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1.  Progressive familial intrahepatic cholestasis 
Progressive familial intrahepatic cholestasis (PFIC) refers to heterogeneous group of autosomal recessive disorders of childhood that disrupt bile formation and present with cholestasis of hepatocellular origin. The exact prevalence remains unknown, but the estimated incidence varies between 1/50,000 and 1/100,000 births.
Three types of PFIC have been identified and related to mutations in hepatocellular transport system genes involved in bile formation. PFIC1 and PFIC2 usually appear in the first months of life, whereas onset of PFIC3 may also occur later in infancy, in childhood or even during young adulthood. Main clinical manifestations include cholestasis, pruritus and jaundice. PFIC patients usually develop fibrosis and end-stage liver disease before adulthood. Serum gamma-glutamyltransferase (GGT) activity is normal in PFIC1 and PFIC2 patients, but is elevated in PFIC3 patients. Both PFIC1 and PFIC2 are caused by impaired bile salt secretion due respectively to defects in ATP8B1 encoding the FIC1 protein, and in ABCB11 encoding the bile salt export pump protein (BSEP). Defects in ABCB4, encoding the multi-drug resistant 3 protein (MDR3), impair biliary phospholipid secretion resulting in PFIC3.
Diagnosis is based on clinical manifestations, liver ultrasonography, cholangiography and liver histology, as well as on specific tests for excluding other causes of childhood cholestasis. MDR3 and BSEP liver immunostaining, and analysis of biliary lipid composition should help to select PFIC candidates in whom genotyping could be proposed to confirm the diagnosis. Antenatal diagnosis can be proposed for affected families in which a mutation has been identified. Ursodeoxycholic acid (UDCA) therapy should be initiated in all patients to prevent liver damage. In some PFIC1 or PFIC2 patients, biliary diversion can also relieve pruritus and slow disease progression. However, most PFIC patients are ultimately candidates for liver transplantation. Monitoring of hepatocellular carcinoma, especially in PFIC2 patients, should be offered from the first year of life. Hepatocyte transplantation, gene therapy or specific targeted pharmacotherapy may represent alternative treatments in the future.
PMCID: PMC2647530  PMID: 19133130
2.  Morphologic Findings in Progressive Familial Intrahepatic Cholestasis 2 (PFIC2): Correlation With Genetic and Immunohistochemical Studies 
Progressive familial intrahepatic cholestasis, type 2 (PFIC2), characterized by cholestasis in infancy that may progress to cirrhosis, is caused by mutation in ABCB11, which encodes bile salt export pump (BSEP). We correlated histopathologic, immunohistochemical, and ultrastructural features in PFIC2 with specific mutations and clinical course. Twelve patients with clinical PFIC2 and ABCB11 mutations were identified, and 22 liver biopsy and explant specimens were assessed. All had hepatocellular cholestasis; most had canalicular bile plugs. At least 1 specimen from every patient had centrizonal/sinusoidal fibrosis, often with periportal fibrosis. Neonatal hepatitis-like features (inflammation, giant cells, necrosis) varied. In 2 of the 5 patients with paired specimens obtained > 6 months apart, lobular and portal fibrosis worsened. Transmission electron microscopy (EM) in all 9 patients studied showed canalicular dilatation, microvilli loss, abnormal mitochondrial internal structure, and varying intra-canalicular accumulation of finely granular bile. Canalicular staining for BSEP was absent in 10 patients and present in 2 patients, 1 of whom had intermittent symptoms. ABCB11 sequencing of all patients identified 6 novel and 10 previously described mutations, with nonsense, missense, and/or noncoding mutations in the 10 patients without immunohistochemically demonstrable BSEP. Missense and/or noncoding mutations were identified in the 2 patients with demonstrable BSEP, whose clinical course was more indolent. Mutations ending ABCB11 transcription appear linked, through hepatocellular necrosis and fibrosis, to worse outcome. In conclusion, light microscopy and electron microscopy findings in clinical PFIC2 can support diagnosis, but are variable and nonspecific. Therefore, no correlation between specific mutations and histopathology is yet possible.
PMCID: PMC3416050  PMID: 21490445
PFIC2; progressive familial intrahepatic cholestasis; bile salt export pump; BSEP; ABCB11; neonatal hepatitis
3.  ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained 
Gastroenterology  2008;136(3):1060-1069.
Background & Aims
Progressive Familial Intrahepatic Cholestasis 1 (PFIC1) results from mutations in ATP8B1 (also known as FIC1), a putative aminophospholipid flippase. However conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1-deficiency produces cholestasis by altering the activity of the nuclear receptor FXR or by impairing the structure of the canalicular membrane
ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes, and Caco2 cells using adenoviral and oligonucleotide siRNAs.
ATP8B1 mRNA and protein expression was greatly reduced in human and rat hepatocytes and Caco2 cells. In contrast, FXR expression and several FXR dependent membrane transporters (BSEP, MRP2) were unchanged at mRNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells as evidenced by 6-ECDCA mediated induction of SHP, BSEP and MDR3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid, CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine consistent with Atp8b1’s function as an aminophospholipid flippase.
ATP8B1- deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane, but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated to bile acid accumulation.
PMCID: PMC3439851  PMID: 19027009
4.  The Membrane Protein, ATP8B1, Signals Through Protein Kinase C-Zeta to Activate the Farnesoid X Receptor 
Hepatology (Baltimore, Md.)  2008;48(6):1896-1905.
Prior loss-of-function analyses revealed that ATP8B1 (FIC1) post-translationally activated the Farnesoid X-Receptor (FXR).
Mechanisms underlying this regulation are elaborated upon by these gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild type and mutated FIC1 expression constructs using a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques.
FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler-disease FIC1 mutants did not activate BSEP, while benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent upon the presence of the FXR ligand, chenodeoxycholic acid. The FIC1 effect on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C (PKC) ζ inhibitors (pseudosubstrate or siRNA silencing). Recombinant PKCζ directly phosphorylated immunoprecipitated FXR. Mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, while the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKCζ in Caco-2 cells resulted in activation of the human apical sodium dependent bile acid transporter promoter.
These results demonstrate that FIC1 signals to FXR via PKCζ. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. BRIC emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.
PMCID: PMC2774894  PMID: 18668687
nuclear receptor; cholestasis; liver; ileum; bile acid
5.  The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II 
PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II–associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep.
PMCID: PMC151156  PMID: 12370274
6.  Intractable itch relieved by 4-phenylbutyrate therapy in patients with progressive familial intrahepatic cholestasis type 1 
Progressive familial intrahepatic cholestasis type 1 (PFIC1), an inherited liver disease caused by mutations in ATP8B1, progresses to severe cholestasis with a sustained intractable itch. Currently, no effective therapy has been established for PFIC1. Decreased function of the bile salt export pump (BSEP) in hepatocytes is suggested to be responsible for the severe cholestasis observed in PFIC1. We found a previously unidentified pharmacological effect of 4-phenylbutyrate (4PB) that increases the expression and function of BSEP. Here, we tested 4PB therapy in three patients with PFIC1.
The therapeutic potency of 4PB in these patients was tested by oral administration of this drug with gradually increasing dosage (200, 350, and 500 mg/kg/day) for 6 months. Biochemical, histological, and clinical data were collected.
4PB therapy had no beneficial effect on the patients’ liver functions, as assessed by biochemical and histological analyses, despite an increase in hepatic BSEP expression. However, therapy with 4PB at a dosage of 350 or 500 mg/kg/day significantly relieved the intractable itch. Serum levels of potential pruritogens in cholestasis were much higher than the reference ranges during the 4PB therapy.
4PB therapy may be a new medication for patients with intractable cholestatic pruritus and may improve quality of life for patients and their families.
PMCID: PMC4105841  PMID: 25022842
Pediatric liver disease; Cholestasis; PFIC1; Pruritus; 4PB
7.  Bile composition in Alagille Syndrome and PFIC patients having Partial External Biliary Diversion 
BMC Gastroenterology  2008;8:47.
Partial External Biliary Diversion (PEBD) is a surgical intervention to treat children with Progressive Familial Intrahepatic Cholestasis (PFIC) and Alagille syndrome (AGS). PEBD can reduce disease progression, and examining the alterations in biliary lipid composition may be a prognostic factor for outcome.
Biliary lipid composition and the clinical course of AGS and PFIC patients were examined before and after PEBD.
Pre-PEBD bile from AGS patients had greater chenodeoxycholic/cholic acid (CDCA/CA), bile salt, cholesterol and phospholipid concentrations than PFIC patients. AGS patients, and PFIC patients with familial intrahepatic cholestasis 1 (FIC1) genotype, responded better to PEBD than PFIC patients with bile salt export protein (BSEP) genotype. After successful PEBD, AGS patients have higher biliary lipid concentrations than PFIC patients and PEBD also increases biliary phospholipid concentrations in FIC1 patients.
Both AGS and FIC1 patients can benefit from PEBD, and preserved biliary phospholipid concentrations may be associated with better outcomes post-PEBD.
PMCID: PMC2585081  PMID: 18937870
8.  A C-terminal tyrosine-based motif in the bile salt export pump directs clathrin-dependent endocytosis 
Hepatology (Baltimore, Md.)  2012;55(6):1901-1911.
The liver specific bile salt export pump (BSEP) is crucial for bile-acid dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related ATP-Binding Cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and 2 large cytoplasmic nucleotide binding domains (NBD). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface ELISA endocytosis reporter system using a chimera of the interleukin 2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif 1310YYKLV1314 (Yxx∅). When expressed in HEK293T cells TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y1310Y1311 amino acids in TacCterm and in full length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity.
Our results indicate constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP.
PMCID: PMC3319652  PMID: 22161577
bile acid transporter; trafficking motif
9.  Bile Salt Export Pump is Dysregulated with Altered Farnesoid X Receptor Isoform Expression in Patients with Hepatocellular Carcinoma 
Hepatology (Baltimore, Md.)  2013;57(4):1530-1541.
As a canalicular bile acid effluxer, bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies showed that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent non-tumor tissues. In contrast to mouse, human BSEP was regulated by farnesoid x receptor (FXR) in an isoform-dependent manner. FXRα2 exhibited a much more potent activity than FXRα1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXRα1 and FXRα2. The FXRα1/FXRα2 ratios were significantly increased with undetectable FXRα2 expression in one third of the HCC tumor samples. Similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh 7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were significantly elevated in HCC tissues. Treatment of Huh 7 cells with IL-6 and TNF-α resulted in a marked increase in the FXRα1/FXRα2 ratio concurrent with a significant decrease in BSEP expression. In conclusion, BSEP expression was severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation, and the restoration of BSEP expression through suppressing inflammation in the liver may re-establish the bile acid homeostasis.
PMCID: PMC3608797  PMID: 23213087
BSEP; FXR; HCC; Bile acids; Gene regulation
10.  Secondary Mitochondrial Respiratory Chain Defect Can Delay Accurate PFIC2 Diagnosis 
JIMD Reports  2013;14:17-21.
Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and often result in hepatic failure. There is no gold-standard test for diagnosing mitochondrial disease, and the current diagnosis relies on establishing a consistent pattern of evidence from clinical data, neuroimaging, tissue biopsy, and biochemical investigations. In some patients, the mitochondrial respiratory chain defect (MRCD) diagnosis is confirmed by genetic investigations. In most cases, genetic investigations are not informative and a number of cases remain unexplained.
Here, we report on two children presenting with liver disease in whom first investigations suggested MRCD, due to decreased liver respiratory chain activities and decreased mitochondrial DNA copy number. However, sequencing of the genes known to be associated with mitochondrial DNA instability did not identify any pathogenic mutations. Further investigations including exome analysis, biliary bile salt analysis, and/or BSEP immunostaining detected a defect in the bile salt export pump (BSEP). Diagnosis of progressive familial intrahepatic cholestasis type 2 (PFIC2), a hereditary disorder in bile formation due to BSEP deficiency was confirmed by ABCB11 gene sequencing. Deleterious mutations were identified in both patients: one harboring compound heterozygous mutations (p.Arg470*/c.1308+2T>A) and the other homozygous nonsense mutation (p.Tyr354*). This report increases awareness of a possible secondary mitochondrial respiratory chain defect in the liver tissue associated with other underlying causes such as PFIC2.
PMCID: PMC4213329  PMID: 24214725
11.  The Bile Salt Export Pump: Clinical and Experimental Aspects of Genetic and Acquired Cholestatic Liver Disease 
Seminars in liver disease  2010;30(2):125-133.
The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.
PMCID: PMC3008346  PMID: 20422495
Cholestasis; progressive familial intrahepatic cholestasis; bile salt export pump (BSEP) mutations; polymorphisms; ATP-binding cassette (ABC) transporters; trafficking; recycling; ubiquitination
12.  The feline bile salt export pump: a structural and functional comparison with canine and human Bsep/BSEP 
The bile salt export pump (BSEP/ABCB11) is the primary transporter for the excretion of bile acids from hepatocytes into bile. In human, inhibition of BSEP by drugs has been related to drug-induced cholestasis and subsequent cytotoxic effects. The role of BSEP in canine and feline liver diseases has not been studied in detail, but the same mechanism of inhibition by drugs as in humans could play a role in veterinary medicine. The aim of this study was to investigate the functional characteristics of feline Bsep in comparison with canine and human Bsep/BSEP with respect to substrate affinities and inhibitory potential of model drugs. Orthologs of all three species were cloned and cell membrane vesicles overexpressing feline, canine and human Bsep/BSEP were prepared for functional analyses.
The cDNA sequences of the open reading frames of feline, canine and human Bsep/BSEP showed a high similarity between the species. Functional studies demonstrated for all species a tendency to a higher affinity of BSEP/Bsep for the conjugated bile acid taurocholic acid (TCA) than glycocholic acid (GCA), and a higher affinity for GCA than for the unconjugated cholic acid (CA). The inhibitory potency of the model inhibitors cyclosporine A, troglitazone and ketoconazole was characterized against TCA uptake into BSEP/Bsep containing membrane vesicles. All three substances potently inhibited TCA uptake without significant species differences.
Structure and functional characteristics of cat, dog and human Bsep/BSEP appeared to be very similar, indicating that the properties of this transporter have been highly preserved among the different species. Therefore, inhibition of BSEP by drugs could also be a mechanism in cholestasis and liver disease in veterinary relevant animal species. This model could be used to predict drug-induced liver injury caused by BSEP inhibition at an early stage in veterinary drug development.
PMCID: PMC3900472  PMID: 24359682
BSEP; ABCB11; Transporter; Cat; Dog; Liver; Drugs; Toxicity; Bile acids; Inhibitor
13.  Novel ABCB11 mutations in a Thai infant with progressive familial intrahepatic cholestasis 
Progressive familial intrahepatic cholestasis (PFIC) type 2 is caused by mutations in ABCB11, which encodes bile salt export pump (BSEP). We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum γ-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type 2.
PMCID: PMC2744194  PMID: 19750581
ABCB11; Bile salt export pump; Progressive familial intrahepatic cholestasis
14.  Degradation of the Bile Salt Export Pump at Endoplasmic Reticulum in Progressive Familial Intrahepatic Cholestasis Type II (PFIC II) 
Hepatology (Baltimore, Md.)  2008;48(5):1558-1569.
The bile salt export pump (Bsep) represents the major bile salt transport system at the canalicular membrane of hepatocytes. When examined in model cell lines, genetic mutations in the BSEP gene impair its targeting and transport function, contributing to the pathogenesis of PFIC II. PFIC II mutations are known to lead to a deficiency of BSEP in human hepatocytes, suggesting that PFIC II mutants are unstable and degraded in the cell. To investigate this further, we have characterized the impact of several PFIC II mutations on the processing and stability of rat Bsep. G238V, D482G, G982R, R1153C and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Except for R1153C, the PFIC II mutants are degraded with varying half-lives. G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. The proteasome provides the major degradation pathway for the PFIC II mutants, while the lysosome also contributes to the degradation of D482G. The PFIC II mutants appear to be more heavily ubiquitinated compared with the wild-type (wt) Bsep, and their ubiquitination is increased by the proteasome inhibitors. Overexpression of several E3 ubiquitin ligases, which are involved in ER-associated degradation (ERAD), lead to the decrease of both mutant and wt Bsep. Gene knockdown studies revealed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, while HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (ΔGly). Furthermore we present evidence that G982R weakly associates with various components of the ER quality control system. These data together demonstrate that the PFIC II mutants and ΔGly are degraded by the ERAD pathway.
PMCID: PMC2587109  PMID: 18798335
15.  ATPase Class I Type 8B Member 1 and Protein Kinase C-ζ Induce the Expression of the Canalicular Bile Salt Export Pump in Human Hepatocytes 
Pediatric research  2010;67(2):183.
The exact molecular mechanism(s) of the disease that results from defects in the ATPase Class I Type 8B Member 1 gene remains controversial. Prior investigations of human ileum and in intestinal and ovarian cell lines have suggested that Familial Intrahepatic Cholestasis 1 (FIC1) activates the Farnesoid X-Receptor (FXR) via a pathway involving Protein Kinase C ζ (PKCζ). Translational investigations of human liver from individuals with FIC1 disease have been confounded by secondary affects of progressive cholestatic liver disease and limited numbers of samples for analysis. These studies, performed in primarily derived human hepatocytes, circumvent this issue. The canalicular bile salt export pump (BSEP) served as a downstream target of FXR. siRNA mediated silencing of FIC1 in human hepatocytes led to a reduction in both human BSEP promoter activity and BSEP protein expression, which correlated with a reduction in FXR expression and redistribution of its localization from the nucleus to the cytoplasm. These changes in BSEP expression could be reproduced by altering the expression of PKCζ; with a positive correlation of PKCζ activity and BSEP expression. Overall, these findings support the hypothesis that FIC1 enhances FXR signaling via a PKCζ dependent signaling pathway.
PMCID: PMC2819672  PMID: 19809379
16.  Genetic evidence of heterogeneity in intrahepatic cholestasis of pregnancy 
Gut  2003;52(7):1025-1029.
Background and aims: The aim of this study was to investigate the genetic aetiology of intrahepatic cholestasis of pregnancy (ICP) and the impact of known cholestasis genes (BSEP, FIC1, and MDR3) on the development of this disease.
Patients and methods: Sixty nine Finnish ICP patients were prospectively interviewed for a family history of ICP, and clinical features were compared in patients with familial ICP (patients with a positive family history, n=11) and sporadic patients (patients with no known family history of ICP, n=58). For molecular genetic analysis, 16 individuals from two independently ascertained Finnish ICP families were genotyped for the flanking markers for BSEP, FIC1, and MDR3.
Results: The pedigree structures in 16% (11/69) of patients suggested dominant inheritance. Patients with familial ICP had higher serum aminotransferase levels and a higher recurrence risk (92% v 40%). Both segregation of haplotypes and multipoint linkage analysis excluded BSEP, FIC1, and MDR3 genes in the studied pedigrees. Additionally, the MDR3 gene, previously shown to harbour mutations in ICP patients, was negative for mutations when sequenced in four affected individuals from the two families.
Conclusions: These results support the hypothesis that the aetiology of ICP is heterogeneous and that ICP is due to a genetic predisposition in a proportion of patients. The results of molecular genetic analysis further suggest that the previously identified three cholestasis genes are not likely to be implicated in these Finnish ICP families with dominant inheritance.
PMCID: PMC1773695  PMID: 12801961
intrahepatic cholestasis of pregnancy; obstetric cholestasis; linkage analysis
17.  Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris 
PLoS ONE  2013;8(4):e60620.
The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.
PMCID: PMC3617136  PMID: 23593265
18.  The Association between Bile Salt Export Pump Single-Nucleotide Polymorphisms and Primary Biliary Cirrhosis Susceptibility and Ursodeoxycholic Acid Response 
Disease Markers  2014;2014:350690.
Background. Primary biliary cirrhosis (PBC) is a chronic and progressive cholestasis liver disease. Bile salt export pump (BSEP) is the predominant bile salt efflux system of hepatocytes. BSEP gene has been attached great importance in the susceptibility of PBC and the response rate of ursodeoxycholic acid (UDCA) treatment of PBC patients. Methods. In this study, TaqMan assay was used to genotype four variants of BSEP, and the Barcelona criteria were used for evaluating the response rate of UDCA treatment. Results. Variant A allele of BSEP rs473351 (dominant model, OR = 2.063; 95% CI, 1.254–3.393; P = 0.004) was highly associated with PBC susceptibility. On the contrary, variant A allele of BSEP rs2287618 (dominant model, OR = 0.617; 95% CI, 0.411–0.928; P = 0.020) provided a protective role and Barcelona evaluation criterion indicated that the frequency of variant allele at BSEP rs2287618 was significantly decreased in UDCA-responsive PBC patients (P = 0.021). Conclusion. These results suggested that BSEP rs473351 was closely associated with the susceptibility of PBC and if people with BSEP rs2287618 were diagnosed as PBC, the UDCA treatment was not satisfactory. Larger studies with mixed ethnicity subjects and stratified by clinical and subclinical characteristics are needed to validate our findings.
PMCID: PMC4216684  PMID: 25392597
19.  Early Identification of Clinically Relevant Drug Interactions With the Human Bile Salt Export Pump (BSEP/ABCB11) 
Toxicological Sciences  2013;136(2):328-343.
A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug-induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a data set of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, whereas positive molecular charge was associated with a lack of inhibition. All approved drugs in the data set (n = 182) were categorized according to DILI warnings in drug labels issued by the Food and Drug Administration, and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including 9 drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich-cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport, and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux, whereas BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.
PMCID: PMC3858191  PMID: 24014644
biliary excretion; drug induced liver injury; hepatocytes; in vitro and alternatives; disposition; risk assessment; alternatives to animal testing; predictive toxicology.
20.  The multiple facets of ABCB4 (MDR3) deficiency 
Opinion Statement
ABCB4 (MDR3) is a lipid translocator that moves phosphatidylcholine from the inner to outer leaflet of the canalicular membrane for extraction into the lumen by bile salts. Genetic mutations of ABCB4 lead to 3 distinct but related hepatobiliary diseases. Progressive familial intrahepatic cholestasis (PFIC) type 3 is a chronic cholestatic syndrome characterized by a markedly elevated GGTP. Patients present later in life than other types of PFIC with jaundice, pruritis and hepatosplenomegaly. Peri-portal inflammation progresses to biliary cirrhosis and eventually causes portal hypertension. Ursodeoxycholic acid (UDCA), which alters biliary bile composition, normalizes liver function tests in approximately half of treated PFIC type 3 patients. Partial or non-responders will eventually require liver transplantation. Gallstone patients with ABCB4 mutations may have Low Phospholipid Associated Cholelithiasis Syndrome. This is characterized by cholesterol gallstones and intrahepatic microlithiasis, along with recurrent biliary symptoms, despite cholecystectomy. A distinct group of patients with ABCB4 mutations may also develop intrahepatic brown pigment stones. The current treatment for these two diseases is UDCA, which may improve biliary symptoms even before the dissolution of stones occurs. Additional therapies such as Farnesoid X receptor ligands/agonists and benzfibrates show promise as future therapeutic modalities. Intrahepatic cholestasis of pregnancy affects pregnant women with abnormal ABCB4 variants. These women suffer from disabling pruritis and may experience steatorrhea as well. Their fetuses are at high risk for prematurity and still births. The definitive treatment for this condition is delivery of the baby. In the interim, limited fat intake, fat soluble vitamin supplementation and UDCA, with or without S-adenosylmethionine can provide symptomatic relief. In the future, additional hepatobiliary diseases related to ABCB4 mutations are likely to be identified. It is likely that this will result in the discovery of additional therapies for PFIC Type 3, gallstones and intrahepatic cholestasis of pregnancy.
PMCID: PMC3888315  PMID: 18221610
21.  NF-E2- related factor 2 (Nrf2) is a positive regulator of human bile salt export pump (BSEP) expression* 
Hepatology (Baltimore, Md.)  2009;50(5):1588-1596.
The bile salt export pump (BSEP, ABCB11) is the major determinant of bile salt dependent bile secretion and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor (FXR). However, BSEP expression is retained in the liver of the Fxr−/− mice although reduced, indicating that there may be additional transcriptional factors that regulate its expression. The NF-E2-related factor-2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant-responsive elements (ARE) that regulate many hepatic Phase I and II enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two Maf recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study we examine if Nrf2 plays a role in human BSEP expression and whether this might be mediated by the MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP mRNA expression by ~ 7-fold in HepG2 cells and protein by ~ 70% in human hepatocytes. siRNAs lowered NRF2 expression in HepG2 cells and prevented the up-regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose-dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. ChIP assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter.
Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury.
PMCID: PMC3013376  PMID: 19821532
ATP-binding cassette (ABC) transporters; antioxidant-responsive element (ARE); oltipraz; bile secretion; gene regulation
22.  Hepatocellular Carcinoma in a Boy with Progressive familial Intrahepatic Cholestasis Type II: Challenging Identification: Case report 
Hepatocellular carcinoma (HCC) is rare in children. Progressive familial intrahepatic cholestasis type II (PFIC2 and also called BSEP ((Bile Salt Export Pump)) deficiency) is an inherited disease that initiates end-stage liver cirrhosis which can predispose to HCC. HCC can occur in 15% of patients with PFIC2. In this case report, an 11-month-old boy with PFIC 2 was admitted for liver transplant work up. The finding of HCC was made incidentally by histopathology on the explanted liver after suspiciously gross examination. In this article, we found that the radiology (US) alone is not enough to exclude HCC. Finally, we conclude that any case of PFIC 2 (male or female) needs routine screening of serum AFP concentration, advanced radiological examination (CT, MRI) as well as careful macroscopic examination of their explanted liver (triple assessment) to exclude HCC.
PMCID: PMC3883614  PMID: 24421753
Progressive familial intrahepatic cholestasis type 2; hepatocellular carcinoma; liver; triple assessment; MRI & CT scan
23.  Polymorphic variants of the human bile salt export pump (ABCB11): functional characterization and interindividual variability 
Pharmacogenetics and genomics  2010;20(1):45-57.
Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11) and to assess interindividual variability in BSEP expression.
We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method. BSEP expression was assessed by real-time mRNA analysis, western blot analysis, and immunofluorescence confocal microscopy.
For the most part, polymorphisms were rare and ethnicity dependent. In-vitro functional studies revealed several rare variants, including 616A>G, 1674G>C, 1772A>G, and 3556G>A, to be associated with significantly impaired taurocholate transport activity while the 890A>G variant trended towards impaired function but was not statistically significant. The 3556G>A variant was associated with reduced cell surface – total protein expression compared with wild-type BSEP. Expression of BSEP by mRNA and protein analysis was determined from a bank of human liver samples. Wide interindividual variability was noted in both mRNA (19-fold) and protein (31-fold) expression levels. The common variant 1331T>C was associated with significantly reduced hepatic BSEP mRNA levels.
Accordingly, our study indicates that there are functionally relevant polymorphisms in ABCB11, which may be of potential relevance in the predisposition to acquired liver disorders such as drug-induced cholestasis.
PMCID: PMC2883163  PMID: 20010382
ABCB11; bile acid; bile salt export pump; cholestasis; pharmacogenetics; polymorphism; transporter
24.  Novel ATP8B1 mutation in an adult male with progressive familial intrahepatic cholestasis 
Progressive familial intrahepatic cholestasis type 1 is a rare disease that is characterized by low serum γ-glutamyltransferase levels due to mutation in ATP8B1. We present a 23-year-old male who experienced persistent marked pruritus for eighteen years and recurrent jaundice for thirteen years, in addition to cholestasis that eventually became fatal. Genetic sequencing studies of the entire coding (exon) sequences of ATP8B1 and ABCB11 uncovered a novel heterozygous missense 3035G>T mutation (S1012I) and a synonymous 696T>C mutation in ATP8B1. The patient’s progression was associated with not only impaired familial intrahepatic cholestasis 1 (FIC1) function but also impaired bile salt export pump expression due to the impaired FIC1 function. Our findings show that patients with intermittent cholestasis can develop progressive liver disease even after several decades and require regular follow up.
PMCID: PMC3508648  PMID: 23197899
ATP8B1; Bile salt export pump; Novel mutation; Progressive familial intrahepatic cholestasis type 1; Intermittent cholestasis
25.  Genetic Factors in the Pathogenesis of Cholangiocarcinoma 
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).
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.
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).
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.
PMCID: PMC3696362  PMID: 21691113
Cholangiocarcinoma; Genetics; ABCB11; ABCB4; ABCC2; ATP8B1; NR1H4

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