inhibitor of Niemann-Pick C1-like 1 (NPC1L1) protein. Enzyme replacement therapy is being developed. Success with liver transplantation has been reported. End-stage renal disease has developed in one LT patient.

Rat and mouse models are available to study experimental therapies of acid lipase deficiency. In vitro correction of fibroblasts in the two human diseases was successful.

The adrenal glands in Wolman disease are grossly enlarged, hard and bright yellow. They cut with a gritty sensation and have a yellow cortex and an inner calcified zone. The surface of the small intestine, particularly the duodenum and ileum, has a yellow velvety appearance.

In Wolman disease, all affected organs, particularly the liver, spleen, adrenals, haemopoietic system and the intestines, are infiltrated by numerous foamy macrophages that contain cholesterol and/or cholesterol esters. Stains for lipid (Oil Red O, Sudan Black) are positive, as is the Schultz modification of the Lieberman–Burchard reaction for cholesterol ; these stains must be performed on frozen sections, either of fresh or formalin-fixed tissue. Frozen sections examined by polarizing microscopy reveal numerous anisotropic acicular crystals in the foamy histiocytes. In the liver, cholesterol and cholesterol ester are mainly stored in Kupffer cells and portal macrophages, while hepatocytes contain increased neutral lipid. Reticuloendothelial cells may also contain free fatty acids. There may be marked pericellular fibrosis and varying degrees of periportal cholangiolar proliferation and fibrosis. Ultrastructurally, the enlarged Kupffer cells contain peripheral vacuoles, sometimes within lysosomes, and large central crystal clefts of cholesterol ester. Lake and Patrick observed that the crystals are membrane bound. Hepatocytes contain many lipid droplets but only occasional crystal clefts. It has been suggested that the cholesterol esters are discharged from the hepatic parenchymal cells in an insoluble form and then are taken up by the Kupffer cells, where crystallization occurs.

The liver in CESD is enlarged, yellow–orange and greasy ( Fig. 3.103 ). The light microscopic and ultrastructural changes in the liver of patients with CESD are similar to those of Wolman disease. A highly characteristic feature of CESD is the presence of greatly hypertrophied Kupffer cells and portal macrophages with a foamy, tan-coloured cytoplasm that stains strongly with PAS ( Fig. 3.104 A and B ). The birefringent cholesterol crystals are shown in Fig. 3.104 C . Periportal fibrosis of varied degree is present in most cases. A recent literature review by Bernstein et al. has shown advanced-stage fibrosis in about one-third of patients. Immunohistochemistry for lysosomal proteins, including cathepsin D, lysosomal-associated membrane protein 1 (LAMP1), LAMP2 and lysosomal integral membrane protein 2 (LIMP2), has been proposed for the diagnosis of CESD on paraffin sections. Ultrastructurally, triglyceride droplets are noted in abundance in hepatic and reticuloendothelial cells; most are surrounded by a single membrane. Many of the lysosomal lipid droplets have a ‘moth-eaten’ appearance caused by the inclusion of cholesterol within them ( Fig. 3.105 ). Cholesterol crystals are also seen lying free in the cytoplasm, as demonstrated in a case of Wolman disease ( Fig. 3.106 ). A recent study of both liver and intestinal biopsy specimens in Wolman and CESD diseases is re-emphasizing the diagnostic role of morphological findings.

Figure 3.103

Cholesterol ester storage disease. This section of liver has a yellow colour and is faintly nodular.

Figure 3.104

Cholesterol ester storage disease. A, Kupffer cells are greatly hypertrophied and have a foamy, light-tan cytoplasm; their nuclei are pyknotic. Hepatocytes show microvesicular steatosis. (H&E stain.) B, Foamy Kupffer cells in the perivenular zone are PAS positive (PAS after diastase digestion). C, Cholesterol crystals display brilliant silvery birefringence. There is a large amount of fat (red) in liver cells. (Oil Red O stain of frozen section.) D, CD68 immunostain of macrophages highlights negatively stained lipids and needle-shaped cholesterol crystals (PAP technique).

Figure 3.105

Cholesterol ester storage disease. Electron micrograph of liver cell containing triglyceride droplets of varied size. ‘Moth-eaten’ appearance of several droplets is caused by presence of cholesterol crystals within them. (×10,000.)

Figure 3.106

Wolman disease. Electron micrograph demonstrating cholesterol crystals in the cytoplasm of a liver cell. (×11,000.)

(Courtesy of Dr. J.E. Dimmick, Vancouver, Canada.)


Gangliosides are glycolipids found mainly in the neuronal membrane. Gangliosidoses are caused by defects in ganglioside catabolic pathways and abnormal accumulation of gangliosides (GM1, GM2) resulting in progressive neurodegenerative disorders. The degree of accumulation of gangliosides in visceral organs, including the liver, is variable and depends on the disease subtype. Disorders related to defect of ganglioside synthesis are very rare.

GM1 gangliosidoses

Three subtypes of GM1 gangliosidosis are recognized, depending on age at clinical presentation.

Type I (infantile)

Type I, the infantile form of GM1 gangliosidosis, manifests during the first 6 months of life. Patients present with progressive psychomotor retardation, seizures, hepatosplenomegaly, oedema of the extremities and failure to thrive. The appetite is poor and sucking is weak, in part because of the infant’s hypotonia. By the first year of life, patients are blind, deaf and in decerebrate rigidity. Death, usually from bronchopneumonia, occurs within 2 years of age.

Facial abnormalities include frontal bossing, a depressed nasal bridge, large low-set ears, an increased distance between the nose and upper lip and downy hirsutism of the forehead and neck. The gums are hypertrophied, and there is mild to moderate mac­roglossia. The corneas are clear. Cherry-red spots are present in the macula in half the patients, and dermal melanosis may be prominent.

Gangliosides accumulate in neurons, liver, spleen and in histiocytes in other tissues, as well as in the epithelial cells of the kidney. This is a result of a deficiency in all three isoenzymes of lysosomal β-galactosidase-1 (A, B and C), located on chromosome 3p21. The mutations in the infantile form of GM1 gangliosidosis interfere with phosphorylation of precursor β-galactosidase, resulting in its secretion instead of its further processing in the lysosomes.

Foam cells are seen in bone marrow preparations. Lymphocytes are vacuolated. Mild radiological abnormalities consist of inferior beaking of the lumbar vertebrae and proximal pointing of the metacarpal bones. Neurons are ballooned and contain cytoplasmic membranous inclusions similar to those of Tay–Sachs disease. Lipid-laden histiocytes are found throughout the reticuloendothelial system. Renal glomerular epithelial cells, hepatocytes and Kupffer cells are finely vacuolated. Ultrastructurally, the vacuoles in affected cells in GM1 gangliosidosis are empty or contain fibrillar or granular material. Kupffer cells may also contain membrane-bound fibrillary structures ( Fig. 3.107 ). Most affected systemic cells, including hepatocytes and phagocytic cells, bind WGA lectin, and to a lesser extent other lectins such as ConA, S-WGA, DSA and BS-1. Enzyme analysis indicates almost complete absence of β-galactosidase A, B and C in skin fibroblasts and leukocytes. Membrane-bound inclusions are also seen in cholangiocytes.

Figure 3.107

GM1 gangliosidosis type I. Electron micrograph showing the cytoplasm of a Kupffer cell replaced by single membrane-bound vacuoles. Some of the vacuoles contain amorphous material, whereas others are filled with irregularly arranged fibrillar material. (×300.)

(Courtesy of Dr. M. Petrelli).

Type II (juvenile)

Type II, the juvenile form of GM1 gangliosidosis, presents at about 1 year of age. Clinical findings are primarily neurological and include seizures, spasticity, ataxia and mental retardation. Radiological changes are minimal. Facial abnormalities, visceromegaly and macular cherry-red spots are absent. Death occurs between 3 and 8 years of age. This disease is also caused by a mutation in the β-galactosidase-1 gene, but only isoenzymes B and C are deficient. Successful bone marrow engraftment in a presymptomatic patient with type II GM1 normalized white cell β-galactosidase levels, but did not influence long-term clinical outcome. In common with type I, there is neuronal storage and foamy histiocytes are present in various organs ( Fig. 3.108 ). Hepatocytes are only slightly vacuolated, and Kupffer cells stain intensely with the PAS reagent ( Fig. 3.109 ). Ultrastructurally, the Kupffer cells contain a distinctive granulofibrillar material ( Fig. 3.110 ), or membrane-bound inclusions may be seen in both Kupffer cells and hepatocytes. Two canine models of GM1 gangliosidosis have been described.

Figure 3.108

GM1 gangliosidosis type II. Clusters of pale-staining foamy Kupffer cells that have eccentric nuclei. (H&E stain.)

Figure 3.109

The same case as illustrated in Fig. 3.108 . Ganglioside-laden Kupffer cells are intensely PAS positive. (PAS stain after diastase digestion.)

Figure 3.110

GM1 gangliosidosis type II. Electron micrograph of a Kupffer cell containing several angulated and focally membrane-bound inclusions. The inclusions are composed of granulofibrillar material and a few osmiophilic granules. (×13,500.)

(Courtesy of Dr. M. Petrelli.)

Type III (adult)

Type III, the adult form of GM1 gangliosidosis, is much less severe than the other two forms and is associated with little or no visceromegaly. Mutations in the β-galactosidase-1 gene are described, but the cause of the phenotypic differences from types I and II is as yet undetermined.

GM2 gangliosidoses

The GM2 gangliosidoses have primarily neurological manifestations. These disorders result from defects in the lysosomal enzyme hexosaminidase, leading to the accumulation of glycolipids, particularly GM2 gangliosidase. Hexosaminidase consists of two major isoenzymes, hexosaminidase A, composed of an α- and a β-subunit, and hexosaminidase B, composed of two β-subunits. The α-subunit has been localized to chromosome 15 and the β-subunit to chromosome 5. A lysosomal enzyme, GM2 activator protein, also localized to chromosome 5, complexes with the lipid substrate for presentation to hexosaminidase for cleavage hydrolysis. Inheritance of all the variants is autosomal recessive. Prenatal diagnosis is available for all types.

Hexosaminidase α-subunit defect or deficiency (Tay–Sachs disease; infantile Tay–Sachs disease)

These patients present at 3–6 months of age with motor weakness, apathy and feeding problems. The most common initial sign is the startle response to sound, characterized by upper- and lower-extremity extension, often associated with a myoclonic jerk. Progressive weakness and hypotonia develop, with definite neurological regression by 10–12 months of age. The mental and motor deterioration continue, with death from bronchopneumonia, usually by 4 years of age. The typical, but not specific, cherry-red spot is observed in the macula during the early stages of the disease. Degeneration of the optic nerve is common and results in blindness. Megalocephaly may develop during the second year of life.

Tay–Sachs disease has a carrier frequency of 1 in 30 for Ashkenazi Jews and 1 in 300 for non-Jewish persons. The diagnosis is based on the absence or near-absence of hexosaminidase A in serum or fibroblast cell cultures. The deficiency allows massive accumulation of GM2 ganglioside and asialo-GM2 in enlarged cerebral neurons, retinal ganglion cells and autonomic ganglia. EM shows concentrically laminated inclusions or membranous cytoplasmic bodies in affected neurons, and somewhat more pleomorphic inclusions in the glia. Although hepatocytes appear normal by light microscopy, they may contain similar bodies by EM examination.

More than 70 mutations in the hexosaminidase A gene (located on chromosome 15) have been described. Prenatal diagnosis is performed in most centres.

Juvenile GM2 gangliosidosis

This disorder presents with motor ataxia between 2 and 6 years of age, followed by progressive dementia. Optic atrophy and retinitis pigmentosa occur late in the disease course without cherry-red spots. Decerebrate rigidity is present by 10–12 years of age, and death from infection occurs between 10 and 15 years of age. Patients have a form of hexosaminidase A which cannot be activated.

Hexosaminidase β-subunit deficiency or defect (Sandhoff disease; infantile Sandhoff disease)

Previously, infants with Sandhoff disease were misdiagnosed as having Tay–Sachs disease because the clinical features and course are similar. In the first few months of life, subtle signs of delayed motor development are noted. Cardiovascular symptoms may be observed early in the course of the disease along with minimal hepatosplenomegaly. During the second half of the first year, little if any progress is made in motor or mental development. Cherry-red spots and early optic atrophy are evident by ophthalmoscopic examination. By 12 months of age the patients no longer use pincer grasp; they develop bilateral pyramidal tract abnormalities, including increased deep tendon reflexes, spasticity and positive Hoffman and Babinski signs. In addition, there is regression in sitting, smiling and laughing appropriately. The psychomotor deterioration is progressive, with death resulting from aspiration pneumonia, usually between 22 and 36 months of age. A juvenile form of this disease exists, but it lacks hepatic involvement.

There are mild skeletal abnormalities in Sandhoff disease, and electrocardiogram (ECG) changes range from mild left ventricular hypertrophy to severe abnormalities compatible with endocardial fibroelastosis. Vacuolated lymphocytes may be present in the peripheral blood. Greatly increased levels of globoside in the urinary sediment and plasma differentiate Sandhoff from Tay–Sachs disease.

Sandhoff disease results from the deficient activity of both hexosaminidase A and B. Decreased activities of both enzymes are found in biopsy specimens, peripheral leukocytes, platelets, cerebrospinal fluid, cultured skin fibroblasts and plasma. There is neural and visceral deposition of GM2 ganglioside, its asialo derivative, and tetrahexosyl ceramide in affected infants. The gene is found on chromosome 5q11. Only the hexosaminidase B gene may be defective in the disease; the requirement for shared subunits between A and B may result in the complete absence of hexosaminidase activity. Heterozygosity in parents is more reliably established by examination of tissue specimens than of serum.

Sandhoff disease shows no ethnic predilection. The diagnosis can be made in utero by demonstration of deficient hexosaminidase levels in amniotic fluid. Patients with Sandhoff disease were previously missed at autopsy because routine formalin fixation obscured the visceral lipid accumulation. However, 1-mm sections of Epon-embedded material reveal the lipid deposition when studied by light microscopy. Lysosomal lipid accumulation in the liver progresses with age; the lysosomes are twice the normal size at 3 months, and by 1 year, many are distended by membranous lipid deposits to a diameter equal to that of the liver cell nucleus ( Fig. 3.111 ). The extent, size and variation of the membranous deposits within lysosomes are characteristic but not pathognomonic of Sandhoff disease. Kupffer cells are also involved and stain positively with the PAS reagent. Periportal fibrosis has been reported. The acinar cells of the pancreas are also deeply involved. EM reveals membranous and ‘zebra body’ hybrids.

Figure 3.111

Sandhoff disease. Electron micrograph of a large cytoplasmic lysosome containing single and laminated membranous structures within a finely particulate matrix. Note the size of the lysosome in comparison to the other cytoplasmic organelles. (×13,500.)

α-Galactosidase A deficiency (Fabry disease)

Fabry disease is an X-linked disorder of glycosphingolipid metabolism. Hemizygous males are variably affected, whereas heterozygous females are often asymptomatic but may develop significant clinical features with advancing age. Fabry disease is caused by a defect in the lysosomal hydrolase α-galactosidase A (α-GLA), which results in accumulation of globotriaosylceramide in tissue lysosomes throughout the body. Complete absence or reduced activity of α-GLA results in more severe or milder forms. The gene localizes to chromosome Xq22. The α-galactosidase gene has been cloned, and several mutations are associated with Fabry disease.

The prevalence of Fabry disease is 1 in 40,000, although it may occur more frequently in geographically isolated communities. Clinical disease usually begins during childhood or adolescence with pain and paraesthesias in the extremities secondary to vascular substrate accumulation that affects peripheral nerves. Nausea, vomiting and diarrhoea are common. Hypohidrosis results from vascular lesions in the autonomic nervous system. Skin signs of the disease are angiokeratomas. Renal lipid deposition leads to proteinuria, isosthenuria and gradual deterioration of renal function. Hypertension, left ventricular hypertrophy, myocardial ischaemia or infarction and cerebrovascular disease may develop. Some patients have lympho­edema and mild anaemia. Death is usually from renal or cardiovascular complications. Atypical variants affecting predominantly the heart or the kidneys are well recognized. The diagnosis may be suspected by the combined findings of corneal dystrophy, lipid-laden and PAS-positive macrophages in the bone marrow, and birefringent Maltese crosses in the urinary sediment. Measurement of α-GLA enzyme activity in blood leukocytes is confirmatory. Examination of urinary sediment can be used as a noninvasive method for early diagnosis and monitoring the effect of therapy.

Phenytoin or carbamazepine may be administered for the symptomatic relief of the neurological problems. Kidney failure may necessitate renal transplantation. Two recombinant enzymes, agalsidase-β and agalsidase-α, have been used clinically with significant long-term benefit, particularly when started before permanent tissue damage has occurred.

The substance that accumulates in the blood vessels, myocardium, kidney, liver, spleen and other organs is globotriaosylceramide (GL-3). Pathological findings in Fabry disease have been reviewed in detail. In the liver there is accumulation of GL-3 and cholesterol in Kupffer cells, portal macrophages and the endothelial cells of blood vessels. The cells are swollen and light tan in H&E preparations ( Fig. 3.112 ). They contain birefringent crystals in frozen sections, and the Schultz modification of the Lieberman–Burchard reaction is moderately positive. The stored material is intensely PAS positive and resists diastase digestion ( Fig. 3.113 ). Ultrastructurally, the stored material in Fabry disease consists of concentrically laminated lysosomal inclusions with a periodicity of 5–6 nm ( Figs 3.114 and 3.115 ). Lipid accumulations consisting of amorphous material as well as stacks of lamellar leaflets may be seen in hepatocytes, Kupffer cells and portal tract macrophages. Two peroxidase-labelled lectins are found to be strongly reactive with the storage material of Fabry disease. This observation has diagnostic implications because lectin binding is more specific than any other histochemical method. Lectin histochemistry with enzyme digestion enabled Kanda et al. to detect α-galactosyl, β-galactosyl and glucosyl sugar residues in the lysosomal deposits at the ultrastructural level in Fabry disease. This technique has potential for the investigation of other glycolipid and glycoprotein storage diseases. The stored material is demonstrable by light and electron microscopy using a mouse monoclonal anti-GL-3 antibody.

Figure 3.112

Fabry disease. Several portal macrophages are hypertrophied and have a tan-coloured cytoplasm. (H&E stain.)

Figure 3.113

Fabry disease. Kupffer cells contain a granular material that stains intensely with PAS after diastase digestion.

Figure 3.114

Fabry disease. Electron micrograph showing multiple dense and often laminated inclusions in hepatocytes and in a Kupffer cell. (×8800.)

Figure 3.115

Fabry disease. Electron micrograph showing concentric lamination of inclusion material, with periodicity of 5–6 nm. (×170,000.)

Sulphatide lipidosis (metachromatic leucodystrophy)

Metachromatic leucodystrophy (MLD) is a lysosomal lipid storage disorder caused by mutations in the gene for arylsulphatase A (ASA), an enzyme involved in the degradation of the sphingolipid 3-O-sulphogalactosylceramide (sulphatide). Sphingolipid storage results in progressive demyelination and severe neurological symptoms. The ASA gene, localized to chromosome 22q13, has been cloned, and multiple mutations which cause MLD have been described.

Clinically, MLD is a group of disorders, differentiated by the time of onset of the symptoms. Symptoms are worse the younger the presentation. Late infantile MLD presents between 1 and 2 years of age with developmental delay. Signs of neurological deterioration include progressive mental retardation, optic atrophy, loss of speech, hypertonic quadriplegia, ataxia and absent tendon reflexes. Pain in the extremities and unexplained fever are common. In the terminal stages of the disease the patient loses sensory contact with the surroundings. Death results from infection or hyperpyrexia.

Early and late juvenile MLD present between 4 and 6 and 6 and 12 years, respectively. The neurological progression previously described proceeds at a slower pace. The adult form of MLD presents after age 16 years (average, 29 years). Mean duration of the disease is 15 years. Most patients present with psychological problems, dementia or paralysis. Progressive neurological abnormalities develop during the course of the disease and include incoordination, ataxia, spastic paresis, visual disorders, tremors, skeletal muscle rigidity, athetotic movements and spastic dysuria. Hydrocephalus has been reported. Death occurs from cachexia or pneumonia. BMT has produced minor, if any clinical improvement, in selected patients.

Diagnosis is made by detection of sulphatide in peripheral nerves and urinary sediment. Deficiency of ASA can be demonstrated in leukocytes, cultured skin fibroblasts and amniotic cells. ASA deficiency was used as the basis for prenatal diagnosis, but the presence of a pseudodeficiency allele of ASA made diagnosis difficult by this method.

Histologically, sulphatide accumulation has been demonstrated in the CNS, liver, gallbladder, pancreas, kidneys, adrenals and other organs. The gallbladder is small and fibrotic and may show multiple polyps or papillomas and contain calculi. Microscopic examination reveals large, foamy macrophages in the gallbladder tunica propria ( Fig. 3.116 ) that stain positively with cresyl violet, toluidine blue, PAS reagent and Hirsch–Pfeiffer stain ( Fig. 3.117 ). In frozen section these contain sudanophilic material that is anisotropic and can also be stained with the Schultz modification of the Lieberman–Burchard reaction due to simultaneous accumulation of cholesterol. Metachromatic granules can be demonstrated in epithelial cells of the gallbladder and to a lesser extent in those of the intrahepatic bile ducts. In the liver, metachromatic granules may be seen in some portal macrophages and less often in Kupffer cells and hepatocytes ( Fig. 3.118 ). Ultrastructurally, prismatic lysosomes are found in epithelial cells of the gallbladder and are composed of periodic leaflets which appear tubular in cross section.

Sep 29, 2019 | Posted by in NEPHROLOGY | Comments Off on Developmental and Inherited Liver Disease
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