Update on Progressive Familial Intrahepatic Cholestasis

Abstract

Three distinct forms of familial intrahepatic cholestasis are the result of mutations in the ATP8B1, ABCB11, and ABCB4 genes. The pathophysiologies of the latter 2 of these diseases are well characterized and are the result of abnormalities in canalicular excretion of bile acids and phospholipids, respectively. The molecular pathophysiology of the systemic disease associated with mutations in ATP8B1 remains unclear. In all of these diseases, wide variations in clinical phenotypes have been observed. The variability can be ascribed at least in part to predicted genotype:phenotype correlations. Disease- and genotype-specific prognoses and therapeutic approaches may exist, although much more information needs to be ascertained before clinicians can confidently make decisions based on genetic information.

Bile is a complex mixture of bile acids, organic anions, phospholipids, cholesterol, and other ions. Specific transport mechanisms exist for each of the components of bile, and defects in many of these transporter systems lead to a discrete form of inherited liver disease. Cholestasis is defined as an impairment of normal bile flow secondary to structural and molecular abnormalities of the liver and/or biliary tree. Three specific disorders will be reviewed here: familial intrahepatic cholestasis 1 (FIC1) disease, whose pathophysiology is still under investigation; bile salt export pump (BSEP) disease, which results from a defect in canalicular bile acid excretion; and multidrug resistance protein 3 (MDR3) disease, which results from a defect in canalicular phospholipid excretion. These disorders are also called progressive familial intrahepatic cholestasis (PFIC) types 1, 2, and 3, respectively. The major differences between these diseases are summarized in Table 1. Bile acids, one of the major components of bile that drives bile formation, are classified into primary bile acids, cholic acid and chenodeoxycholic acid, and secondary bile acids, deoxycholic acid, and lithocholic acid. Synthesis of bile acids is a complex multistep process using cholesterol as a main nucleus and requires 17 enzymes that are expressed in the liver. Defects in bile acid biosynthesis can lead to a spectrum of other types of intrahepatic cholestasis. Secondary bile acids are formed as the result of bacterial metabolism in the colon. Cholic acid is the main primary bile acid and accounts for approximately 70% of the circulating pool, whereas chenodeoxycholic acid accounts for 30%. Both cholic acid and chenodeoxycholic acid undergo conjugation with taurine and glycine, forming conjugated bile salts, which have increased solubility in the intestine. Flux of bile acids across hepatocytes from blood to bile is a major driving force for bile formation. Bile salts are secreted from the liver into the bile against a concentration gradient by an adenosine triphosphate (ATP)-dependent transporter, the BSEP, located at the canalicular membrane of hepatocytes. Once in the intestine, bile salts play an important role in activating digestive enzymes and emulsify dietary lipids, cholesterol, and fat-soluble vitamins. Approximately 95% of bile acids are recovered in the gut during each cycle of the enterohepatic circulation, and the 5% that are lost are replaced by new synthesis in the liver. The majority of intestinal bile salts are reabsorbed in the terminal ileum by the apical sodium-dependent bile acid transporter ^(1–6).

TABLE 1: Comparison of 3 forms of PFIC

FIC1 DISEASE

FIC1 disease is caused by mutation of the FIC1 protein encoded by the ATP8B1 gene (initially named FIC1 gene) located on chromosome 18q21-q22 ⁽⁷⁾. FIC1 is the first human member of the P4 subfamily of P-type ATPases; members of this family are thought to play an important role in the transport of aminophospholipids across the cell membrane ⁽⁸⁾. FIC1 is expressed in a wide variety of tissues, including the liver (canalicular surface of hepatocytes and apical membrane of cholangiocytes), the intestine, and the pancreas. It is worth noting that the expression of FIC1 mRNA is stronger in the intestine than in the liver ^(9,10). Diarrhea is a symptom in FIC1 patients that can become intractable after liver transplant, suggesting a role for FIC1 in intestinal fluid and nutrient handling ⁽¹¹⁾. Pancreatic expression of FIC1 may be relevant in light of the finding of the recurrent pancreatitis that has been described in some patients with FIC1-related disease ^(12,13).

Function and Regulation

PFIC1 and benign recurrent intrahepatic cholestasis (BRIC) were once considered to be 2 separate diseases. In 1998 FIC1 was identified as the gene mutated in both PFIC1 and BRIC ⁽¹⁴⁾. With this finding, these diseases are now broadly referred to by some as FIC1 disease. Intermittent disease that evolves into a more progressive and persistent form has been described, substantiating that there is a spectrum of FIC1 disease from progressive FIC1 (former PFIC1) to benign recurrent FIC1 disease (former BRIC). The protein encoded by this gene is termed FIC1. FIC1 does not seem to function directly as a bile acid transporter, nor does it directly affect transporter function. FIC1 is proposed to function as an aminophospholipid flippase, flipping phosphatidylserine from the outer to the inner hemileaflet of the plasma membrane, thereby playing an important role in maintaining aminophospholipid asymmetry between the inner and outer hemileaflets of plasma membranes. Linking predicted changes in membrane composition to the clinical phenotype of FIC1 disease has been difficult.

Whether intestinal FIC1 protein has a role in bile salt absorption is still not clear and requires further evaluation. The intestinal bile acid transporter, apical sodium-dependent bile acid transporter, was found to be upregulated in 3 children with FIC1-related disease relative to 2 children with other forms of intrahepatic cholestasis ⁽¹⁵⁾. This finding was recapitulated in an in vitro model of FIC1 disease and may be linked to alterations in the function of the farnesoid X receptor (FXR), a key regulator of bile acid homeostasis. The link to FXR could be extrapolated to alter the expression of BSEP, the canalicular bile acid transporter. It has been hypothesized that in FIC1 disease, FXR signaling is reduced, leading to diminished BSEP expression. Two small studies of human liver have substantiated this hypothesis, whereas an additional study has not supported this concept ^(16–18). An abnormality of the enterohepatic circulation of bile acids was further supported by the findings of Groen et al ⁽¹⁹⁾; a significant increase of serum bile salt concentration was noted in Atp8b1 (−/−) mice in comparison with wild-type mice when they were fed bile salts. Interestingly, in that same model there was no difference in intestinal apical sodium-dependent bile acid transporter expression, which indicates that intestinal absorption of bile salts does not explain the elevation of serum bile salt concentration in the FIC1 mouse model. Alterations in the stability of canalicular membrane proteins caused by changes in membrane composition is also hypothesized to explain the cholestasis observed in FIC1 disease ⁽²⁰⁾.

Mutations of FIC1

A comprehensive description of ATP8B1mutations in patients with FIC1 disease and BRIC was performed by Klomp et al ⁽²¹⁾, who screened 180 PFIC1 and BRIC families and identified 54 different mutations. Different types of mutations were reported, including splicing mutations, nonsense, small insertions or deletions, frameshift, large deletion, small in-frame deletion, and missense mutations. Both homo- and heterozygote patterns were described. ATP8B1 mutations were present in 30% and 40% of PFIC1 and BRIC patients, respectively. There seemed to be a correlation between mutation type and disease severity. Mutations that were predicted to affect expression or function of the FIC1 protein to a greater extent, such as frameshift, nonsense, and large deletions were detected more frequently in the patients with progressive disease, whereas missense mutations were more common in BRIC patients. Nonsense mutations in the carboxyterminal portion of the FIC1 protein were more likely to be found in patients with BRIC compared with nonsense mutations found in the aminoterminal of the protein. This is logical, inasmuch as carboxyterminal truncations of FIC1 may still yield a partially functional protein. Inasmuch as the specific function of FIC1 is not known, it has not yet been possible to directly test the functional consequence of these mutations on ATB8B1. Preliminary studies indicate that BRIC mutations lead to a partially functional FIC1 protein ⁽²²⁾. Some mutations were more common in certain populations than others, and some were clustered in certain families, findings that suggest a founder effect. For example, the missense mutation I661T was more common in BRIC patients of European descent, G308V in the original Byler Amish kindred, and D554N in the Greenland Inuit. Mutations G1040R and E665X were detected in families of Dominican and Saudi descent. Reduced penetrance may explain the presence of homozygote I661T mutation that was found in asymptomatic parents of patients with BRIC disease ^(14,21).

Clinical Presentations

Byler disease was first described in an Amish kindred in 1965 and then in 1969 ^(23,24). Patients with a clinical disorder similar to Byler disease but not of Amish descent have been said to have Byler syndrome ⁽²⁵⁾. The disease was also described as Greenland familial cholestasis in the Inuit population in Greenland and Canada ⁽²⁶⁾. PFIC1 and BRIC have different clinical presentations and used to be considered 2 different diseases until the discovery by Bull et al ⁽¹⁴⁾ that both diseases share the same gene and have been named FIC1 disease accordingly. In general, FIC1 disease can be classified instead into progressive/severe and benign recurrent forms. Intermediate phenotypes have also been described ⁽²⁷⁾. In all forms, the diseases are inherited in an autosomal recessive manner and are characterized by low γ-glutamyl transpeptidase (GGT; in relation to the degree of cholestasis).

Patients with progressive/severe FIC1 disease (formerly PFIC1) often present in the first year of life with intense pruritus that may be disproportionate to the degree of hyperbilirubinemia. The intense pruritus can lead to excoriation or even self-mutilation; the absence of hyperbilirubinemia has led to this disease being miscategorized as a dermatological condition. Other common early symptoms include gallstones and complications related to fat malabsorption and fat-soluble vitamin deficiencies. Some patients have respiratory symptoms, including cough and wheezing, that was thought to be due to asthma but resolved with successful therapy (either by liver transplantation or by partial biliary diversion) ⁽²⁸⁾. Failure to thrive and chronic diarrhea are common symptoms in these patients, starting early in life. Diarrhea may become more prominent after liver transplantation, which could be due to lack of FIC1 protein in the intestinal epithelium ⁽¹¹⁾. Other symptoms include epistaxis (in the absence of thrombocytopenia or coagulopathy), pancreatitis, cholecystitis, sensorineural hearing loss, delayed sexual development, and amenorrhea ^(12,28,29).

The benign form of FIC1 disease, BRIC, was first described in 1959 ⁽³⁰⁾. As the name suggests, this form of the disease is characterized by recurrent episodes of cholestasis with periods of relative well-being in the intervening periods. The pruritus associated with the episodes is not benign to the affected individual, but the general absence of histological progression of disease has led to the designation of “benign” disease. These patients vary in the age at first presentation and in the frequency and duration of cholestatic episodes. Age at presentation ranges from infancy to late adulthood. The number of discrete cholestatic episodes varies from a few episodes per year to 1 episode per decade; each episode could last several weeks to months ⁽¹³⁾. The pruritus can be intense, can have a profound impact on sleep and daily activities, and can even drive patients to become suicidal or to choose liver transplantation to improve their quality of life. The hallmark of this disease type is the intermittent nature of cholestatic episodes with clinically and biochemically asymptomatic periods in between. With pruritus, patients may have jaundice, anorexia, fatigue, and diarrhea. Cholecystitis, gallstones, pancreatitis, and renal stones have been described occasionally. Seasonal variation, exacerbation with pregnancy or contraceptive pills, and onset after infection have been described in this disease ⁽³¹⁾. Overall, it is unclear what triggers an episode of cholestasis and/or whether there are means of preventing episodes.

Laboratory findings in FIC1 disease are significant for normal GGT, mild direct hyperbilirubinemia, mild to modestly elevated alanine aminotransferase and aspartate aminotransferase, coagulopathy (initially due to vitamin K deficiency), fat-soluble vitamin deficiencies, markedly elevated serum bile acids (the sine qua non of the cholestasis in these patients), and normal cholesterol levels ⁽²⁸⁾. Occasionally, abnormal sodium and chloride sweat concentrations are measured in the absence of pancreatic insufficiency ^(32,33). Radiological evaluation does not show obstructive pathological changes. In the recurrent form, laboratory test results between the episodes are generally normal ⁽³⁴⁾.

Histology

Liver pathologic changes vary with the age at which the biopsy is done and is variable in severity. In the benign recurrent form (BRIC1), the liver histology can be normal during the noncholestatic interludes ⁽³⁴⁾. A clue to the diagnosis can be adduced by the small hepatocytes on the early life biopsy (Fig. 1A). Bland canalicular cholestasis is the rule, with minor and varying degrees of cholemic effect, pseudoacinar rosettes, hepatocellular ballooning, and giant cell transformation in the centrilobular areas. The histological picture of “neonatal hepatitis” is more characteristic of BSEP disease. Immunostaining for the canalicular enzymes will show a diffuse and variable reduction in canalicular expression of CD13, GGT, and pCEA although staining for BSEP and MDR3 is retained. Staining for CD10 is not useful at this age (34a). Most characteristic are the ultrastructural findings. Electron microscopy reveals coarse, particulate, and granular bile, commonly referred to as Byler bile (Fig. 2A). This feature is not seen in BSEP or MDR3 disease. Less specific are the loss of canalicular microvilli and the thickened wall of pericanalicular microfilaments that may be seen in most instances of chronic cholestasis. Histopathology later in the course, best seen at hepatectomy, is highlighted by increasing portal fibrosis with eventual bridging in the presence of canalicular cholestasis (Fig. 1B). Frank cirrhosis may eventuate. The bile retains its characteristic Byler bile features ⁽³⁵⁾.

FIG. 1: Histological features of early and late biopsy specimens in FIC1, BSEP, and MDR3 diseases (hematoxylin and eosin in all views). A, An early biopsy of FIC1 disease liver (10 weeks old) is notable only by the apparently small hepatocytes and a canalicular bile plug. B, Hepatectomy in a 13-year-old patient showed focal bridging fibrosis and mild nonobstructive biliary prominence. C, Early biopsy of a BSEP-deficient liver in a 9-months-old infant shows a nonobstructive giant cell pattern. The cholestasis is not obvious. D, Hepatectomy in a 6-year-old patient shows delicate bridging fibrosis, loss of giant cell appearance, and ductal cholestasis. E, Early biopsy of MDR3 in a 9-month-old patient shows canalicular bile in small amounts and mild bile ductular proliferation without giant cell changes.F, Hepatectomy at 13 years reveals a densely fibrotic liver without obstructive biliary changes and, in this instance, an inflammatory portal component.

FIG. 2: Electron microscopic characteristics of canalicular bile in FIC1 and BSEP disease. A, FIC1 disease: electron microscopy of 1A above reveals canalicular bile that is coarsely particulate, and the microvillus surface is attenuated. B, BSEP disease: electron microscopy reveals dense and amorphous bile without the particulate features seen in FIC1. Loss of microvillus pattern is common to all.

Treatment and Outcome

Progressive FIC1 disease can lead to death secondary to complications of end-stage liver disease (typically in the second decade of life), whereas in general the recurrent benign form does not carry a risk of chronic or progressive liver disease. Treatment options include medical and surgical interventions. Supplementation with medium-chain triglycerides and appropriate forms of fat-soluble vitamins is crucial for these patients. Initial descriptions of early mortality in FIC1 disease were primarily related to hemorrhage related to vitamin K deficiency. Ursodeoxycholic acid (UDCA) failed to prevent episodes of cholestasis in patients with benign recurrent FIC1 disease ^(31,36). Partial improvement of pruritus and abnormal liver biochemistries was observed in some patients with the progressive FIC1 disease after they had received UDCA for 12 months ⁽³⁷⁾. Early reports from Jacquemin et al ⁽³⁸⁾ showed a response in some PFIC patients with normal GGT to UDCA; although these findings preceded the current genetic classification and most likely represent a mixture of patients with various forms of intrahepatic cholestasis. Neither phenobarbital nor cholestyramine typically leads to clinical improvement ⁽³¹⁾. Although there is limited experience with rifampin in children with FIC1 disease, it did decrease pruritus and cholestasis in some children with progressive FIC1 disease and may have prevented further cholestatic episodes in patients with the benign recurrent form ^(39,40). In general, these responses to medical therapy are of limited duration.

Nontransplant surgical intervention can be effective in many patients with FIC1 disease. The hypothesis underlying these procedures is that interruption of the enterohepatic circulation of bile acids would modify the disease course, presumably by diminishing the accumulation of toxic bile salts. The 2 primary forms of surgical intervention that are directed at interrupting the enterohepatic circulation of bile acids are partial external biliary diversion (PEBD) and ileal exclusion. In PEBD (cholecystojejunocutaneostomy), initially described in 1988 by Whitington and Whitington ⁽⁴¹⁾, a short jejunal segment is anastomosed to the dome of the gallbladder, terminating as an end stoma to permit discarding of bile. Approximately 30% to 50% of secreted bile drains through this diversion. A subsequent report in 1995 by Emond and Whitington ⁽⁴²⁾ showed that when biliary diversion is performed early in the disease course (ie, before the development of cirrhosis) in patients with progressive FIC1 disease, clinical and biochemical improvement is more likely. In 2003 Kurbegov et al ⁽⁴³⁾ extended these observations and described pre-PEBD and post-PEBD changes in FIC1 patients. These changes included improvement of clinical symptoms, growth parameters, liver histology including fibrosis (in a patient as early as 9 months after the surgery), and electron microscopy, and significant improvement (or even normalization) of liver function test results. In 1 patient for whom preoperative and postoperative bile analysis was available, there was normalization of the bile acid profile with predominance of chenodeoxycholic acid. A potentially promising and less invasive laparoscopic approach of PEBD showed good postoperative results, including clinical and biochemical improvement in the short-term 2 month follow-up period reported after the surgery ⁽⁴⁴⁾. Endoscopic nasobiliary drainage has been used in cases of obstructive jaundice of various causes. This approach resulted in the long-lasting disappearance of pruritus and normalization of serum bile salts within 24 hours in 3 patients with benign recurrent FIC1 disease ⁽⁴⁵⁾.

In ileal exclusion (bypass), bile acid reabsorption is diminished by bypassing approximately 15% of the small intestine, at the terminal ileum, without the need for creating an external fistula, as in PEBD. An anastomosis is created between the divided proximal ileum and the cecum, bypassing the terminal ileum and ileocecal valve. This procedure showed promising results in FIC1 disease with resolution of pruritus and improvement of liver biochemical test results. There was histological improvement in 1 patient who underwent repeat liver biopsy after surgery ⁽⁴⁶⁾. After 12 to 48 months of follow-up of PEBD or ileal bypass, recurrence of symptoms and increase of serum bile acids was encountered in some patients who underwent ileal bypass surgery. This may indicate that PEBD is a better option for patients with FIC1 disease ⁽⁴⁷⁾.

Liver transplantation remains an option for patients with a progressive form of the disease, although it may be less desirable than the aforementioned surgical approaches. It is especially useful in children with evidence of cirrhosis and portal hypertension, given that PEBD and ileal exclusion can be problematic in that setting. It must be remembered that FIC1 disease is systemic, and thus liver transplantation is not “curative” ⁽⁴⁸⁾. Liver transplantation, as opposed to the nontransplant surgical approaches, is associated with a finite and real risk of serious morbidity and mortality. In light of the systemic nature of FIC1 disease, there is a potential risk for the development or worsening of diarrhea after liver transplantation in some FIC1 patients ^(11,49). Posttransplant steatohepatitis is another condition that may be ascribed to the systemic expression of the FIC1 protein ⁽⁴⁹⁾.

BSEP DISEASE

Synthesized and recycled bile acids are transported from within hepatocytes across the canalicular membranes against a concentration gradient driven by an ATP-dependent pump. This step is thought to be rate limiting for bile acid–dependent bile flow. This ATP-dependent transporter is encoded by the gene ABCB11 and is also known as the bile salt export pump (BSEP). Mutations in the ABCB11 gene lead to an isolated defect of the BSEP transporter, which is responsible for 1 subtype of progressive familial intrahepatic cholestasis, that being PFIC2 (designated henceforth in this review as BSEP disease) ⁽⁵⁰⁾.

In 1995, a gene (then named the sister of p-glycoprotein (spgp)) was discovered by Childs et al ⁽⁵¹⁾ in pig liver. It was so named because of its close similarity to the p-glycoprotein 170 (MDR1) gene that transports organic cations into bile and serves as drug efflux pump. At the time of its initial description it was not known to be a bile acid transporter. The true function of spgp as a canalicular ATP-dependent BSEP of the liver became apparent as a result of both biochemical and genetic investigations ^(52,53). BSEP is a member of the B family of the ATP-binding cassette (ABC) superfamily of transporters and was identified in humans as ABCB11⁽⁵²⁾. Expression of BSEP in insect cells demonstrated its capacity for ATP-coupled transport of bile acids. The gene encoding BSEP (ABCB11) has been mapped on chromosome 2q24-31 and was found to be mutated in several patients with low GGTP (gamma-glutamyl transpeptidase) cholestasis ⁽⁵³⁾. BSEP is expressed primarily in the liver, but it is also expressed at lower levels in other organs like human testis ⁽²⁵⁾, rat brain cortex, placenta ⁽⁵⁴⁾, and small and large intestines ⁽⁵⁵⁾. The functional role of expression in these nonhepatic tissues is not completely clear.

Function and Regulation

Excretion of bile salts from the liver is important for viability of hepatocytes because intracellular accumulation can lead to necrosis and apoptosis of hepatocytes ⁽⁵⁶⁾. BSEP is considered to be the main BSEP, a supposition that is supported by the liver disease that ensues in humans when it is mutated. Interestingly, as observed for mouse models of FIC1 disease, mutation of BSEP in mice did not lead to significant liver disease ⁽⁵⁷⁾. This unexpected finding could be attributable to the ability of the knockout mice to detoxify hydrophobic bile acids and to use an alternative, as yet undescribed, system to transport bile acids across canalicular membrane. Feeding a diet supplemented with cholic acid to these knockout mice caused severe cholestasis, liver necrosis, high mortality, and elevation of bile acid output and flow. This last finding may further support the possibility of the alternative bile salt transport system ⁽⁵⁸⁾. BSEP is an ATP-dependent transporter, located at the canalicular membrane of hepatocytes. It has a crucial role and function as the main pump of taurine- and glycine-conjugated bile salts against the concentration gradient from inside hepatocytes into the bile canaliculus ⁽⁵²⁾. Bile salt transport is influenced by different factors, including the bile salt pool. Decreased secretion was related to pool size because it was diminished after prolonged biliary drainage ⁽⁵⁹⁾. This finding was also supported by the finding that recovery of bile flow after liver transplant was paralleled by increased BSEP expression ⁽⁶⁰⁾.

The FXR, a nuclear receptor for bile acids, acts as an intracellular bile salt sensor in hepatocytes and upon activation binds with retinoid X receptor (RXR) to the BSEP promoter, leading to upregulation of BSEP expression and consequently to increased bile salt secretion. FXR/RXR has been considered a major transcriptional factor controlling BSEP expression ⁽⁶¹⁾. Higher levels of hepatobiliary export proteins, including BSEP, MDR3, and MRP4 were found in patients with gallstones treated with UDCA, which may indicate a role of UDCA in regulating BSEP ⁽⁶²⁾.

Inhibition of BSEP leads to reduced bile salt secretion, reduced bile flow, and cholestasis. Several medications known to cause cholestasis were found to inhibit BSEP expression; they include estrogen, cyclosporine A, estradiol, glybenclamide, rifampicin, and rifamycin ⁽⁶³⁾. It is possible that high estrogen levels during pregnancy could contribute to intrahepatic cholestasis of pregnancy via BSEP inhibition. Also bacterial lipopolysaccharide and obstructive cholestasis have been linked to inhibition of BSEP expression, which was reversible in the second case after successful restoration of bile flow ^(64,65).

Mutations of BSEP

BSEP disease is caused by genomic alterations of the ABCB11 gene, which results in functional defects of BSEP-mediated canalicular bile salt secretion. A large number of genetic mutations have been described, including missense, nonsense, and deletions presenting as either homozygous or compound heterozygous defects ^(2,57,66). Jansen et al ⁽⁶⁷⁾ originally discovered 10 mutations in 25 families from several distinct populations: in 16 patients the mutations were homozygous. They found a high correlation between BSEP mutations and immunohistochemical absence of BSEP protein expression in liver. Biliary secretion of bile salts was markedly reduced in BSEP-deficient patients who did not have an identifiable mutation but had an absence of BSEP antigen in liver. E297G is the most common mutation in individuals of European descent, accounting for approximately 30% of BSEP mutations in European series. Other common mutations include R575X, R1057X, G982R, C336S, R1153C, D482G, K461E, R1153C, R1268Q, R1090X, G238V, S114R, S593R, del 695, and del 3213 ^(66,67). A few mutations of BSEP have also been found in patients with acquired forms of cholestasis, including primary biliary cirrhosis and primary sclerosing cholangitis ⁽⁶⁸⁾. The interested reader is referred to Pauli-Magnus et al ⁽²⁾ for a more detailed accounting of genetic mutations in PFIC diseases. Individuals with low expression levels of BSEP could be at risk for the development of acquired forms of cholestasis, like drug-induced cholestasis and intrahepatic cholestasis of pregnancy ⁽⁵⁰⁾. The E297G and D482G mutations may yield proteins that are functional but do not traffic appropriately to the canalicular membrane. As such, these mutations may be associated with a milder form of disease compared with the other nonfunctional mutants. Even more subtle mutations of ABCB11 were described in patients with an intermittent form of disease (BRIC2; see below), these mutations include E186G, A570T, T923P, A926P, R1050C, R1128H, V444A, and E297G. The E297G mutation was previously described in patients with BSEP disease, and V444A was also found in intrahepatic cholestasis of pregnancy ^(69,70). More specific genotype-phenotype correlations require continued in-depth investigation of the clinical course of patients with intrahepatic cholestasis and careful examination of other factors that may influence the disease.

Clinical Presentations

Depending on the clinical course of this syndrome, 2 somewhat distinct clinical entities of the BSEP deficiency syndrome are known: a mild form also called benign recurrent intrahepatic cholestasis type 2 (BRIC2) and a severe form also called progressive familial intrahepatic cholestasis type 2 (PFIC2). These 2 diseases represent a heterogeneous group of cholestatic conditions, and benign disease may evolve into a more progressive form ⁽⁷¹⁾. Thus, a spectrum of disease is associated with mutations in BSEP, and as such some refer to them in general as BSEP or ABCB11 disease. Patients with BRIC2 have recurrent episodes of intense pruritus that usually are preceded by jaundice, pruritus with excoriations, steatorrhea, possibly nausea, vomiting, anorexia, right upper quadrant pain, and weight loss. Hepatomegaly may develop, although splenomegaly is usually absent. Cholestatic episodes may last for months; between episodes these patients are asymptomatic and have normal biochemical test results. A distinctive finding of BRIC2 compared with BRIC1 (disease related to defects in FIC1; see above) is the relatively common development of cholelithiasis (which could be related to supersaturating the bile with cholesterol) and the absence of extrahepatic manifestations like pancreatitis in BRIC2 patients ⁽⁶⁹⁾. Laboratory testing during episodes reveals elevated bilirubin, alkaline phosphatase, and serum bile acids. Alanine aminotransferase, aspartate aminotransferase, and GGTP are normal to mildly elevated.

Patients with more severe defects in BSEP have progressive disease characterized by jaundice, pruritus, failure to thrive, hepatomegaly, and splenomegaly. Cirrhosis has been noted as early as in the neonatal period. Jaundice can be intermittent early in the disease course and then becomes progressive. Pruritus is usually intense, can present as irritability in infants who cannot scratch, and is typically resistant to medical therapy. Life-threatening hemorrhage secondary to cholestasis-related vitamin K deficiency can be a dramatic presentation. Laboratory findings include direct hyperbilirubinemia, elevated alanine aminotransferase and aspartate aminotransferase, normal GGTP, elevated serum bile acids, and decreased biliary bile salts ⁽⁶⁷⁾. Imaging studies show a normal biliary tree.

Histology

Early biopsy in infancy reveals a “neonatal hepatitis-like” pattern with hepatocellular swelling and giant cell transformation, mild inflammatory presence, and canalicular cholestasis (Fig. 1C). All patients with BSEP mutations fail to demonstrate immunostaining for BSEP protein even though other canalicular enzymes such as pCEA and MRP2 are demonstrable ⁽⁶⁷⁾. Electron microscopy reveals compact bile plugs without features of Byler bile (Fig. 2B). The benign recurrent variant form of ABCB11 mutation, BRIC2, showed preservation of BSEP immunostaining in the liver ⁽⁶⁹⁾. Later biopsy specimens or hepatectomy specimens reveal canalicular cholestasis without the giant cell component but with varying degrees of ductular proliferation and fibrosis (Fig. 1D). Fibrosis and nodular hyperplasia can vary in extent, and hepatocellular carcinoma (HCC) develops on this background.

Treatment and Outcome

The therapeutic approach for BSEP disease includes both medical and surgical interventions, although the latter seem to be more effective. Several medications have been given to these patients with variable results, including cholestyramine, phenobarbital, rifampin, and UDCA. Before the current PFIC classification, a study published in 1997 by Jacquemin et al ⁽³⁸⁾ showed improvement of clinical symptoms and biochemical abnormalities in approximately 40% of patients with high and low GGT PFIC who were treated with long-term UDCA. Some patients even showed histological improvement on sequential liver biopsies. UDCA, a hydrophilic bile acid that is widely used in patients with cholestatic liver disease, may provide its therapeutic effects by reversing the potential hepatotoxicity of endogenous bile acids that accumulate during cholestasis. It also may increase the hepatocyte excretion of endogenous bile acids and inhibits their intestinal absorption. Although few data supports the use of medical therapy in patients with genotypically well-defined BSEP disease, many clinicians will try it as a first and relatively benign therapeutic approach. PEBD may result in amelioration of disease in some patients, especially those with functionally milder mutations. Liver transplantation is the ultimate therapy and is indicated in cases of well-defined cirrhosis, failed medical and surgical approaches, or intractable pruritus.

BSEP disease is typically a progressive disease, with worsening hepatic function and ultimate development of cirrhosis, which requires liver transplantation during late childhood or early adolescence; although it can be required as early as the first year of life ⁽⁶⁷⁾. BSEP disease may represent a significant risk for the development of liver cancer in children. Richter et al ⁽⁷²⁾ reported a 2-year-old patient with BSEP disease who was found to have hepatoblastoma in the explanted liver at the time of transplant. Knisely et al ⁽⁷³⁾ described childhood HCC and cirrhosis in 11 patients. Genotypically defined BSEP disease was demonstrated in 9 of 11 children. No association was found between HCC progression and a particular mutation in ABCB11. In earlier literature, other cases of HCC were reported in children with “giant-cell or neonatal hepatitis” ^(74,75). It is clearly possible that these children may have had BSEP disease, which could further support the recent association of liver malignancy and BSEP disease. Other than tyrosinemia and hepatitis B, no other childhood liver diseases have such a high apparent prevalence of HCC. Therefore; surveillance for HCC in children with BSEP disease may be warranted. Cholangiocarcinoma has also been described in 2 children with BSEP disease ⁽⁷⁶⁾. It is unclear why there is such a predisposition to hepatobiliary cancer in children with BSEP disease.

MDR3 DISEASE

MDR3 disease is caused by mutation of the MDR3 glycoprotein, which is coded by the ABCB4 gene (member of the ABC transporters) and has been mapped to chromosome 7q21. Although mRNA transcripts of MDR3 have been detected in several tissues like the placenta, terminal ileum, tonsils, spleen, muscles, and adrenal gland, the MDR3 protein itself is expressed primarily within hepatocytes ^(54,77).

Function and Regulation

MDR3 is located in the canalicular membrane of hepatocytes. Its main role is to transport phosphatidylcholine from inside hepatocytes into the bile canaliculus. The protein functions as a phosphatidylcholine floppase, transferring this lipid from the inner to the outer hemileaflet of the lipid bilayer of the canalicular membrane. The exact mechanism of transferring the phosphatidylcholine from the outer hemileaflet into bile is not completely clear. An initial model of this process included the generation of a unilamellar vesicle of the canalicular hemileaflet that budded into the canaliculus, transferring phosphatidylcholine and cholesterol into bile ⁽⁷⁸⁾. Adding phosphatidylcholine to bile salts micelles reduces their detergent activity and therefore protects the cholangiocytes that make up the intrahepatic bile ducts. The function of mdr2 (rodent ortholog of human MDR3) was first discovered in 1993 by Smit et al ⁽⁸⁰⁾, who generated mdr2-deficient mice using knockout technologies. The homozygote mdr2 (−/−) mice were found to have direct hyperbilirubinemia and elevated liver enzymes. There was no significant difference in these biochemistries between the heterozygote mdr2 (−/+) and the wild-type (+/+) mice. Liver tissue showed cholestasis and inflammation accompanying ductal proliferation in the homozygotes (−/−) mice, which was not present in the heterozygote or wild-type mice. Bile analysis in the mdr2 (−/−) was significant for undetectable concentrations of biliary phospholipids and low cholesterol. The phospholipid concentration in the heterozygotes was 60% of that in the wild type, but the cholesterol concentration did not differ. Liver disease in homozygote mice was attributed to the detergent action of phospholipid deficient bile ⁽⁷⁹⁾. This work was further confirmed by the finding of normal concentrations of phospholipids in bile and the absence of inflammatory changes in the liver in mdr2 (−/−) transgenic mice which expressed human MDR3.

Clinical Presentations

MDR3 deficiency has been documented in several disease phenotypes, including intrahepatic and gallbladder cholesterol lithiasis, intrahepatic cholestasis of pregnancy, and progressive familial intrahepatic cholestasis type 3 (PFIC3). Multiple features of MDR3 disease have been seen in some patients, including a patient described by Lucena et al ⁽⁸¹⁾, who reported all 3 disease entities in a 47-year-old woman.

Cholelithiasis

Biliary cholesterol solubilization depends on balanced concentrations of sterols, bile salts, and phospholipids. Imbalance due to abnormal concentration of any component, like phospholipids, can contribute to stone formation ⁽⁸²⁾. This is the likely explanation for the finding of intrahepatic and gallbladder cholesterol stones in patients with MDR3 deficiency. Intrahepatic lithiasis is generally an uncommon clinical problem. The first report of an association between MDR3 deficiency and cholelithiasis was in 2001 by Rosmorduc et al ⁽⁸³⁾, who described 6 adult patients who presented with biliary colic, pancreatitis, or cholangitis; chronic cholestasis; recurrence of symptoms after cholecystectomy; and bile duct stones. Bile composition showed high cholesterol/phospholipid ratios, cholesterol crystals, and low phospholipid concentration. Four of these patients had mutations (missense and insertion) of the MDR3 gene. These patients had remarkable responses and remission induced by UDCA. Interestingly, the finding of intrahepatic lithiasis has not been described in mdr2 (−/−) mice, with 1 possible explanation for this finding being the low secretion of biliary cholesterol ⁽⁸⁴⁾.

Intrahepatic Cholestasis of Pregnancy

This uncommon disorder presents during the second or, more commonly, third trimester of pregnancy with intense pruritus, which becomes more severe with advancing gestation and cholestasis. The disease carries a risk of fetal distress, prematurity, stillbirth, and recurrence with subsequent pregnancies. Laboratory test results are significant for elevated serum bile acids (which may be the first biochemical abnormality), aminotransferases, alkaline phosphatase, and bilirubin. The GGTP is usually normal or mildly elevated. The cause of this disease is still unknown, but elevated estrogen levels are likely to play a pathophysiological role. Intrahepatic cholestasis of pregnancy typically occurs during the third trimester and in multiple pregnancies, when serum concentrations of estrogen reach their peak. Patients with intrahepatic cholestasis of pregnancy have spontaneous and progressive disappearance of cholestasis after delivery ^(85,86). Although the pathogenesis of intrahepatic cholestasis of pregnancy is multifactorial, there is strong evidence of a genetic predisposition. Numerous studies have investigated the association of mutations of genes implicated in cholestasis such as FIC1, BSEP, and MDR3 in these patients ^(68,87). Recent reports show that the role of MDR3 mutation in these patients can be as high as 20% ^(87,88).

Progressive Familial Intrahepatic Cholestasis Type 3

MDR3 disease (PFIC3) was first identified in 1996 by Deleuze et al ⁽⁸⁹⁾, who described 2 children with cholestasis and elevated gGTP. MDR3 mRNA was not detected in liver tissues of either patient using Northern blot, and 1 patient had low biliary phospholipid. Two years later, de Vree et al ⁽⁹⁰⁾ described 2 children with cholestasis, elevated GGT, absence of immunohistochemical staining of canalicular MDR3, and specific nonsense and deletion mutations of the MDR3 gene.

Mutations of MDR3

The disease generally follows an autosomal recessive pattern of inheritance. A wide variety of distinct genetic mutations have been described and occur as either homozygous or compound heterozygous mutations. Interestingly; known heterozygote family members of affected children can experience manifestations related to MDR3 deficiency, most specifically intrahepatic cholestasis of pregnancy (akin to fatty liver disease of pregnancy described in carriers of fatty acid oxidation defects) ^(86,91). Mutations leading to premature truncation of the protein and missense mutations have been described. In the report by Jacquemin et al 16 mutations were described in 17 patients: 6 led to premature protein truncation ⁽⁹¹⁾. The missense mutation R625G was the most common, was identified in 5 patients, and showed a good response to UDCA. Children with the missense mutation had relatively less severe disease than did those with truncated protein mutation. They had later disease onset, slower progression to liver failure, and good response to UDCA, which could delay or prevent the need for liver transplantation. This could be explained by the presence of residual transport activity. UDCA may also alter the bile salt pool to one that is more hydrophilic and less injurious to cholangiocytes. This hypothetical mechanism could be augmented by another possible explanation: that is, the potential for upregulation of residual MDR3 by UDCA, as has been shown in mice ⁽⁹²⁾.

Clinical Presentations

Jacquemin et al reported 31 patients with MDR3 disease presenting between the ages of 1 month (initial presentation was jaundice) and 20.5 years (initial presentation was hepatosplenomegaly and gastrointestinal bleeding) ⁽⁹¹⁾. Approximately half of these patients presented during infancy. The most common presentations described were jaundice, pale stools, pruritus, hepatomegaly, and splenomegaly (as a manifestation of portal hypertension). Pruritus was reported in these infants within the first few months of life (as young as 1 month old), although it may be difficult to assess pruritus at this young age unless this was an interpretation of irritability. Interestingly; 2 patients initially presented with recurrent episodes of itching that became persistent after 1 year of age. Two other patients had jaundice, which resolved within the first year of life. Splenomegaly was present in 27 patients at a mean age of 5.5 years (range 8 months–20.5 years). Esophageal varices developed in 19 patients at a mean age of 9 years (range 1.5–20.5 years). The mean ages for gastrointestinal bleeding and liver failure were 11.5 and 7.5 years, respectively. The earlier presentation of liver failure likely represents a more severe functional defect in the MDR3 protein, leading to synthetic failure before variceal hemorrhage occurs.

Liver function tests showed elevated aminotransferases, alkaline phosphatase, GGTP, total bile acid concentration and conjugated hyperbilirubinemia. As expected with deficiency of this transporter, bile analysis revealed low concentrations of phospholipids and elevated ratios of bile salt to phospholipid and cholesterol to phospholipid. As demonstrated by published experiences, the disease can progress to cirrhosis, portal hypertension, and hepatic failure requiring liver transplantation. Immunohistochemical staining for MDR3 using polyclonal antibody α-REG1 and monoclonal antibody P3II26 showed complete absence of canalicular staining in these patients, especially those with severe mutations that lead to absent or unstable MDR3 protein ^(89–91).

Histology

Biopsy pathology varies with age at diagnosis. Early life biopsy is characterized by cholestasis with “biliary” features, mildly expanded portal areas with ductular proliferation (Fig. 1E). MDR3 (MRP2) immunostaining may be selectively lost in the presence of CD10, CD13, pCEA, and BSEP staining in some but not all instances of MDR3 mutations ^(90,93). Mutations that lead to expression of a truncated protein are associated with complete loss of immunostaining. Biopsy results later in life are also variable (Fig. 1F). Cholestasis of pregnancy may have a bland canalicular cholestatic appearance. There is an adult form of biliary cirrhosis that is also associated with MDR3 mutation and loss of immunostaining for MDR3 in most ⁽⁹¹⁾. Electron microscopy has not yielded diagnostically useful clues in this condition other than to exclude Byler-type bile.

Treatment and Outcome

In the same case series previously reported by Jacquemin et al ⁽⁹¹⁾, 58% of patients underwent liver transplants at a mean age of 7.5 years (range 2.5–16 years), and none of the transplanted patients had malignancy at the time of surgery, despite the finding of liver cancer in mdr2−/− mice. UDCA was given to 24 patients with variable results: complete normalization of liver function test results in 41%, negative response (no change in liver function test results) in 37%, and partial improvement in 20%. Heterozygous patients had a better prognosis with response to UDCA, documented in 4 of 5 patients; the 1 patient who did not show response underwent liver transplant.

SUMMARY

Three distinct forms of familial intrahepatic cholestasis are the result of mutations in the ATP8B1, ABCB11, and ABCB4 genes. The pathophysiologies of the latter 2 of these diseases are well characterized and result from abnormalities in canalicular excretion of bile acids and phospholipids, respectively. The molecular pathophysiology of the systemic disease associated with mutations in ATP8B1 remains unclear. In all of these diseases, wide variations in clinical phenotypes have been observed. The variability can be ascribed at least in part to predicted genotype:phenotype correlations. Disease- and genotype-specific prognoses and therapeutic approaches may exist, although much more information needs to be ascertained before clinicians can confidently make decisions based on genetic information. Ongoing prospective multicentered investigations sponsored by the National Institutes of Health (http://rarediseasesnetwork.epi.usf.edu/clic/about/index.htm.) are expected to provide additional insights into this complex and evolving area of pediatric gastroenterology.

Acknowledgments

The authors thank Prof E. Cutz, Toronto, for supplying material through A. Knisely, a member of Dr Richard Thompson's working group at King's College Hospital, London; and Dr Richard Thompson for supervision of the mutational analysis.