Cholestasis is defined as a pathologic state of reduced bile formation or flow. Most cholestatic conditions can be classified as either obstructive or hepatocellular in origin and result in the retention of substances normally excreted into the bile, such as bilirubin, bile acids, or cholesterol, with consequent cell injury. Obstructive cholestasis results from an anatomic or functional obstruction of the biliary system. This can be at the level of the large or extrahepatic bile ducts (i.e., biliary atresia or cholelithiasis) or smaller intrahepatic ducts (i.e., bile duct paucity associated with Alagille syndrome). Hepatocellular cholestasis results from impairment of mechanisms of bile formation and implies defective function of most or all hepatocytes. This chapter discusses the most common cholestatic diseases that have a defined genetic etiology. The function and distribution of the specific genes involved in any of these conditions will dictate whether a defect in the gene results in an isolated cholestatic liver disease (i.e., progressive familial intrahepatic cholestasis type 3 [PFIC-3]) or a systemic disease (i.e., cystic fibrosis or Alagille syndrome). The diseases are categorized mechanistically according to where their associated genetic defect affects bile formation or flow. Using the information from Chapter 3 regarding bile acid physiology as a background, the various necessary components of bile production may be divided into the following: (1) bile acid production, (2) hepatocellular transporters that facilitate bile flow, and (3) membranes and organelles that participate in bile flow. In most clinical forms of hepatocellular cholestasis, the molecular mechanism is a result of impaired bile flow secondary to a defect in membrane transport, embryogenesis, mitochondrial function, or bile acid biosynthesis ( Table 70-1 ).
|Defect in||Cholestatic Disease|
|Bile acid biosynthesis||Disorders of bile acid synthesis|
Defects in Bile Acid Production
Bile Acid Synthetic Defects
Bile acid synthetic defect (BAD) diseases constitute the first general category of genetic cholestatic diseases in which the mechanism is impairment in bile acid production. The bile acids are produced in the hepatocyte and drive more than 60% of bile flow. They are synthesized from cholesterol by 14 enzymatic steps, all of which are coded for by a specific gene. There are seven known gene defects in this pathway, which are described in Chapter 3 . These diseases cause hepatocellular cholestasis due to toxicity of retained abnormal bile acid intermediates and low production of normal bile acids with resultant insufficient bile flow for normal function. Progressive liver damage is then inevitable. Clinical presentation varies among the seven disorders; however, jaundice, cholestasis, elevated transaminases, fat-soluble vitamin deficiency associated with low γ-glutamyl transferase (GGT), and low serum bile acids are the hallmarks of the disease. Diagnosis of BAD is made by testing the urine for normal and abnormal bile acid species with use of fast atom bombardment spectroscopy (FABS), which can identify the “fingerprint” of the inborn error by the pattern of bile acids present.
Defects in Membrane Transporters
The normal mechanisms of bile formation are described in Chapter 3 . Specifically, bile formation is dependent on the interaction of the bile acid transporters and solute carrier systems on the basolateral membrane of the hepatocyte and the mostly ATP-dependent transporters (ABC transporters) located on the canalicular membrane ( Figure 70-1 ). Bile flow begins at the basolateral membrane with uptake and exchange of solute from the portal blood, which does not require active transport. The basolateral transporters include the superfamily of organic ion transport proteins (OATPs), which allow organic anion uptake in exchange for bicarbonate or glutathione. The sodium-dependent taurocholate cotransporter (NTCP) transports only bile acids coupled with sodium. The multidrug resistance-associated proteins—MRP3(Abcc3) and MRP4(Abcc4)—function as basolateral efflux pumps and are upregulated under cholestatic conditions to export bilirubin conjugates (by the former) and bile acids and glutathione (by the latter). There are no described genetic disorders of cholestasis related to basolateral membrane transporters.
The canalicular membrane transporters reside in the canalicular membrane, which is rich in cholesterol and sphingomyelin (see Figure 70-1 ). This membrane is very metabolically active, containing many ATP-dependent solute transport proteins. It also houses ion and water exchangers, vesicle fusion proteins (i.e., soluble N -ethylmaleimide-sensitive-factor (NSF) attachment protein (SNAP) or soluble N -ethylmaleimide-sensitive-factor (NSF) attachment protein receptor (SNARE), skeletal proteins (i.e., villin), and tight-junction proteins. Of particular importance are FIC1 ( ATP8B1 ), which is an aminophospholipid translocase; the bile salt export pump (BSEP) ( ABCB11 ), which mediates conjugated bile acid transport; and the multidrug resistant protein MDR3 ( ABCB4 ), a flippase of phosphatidylcholine. Defects in each of these transporters have been linked to inherited cholestatic diseases that were commonly identified as progressive familial intrahepatic cholestasis types I, II, and III, respectively ( Table 70-2 ).
|18q21-22||ATP8B1/FIC1||ATP-dependent amino-phospholipid transport||Normal|
|2q24||ABCB11/BSEP||ATP-dependent bile-acid transport||Normal|
|PFIC-3||7q21||ABCB4/MDR3||ATP-dependent translocation of phosphatidylcholine (one word)||High|
Progressive Familial Intrahepatic Cholestasis (PFIC)
Our understanding of the family of conditions that make up PFIC and the genotypic and phenotypic differences among them is the result of functional and genetic mapping studies that have identified the genes and their functions over the last 15 years. Initially PFIC was clinically identified by the presence of hepatocellular cholestasis, low serum levels of GGT activity and autosomal recessive inheritance. PFIC has now been redefined into five separate and distinct diseases with their own specific gene defects and distinct clinical profiles. Each of these genes codes for a canalicular transporter involved in bile export (see Table 70-2 ).
PFIC has evolved from its origins as a constellation of symptoms seen in the Amish who were descended from a single ancestor, Jacob Byler, when it was labeled “Byler’s disease.” Subsequently, numerous phenotypically similar non-Amish patients were reported, and then the term Byler syndrome was used to describe these patients. Later, the term PFIC was applied to all Byler-like patients—however, the patients were sorted into two distinct subtypes: [low-GGT PFIC (PFIC-1 and PFIC-2) and high-GGT PFIC (PFIC-3)]. It is now the custom to refer to these diseases by their gene defect, that is, PFIC-1 as FIC1 disease or by the transporter defect (see Table 70-2 )— ATP8B1 (FIC1 ), ABCB11 (BSEP) , and ABCB4 (MDR3) . In all three cases, the involved gene has two unique identifying names. Despite their genetic distinctness, there are many clinical similarities between the PFIC subtypes, especially between PFIC-1 and PFIC-2. PFIC is clinically characterized by chronic cholestasis that begins in early childhood and usually progresses to cirrhosis within the first decade of life. The average age at onset is 3 months, and the disease may progress rapidly and result in cirrhosis during infancy or be slowly progressive with minimal scarring well into the teenage years. Pruritus is the dominant feature of cholestasis in the majority of patients. The pruritus is often misdiagnosed as skin disease because of the intense itching, which is unexplained; liver disease is not considered because of the disproportionately low level of jaundice in this condition. Patients begin to present with generalized mutilation of skin, usually most severe on the extensor surfaces of the arms and legs and on the flanks of the back, due to the disabling pruritus, which does not usually respond to medical therapies.
Severe episodes of recurrent epistaxis, perennial asthma-like disease, and growth failure are common associated problems. PFIC patients are described as “stocky” because of a high prevalence of short stature (95% of patients have stature below the fifth percentile), with often normal weight for height. Without treatment, there is often delayed onset of puberty and sexual development. Intellectually, these patients are equal to their peers if their pruritus is treated. Without treatment, their scholastic achievement can be compromised by inability to focus or concentrate and loss of sleep due to constant pruritus. Complications of cholestasis such as fat-soluble vitamin deficiencies are prevalent in untreated patients discussed elsewhere in the text. Most patients will develop hepatic fibrosis and eventually cirrhosis, which is associated with the findings of hepatomegaly and sometimes splenomegaly. Unlike other cholestatic disease such as ALGS, PFIC patients do not develop xanthomas. In a recent clinical study comparing the presentation and course of PFIC-1 and PFIC-2 patients, several key differences were noted, which included that PFIC-2 (BSEP) patients exhibited more severe hepatobiliary disease compared to PFIC-1 (FIC-1) patients, whereas PFIC-1 patients had greater evidence of extrahepatic disease with diarrhea, pancreatic disease, pneumonia, abnormal sweat tests, hearing impairment, and poor growth. Patients with PFIC-1 are more likely to have associated watery diarrhea, some of which is severe. This secretory diarrhea may persist after liver transplantation.
Hallmark laboratory findings in PFIC-1 and PFIC-2 are low GGT and normal or near-normal serum cholesterol, but markedly elevated levels of serum bile acids. In contrast, the GGT in PFIC-3 is elevated. Other serum values of liver-related enzymes such as alkaline phosphatase, aminotransferases, bilirubin, and bile salts are not distinct from those seen in several other cholestatic disorders.
The histopathologies of PFIC-1 and PFIC-2 are similar at the light microscopy level ( Figures 70-2 and 70-3 ). Uniformly there is the presence of hepatocellular and canalicular cholestasis with pseudoacinar transformation consistent with cholate injury. The presence of multinucleated giant cell transformation is most commonly seen in infancy and has been recently reported to be more commonly seen in PFIC-2 (BSEP-deficiency) than PFIC-1 (FIC-1 deficiency). Degeneration of bile ducts may be seen with apoptotic changes of the biliary epithelium consisting of pyknotic nuclei (small and hyperchromatic) and attenuated cytoplasm and loss of duct lumina. Inflammation is absent. Bile duct paucity develops in 70% of older children because of these changes. In advanced fibrosis, there may be bile ductules at the edge of the portal tract. Lacy lobular fibrosis typically develops early and progresses to portal to central bridging and eventually to cirrhosis (see Figures 70-2 and 70-3 ). The rate of progression of the fibrosis is highly variable, but correlates loosely with the severity of the clinical disease. The major distinguishing histologic finding is seen at the level of electron microscopy with the presence of coarse granular bile in canalicular spaces of PFIC-1 patients, labeled “Byler’s bile.” PFIC-2 patients have a more filamentous morphology than the bile seen in the canalicular spaces. PFIC-2 (BSEP disease) has been associated with multiple cases of gallstones and hepatocellular carcinoma.
In PFIC-1 and PFIC-2, there is also an abnormal distribution both quantitatively and qualitatively of bile acids in serum and bile. Total serum bile acid concentrations are markedly elevated (usually greater than 200 µmol/L, normal less than 10) with an elevated ratio of chenodeoxycholic acid to cholic acid conjugates, usually greater than 10:1. The total biliary bile acid concentrations are low (0.1 to 0.3 mmol/L, normal above 20) even in comparison to other cholestatic syndromes such as ALGS, with a predominance of cholic acid conjugates.
As indicated by its name, PFIC is a progressive disease that culminates in cirrhosis and end-stage liver disease in the majority of patients. Medical treatment has consisted of the usual supportive care of cholestatic disease with fat-soluble vitamin supplementation and the use of ursodeoxycholic acid (ursodiol, 20 to 30 mg/kg/day). Although there is evidence that ursodiol may enhance bile flow, there is no evidence that it alters disease progression overall.
Surgical therapy consisting of the partial external biliary diversion (PEBD) has been used for the last two decades to treat PFIC and ALGS. PEBD involves the surgical placement of an enteric conduit between the gallbladder and the skin through which bile flow is partially diverted away from the enterohepatic circulation. It typically results in an approximately 50% diversion of bile flow, which amounts to ~30 to 120 mL of bile per day that drains into the ostomy bag and is discarded. PEBD has been effective in improving chronic cholestasis and its associated complications in both ALGS and PFIC. PEBD has become a standard intervention for PFIC and can slow or halt the progression of liver disease in this condition, which usually progresses to cirrhosis and end-stage liver disease if untreated. After diversion, the bile salt pool converts to predominantly cholic acid conjugates, which has been associated with histologic and clinical improvement of the liver disease. PEBD may not always affect the natural progression of either ALGS or PFIC, and at present, no clinical parameters have been defined that predict patients who are likely to respond to biliary diversion procedure. Although PEBD has been shown in some cases to halt or reverse disease progression, it is generally ineffective in patients with established cirrhosis. Rare patients develop “watery” bile output after PEBD, with severe electrolyte losses that need to be monitored and replaced. Twenty years of experience with PEBD and PFIC has demonstrated variable relief of pruritus, improvement in liver histology, improved growth, and improvement in bile acid content of bile. A variation of PEBD is the limited ileal diversion, in which the distal 20% to 25% of the ileum is removed from the intestinal mainstream and made into a self-emptying blind loop, which results in loss of bile salts similar to PEBD. Ileal diversion is usually reserved for patients who have had a cholecystectomy, as it tends to become less effective over time. In some cases, PEBD or ileal diversion does not significantly affect the progression to cirrhosis, and liver transplantation becomes necessary.
PFIC-1: FIC1 Disease
The gene for PFIC-1 (Byler’s disease), FIC-1 , has been mapped to a 19 cM region of 18q21-q22 by the detection of a preserved haplotype in affected members of the Byler pedigree. FIC-1 codes for an ATP-binding cassette (ABC), which is an aminophospholipid translocase that flips phosphatidylserine and phosphatidylethanolamine from the outer to the inner layer of the canalicular membrane.
PFIC-2: BSEP Disease
The PFIC-2 gene is located at chromosome 2q24. It codes for an ABC bile salt transporter also called the BSEP. The PFIC-2 gene is analogous to the rat sister gene of p-glycoprotein (S-PGP), which, in rats, has been shown to be important in bile salt transport. Studies of liver tissue from patients with mutation of BSEP have revealed lack of canalicular BSEP expression by immunohistochemistry ( Figure 70-4 ). This finding has clarified that PFIC-2 patients may have limited or no BSEP protein and therefore have a primary inability to transport bile salt.
PFIC-3, Multidrug Resistance Gene-3 ( MDR-3 ) Deficient Disease
PFIC-3 is distinct from the previous two disorders primarily in that high serum GGT level is present. This condition shares the pattern of the first two disorders in that it is familial, recessive, and begins as intrahepatic cholestasis in the first year and progresses toward hepatic failure in the first few years of life. A major distinction, besides the high GGT, is the histopathology, which has more of an “obstructive” pattern. Liver biopsies show expanded portal areas with proliferation of interlobular bile ducts plugged with bile. Analysis of PFIC-3 bile revealed very low concentrations of phospholipids and led to investigations of the human analog of the Mdr-2 knockout mouse, which had a similar phenotype of no phospholipids in the bile and obstructive findings on liver biopsy. It was thereby discovered that PFIC-3 is caused by mutations in an export pump of the ABC transporter family called multidrug resistance 3 (MDR-3) that is expressed on the canalicular membrane. It functions in the translocation of phosphatidylcholine across the canalicular membrane. Mdr-2–deficient mice made transgenic by expression of the human homolog of mdr-2, MDR-3, recover function and excrete phospholipid in their bile. This finding confirms the functional homology between the mouse and human genes and further suggests that phospholipid excretion is limited by the amount of MDR-3 or mdr-2 present. The MDR-3 gene has been mapped to 7q21. The absence of phospholipids in this condition is felt to destabilize the formation of micelles due to insufficient phospholipids to solubilize the cholesterol. The imbalanced micelles likely promote lithogenic bile with crystallized cholesterol, which could produce small bile duct obstruction.
There have been reports of several families with clinical and biochemical features consistent with PFIC who do not have mutations in FIC-1, BSEP, or MDR-3 . Four children from an Amish kindred have recently been described with a defect in the sinusoidal uptake of bile salts. Therefore, there may be a wider spectrum of disease in PFIC yet to be described.
Benign Recurrent Intrahepatic Cholestasis
The condition benign recurrent intrahepatic cholestasis (BRIC) presents very similarly to PFIC, with cholestasis, pruritus, low GGT level, and high serum bile acid level; however, the hallmark of this condition is intermittent episodes of cholestasis without progression to liver failure and later onset than PFIC. Patients are totally asymptomatic, both clinically and biochemically, in-between the episodes of cholestasis. BRIC shares the same locus with PFIC-1 ( ATP8B1 mutation) and PFIC-2 [ ABCB11 (BSEP)], but the mutations cause only partially impaired protein synthesis (see Table 70-2 ). The cholestasis episodes have been treated by temporary biliary diversion using nasobiliary tube drainage of bile during the episode with some success in relieving the pruritus. However, in select cases, surgical biliary diversion has also been used for cases of BRIC with frequent debilitating attacks, or when there appears to be progression to permanent cholestasis. The clinically more severe disease identified as Greenland Eskimo infantile cholestasis is seemingly also a variant of FIC1 disease.
Hereditary Cholestasis with Lymphedema: Aagenaes Syndrome
Aagenaes syndrome is identified as a genetic form of cholestasis associated with lymphedema that is mapped to chromosome 15q. It was initially reported in Norwegian patients; however, subsequently, there have been reports in Italian children, in Japanese children, and in siblings with French/German heritage. The inheritance appears to be autosomal recessive. Studies are underway to identify the gene locus for this disease using linkage disequilibrium.
Clinically, jaundice in the first weeks of life with acholic stools may be the first manifestation of the disease. Overall, the cholestatic liver disease tends to improve with age such that bilirubin and aminotransferase levels may be normal by school age. Cholestasis occurs episodically in older children with cholestatic periods lasting 2 to 6 months. The liver disease tends to be mild in most patients, but several older children and adults have progressed to cirrhosis. Both puberty and pregnancy have been associated with transient increases in cholestasis.
The liver histopathology in early childhood shows massive giant cell transformation of hepatocytes and intracellular retention of bile pigment. Patients in clinical remission may have liver morphology close to normal. Some patients may have bile plugs and a slight increase in portal fibrosis. Four of 26 patients reported by Aagenaes et al. have developed biopsy-proven cirrhosis. Treatment is that of cholestasis, utilizing fat-soluble vitamin supplementation and choleretic agents. The lymphedema usually appears in the lower extremities in early childhood and has been attributed to lymphatic vessel hypoplasia. The greater clinical problem tends to be the lymphedema, which can become disabling. Patients are offered physical therapy and restrictive wraps to limit the fluid accumulation and prevent skin breakdown.
Arthrogryposis Multiplex Congenita, Renal Dysfunction, and Cholestasis Syndrome
Arthrogryposis multiplex congenita, renal dysfunction, and cholestasis syndrome (ARC) is an autosomal recessive multisystemic disorder associated with the germline mutation VPS33B that is mapped to chromosome 15q26. In the largest study of ARC patients published to date involving 66 patients, the most prevalent clinical features described were failure to thrive, the presence of neonatal cholestasis with low GGT level, platelet dysfunction with high risk of hemorrhage with liver biopsy and spontaneous bleeding, renal tubular leak, and hypotonia with arthrogryposis. Less frequent clinical features include small for gestational age, dysmorphic features (lax skin, low-set ears, high arched palate, and cryptorchidism), ichthyosis, metabolic acidosis, nephrogenic diabetes insipidus, recurrent infections, recurrent febrile illnesses, and diarrhea. Survival beyond a year of age is unusual. If the patient survives infancy, cerebral manifestations including severe developmental delay, hypotonia, nerve deafness, poor feeding, microcephaly, and defects of the corpus callosum may become evident.
VPS33B is a vacuolar sorting protein involved in the regulation of vesicular membrane fusion and protein sorting by interacting with SNARE protein on membranes such as the canalicular membrane. Patients with ARC have evidence of abnormal polarized membrane protein trafficking by immunostaining of renal and liver biopsies that show mislocalization of several apical membrane proteins in the liver and kidney ( Figure 70-5 ). In the zebrafish knockdown of the vsp33b ortholog, bile duct paucity and impaired intestinal lipid absorption were observed. These findings phenocopied the digestive disease seen in ARC patients and suggest that VPS33B may play a role in primary bile duct development. VPS33B is also required for megakaryocyte and platelet alpha-granule biogenesis. Defects in platelets of patients with ARC have been documented and are associated with increased platelet size, a pale appearance in blood films, and a decrease or absence in alpha-granules. This defect causes platelet dysfunction that clinically has been manifested as increased incidence of bleeding with liver biopsy and spontaneous bleeding.
Severe cholestasis may occur in association with arthrogryposis multiplex congenita and renal disease. The cholestatic liver disease is usually present at birth, and paucity of intrahepatic bile ducts and multinucleate transformation of hepatocytes are the predominant features. Lipofuscin disposition has been described in several cases of ARC, and pigmentary change, bile duct paucity, and giant cell transformation may coexist in some patients. Patients with ARC rarely survive long enough to develop cirrhosis. Causes of death have been reported as infection, bleeding complications, or metabolic derangements.
Neonatal Ichthyosis–Sclerosing Cholangitis Syndrome
Many human and animal cholestatic disorders are associated with changes in hepatocyte cytoskeleton and tight junctions (TJs). Neonatal ichthyosis–sclerosing cholangitis syndrome (NISCH) is identified in neonates by the presence of ichthyosis and sclerosing cholangitis. The liver histology initially reveals cholestasis that rapidly progresses to the classical fibrous obliteration of small bile ducts and “onion-skinning” periductal fibrosis ( Figure 70-6 ). Patients with the same mutation in the claudin-1 (CLDN1) gene as the originally described cohort may also show congenital paucity of the bile ducts. NISCH is caused by a mutation in the claudin-1 gene located on chromosome 3q27-q28. Claudin-1 is a tight junction (TJ) protein. In the liver, TJs separate bile flow from plasma and are composed of strands of claudins and occludin. This mutation results in total absence of the claudin-1 protein in the liver and skin of affected patients. The lack of the claudin-1 protein has been shown to be associated with increased paracellular permeability between polarized hepatic cell lines, supporting the hypothesis that bile leakage through deficient TJs is involved in the liver pathology in NISCH patients.
North American Indian Childhood Cirrhosis
North American Indian childhood cirrhosis (NAIC) is a nonsyndromic form of autosomal recessive cholestatic disease in Ojibway-Cree children from northwestern Quebec. It classically involves a child who had apparent transient neonatal jaundice who then progresses to biliary cirrhosis. The histopathologic findings show bile duct damage and severe fibrosis. The condition has been mapped to a mutation in the cirhin gene, which is on chromosome 16q22. Cirhin is found in embryonic liver, is predicted to localize to mitochondria, and has a structural motif.
Cystic fibrosis (CF) is fully described in Chapter 82 . CF, however, deserves to be mentioned briefly here as an inherited disease of cholestasis that is known to be caused by a disorder of membrane transport. CF is due to a mutation in the gene CFTR , which codes for a chloride exchange channel that is expressed in tubular epithelium, particularly in the lung and biliary tract. The mutated gene results in impaired chloride exchange, resulting in thickened secretions in the airways as well as the biliary system. The thickened secretions result in “inspissated bile,” which leads to plugging of the small bile ducts and eventually biliary cirrhosis.
Disorders of Embryogenesis: Alagille Syndrome
Alagille syndrome (ALGS; OMIM #118450), or arteriohepatic dysplasia, is an autosomal-dominant disorder characterized by paucity of intrahepatic bile ducts, cholestasis, congenital heart defects, distinct facial appearance, and skeletal and eye anomalies. In addition, renal and vascular system involvement is present in a significant number of patients with ALGS. Overall, the incidence of ALGS is at least 1:70,000 live births, but the disease is likely underdiagnosed because of the variability in clinical presentation, even within the same family. Chronic cholestatic liver disease in ALGS is a significant cause of morbidity, leading to significant pruritus, malabsorption, and xanthomas. In some patients, cholestasis improves over time, whereas in others it may progress to portal hypertension or liver failure. It is estimated that 20% to 40% of ALGS patients will eventually require liver transplantation. In 1997, mutations in the JAG1 gene, which encodes a ligand in the Notch signaling pathway, were shown to cause ALGS. The discovery that JAG1 is a disease gene for Alagille syndrome, a disorder with paucity of intrahepatic bile ducts as one of its major features, identified JAG1 and the Notch signaling pathway as crucial for the development of liver, bile ducts, and other organs affected in this multisystem disorder. The advent of molecular testing for ALGS has led to improved insight into the spectrum of JAG1- mutation associated disease and has advanced understanding of the role of the Notch pathway in organogenesis.
In their early report of arteriohepatic dysplasia, Watson and Miller suspected that the disorder was inherited in an autosomal dominant fashion. It was later discovered that about 5% of patients carried cytogenetically visible deletions on chromosome 20, and studies of multiple patients and families with deletions and balanced translocations allowed narrowing of the critical region to a small area on chromosome 20p12. With current techniques, JAG1 mutations, 60% of which are de novo, can now be identified in up to 94% of patients who meet clinical criteria for ALGS. The majority of the mutations (72%) are protein truncating, whereas about 15% occur in splice sites and 13% are missense mutations. Despite the differences in JAG1 mutation types, no genotype–phenotype correlation has been identified in ALGS. In fact, related patients who carry the same mutation may have widely variable clinical phenotypes, suggesting that genetic modifiers may play a role. NOTCH2 mutations have been identified in a small group of JAG1- negative individuals, and a small percentage of ALGS patients remain without a molecular diagnosis.
The Notch pathway is an evolutionarily conserved intercellular signaling mechanism involved in cell fate determination in multiple organ systems. The pathway was first described in Drosophila , where the Notch transmembrane receptor interacts with its ligands, Delta and Serrate, to govern cell differentiation. To date, four Notch receptor genes have been identified in vertebrates ( Notch1 , Notch2 , Notch3 , and Notch4 ), which signal to five ligands ( Jag1 , Jag2 , Dll1 , Dll3 , Dll4 ). It is generally accepted that the Notch receptors are activated by ligand binding to the extracellular domain. The intracellular domain is then proteolytically cleaved and translocated to the nucleus, where it interacts with nuclear proteins to activate a cascade of downstream transcription factors.
JAG1 and the Notch receptor genes are widely expressed during development, especially in organs affected in ALGS, such as liver, heart, vasculature, and kidney. Multiple reports have demonstrated JAG1 expression in vascular structures in the developing liver. JAG1 and NOTCH2 are both expressed in the ductal plate during bile duct specification in embryonic liver. JAG1 is also expressed extensively in the developing heart, especially in the pulmonary artery, aorta, and developing valves, correlating with cardiovascular phenotypes in ALGS. Multiple mouse models have shed light on the critical roles of JAG1 and NOTCH2 in the development of the organs affected in ALGS. Organ-specific expression patterns and Notch pathway functions are discussed in the sections on clinical manifestations.
In 1975, Alagille and colleagues published a case series of 15 patients with bile duct hypoplasia and characteristic features including distinctive facies, vertebral anomalies, cardiac murmur, and growth failure. To this day, the clinical diagnosis of Alagille syndrome follows the same guidelines of bile duct paucity plus three of five clinical criteria including cholestasis, cardiac murmur or heart disease, skeletal anomalies, ocular findings, and characteristic facial features. With the advent of molecular diagnosis, it has become clear that not every individual carrying a damaging mutation in JAG1 would be diagnosed with ALGS on a clinical basis. Kamath and colleagues studied 53 mutation-positive relatives of 34 ALGS probands and found that only 21% had clinical features that would have led to a diagnosis of ALGS. Thirty-two percent had mild clinical features, and 45% did not meet clinical criteria. In stark contrast to the high penetrance of clinical features identified in studies of probands ( Table 70-3 ), this study demonstrated the clinical variability in individuals carrying JAG1 mutations. This information has led some investigators to suggest revising diagnostic criteria, taking into account family history and molecular testing.
|Feature, % (n)||Alagille,1987 |
|Hoffenberg et al.,1995 (Ref. )||Emerick et al.,1999 (Ref. )||Quiros-Tejeira et al.,1999 (Ref. )||Weighted %|
|Bile duct paucity||100 (80)||81 (22)||80 (20/25)||85 (69/81)||83 (34/41)||89|
|Cholestasis||91 (73)||93 (25)||100 (26)||96 (88)||100 (43)||95|
|Cardiac murmur||85 (68)||96 (26)||96 (24/25)||97 (90)||98 (42)||94|
|Vertebral anomalies||87 (70)||33 (6/18)||48 (11/23)||51 (37/71)||38 (12/32)||61|
|Facies||95 (76)||70 (19)||92 (23/25)||96 (86)||98 (42)||92|
|Ocular findings||88 (55/62)||56 (9/16)||85 (17/20)||78 (65/83)||73 (16/22)||80|
|Renal||73 (17/23)||19 (5)||40 (28/69)||50 (15/30)||44|
|Intracranial event or vascular anomaly||15 (4)||14 (13)||12 (5)|
|Pancreatic insufficiency||41 (7/17)|
|Growth retardation||50 (40)||90 (24)||87 (27/31)||86 (37)||71|
|Developmental delay||52 (14)||2 (2)|
|Mental retardation||16 (13)||16 (15)|
Clinical Manifestations of Alagille Syndrome
Hepatic Manifestations of ALGS
The most common clinical presentation of ALGS is neonatal cholestasis, which can be difficult to distinguish from other causes of obstructive cholestasis, especially biliary atresia. Typically, the initial biochemical abnormalities consist of conjugated hyperbilirubinemia, modestly elevated liver enzymes, and high alkaline phosphatase and GGT levels. Hepatomegaly is almost universally present. In the cholestasis evaluation, nuclear medicine scintiscan does not help to differentiate ALGS from biliary atresia. In one report, 61% of ALGS infants had no tracer excretion at 24 hours. Liver biopsy may also be nondiagnostic in the neonatal period, because of evolution of bile duct paucity over time ( Figure 70-7 ). In one large clinical study, paucity was present in 95% of biopsies after 6 months of age, but in only 60% of biopsies obtained earlier than 6 months. Infants presenting with bile duct proliferation may undergo intraoperative cholangiogram to rule out biliary atresia. However, abnormalities of the intrahepatic and extrahepatic biliary tree are common in ALGS at the time of cholangiogram, with the most frequent abnormalities being nonvisualization of the intrahepatic biliary tree or hypoplasia of the extrahepatic system. In most published series, a small number of ALGS infants have undergone the Kasai portoenterostomy, for a presumed diagnosis of biliary atresia (BA) at the time of cholangiogram. The Kasai operation is not indicated in ALGS, and most published reports suggest that these infants may have a worse hepatic outcome and be more likely to require liver transplantation.
Chronic Cholestasis and Natural History.
Chronic cholestasis is a nearly universal feature of ALGS, which may be mild or severe. As in infancy, biochemical abnormalities typically include elevations in bilirubin and serum bile acid levels and a high GGT level out of proportion to alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Total protein and albumin are usually normal, as are indicators of synthetic function such as the ratio of prothrombin time (PT) to the international normalized ratio (INR) and partial thromboplastin time (PTT), in the presence of adequate vitamin K stores. As cholestasis progresses, the cholesterol level may rise to the thousands of mg/dL, with the appearance of skin xanthomas at levels over about 500 mg/dL. Xanthomas typically appear on the extensor surfaces of the fingers, the palmar creases, popliteal fossae, and inguinal creases. Depending on their location, xanthomas can be debilitating and can impair movement and function. Pruritus is usually not apparent in early infancy, but it will progress over the first few years of life in cases of significant cholestasis. The pruritus in ALGS can be extremely severe, interrupting sleep and daily activities, and may require multiple medical interventions. In cases of refractory to medical therapy, severe cholestasis, pruritus, and xanthomas may respond to biliary diversion. A minority of ALGS patients develop progressive liver disease leading to cirrhosis and portal hypertension. Hepatocellular carcinoma has also been reported rarely in this disorder and may occur in young children in the absence of cirrhosis.
The largest study of liver disease outcome in ALGS is a report of 163 patients from France, 132 of whom presented with neonatal cholestasis and 31 of whom presented later with signs of cholestatic liver disease. At the study end point, the patients who had presented with neonatal jaundice were much more likely to remain jaundiced, to have persistent pruritus and xanthomas, and to have ongoing hepatosplenomegaly. Jaundice and cholestasis eventually resolved in the majority of patients with presentation in childhood, and in about 15% of the neonatal cholestasis group. Bile duct paucity in ALGS appears to evolve during the postnatal period in many cases and is identified in only 60% of liver biopsies performed before 6 months of age.
In general, medical management of cholestasis in ALGS is similar to that of other cholestatic disorders. Adequate nutrition is crucial; a high-calorie diet with a high proportion of fat from medium-chain triglycerides is recommended in the neonatal period. Many older patients also have poor weight gain requiring nutritional supplements. In addition, many patients are deficient in fat-soluble vitamins. The majority of ALGS patients with prolonged cholestasis require medical therapy for pruritus, such as ursodeoxycholic acid, antihistamines, bile-acid binding resins, rifampicin, or naltrexone. A detailed discussion of treatment of cholestasis is outside the scope of this chapter, but further information is provided elsewhere in the text.
Requirement for liver transplantation in ALGS varies among the major clinical studies, ranging from 21%, to 31%, to 47% as reported by Quiros-Tejeira and colleagues. In a study of liver disease outcome in 163 ALGS children, the overall calculated survival with native liver was 51% at 10 years and 38% at 20 years. The most common indications for liver transplantation in the ALGS population were unremitting cholestasis leading to severe pruritus and xanthomata, recurrent and poorly healing bone fractures, end-stage liver disease, and portal hypertension with gastrointestinal bleeding. Several retrospective studies have reported outcome of liver transplantation in ALGS patients. One-year graft and patient survival have been reported as 87.5% and 91.7%, respectively, which is comparable to transplantation outcomes for other diagnoses. In a recent retrospective study utilizing the United Network for Organ Sharing (UNOS) database, comparing transplantation outcomes for ALGS to biliary atresia, the ALGS group had lower overall 1- and 5-year patient and graft survival and higher rates of graft failure than the BA group. As would be expected, the ALGS patients also had higher rates of cardiac and neurologic complications leading to mortality posttransplantation. In another recent study, the authors performed a retrospective analysis of the Studies in Pediatric Liver Transplantation (SPLIT) database and reported the outcomes of 91 ALGS patients and 236 age-matched BA patients undergoing liver transplantation. The 1-year patient survival rates were 87% for the ALGS group and 96% for the BA patients. Most of the deaths in the ALGS group occurred in the first 30 days, and associated factors included biliary, vascular, central nervous system, and renal complications. In addition, the ALGS patients were more likely to develop renal insufficiency posttransplantation. Liver transplantation can be accomplished successfully in ALGS, with outcomes similar to other indications, but the multisystem nature of this disorder leads to increased risk of complications in other organ systems. Significant cardiac disease is a major cause of morbidity and mortality in this population, and a detailed cardiac evaluation is necessary even in the absence of severe structural heart disease. Renal evaluation before transplantation is also advisable and may indicate that kidney-sparing immunosuppressive regimens should be used.
Cardiac murmur is a highly penetrant feature of ALGS, with incidence ranging from 85% to 98% in major clinical studies (see Table 70-3 ). Overall, by far the most common abnormality is stenosis at some level in the pulmonary arterial tree, detected in 67% (49 of 73) of patients evaluated in a large study from the Children’s Hospital of Philadelphia. In this study, 22 (24%) of 92 patients had structural heart disease, with the most common structural lesion being tetralogy of Fallot (n = 10; 4 with associated pulmonary atresia). Other common heart lesions included ventricular septal defects, many of which were also associated with pulmonary atresia or pulmonic stenosis. Mortality was dramatically higher in the group with structural heart disease compared to those without, with a predicted 20-year survival of only 40%.
The largest published study of cardiovascular phenotype in ALGS is a retrospective analysis of 200 individuals with either a JAG1 mutation or a clinical diagnosis of ALGS. In this group, cardiovascular anomalies were identified by imaging in 75%, and 19% had a murmur consistent with peripheral pulmonic stenosis with either a normal echocardiogram or no imaging. Of the patients with identified anomalies, 82% were right sided and 15% left sided; 8% of patients had both right- and left-sided defects. The most common abnormality was stenosis or hypoplasia of the branch pulmonary arteries, with the most common structural anomaly being tetralogy of Fallot, present in 15%. Of interest, a specific JAG1 mutation has been associated with familial tetralogy of Fallot in the absence of hepatic or other ALGS clinical manifestations.
Vertebral arch defects were identified in 8 (53%) of 15 patients in one of the earliest reports of the syndrome in 1975. The typical finding of butterfly vertebrae seems to be one of the least penetrant features, reported in 33% to 87% of patients in the major case series (see Table 70-3 ). Other minor skeletal abnormalities identified in ALGS patients include a decreased interpedicular distance in the lumbar spine, seen in 53% (23 of 43) of patients in a large study by Alagille and colleagues. Shortened distal phalanges in the hands have also been reported. Supernumerary digital flexion creases have been identified in 35% of ALGS probands in one study, whereas they are found in only 1% of the general population. Collectively, these musculoskeletal features may be useful in determining a clinical diagnosis of Alagille syndrome, but in general, they are not clinically significant.
In contrast, risk of recurrent and poorly healing bone fractures in ALGS patients is a significant source of morbidity in this population and may even become an indication for liver transplantation in severe cases. ALGS patients are recognized to have deficits in size-adjusted bone mass as measured by dual-energy X-ray absorptiometry (DXA). In one report, ALGS children were small for age and had decreased bone area and bone mineral content, adjusted for both age and height z-score, when compared with controls. In another recent study, the estimated incidence rate of femur fracture in ALGS was 50 times that seen in the general population.
Ocular abnormalities are extremely common in children and adults with ALGS. The most well-known ocular features are deep-set hyperteloric eyes and the finding of bilateral posterior embryotoxon. The latter finding was first noted in 1979 and has subsequently become a major feature of the disease. Posterior embryotoxon is thought to represent a prominent thickened or hypertrophied Schwalbe’s line that is anteriorly displaced, visible through a clear cornea as a sharply defined, concentric white line, or opacity anterior to the limbus ( Figure 70-8 ). Whereas it may be found in 90% to 95% of patients with ALGS and is found in parents of patients with ALGS, it is present in the normal population at a frequency between 8% and 15%. Other common ocular findings in ALGS include posterior segment eye changes in 90% of patients, a variety of optic disk findings in 76%, anomalous retinal vasculature in 29%, alteration of the chorioretinal pigment in 76%, diffuse hypopigmentation of the fundus, and the presence of optic disk drusen on B scan. Despite the high prevalence of optic abnormalities in these patients, visual acuity does not appear to be adversely affected, and the changes do not appear to be progressive, although longitudinal studies will be required.