Developmental and Inherited Liver Disease

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, HT1, α1-AT deficiency, mitochondrial disorders, inherited disorders associated with intrahepatic cholestasis and NP disease type C may progress rapidly to advanced fibrosis. In CF 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 (CHF).

Despite the presence of fibrosis, some metabolic disorders (e.g. WD, HT1) may present with acute 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 a contraindication for liver transplantation (LT).

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 WD. Paediatric obesity and associated fatty liver disease may act as a confounding factor. WD should always be considered, even in obese children and in the absence of histochemically demonstrable copper or copper-binding protein. Poor nutrition (as in CF) or a metabolic crisis can contribute to the development, severity and distribution of macrovesicular steatosis. 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 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 idiosyncratic 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 (ER). 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), immunodeficiency and autoinflammation due to variants in RBCK1 , hypofibrinogenaemia, as well as in drug-induced hepatocellular injury and chronic hepatitis B. In adults, fibrinogen-associated ground-glass changes do not always indicate genetic disorder of fibrinogen production, as some cases result from the reactive accumulation of mixed proteins in cytoplasmic inclusions, which include but are not exclusively fibrinogen.

Diastase-resistant PAS-positive (D-PAS+) globules in periportal hepatocytes usually indicate α1-AT deficiency, and typically patients with the PI*Z allele, due to retention of the polymerized abnormal enzyme within the rough ER (RER). 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-ACT deficiency, and D-PAS-negative/α1-AT-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, WD or copper toxicosis. In the early stage of WD, hepatocellular copper is intracytoplasmic rather than lysosomal and therefore not routinely 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 for full discussions of inheritable iron disorders.) Children with genetic haemochromatosis are asymptomatic. Juvenile haemochromatosis, associated with mutations in the gene encoding hemojuvelin ( HJV ) or encoding hepcidin antimicrobial protein (HAMP), usually presents clinically in the second decade of life, mainly as a cardiac disorder. Siderosis may also be observed in children with peroxisome disorders (e.g. Zellweger syndrome).

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 (ALGS). 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 CHF, 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 characterized 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 (lysosomal acid lipase [LAL] deficiency) affects the liver, Gaucher disease affects liver and reticuloendothelial organs, and mucopolysaccharidosis (MPS) affects multiple organs including the central nervous system (CNS). 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 LAL deficiency (Wolman disease and cholesterol ester storage disease). Sphingomyelin accumulation in Kupffer cells and hepatocytes is characteristic of NP disease types A and B.

In contrast, other disorders affect reticuloendothelial cells only and spare hepatocytes. In NP disease type C, accumulation of esterified cholesterol and other lipids affects very few Kupffer cells, often on 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, MLD, 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.

Clinical features

The common clinical findings in ALGS 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 vertebrae (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 ALGS. 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 LT 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 LT. ^, Skin changes relate mainly to the formation of xanthomas, which regress after successful LT.

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 which may result in a clinical overlap with BA. ^, 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. ^, Inhibition of the ileal bile acid transporter (IBAT) has been proposed for the treatment of ALGS.

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 LT and 60% for those who required transplantation. LT was reserved for patients with chronic liver failure, intolerable pruritus unresponsive to medical treatment and severe growth failure and was necessary for hepatic decompensation 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 transplantation (two succumbing to end-stage liver disease before the advent of transplantation). Overall survival in this series was 62% at 20 years and seemed unaffected by the 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. Liver transplantation is an option for those in end-stage liver failure, or with severe chronic cholestasis or intractable pruritus; however, the 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 transplantation is variable. ^, The long-term complications of ALGS not requiring transplantation have included renal failure and intracranial bleeding or stroke. ^{, ,} HCC has been reported in children and adults with ALGS. Although rare, hepatic malignancy may occur in ALGS infants.

Pathology

The histopathological aspects of ALGS 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. Its expression is lost 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 ). Large, often centrally located, regenerative nodules may also develop. Portal inflammation and periportal ductular reaction, when present, are seen mainly in early infancy and can suggest the presence of distal duct obstruction. ^{, ,} In rare ALGS cases, the extrahepatic bile is narrowed; however, typically it is not. Misdiagnosis of ALGS as BA leading to portoenterostomy in the ALGS patient risks a failed portoenterostomy ^, and consequent early LT.

Ultrastructural studies of the intrahepatic bile ducts in ALGS have been reported. ^{, , ,} 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.

Molecular aspects

ALGS 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 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 the 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. Transforming growth factor-β (TGF-β) signalling drives hepatocyte transdifferentiation and de novo bile duct formation. 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.

Primary disorders of cilia development or cystogenesis

Most studies on the 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 cilia 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 consequently develop: they involve 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 the formation of papillary projections and overexpression of Ki-67 and proliferating cell nuclear antigen (PCNA), 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 germline 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 evidence that HNF1β localized in bile duct epithelium regulates expression of PKHD1, together with other ‘cystoproteins’ that co-localize into the cilium. A variant in DNAJB11, a cofactor to the chaperone protein BiP, has been associated with a clinical overlap of autosomal dominant polycystic liver disease (ADPLD) with autosomal dominant tubulointerstitial kidney diseases (ADTKDs).

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 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. The 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 TGF-β1 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 CHF-like picture. Its roles in liver and kidney appear 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 incidence 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 forms of ARPKD map to the same locus. ^, The gene encodes a 4074-amino acid protein, called either 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, incidence in males and females is equal. 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. Subsequent experience has found a spectrum of severity such that not all cases fit into these sharply defined subgroups. ARPKD has been reviewed by a number of authors. ^{, ,}

The majority of ARPKD patients present in the perinatal period with enlarged kidneys and signs of respiratory distress related to pulmonary hypoplasia. Those with extensive renal cystic change affecting at least 90% of renal parenchyma typically die in the perinatal period. Some ARPKD patients present later in childhood with increasing renal insufficiency and portal hypertension. In one series of 42 children with ARPKD from 20 sibships, 12 patients presented in the perinatal period, 9 in the neonatal period, 13 in the infantile period, and 8 in the juvenile period; the presentation and course of the disease were 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.

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. 3.26A ). They appear to branch or ‘anastomose’ and often show polypoid projections ( Fig. 3.26B ). 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.

Juvenile and adult presentation: 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. 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. The 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.

A number of malformation syndromes, characterized by hepatic morphological changes that resemble those of CHF (DPM), can be differentiated by the associated findings. Most 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 – CHF , 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, MPI-CDG), ^, sometimes with protein-losing enteropathy.

• Joubert syndrome, with cerebellar vermis hypoplasia/aplasia, colobomata and psychomotor retardation ; sometimes 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 CBD. Liver enzymes are generally normal except during episodes of biliary obstruction. The diagnosis is established by a variety of imaging modalities (e.g. ERCP, sonography, 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%. HCC occurs rarely. Medical treatment consists of symptomatic or prophylactic treatment of cholangitis and promotion of bile flow with ursodeoxycholic acid. 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. More recent experience has been reported. ^,

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 CHF (in which case it is termed Caroli syndrome ), rarely with infantile polycystic disease (ARPKD) and even adult polycystic disease (ADPKD).

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 1:1000 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 locus, 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 7 were PKD2 (12%). The authors found that the position of the mutation in PKD1 is predictive for the development of intracranial aneurysms (59 mutations are more commonly associated with vascular disease) and is therefore of prognostic importance. A de novo WT1 splice site variant was identified in addition to a PKD1 missense variant in a patient with a maternal history of ADPKD.

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 adolescence.

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, treatment is 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. When compared with patients with PCLD without kidney abnormalities, young ADPKD female patients showed a more severe phenotype expressed as height-adjusted total liver volume. 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 contributes to making diagnosis of cyst infections more readily and more accurately. This may allow early treatment with appropriately selected antibiotics; 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. ^, In the absence of molecular genetic diagnosis, the criteria for identification of this disorder in children include 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 a 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.

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.

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 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 ). CHF 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.

Polycystic liver disease without kidney abnormalities

Isolated 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 , segregates in some families with PCLD. The protein is widely distributed in tissues and predicted to be an ER 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 the 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 ER (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 SEC63p 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 does not seem to be implicated in PCLD. 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 (LDL) receptor-related protein 5 ( LRP5 ) gene in patients with PCLD. Other genes associated with PCLD are GANAB , ALG8 and SEC61B .

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 sonography, CT or other imaging modalities. Solitary bile duct cysts involve the right lobe twice as often as the left. Rarely, they 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.

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.

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. ^,

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; however, the serum concentration of the natural substrate, unesterified 7α-hydroxy-cholesterol, was elevated. 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 UDCA. The challenge here is to distinguish 3βHSD (and 5α-reductase) deficiency from bile canalicular transporter disorders, especially when the presence or absence of pruritus does not provide a clinical distinction. In both of 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 aetiology 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 5β-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 UDCA is unclear, except that treatment may decrease 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 hyperbilirubinaemia and low GGT and whose liver disease had already advanced to cirrhosis. Serum levels of 27-hydroxycholesterol 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 (R388*) as a heterozygous defect. There was no response to either UDCA or cholic acid, either by clinical indicators or by 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 patients had cirrhosis with multinucleated giant cells on liver biopsy. A single case of successful treatment with chenodeoxycholic acid 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. Molecular genetic diagnosis may be critically important in doubtful cases.

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 and 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, encoded by BAAT ). Some patients with defective amidation present in early infancy with conjugated hyperbilirubinaemia, occasionally severe enough to resemble BA. 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 a 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 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. 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 ER (SER) 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 SER, 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. UDCA 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 21 years, Gonzales et al. found that serum aminotransferases normalized and liver histology improved, including in three patients with apparent regression of cirrhosis. The recently reported American long-term experience was also favourable.

For CTX, a combination of chenodeoxycholic acid and pravastatin (an inhibitor of 3-hydroxy-3-methylglutaryl CoA, 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. LDL apheresis has been added to the regimen in a few patients.

LT 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.

Two comprehensive reviews of diseases of bile acid synthesis are recommended for further reading. ^,

FIC1 (ATP8B1) 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 some members of the Amish Byler family suffer. ^,

The function of the FIC1 protein, a P-type ATPase, has been unclear. ^, It may play a role in maintaining the functional integrity of the bile canalicular membrane. 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 FIC1 deficiency, although in the absence thus far of a specific antibody available for formalin-fixed paraffin-embedded (FFPE) tissue. In patients with FIC1 deficiency, the 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 from bleeding or infections between ages 6 weeks and 3 years. 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 liver biopsies from 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 BSEP (known as BSEP or ABCB11). ^, 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. In the largest multicentre retrospective study to date on 264 patients, genotypic severity was graded into three categories (BSEP1, 2 and 3), the highest associated with the lowest predicted residual BSEP function. This classification predicted long-term native liver survival, risk of developing of HCC and the effect of surgical biliary diversion. 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 HCC 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.

The histological features of BSEP deficiency are not specific. In an attempt to improve this, IHC 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 or de novo expression (retargeting) has been described in children treated with UDCA and biliary diversion.

Genetic liver diseases in which the gene is only expressed in the liver have generally been thought to be ideal candidates for LT. 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 ‘novel’ 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.

Impaired expression of BSEP may occur as a consequence of other genetic abnormalities. In cholestasis associated with mutations in MYO5B , BSEP may be relatively underexpressed. With mutations in NR1H4 encoding the farnesoid X receptor (FXR), BSEP is undetectable. These disorders must be differentiated from BSEP deficiency due to ABCB11 abnormality.

Multidrug-resistance protein 3 deficiency

This entity, previously termed ‘type 3 PFIC’, has been shown to be caused by a deficiency in 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 due to this toxic bile, in particular the damage it causes to the biliary epithelium resulting in a cholangiopathy. Lipid clefts may be identified in the lumen of bile ducts. In severe cases the features seen include portal inflammation, bile ductular reaction and subsequent fibrosis ( Fig. 3.51A ). 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 BA 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 WD. In the somewhat older child, the cardinal diagnostic finding is that cholangiography discloses normal biliary architecture despite the appearance of bile duct obstruction.

The result of partial loss of function of MDR3 may have more widespread clinical importance than the neonatal disease. Degiorgio et al. have shown that a substantial proportion of adults with cholestatic 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 an ABCB4 , c.2362C>T (p.Arg788Trp). Heterozygosity for ABCB4 mutations has 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. 3.51B ), 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 LT.

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

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 bile canalicular transporter disorders. 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 findings consistent with intrahepatic cholestasis, notably elevated serum GGT and total serum bile acids; and 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. More recently variants in ABCB11 were suspected in instances of cholestasis in young men taking bodybuilding supplements. 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).

North American Indian childhood cirrhosis

NAIC is one of several disparate disorders identified as ribosomopathies, of which Blackfan–Diamond syndrome is perhaps the best known. Weber et al. studied 14 children, all from the Cree and Ojibwa–Cree tribes, with familial occurrence of neonatal hepatitis syndrome. Conjugated hyperbilirubi­naemia 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 serum 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. In some patients, LT has been required. Initial ultrastructural and immunohistochemical studies suggested that this group of children might represent a human model of microfilament dysfunction-induced cholestasis.

Investigation of the molecular genetics of NAIC has clarified its pathogenesis. 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, is a nucleolar protein. Cirhin, also known as human Utp4, is prominent in ribosomal biogenesis. It interacts with the nucleolar protein NOL11, and the complex has a role in ribosomal 18S rRNA maturation. The common mutation found in patients with NAIC disrupts that interaction. A zebrafish model suggests a role in biliary development mediated via a p53-dependent process. Pathogenetic mechanisms involving p53 are implicated in some other ribosomopathies. Cirhin may have other actions in hepatocytes leading to cell damage.

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.

Truncating mutations in TJP2 can produce a much more severe clinical phenotype. Affected children present in the first 3 months of life with severe cholestatic liver disease and low serum GGT (without mutations in ATP8B1 and ABCB11 ). They were found to have homozygous mutations on 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: the majority have required LT.

Recent studies have shown that pathogenetic variants of USP53 encoding ubiquitin carboxyl-terminal hydrolase 53, a protein which interacts with TJP2, may be associated with a low-GGT cholestatic disorder. In one report, affected individuals presented with jaundice before the age of 7 months; treatment included UDCA and cholestyramine; jaundice resolved by age 2 years. Other observations indicate the potential for chronic liver disease. Some children had deafness. Hearing loss was reported in another affected kindred.

Likewise, BAAT mutations may be associated with more severe liver disease, classified as an amidation disorder of bile acid synthesis (see earlier).

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 uridine diphosphate (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 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, LT is the required intervention.

The genetic basis of NSC is increasingly being characterized. In neonatal ichthyosis-sclerosing cholangitis (NISCH) 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 CLDN1 (found on chromosome 3q27-q28), which encodes claudin-1, a tight junction protein. Biliary damage may be caused by disturbed regulation of hepatic paracellular permeability. The severity of the neonatal liver disorder may resemble biliary atresia BA. Serum GGT level is increased. Liver biopsy shows features of duct obstruction. Seven of 24 NSC patients were found to harbour truncating variants in the doublecortin domain containing 2 gene ( DCDC2 ), encoding a protein expressed in cholangiocyte cilia. Lack of primary cilia and expression of DCDC2 and the ciliary protein acetylated α-tubulin (ACALT) were shown by EM and IHC. More recently, likely deleterious variants in KIF12 , an orphan member of the kinesin family of molecular motors, were found in three families with a clinical picture of NSC. Of note, KIF12 function, together with PKHD1 , is regulated by HNF1β.

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, 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. The prognosis for patients with ARC syndrome is generally poor, with limited survival beyond infancy. Cirrhosis may develop. Milder ARC syndromes with longer survival have been described.

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 conjugated hyperbilirubinaemia. Despite the severity of presentation, which can resemble BA 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 lesions have been observed in MAS patients, including inflammatory hepatocellular adenomas, atypical focal nodular hyperplasia and hepatoblastoma.

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 LT. HCC 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. Importantly, MYO5B disorder can be associated with cholestasis without MVID being present. ^, The cholestatic disorder shows low/normal γGT. Trafficking of ABC transporters such as BSEP and MDR3 from trans-Golgi to the canalicular membrane via endosome depends on the interaction between MY05B and RAB978-0-7020-8228-3. MY05B deficiency thus impairs canalicular bile secretion. ^, In patients with MVID, the degree of cholestasis may worsen after intestinal transplantation.

Other rare genetic causes of intrahepatic cholestasis

Very rare patients with defects in NR1H4 encoding for farnesoid X receptor (FXR) have presented neonatally with a clinical and histological picture similar to BSEP (ABCB11) deficiency. ^, Bile acids are the natural ligand for FXR, and in turn FXR regulates ABCB11 . The disorder shows low γGT and undetectable BSEP; severe coagulopathy may reflect the role of FXR in regulating coagulation. Implicated mutations in NR1H4 result in loss of FXR function.

A recent study has shown two other potential candidate genes, WDR83OS and lipolysis-stimulated lipoprotein receptor ( LSR ). The patient with a novel variant of the LSR gene also had low GGT. Mutations in SCYL1 were found in children with episodes of low-GGT cholestasis triggered by infection (characterized as ‘ALF’) and progressive neurodegeneration: hepatic involvement was mainly found after the neonatal period. Histological changes included advanced fibrosis, microvesicular steatosis and depletion of SCYL1 by IHC. TEM showed enlarged Golgi cisternae. The extensive diversity within the genetic basis for inherited cholestatic disorders is thus becoming apparent.

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 greater than 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 estimated incidence of 1 per 1 million general population. Inheritance is autosomal recessive. Patients lack the enzyme 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. LT 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. Of 15 UGT1A1 c.222C>A homozygotes (median age, 14.6 years; range, 4.4–23.6 years), 9 had mild to severe fibrosis during long-term follow-up.

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. Serum 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 White populations. ^, 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 SER.

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 minutes over those at 45 minutes after intravenous (IV) 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. Disease onset may occur during pregnancy or in relation to taking oral contraceptives. 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 cMOAT. The ABCC2 gene is localized to chromosome 10q24. It was cloned in humans in 1996. Human multidrug-resistance protein 2 ( MRP2 ), is the same gene with 32 exons. Multiple gene mutations have been documented and can be detected in fibroblasts. ^{, ,} On liver biopsy, IHC for cMOAT (MRP2 protein, also called cMOAT for multidrug organic anion transporter or ABCC2 for ATP-binding cassette subfamily C member 2) reveals a complete absence of staining ( Fig. 3.56C ). 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.

Accumulation of a coarsely granular brown pigment, called lipomelanin , in liver cells is characteristic of the Dubin–Johnson syndrome ^, ( Fig. 3.56A ). 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. 3.56B ), 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 ultrastructurally 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 and related to homozygous variants in both the SLCO1B1 and SLCO1B3 genes on chromosome 12, encoding for organic anion transporting polypeptide 1B1 and 1B3 ( OATP1B1 , OATP1B3 ). These proteins mediate reuptake across the sinusoidal hepatocellular membrane of several compounds including bilirubin glucuronide. ^, Hepatobiliary scanning reveals either no or minimal visualization of the liver, a clue to the diagnosis. 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.