Wilson Disease and Other Inherited Metabolic Diseases of Liver

Lizhi Zhang, MD

18.1 INTRODUCTION

Metabolic dysfunction of liver can be due to several causes. Congenital deficiency of a specific enzyme or enzymes is the most important etiology in this group of diseases. Inherited disorders of liver metabolism can essentially involve any metabolic pathway, which results in abnormalities in the synthesis or catabolism of carbohydrates, proteins, or lipids; the metabolism of copper, iron, amino acids, or vitamins; bile acid synthesis and metabolism; detoxification; coagulation cascade; and urea cycle. These diseases can lead to hepatocyte injury and liver structural damage, but some may present with severe functional abnormalities without obvious structural changes. Abnormal accumulations of metabolites in the liver are often revealed by histopathologic examination and may provide diagnostic clues. These changes are summarized in Table 18.1 for quick reference.

In this chapter, Wilson disease and other inherited metabolic diseases are discussed, with a focus on entities that are mostly commonly encountered in the practice of surgical pathology. Of note, there are many additional genetic diseases that can involve liver but are not covered in this chapter due to limited space. Comprehensive reviews of the rare entities that are not covered in this chapter are available in most cases through a PubMed search.

18.2 DISORDERS OF COPPER METABOLISM

Wilson disease

Clinical features

The estimated prevalence of Wilson disease is 1 in 30,000, with a corresponding carrier frequency of 1 in 90 in most populations.¹ Neurologic and psychiatric signs can include movement disorders, rigid dystonia, and depression. Overall, approximately 40% of individuals with Wilson
disease will manifest clinically primarily with liver disease, especially in children and younger adults, typically between 5 to 35 years old. However, initial presentation as severe liver disease has been reported in children <2 years old and in older adults.²,³ The clinical presentation of liver disease varies significantly and may include recurrent jaundice because of acute or chronic hemolysis, acute self-limited hepatitis, autoimmune hepatitis, fulminant hepatic failure, chronic liver disease with portal hypertension, or fatty liver disease. Kayser-Fleischer rings owing to copper deposition in Descemet’s membrane of the cornea can be observed in 50% to 90% cases.

Laboratory findings

Mildly elevated liver transaminase and bilirubin levels are present in most patients but can be significantly elevated in cases of acute liver failure. Alkaline phosphatase levels may be low in individuals presenting with acute liver failure. With disease progression, liver synthetic function can be affected and manifested as low albumin levels or prolonged prothrombin times. The AST (aspartate aminotransferase):ALT (alanine aminotransferase) ratio is commonly >2.2, but this finding is not very specific.

Laboratory tests can be used to evaluate for Wilson disease. Serum ceruloplasmin levels are low (<0.2 g/L) in over 90% of cases, but can be normal in patients with significant active liver inflammation because ceruloplasmin is an acute phase protein. Serum copper levels are usually low because ceruloplasmin, the main copper carrier protein, is also low. In addition, 24-hour urine level above 100 µg confirms Wilson disease, although levels above 40 µg strongly suggest Wilson disease. However, these tests are not entirely specific. For example, elevated urine copper levels sometimes occur in autoimmune hepatitis and cholestatic liver disease.⁴

Quantitative liver copper analysis is also useful in diagnosing Wilson disease, in which the hepatic copper concentration is usually >250 µg/g dry weight. Copper levels can also be elevated in chronic cholestatic liver disease, though usually the levels are considerably lower. Finally, some individuals with early Wilson disease can have moderately elevated copper levels that are less than 250 µg/g dry weight.

Imaging

Imaging findings of liver in Wilson disease vary with the form and degree of liver diseases, but may include fatty change, fulminant liver necrosis, or cirrhosis.

Gross findings

Explanted livers typically show established cirrhosis. The cirrhosis commonly has a micronodular pattern but can be mixed or even macronodular. In cases with fulminant liver failure, the liver may keep its normal shape and surface texture, but in most cases the necrosis will also lead to shrinkage of liver with a wrinkled capsular surface.

Microscopic findings

The pathologic changes in the liver are believed to result from toxic effects of copper accumulation. In precirrhotic livers, the histologic
findings can range from almost normal liver parenchyma to massive necrosis. The histopathologic changes can be categorized into the following patterns:

“Almost normal” liver pattern. In this pattern, the liver shows minimal to mild nonspecific changes. These changes can include variable combinations of minimal to mild portal lymphocytic inflammation, minimal to absent steatosis, rare apoptotic cells, moderate hepatocyte nuclear anisonucleosis, focal hepatocyte nuclear glycogenation, mild Kupffer cell hyperplasia, and no fibrosis (Fig. 18.1).

Acute hepatitis pattern. In this pattern, affected individuals typically present clinically with acute hepatitis and sometimes with liver failure. The liver biopsy shows marked inflammation with hepatocyte necrosis. The portal tracts show marked inflammation composed of lymphocytes and plasma cells. Interface hepatitis can be prominent and there can be a brisk bile ductular proliferation. The lobular parenchyma has significant necroinflammatory activity and variable degrees of necrosis (Fig. 18.2). The hepatocytes are swollen with ballooning degenerations. Copper deposition may be identified in hepatocytes and, when there is massive or submassive necrosis, in the Kupffer cells or portal macrophages.⁵ Macrovesicular steatosis can be present but is not a predominant feature. Mild cholestasis and fibrosis can also be seen. This pattern is often indistinguishable from autoimmune hepatitis.

Fatty liver disease pattern. In this pattern, the findings can range from steatosis to steatohepatitis. Hepatyoctes with nuclear glycogenation are common. When there is steatohepatitis, the biopsy shows macrovesicular steatosis, hepatocytes with ballooning degeneration, Mallory body formation, and occasional apoptotic cells (Fig. 18.3). Fibrosis can also be found in some cases.

Chronic hepatitis pattern. The findings in this pattern are similar to those of chronic hepatitis due to other etiologies (Fig. 18.4). Some changes can mimic autoimmune hepatitis. The portal tracts are expanded by lymphocytes, occasional plasma cells, variable degrees of bile ductular proliferation, and fibrosis. Interface activity can be prominent. The lobules also show variable inflammation with spotty necrosis. Steatosis can be present in variable degrees. Advanced fibrosis can be present.

Cryptogenic cirrhosis pattern. In this pattern, the liver biopsy shows established cirrhosis with minimal or mild inflammatory activity (Fig. 18.5). The fibrotic septa may be thin or wide, containing mild nonspecific inflammation and mild bile ductular proliferation. Mild steatosis can be present and ballooned hepatocytes with Mallory hyaline can be prominent in a subset of cases. The lobules usually lack significant inflammation, besides scattered apoptotic cells. The hepatocytes may be enlarged and show oncocytic changes with prominent granular eosinophilic cytoplasm because of increased number of mitochondria. Giant cell transformation is occasionally seen, especially in the setting of cholestasis. The copper deposits can be readily seen in periseptal hepatocytes using Rhodanine copper stain, but the distribution of copper is variable, with some nodules being loaded with copper and others having only minimal copper or no copper (Fig. 18.6).

Immunohistochemistry and special stains

There are several stains that can be used to identify copper, including Orcein, Timm silver, Rhodanine, Victoria blue, and rubeanic acid. Rhodanine is the most commonly used stain because of its reliability, reproducibility, a linear relationship with tissue copper concentration,
and simple and fast staining techniques. Orcein and Victoria blue stains are not specific for copper, as they detect copper-associated proteins in lysosomes, which may or may not contain copper. The Timm silver sulfide and rubeanic acid stains are more sensitive than Rhodanine but are not widely used. The Timm silver sulfide stain needs a longer (24-hour) deparrafination time, and rubeanic acid requires a 72-hour incubation to reach the best results.⁶,⁷

Copper deposits on the Rhodanine stain are seen as small red-brown granules in the cytoplasm of hepatocytes and tend to have a zone 1 distribution (Fig. 18.6), but the copper deposition can be panlobular when there is marked copper accumulation.⁸ The Rhodanine copper stain is very helpful but is neither entirely sensitive nor specific in isolation for diagnosing Wilson disease. This is because early in the course of Wilson disease, the copper in the hepatocytes is located in the cytosol and not the lysosomes and the Rhodanine stain only detects lysosomal deposits. Therefore, a negative copper stain cannot rule out Wilson disease. If Wilson disease is clinical suspected, quantitative copper analysis should be performed on the liver biopsy.

In addition, the Rhodanine stain is positive in chronic cholestatic liver disease. Copper is normally excreted in the bile and cholestatic conditions can lead over time to copper deposits in the hepatocytes, though the deposits tend to be more focal and milder than seen in Wilson disease. Cirrhotic livers can also have nonspecific copper accumulation, but again the copper deposition tends to be focal and mild.⁹

Ultrastructural findings

On ultrastructural examination, the mitochondria show variable numbers and sizes, dense matrix with occasional vacuolated granules, crystal inclusions, enlarged intercristal spaces, and separation of the outer from the inner membranes. Other alterations include increased numbers of peroxisomes, lipofusion granules, multivesicular bodies, and lipid droplets.⁵,¹⁰

Molecular genetic findings

Wilson disease is inherited in an autosomal recessive manner. Heterozygotes may have mild biochemical abnormalities in copper metabolism but most of them have no clinically significant disease. Although more than 300 disease-causing mutations of ATP7B have been identified, only a small number of mutations are responsible for most cases in a specific population. For example, in Western populations, the H1069Q mutation is present in approximately 50% of cases, but it is rare in Chinese populations, who tend to have the R778L mutation. The mutations can be detected using targeted mutation analysis or whole gene sequencing. Identification of two disease-causing mutations establishes the diagnosis of Wilson disease. However, genetic testing for clinical diagnosis is often difficult because of the large number of mutations and the fact that not all mutations are disease-causing. If disease-causing mutations are detected in an index case, then subsequent targeted mutation analysis can be very helpful for screening the extended family.

Differential diagnosis

There are no specific histopathologic patterns for Wilson disease. Therefore, the possibility of Wilson disease is considered in essentially

all liver biopsies when working up the etiology of unexplained liver disease, especially in children and younger adults. Several patterns can raise suspicion for Wilson disease, including fatty liver disease in younger individuals with no risk factors, cryptogenic cirrhosis in young or middle aged adults with no risk factors, or an acute unexplained hepatitis or acute liver failure in young individuals. Even in cases where the clinical and pathological findings suggest autoimmune hepatitis, clinicians should still be reminded to rule out Wilson disease.

Table 18.1 Inclusions, Deposits, or Crystals in Live of Common Genetic Disorders

Disorders	Deposit component	Location of inclusions	Light microscopic features	Electron microscopic features	Special stains	Mimickers
Wilson disease	Copper	Hepatocytes; rare in Kupffer cells and macrophages	Not visible	Prominent mitochondria with variable number and size, dense matrix, occasional vacuolated granules, crystal inclusions, and separation of the outer from the inner membranes	Rhodanine (most commonly used), Orcein, Timm silver, Victoria blue, and rubeanic acid	Lipofuscin
Hereditary Hemochromatosis	Iron	Hepatocytes; also in Kupffer cells, biliary epithelial and endothelial cells	Dark brown rusty pigments	Electron-dense ferritin cores and siderosomes	Prussian blue stain, with pericanalicular pattern	Lipofuscin, Dubin-Johnson pigments, bile, hemosiderin
Glycogen storage disease (GSD)	Glycogen	Hepatocytes	Swollen hepatocytes with pale-staining cytoplasm and prominent cell membrane; Light eosinophilic ground glass inclusions in type IV	Abundant glycogen rosettes in all types; monoparticulate glycogen in enlarged lysosomes in type II; nonmembrane bound, randomly oriented fibrillary material in type IV	PAS and digested by diastase	Glycogenic hepatopathy; Lafora bodies and hepatitis B inclusions for type IV; Pseudoground glass inclusions
Lafora disease	Insoluable glycogen in the form of polyglucosan	Hepatocytes	Homogeneously eosinophilic or ground glass Lafora bodies with round or oval shape and distinct border surrounded by a halo	Nonmembrane bound fibrillary and granular material	PAS, colloidal iron, Lugol iodine stains	Inclusions in type IV GSD, hepatitis B; Pseudoground ground glass inclusions; Lafora body-like inclusions related to cyanamide or 6-thioguanine
Mucopolysac-charidosis	Glycosamino-glycans	Hepatocytes and Kupffer cells	Hepatocytes and Kupffer cells with rarified cytoplasm or numerous small-to medium-sized cytoplasmic vacuoles.	Membrane bound vacuoles in the hepatocytes and Kupffer cells	Colloidal iron stains with greenish-blue cytoplasmic staining; weakly positive of PAS and PASD	GSD, microvesicular steatosis
α-1-Antitrypsin deficiency	α-1-Antitrypsin	Hepatocytes	Eosinophilic globules with variable size in periportal hepatocytes	Amorphous finely granular or fibrillary deposits in endoplasmic reticulum	PASD; bright red on Trichrome stain; α-1-Antitrypsin immunostain	Megamitochondria, Globular amyloid, fibrin globules
Afibrinogen	Fibrinogen	Hepatocytes	Round or polygonal light eosinophilic inclusions with a dark core, irregular outline, and a clear halo. The cytoplasm filled with fine globules with ground-glass appearance.	Dilated cisterns of rough endoplasmic reticulum filled with densely packed tubular structures arranged in curved bundles with a fingerprint-like pattern	Fibrinogen immunostain; PTAH; PAS	α-1-Antitrypsin globules, hepatitis B ground glass or pseudoground ground glass inclusions
Gaucher disease	Glycocerebroside	Kupffer cells and macrophages	Amphophilic or faintly stained, striated, and wrinkled-paper appearance	Spindle or rod-shaped inclusions surrounded by a limiting membrane with internal tubular structures on cross section	PAS or Trichrome stains	GSD inclusions, Lafora bodies, pseudo-Gaucher cells in patients with hematologic malignancy or anemia
Niemann-Pick disease	Sphingomyelin	Kupffer cells, macrophages, and hepatocytes	Hypertrophied Kupffer cells with foamy or microvesicular cytoplasm in sinusoidal spaces; hepatocytes less foamy than Kupffer cells	Dense mixed lipid lysosomes containing centrically laminated myelin-like inclusions	Oil red O, Luxal fast blue, or Baker acid hematin stains on frozen sections. PAS stain is variable positive or negative	Lipid-laden foamy cells in other disorders. Gaucher cells, cystine crystals
Cholesterol storage disease	Cholesteryl ester	Kupffer cells and hepatocytes; may also in biliary epithelial and endothelial cells	Kupffer cells and macrophages with foamy cytoplasm, marked microvesicular steatosis in hepatocytes, and brilliant silver birefringent crystals in frozen sections.	Peripheral vacules or large central cholesterol ester clefts in Kupffer cells; lipid droplets and occasional crystal clefts in hepatocytes	PASD	GSD, microvesicular steatosis, Gaucher cells, Niemann-Pick disease
Porphyria cutanea tarda	Uroporphyrin	Hepatocytes	Needle-shaped crystals with variable lengths; birefringent under polarized light best seen in unstained paraffin sections	Needle-shaped crystals with alternating areas of differing electron density	Ferric ferricyanide reduction test on unstained paraffin sections	Cystine crystals; may be overlooked in cases with heavy iron overload.
Erythropoietic protoporphyria	Protoporphyrin	Hepatocytes, Kupffer cells, and canaliculi	Dark brown pigments within the biliary canaliculi and macrophages; red to orange Maltese cross shape of birefringent crystalline pigment under polarized light	Crystal-containing vacuoles arranged in a “star-burst” pattern in cytoplasm and canaliculi	N/A	Bile plugs, Dubin-Johnson pigments, large iron deposits
Cystinosis	Cystine	Kupffer cells	Cystine crystals with a brilliant silvery birefringence under polarized light	Hexagonal crystalline structures	N/A	Gaucher cells, Niemann-Pick disease
Dubin-Johnson syndrome	Polymers of epinephrine metabolites	Hepatocytes	Coarsely granular dark brown pigments	Dense particles within lysosomes in a background of more homogenous moderately dense granular material, often associated with lipid droplets	Fontana-Masson	Lipofuscin, hemosiderosis, bile

Prognosis and treatment

If left untreated, Wilson disease tends to become progressively worse and can eventually be fatal. With early detection and treatment, most patients can live relatively normal lives. The main therapy for Wilson disease is chelating agents such as D-penicillamine and trientine to increase the removal of copper from the body or to prevent the absorption of copper from the diet. Liver transplantation is indicated in cases with fulminant liver failure or advanced fibrosis. Liver
transplantation is not recommended in those cases with severe neuropsychiatric illness, as its benefit in this setting is uncertain.

Figure 18.1 Wilson disease. Almost normal liver pattern.

Figure 18.2 Wilson disease. Autoimmune hepatitis-like pattern with marked lymphoplasmacytic infiltrate, interface activity, and necrosis.

Figure 18.3 Wilson disease. Fatty liver changes.

Figure 18.4 Wilson disease. Chronic hepatitis changes.

Figure 18.5 Wilson disease. Cirrhotic liver with no specific morphologic features.

Figure 18.6 Wilson disease. Marked copper accumulation in periportal hepatocytes demonstrated by a Rhodanine stain.

Other copper metabolic disorders

Besides Wilson disease, there are several other copper overload disorders not related to ATP7B mutations, including Indian childhood cirrhosis,
Tyrolean infantile cirrhosis, and idiopathic copper toxiocosis. Most individuals with these diseases present before the age of 2 years with histories of progressive lethargy, increased infections, and hepatomegaly. The etiology of these diseases is still not fully understood and both genetic defects in copper metabolism and excessive copper intake may have roles in the excess copper accumulation. The histologic changes in the liver appear to be similar in these diseases, though published descriptions remain sparse. Essentially all cases are diagnosed at the cirrhotic stage and characteristically have a micronodular pattern composed of very tiny nodules. The lobules typically show marked hepatocyte ballooning degeneration, abundant Mallory body formation, and scattered acidophil bodies. Steatosis is typically absent. The inflammation is mild and composed of lymphocytes, histiocytes, neutrophils, and a few plasma cells. Cholestasis can be prominent. The copper accumulation starts in periportal hepatocytes and then extends to the entire lobules and is typically marked and diffuse by the time the liver is cirrhotic. In addition to cirrhosis, the trichrome stains can show marked central vein fibrosis and marked pericellular fibrosis.

18.3 ENDOPLASMIC RETICULUM STORAGE DISEASES

Endoplasmic reticulum storage diseases are a group of inborn errors of metabolism affecting secretory proteins, resulting in hepatocellular storage and plasma deficiency of the corresponding protein. The abnormal proteins cannot be transferred from the rough endoplasmic reticulum to the smooth endoplasmic reticulum, leading to hepatocellular accumulation. Diseases include α-1-antitrypsin deficiency, afibrinogenaemia or hypofibrinogenaemia, α-1-antichymotrypsin deficiency, and antithrombin III deficiency.

α-1-Antitrypsin deficiency

Clinical features

α-1-Antitrypsin deficiency is an uncommon but not rare disease, and it has been estimated that 1 in 3,000 to 5,000 individuals carry mutations in the SERPINA1gene. α-1-Antitrypsin deficiency is underdiagnosed because not every individual with mutations will develop clinically significant disease.¹¹ The clinical presentation depends on the degree of α-1-antitrypsin deficiency, which is associated with different mutations in the SERPINA1gene. Over 100 alleles of SERPINA1 have been identified. The allelic genotypes have historically been determined by migration of the α-1-antitrypsin protein using gel electrophoresis. The normal phenotype is designated as PiM (Pi = protease inhibitor). The most common deficiency variants are PiS and PiZ. The most prevalent carrier phenotypes (not associated with disease in most cases) are PiMS and PiMZ, whereas the most common deficiency phenotypes (disease causing) are PiSS, PiSZ, and PiZZ, along with other rare deficiency alleles such as Mmalton, Mduarte, and null.¹²,¹³ Most individuals with α-1-antitrypsin deficiency have symptoms that manifest primarily in the respiratory system, with the development of emphysema during their thirties or forties. Cigarette smoking can accelerate disease progression.

Approximately 10% of PiZ individuals develop clinically significant liver disease with variable presentations. There is a distinct bimodal distribution of liver diseases in α-1-antitrypsin deficiency. It is the most common genetic cause of liver disease in neonates and children, which manifests as neonatal hepatitis and cholestatic jaundice. The prognosis of α-1-antitrypsin deficiency is generally excellent during children and adolescence, and most of them recover and have minimal or no liver disease by adulthood. Adults with α-1-antitrypsin deficiency, especially male patients, may have chronic liver disease, typically presenting in their 50s. They may present with asymptomatic abnormal liver enzymes, advanced cirrhosis, or hepatocellular carcinoma.

The association between heterozygosity of A1AT alleles and risk of developing chronic liver disease is controversial. It is generally accepted that heterozygosity of A1AT alleles does not increase the risk of liver disease in childhood, but approximately 10% adults with PiMZ phenotype will develop chronic liver disease.¹⁴

Laboratory findings

Serum A1AT levels are used to screen for α-1-antitrypsin deficiency. The serum A1AT levels correlate broadly with the genotype, but the serum levels alone cannot establish a diagnosis of α-1-antitrypsin deficiency. The serum levels of A1AT are typically as follows: PiMS (80% of normal), PiMZ (60%), PiSS (60%), PiSZ (40%), and PiZZ (15%).

Neonates with PiZ who develop neonate hepatitis and jaundice can have markedly elevated liver enzymes and hyperbilirubinemia. Approximately 50% of clinically well PiZ infants have mildly abnormal liver enzymes in the neonatal period, but most resolve during follow-up, with fewer than 10% of individuals having persistence of mildly abnormal liver enzymes.¹⁵ Adults may present with asymptomatic abnormal liver enzymes which are indistinguishable from other common causes of chronic liver diseases.

Imaging

Imaging studies may show hepatomegaly or changes associated with cirrhosis or hepatocellular carcinoma. Chest imaging studies show changes of emphysema or chronic obstructive pulmonary disease.

Gross findings

In cirrhotic livers removed for transplantation, α-1-antitrypsin deficiency usually shows either a micronodular or a mixed micro- and macronodular pattern of cirrhosis.

Microscopic findings

The finding of intracytoplasmic round or oval eosinophilic globules in periportal hepatocytes is characteristic of α-1-antitrypsin deficiency. It should be noted that the presence of α-1-antitrypsin globules in hepatocytes does not always correlate with clinical deficiency or with the presence of liver disease. The globules are eosinophilic on hematoxylin and eosin (H&E) sections and are bright red on trichrome stain (Figs. 18.7 and 18.8). The globules are best seen using periodic acid-Schiff (PAS) stain with diastase (PASD), which shows bright magenta globules (Fig. 18.9). The globules are only in the hepatocytes and can be patchy in the early phases of the disease. They are not always recognizable on H&E examination, and routine PASD stains are helpful to ensure the globules are not missed (Figs. 18.10 and 18.11). Although they are typically in periportal hepatocytes, the hepatocytes in all the zones can be affected in severe cases. In rare cases with the null phenotype, there are no globules in the hepatocytes because there is no production of the protein and there generally is no liver disease. The globules are absent or difficult to detect in infants less than 3 months of age, and the diagnosis of α-1-antitrypsin deficiency will depend on serum or genetic tests. Infants with clinical disease may exhibit liver injury resembling neonatal hepatitis, cholestatic hepatitis, or
extrahepatic biliary atresia. The liver biopsy can show marked cholestasis, bile ductular proliferation, mild portal and lobular inflammation, periportal steatosis, and occasional giant cell transformation (Fig. 18.12). Variable degrees of fibrosis are often present and occasional cases may have bridging fibrosis or even cirrhosis in the initial biopsy, which is indicative of rapid progression of liver disease.

Figure 18.7 α-1-Antitrypsin deficiency. Round intracytoplasmic α-1-antitrypsin globules in variable sizes seen on H&E section as eosinophilic globules.

Figure 18.8 α-1-Antitrypsin deficiency. Bright red globules on Trichrome stain.

Figure 18.9 α-1-Antitrypsin deficiency. Bright magenta PAS-positive globules with diastase resistance.

Figure 18.10 α-1-Antitrypsin deficiency. The globules may not be recognizable on routine H&E section.

Figure 18.11 α-1-Antitrypsin deficiency. The globules are readily revealed by PASD stain in the same case showed in Figure 18.10.

In adults affected with PiZ, the pathologic findings of liver are usually nonspecific, besides the presence of A1AT globules. The number and size of the globules increases with age. The portal tracts may contain mild inflammation and mild bile ductular proliferation, whereas interface hepatitis and lobular necroinflammatory activity are minimal or absent. Mild steatosis is common. Variable fibrosis is present and cirrhosis can develop in about 15% of patients (Fig. 18.13). Cirrhosis is the major driver of risk for hepatocellular carcinoma in α-1-antitrypsin deficiency, but both cholangiocarcinoma and combined hepatocellular carcinoma-cholangiocarcinoma have been reported in patients with PiZ but without cirrhosis.¹⁶

Figure 18.12 Neonatal α-1-Antitrypsin deficiency. Presented with neonatal giant cell hepatitis with cholestasis, small foci of hematopoiesis, mild portal inflammation, and ductopenia. No α-1-antitrypsin globules present.

Figure 18.13 α-1-Antitrypsin deficiency. Established cirrhosis owing to α-1-antitrypsin deficiency in adults (inset, high magnification showing bright red α-1-antitrypsin globules in hepatocytes).

Immunohistochemistry and special stains

PASD stains are routinely performed on medical liver biopsy specimens. The A1AT globules are strongly PASD-positive. Of note, there are many intracytoplasmic globules or inclusions that can mimic globules, which are discussed in the differential diagnosis section.

Immunostains for A1AT globules can be useful in certain circumstances, such as in infants when A1AT globules have not become apparent or when PASD-positive globules are focal or have unusual shapes or distribution. The A1AT globules are strongly positive by immunostain and are typically present in periportal hepatocytes (Figs. 18.14 and 18.15). The larger globules may show more intense positive staining at their periphery. Normal hepatocytes will also show granular diffuse cytoplasmic staining, so to be positive, there should be strongly staining of distinct globules. There is no correlation between immunostaining patterns and either A1AT serum concentrations or the phenotypes.¹⁷

Ultrastructural findings

Electron microscopy shows characteristic amorphous A1AT deposits primarily in dilated smooth endoplasmic reticulum and also in the rough endoplasmic reticulum. The deposits have a finely granular or fibrillary appearance, but there are no distinct patterns or structures. The findings in early A1AT accumulation can appear as less dense deposits or barely detectable deposits in dilated smooth endoplasmic reticulum.¹⁸

Figure 18.14 α-1-Antitrypsin deficiency. Eosinophilic globulesin periportal hepatocytes.

Figure 18.15 α-1-Antitrypsin blobules. Confirmed by an immunohistochemical stain showing intense cytoplasmic staining and darker globules.

Molecular genetic findings

α-1-Antitrypsin deficiency is inherited in an autosomal codominant manner, caused by mutations in the SERPINA1 gene, which is located on the long arm of chromosome 14. There are more than 100 allelic variations/mutations of the SERPINA1 gene, but the most important is a missense mutation in exon 5 that produces the allele “Z.” The nonmutated SERPINA1 corresponds to the “M” allele and produce normal A1AT. Genetic testing for α-1-antitrypsin deficiency targets the mutated region of SERPINA1using either DNA amplification and sequencing or hybridization by allele-specific oligonucleotides probes.

Differential diagnosis

Of note, PASD-positive globules in hepatocytes are not pathognomonic for clinical disease, as they can be found in genetic A1AT heterozygotes and homozygotes who lack clinical disease. In addition, very rare cases have been reported of individuals with a normal PiM phenotype developing A1AT globules during acute inflammation or severe illness, because of overproduction and high plasma levels of A1AT.¹⁹,²⁰

In addition, there are many eosinophilic inclusions in hepatocytes that can mimic A1AT globules on H&E. Mega-mitochondria are frequently seen in liver biopsies. They tend to be randomly distributed, oval-shaped, single or few numbers in the cells and are PASD-negative (Fig. 18.16). Lipofuscin has fine
granular particles but the granules are smaller than A1AT globules, are not eosinophilic on H&E and are reddish brown color on PASD stains (Fig. 18.17). Macrophages can contain large granules, mimicking A1AT globules on PASD stain (Fig. 18.18). Immunoglobulin globules are round and eosinophilic on H&E staining and can be highlighted by immunoglobulin G (IgG) or immunoglobulin M (IgM) immunostains (Fig. 18.19). In most cases, these result from elevated serum immunoglobulin levels, so are found in autoimmune hepatitis, primary biliary cirrhosis, and other conditions associated with high serum immunoglobulin levels. The fibrin globules seen in fibrinogen storage disease are often larger than A1AT globules and are PASD-negative. They sometimes will contain dark cores and can be highlighted by phosphotungstic acid hematoxylin (PTAH) stains (Fig. 18.20) or immunostains for fibrinogen.

Figure 18.16 Megamitochondria (PASD stain), mimicking α-1-antitrypsin globules.

Figure 18.17 Lipofuscin (PASD stain).

Figure 18.18 Ceroid-laden macrophages (PASD stain).

Figure 18.19 IgG globules (arrows) in macrophages in autoimmune hepatitis (inset, IgG immunohistochemistry).

Figure 18.20 Afibrinogenemia. Cytoplasmic inclusions of abnormal fibrinogens surrounded by a clear halo with a pink core.

Afibrinogenaemia and hypofibrinogenaemia

Afibrinogenemia and hypofibrinogenemia are rare inherited disorders caused by mutations in any one of three fibrinogen genes located on chromosome
4, FGA, FGB, and FGG. In afibrinogenemia, the fibrinogen levels are less than 0.1 g/L and manifests clinically primarily as bleeding, which can range from mild to severe. Patients with hypofibrinogenemia are usually asymptomatic, with no spontaneous bleeding episode, or have mild bleeding. Both can also be associated with thrombosis. Laboratory testing shows abnormal coagulation tests with low or absence of fibrinogen. Replacement of the fibrinogen is the main treatment.

Figure 18.21 Afibrinogenemia. Weaker PASD staining of the inclusions.

Mutations in FGG are associated with storage of fibrinogen in the rough endoplasmic reticulum of hepatocytes, leading to liver disease of variable severity. Affected individuals may have elevated liver enzymes or present with cryptogenic cirrhosis.²¹,²² The cytoplasmic inclusions of abnormal fibrinogen are round or polygonal with irregular outlines (Fig. 18.20). The globules are often surrounded by a clear halo with a dark pink core. The globules are eosinophilic or weakly stained on H&E sections and are either weakly positive or negative on PAS stain (Fig. 18.21). The globules can be detected with PTAH stain (Fig. 18.22) or immunostain for fibrinogen. Electron microscopy shows dilated rough endoplasmic reticulum filled with densely packed tubular structures arranged in curved bundles with a fingerprint-like pattern.²²

18.4 DISORDERS OF CARBOHYDRATE METABOLISM

Glycogen storage diseases are inherited disorders of glycogen metabolism caused by defects in the processing of glycogen synthesis or the breakdown of glycogen within liver, muscles, and other cell types. Most cases are inherited in an autosomal recessive manner. There are at least 11 types of glycogen storage disease, which are classified based on the enzyme deficiency and the affected tissue. The estimated incidence of glycogen storage disease is 1 in 20,000 to 43,000 live births and the most common type is IX.²³

Figure 18.22 Afibrinogenemia. Abnormal fibrinogens stained black with phosphotungstic acid hematoxylin (PTAH) stain (inset, inclusions negative for α-1-antitrypsin immunohistochemistry).

Types I, III, VI, IX, and XI, primarily involve liver with abnormal accumulations of glucose within the hepatocytes. Many of the different glycogen storage diseases have common findings at clinical presentation, including hepatomegaly, hypoglycemia, short stature, and recurrent infections. As an exception, glycogen storage disease types II and IV typically are not associated with hypoglycemia at presentation.

The hepatocytes in glycogen storage disease will show glycogenosis or steatosis or both. The cases with glycogenosis can either show diffuse changes affecting all of the hepatocytes or show a mosaic pattern with admixed hepatocytes that show less striking glycogen accumulation. The affected hepatocytes are swollen and pale-staining with prominent cell membranes and often have prominent glycogenated or pyknotic nuclei. Glycogen storage diseases types III, IV, and VI are the most likely to develop liver fibrosis, but fibrosis can also be seen in types I and IX.²⁴ The abnormal glycogen accumulation can be highlighted by PAS stains, but the diagnosis of glycogen accumulation is based on the H&E findings and not the PAS stain, as even hepatocytes in the normal liver can be strongly PAS-positive. In addition, the diagnosis of glycogen storage disease and the specific subtype cannot be established by histology alone. Instead, a diagnosis is based on the combination of clinical findings, biochemical profiles, enzyme activity assay results, histologic findings, and genetic testing.

Glycogen storage diseases

Glycogen storage disease type 0

Glycogen storage disease type 0 is due to glycogen synthase deficiency. In fact, type 0 is not a true glycogen storage disease because there is a marked decrease in liver glycogen content. Individuals with type 0 glycogen storage disease present typically in the first year of life with fasting hypoglycemia but no hepatomegaly. The liver typically shows macrovesicular steatosis with no glycogenosis. PAS stains can show diminished glycogen in hepatocytes.²⁵

Glycogen storage disease types Ia/b

Type I glycogen storage disease is caused by deficiency of either glucose-6 phosphatase (type Ia) or glucose-6-phoshate translocase (type Ib). In type 1b, the defective translocase doesn’t allow entry of substrate glucose-6-phosphate into the endoplasmic reticulum. Both subtypes present with hypoglycemia and hepatomegaly shortly after birth. Lactic acidosis, hyperlipidemia, hyperuricemia, and slightly elevated liver transaminase levels are common. Type Ib also has distinct features of recurrent infections, neutropenia, neutrophil dysfunction, and the development of inflammatory bowel disease resembling ulcerative colitis or Crohn’s disease.²⁶,²⁷ Microscopically, the hepatocytes are typically swollen with pale-staining cytoplasm, have prominent cell membranes, and have prominent glycogenated nuclei (Figs. 18.23 and 18.24).²⁴ Some cases will also show macrovesicular steatosis. Unusual findings including localized peliosis hepatis and Mallory body formation in the perivenular hepatocytes.²⁸ Portal fibrosis may be present in some cases.

Figure 18.23 Glycogen storage disease type I. Diffusely enlarged hepatocytes with pale-staining cytoplasm and centrally located pyknotic nuclei.

Figure 18.24 Glycogen storage disease type I. PAS stain confirming abundant glycogen.

Hepatic adenomas can develop at any age but typically occur during or after puberty, with a reported prevalence ranging from 22% to 75%.²⁹ Most of the adenomas are of the inflammatory subtype.³⁰ They can also be β-catenin activated and have a risk for malignant transformation.³¹ Hepatocellular carcinoma has been reported in children younger than 1 year of age.³²

Glycogen storage disease type II

Type II glycogen storage disease, also known as Pompe disease, is due to acid maltase deficiency. This type primarily involves the muscular system and the main clinical features are cardiomyopathy and muscular hypotonia. Although the enzyme is also deficient in the liver, hepatomegaly and hypoglycemia usually are not present. The liver typically shows marked glycogenosis with no fibrosis. Of note, electron microscopy in type II glycogen storage disease shows a distinct pattern of glycogen accumulation, with monoparticulate glycogen in enlarged lysosomes.³³

Glycogen storage disease type III

Type III glycogen storage disease, also known as Forbes disease or Cori disease, results from a deficiency of glycogen debranching enzyme. There are four subtypes are IIIa and IIIb and the two major subtypes. Type IIIa (80% of cases) affects both the liver and muscle, whereas type IIIb (15% of cases) affects only the liver. Patients typically present with hepatomegaly, hypoglycemia, and short stature. The liver biopsy shows marked hepatocellular glycogenosis, with rarefied cytoplasm and centrally or eccentrically located pyknotic nuclei. Portal fibrosis is often present and some cases can progress to cirrhosis, increasing the risk for hepatocellular carcinoma.³⁴

Glycogen storage disease type IV

Type IV glycogen storage disease, also known as Andersen disease, is caused by a deficiency of glycogen branching enzyme, leading to the accumulation of amylopectin-like polysaccharides in affected tissues. The clinical presentations vary significantly depending on which tissues develop polysaccharide accumulation. In the classic hepatic form, affected individuals present with failure to thrive and hepatosplenomegaly and the disease can rapidly progress to cirrhosis, which is often present by age 5.³⁵ However, a variant of type IV glycogen storage disease has been reported with liver involvement that is either nonprogressive or slowly progressive. In these cases, there is hepatosplenomegaly and mildly elevated transaminases, but there is no further progression of disease and liver enzymes may return to normal.³⁶

Type IV glycogen storage disease is the only glycogen storage disease with characteristic findings under light microscopy. The hepatocytes show distinctive ground glass type inclusions (Fig. 18.25). The inclusions are PAS-positive and are commonly partially diastase resistant because they are composed of amylopectin-like material, not glycogen (Fig. 18.26). However, the PAS with diastases results will also depend on how aggressively the slide is digested. The inclusions can be digested by pectinase or amylase.³⁷ This cytoplasmic finding is not seen in any of the other glycogens storage diseases, but chronic hepatitis B infection (Fig. 18.27), drug induced glycogen psuedoground glass changes (Fig. 18.28), and Lafora disease (Fig. 18.29) should be excluded. The inclusions in type IV glycogen storage disease are typically found in periportal hepatocytes, but they can also be found in other zones. On ultrastructural examination, the inclusions are composed of nonmembrane bound, randomly oriented fibrillary material with abundant glycogen rosettes. Fibrosis
is common in type IV glycogen storage disease and some cases may develop cirrhosis.

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