Progressive Familial Cholestasis Research Articles

Byler’s disease in a young child (own clinical observation)

Bailer’s disease is an ultra-rare (orphan) pathology associated with a deficiency of the membrane enzyme responsible for the transport of fat-soluble compounds and bile acids through the tubular membrane of the hepatocyte, as a result of which they accumulate in liver cells and have a damaging effect on them, being triggering factors of apoptosis. A clinical case of diagnosis of progressive familial cholestasis in a child aged 2 months with genetic verification of the diagnosis and successful treatment, including liver transplantation, is presented. Two mutations in the heterozygous state were identified: the previously described pathogenic mutation CM033442 and a previously undescribed mutation in the PGM1 gene.

Mutations in Myosin 5B in Children With Early-onset Cholestasis.

Mutations in Myosin 5B (MYO5B) are known to be associated with microvillous inclusion disease (MVID) a genetic cause of neonatal intractable diarrhoea. More recently, they have been reported in children with cholestasis but without typical gastrointestinal symptoms of MVID. We describe our series of children with cholestasis and mutations in MYO5B. Clinical, laboratory, and histological data were collected from patients with cholestasis and pathogenic mutations in MYO5B, found by next generation sequencing (NGS) but with minimal gastrointestinal disease. Six patients (3 boys) were identified. Median age at presentation was 19 months (range, 3-92). Presenting features were jaundice, pale stools, pruritus, and failure to thrive. Patients 5 and 6 had intractable diarrhoea until the age of 3 and 7 years, respectively, but currently are on full enteral diet with no intestinal symptoms. Median values for serum total bilirubin were 55 μmol/L (2-500), alanine aminotransferase 73I IU/L (32-114), γ-glutamyltransferase 7 IU/L (7-10), and serum bile acids 134 μmol/L (18-274). Three patients underwent 1 or more types of biliary diversion for symptom control. Median follow-up was 5 years (2-22). At most recent follow-up, they all reported pruritus while on antipruritics. Patient 1 had a liver transplant. We identified 6 patients, with mutations in MYO5B, early-onset cholestasis and pruritus, with variable response to biliary diversion without typical MVID.

Elevated copper impairs hepatic nuclear receptor function in Wilson's disease.

Wilson's disease (WD) is an autosomal recessive disorder that results in accumulation of copper in the liver as a consequence of mutations in the gene encoding the copper-transporting P-type ATPase (ATP7B). WD is a chronic liver disorder, and individuals with the disease present with a variety of complications, including steatosis, cholestasis, cirrhosis, and liver failure. Similar to patients with WD, Atp7b⁻/⁻ mice have markedly elevated levels of hepatic copper and liver pathology. Previous studies have demonstrated that replacement of zinc in the DNA-binding domain of the estrogen receptor (ER) with copper disrupts specific binding to DNA response elements. Here, we found decreased binding of the nuclear receptors FXR, RXR, HNF4α, and LRH-1 to promoter response elements and decreased mRNA expression of nuclear receptor target genes in Atp7b⁻/⁻ mice, as well as in adult and pediatric WD patients. Excessive hepatic copper has been described in progressive familial cholestasis (PFIC), and we found that similar to individuals with WD, patients with PFIC2 or PFIC3 who have clinically elevated hepatic copper levels exhibit impaired nuclear receptor activity. Together, these data demonstrate that copper-mediated nuclear receptor dysfunction disrupts liver function in WD and potentially in other disorders associated with increased hepatic copper levels.

ABCB4 deficiency is causative for hepatic copper accumulation in a patient with features of Wilson Disease

Clinical Case: A 21-year-old patient presented with mild pruritus and elevated liver enzymes (ALT 181 U/L; AST 106 U/L; γ-GT 212 U/L; AP 220 U/L). Of note, his sister had been diagnosed for Wilson Disease (WD) before. Comprehensive work-up provided no evidence of viral or autoimmune liver disease, primary biliary cirrhosis or sclerosing cholangitis. 24-hour urinary copper excretion was increased (101µmol/day), however, with normal serum copper and coeruloplasmin levels. Kayser-Fleischer rings were not detectable. Liver histology was compatible with chronic hepatitis with mild portal and parenchymal inflammation, mild fibrosis, increased copper staining and regular bile ducts. Quantitative analysis revealed elevated hepatic copper content (102µg/g dry weight). Thus, a diagnosis of probable WD was entertained and chelation therapy started. However, there was no biochemical response and sequencing of the ATP7B gene yielded unremarkable results. Given the histological findings and positive family history, sequencing of the hepatocanalicular phospholipid transporter ABCB4 was carried out revealing a heterozygous mutation (c.139C>T; p.Arg47X). In addition, a second heterozygous mutation (c.959C>T; p.Ser320Phe) was detected previously described in progressive familial cholestasis type 3 (PFIC3). Ursodeoxycholic acid was started with significant biochemical improvement. Discussion: Variable degrees of secondary copper overload have been appreciated in common cholestatic disorders e.g. PBC or PSC. However, adult ABCB4 deficiency with a PFIC3-like phenotype underlying significant hepatic copper deposition with challenging differentiation from WD has only rarely been reported.(1,2) Whether or not ABCB4 plays an active role in hepatic copper homeostasis beyond alterations in cholestasis-related copper excretion is unknown. The reported clinical case underscores the potential of dedicated genetic testing in the clinical assessment of hereditary cholestatic disorders. Literatur: 1. Anheim et al. Clin Genet 2010 2. Ramraj et al. Hepatology 2010

Genetic Factors in the Pathogenesis of Cholangiocarcinoma

Background: Cholangiocarcinoma (CC) is increasing in incidence, but its pathogenesis remains poorly understood. Chronic inflammation of the bile duct and cholestasis are major risk factors, but most cases in the West are sporadic. Genetic polymorphisms in biliary transporter proteins have been implicated in benign biliary disease and, in the case of progressive familial cholestasis, have been associated with childhood onset of CC. In the current study, five biologically plausible candidate genes were investigated: ABCB11 (BSEP), ABCB4 (MDR3), ABCC2 (MRP2), ATP8B1 (FIC1) and NR1H4 (FXR). Methods: DNA was collected from 172 Caucasian individuals with confirmed CC. A control cohort of healthy Caucasians was formed. Seventy-three SNPs were selected using the HapMap database to capture genetic variation around the five candidate loci. Genotyping was undertaken with a competitive PCR-based system. Confirmation of Hardy-Weinberg equilibrium and Cochran-Armitage trend testing were performed using PLINK. Haplotype frequencies were compared using haplo.stats. Results: All 73 SNPs were in Hardy-Weinberg equilibrium. Four SNPs in ABCB11 were associated with altered susceptibility to CC, including the V444A polymorphism, but these associations did not retain statistical significance after Bonferroni correction for multiple testing. Haplotype analysis of the genotyped SNPs in ATP8B1 identified significant differences in frequencies between cases and controls (global p value of 0.005). Conclusion: Haplotypes in ATP8B1 demonstrated a significant difference between CC and control groups. There was a trend towards significant association of V444A with CC. Given the biological plausibility of polymorphisms in ABCB11 and ATP8B1 as risk modifiers for CC, further study in a validation cohort is required.

Progress of the causes of infantile cholestasis

Infantile cholestasis is a syndrome caused by multiple factors with conjugated hyperbilirubinemia for clinical characteristics. The etiologic factors include infection, poisoning, inherited metabolic defects and bile duct abnormalities. Infection, biliary atresia accounts for more than half of cholestasis children. Recently, the new causes have been recognized, for example citrin deficiency, Alagille syndrome,progressive familial cholestasis symptoms caused by different causes are similar to biliary atresia, but the prognosis and treatment are quite different. If misdiagnosed, the condition will exacerbate. Understanding the various causes plays an important role for the early diagnosis and differential diagnosis of infantile cholestasis. Key words: Cholestasis; Infantile; Etiology

P41 Genetic variation in biliary transporters as a susceptibility factor for cholangiocarcinoma

IntroductionCholangiocarcinoma (CC) is increasing in incidence globally but its pathogenesis remains poorly understood. Chronic inflammation of the bile duct and cholestasis are major risk factors but most cases in the...

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

P(4)-ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P(4)-ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P(4)-ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

Investigation of the Lith1 candidate genes ABCB11 and LXRA in human gallstone disease

Genetic susceptibility in the causation of gallbladder diseases was recognized as early as 1937. A major gallstone susceptibility locus (Lith1) was identified in 1995 by quantitative trait locus mapping in mice. Two attractive positional and functional candidate genes in LXRA and ABCB11 are located in this interval. ABCB11 is associated with progressive familial cholestasis. This study was undertaken to investigate LXRA and ABCB11 as candidate genes for gallstone disease in humans. Eight hundred and ten patients who underwent cholecystectomy for symptomatic gallstone disease (median age of onset, 50 years) were compared with 718 sex-matched control individuals. Control individuals were sonographically free of gallstones. Haplotype tagging and all known coding single nucleotide polymorphisms (SNPs) were genotyped for ABCB11 (n=29) and LXRA (n=10). The investigated high-risk patient sample provides a power of greater than 80% for the detection of odds ratios down to 1.55. No evidence of association of the two genes in the single point tagging markers, coding variants or in the sliding window haplotype analysis was detected (all nominal single-point P values>or=.08). In conclusion, in the investigated German sample, no evidence of association of ABCB11 and LXRA to gallstone susceptibility was detected. The gallstone trait is not allelic to progressive familial cholestasis at the ABCB11 locus. Systematic fine mapping of the Lith1 region is required to identify the causative genetic variants for gallstone in mice and humans.

The bile salt export pump: molecular properties, function and regulation

Secretion of bile salts from the hepatocyte into bile is the major driving force for the generation of bile flow. Identification of the bile salt export pump (BSEP, ABCB11) as the main adenosine-triphosphate-dependent bile salt transporter in mammalian liver has led to a greater understanding of the biliary bile salt secretory process and its regulation. The biology and pathobiology of BSEP have been the subject of many recent studies. Thus, it has been recognized that while mutations in the gene encoding BSEP are responsible for a subgroup of progressive familial cholestasis (progressive familial intrahepatic cholestasis subtype 2), a pediatric cholestatic disorder that may progress to cirrhosis, defective expression or function of BSEP may underlie some forms of drug-induced cholestasis. The present review summarizes recent data on the molecular properties and regulation of BSEP, as well as the clinical implications of absent or defective function of this hepatic efflux pump.

Effect of mdr2 mutation with combined tandem disruption of canalicular glycoprotein transporters by cyclosporine A on bile formation in mice

The inhibition of canalicular glycoprotein transporters has been suggested as the cause of familial intrahepatic cholestasis. Mutations in multidrug resistance 3-glycoprotein (MDR3) gene induce progressive familial intrahepatic cholestasis type 3 (PFIC3). Phenotypically, mutation in mdr2 in mice resembles the disruption of MDR3 in human. Secondly, mutation in the bile salt exporting pump (BSEP)/sister of P-glycoprotein (spgp) gene causes progressive familial intrahepatic cholestasis type 2 in human. However, in spgp knock-out mice only a mild persistent cholestasis occurs. The aim of this study is to evaluate the effects of various P-glycoprotein (Pgp) transporters on bile formation and the canalicular transport of taurocholic acid (TCA) in an attempt to understand the combined role of these transporters in the pathogenesis of familial intrahepatic cholestasis. Total bile acid (TBA) and cholic acid secretion rate were decreased in the mdr2 knock-out mice. However, bile flow (BF) and the secretion of muricholic acids were increased. Secretion of cholesterol was negligible and no phospholipids were detected in bile of mdr2 knock-out mice. Treatment with cyclosporine A (CsA) decreased the BF, and the biliary secretion of bile salts (BS) and phospholipids as compared to wild type mice, but after the injection of TCA+CsA, the BF, and the biliary secretion of BS and lipids were increased as compared to the wild type mice treated with CsA alone. In the mdr2 knock-out mice, CsA treatment decreased the BF and the secretion of BS but after the injection of TCA+CsA, the BF and the biliary secretion of BS were increased and the phospholipid secretion was slightly stimulated as compared to the mdr2 knock-out mice treated with CsA alone. Conclusion: Disruption of the mdr2 gene and the inhibition of glycoprotein transporters by CsA induce cholestasis in mice which is characterized by reduced BF, BS and biliary lipid secretion. However, CsA treatment did not significantly increase the cholestatic effect in the mdr2 knock-out mice. The injection of TCA decreased the cholestatic effect in the mdr2 knock-out mice as well as the inhibition of glycoproteins transporters by CsA. These data suggest that mutation in the canalicular mdr2 is an important factor during the development of progressive familial cholestasis.

Hepatic transport systems.

The identification of the genes responsible for various genetic liver disorders lead to a better understanding of basic physiology of hepatic transport systems. In this review we focus on transport systems involved in the generation of bile and in the maintenance of copper homeostasis. Abnormal function of these transporters results in diseases like Wilson's disease, progressive familial cholestasis syndromes, Dubin-Johnson syndrome and cystic fibrosis. Beyond these well defined diseases, functional impairments of transport proteins may predispose to non-genetic diseases ranging from intrahepatic cholestasis of pregnancy to neurodegenerative disorders including Alzheimer's disease.

BSEP: function and role in progressive familial intrahepatic cholestasis.

Secretion of bile acids is the major driving force for bile flow in mammals. The recently described adenosine triphosphate (ATP)-dependent bile acid transporter, bile salt export pump (BSEP), formerly called sister of p-glycoprotein, is responsible for active transport of bile acids across the hepatocyte canalicular membrane into bile. It is now recognized that mutations in the gene encoding this protein (ABCB11) are responsible for a subgroup of infants and children with progressive familial cholestasis (PFIC-2), a cholestatic disorder causing extreme pruritus, growth failure, and progression to cirrhosis in the first decade of life. Understanding the structure and function of BSEP has improved our understanding of the mechanisms underlying bile secretion. Determining genotype/phenotype relationships in patients with mutations in this gene are currently ongoing.