Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing.

UNLABELLED: The gene encoding the human bile salt export pump (BSEP), ABCB11, is mutated in several forms of intrahepatic cholestasis. Here we classified the majority (63) of known ABCB11 missense mutations and 21 single-nucleotide polymorphisms (SNPs) to determine whether they caused abnormal ABCB11 pre-messenger RNA splicing, abnormal processing of BSEP protein, or alterations in BSEP protein function. Using an in vitro minigene system to analyze splicing events, we found reduced wild-type splicing for 20 mutations/SNPs, with normal mRNA levels reduced to 5% or less in eight cases. The common ABCB11 missense mutation encoding D482G enhanced aberrant splicing, whereas the common SNP A1028A promoted exon skipping. Addition of exogenous splicing factors modulated several splicing defects. Of the mutants expressed in vitro in CHO-K1 cells, most appeared to be retained in the endoplasmic reticulum and degraded. A minority had BSEP levels similar to wild-type. The SNP variant A444 had reduced levels of protein compared with V444. Treatment with glycerol and incubation at reduced temperature overcame processing defects for several mutants, including E297G. Taurocholate transport by two assessed mutants, N490D and A570T, was reduced compared with wild-type. CONCLUSION: This work is a comprehensive analysis of 80% of ABCB11 missense mutations and single-nucleotide polymorphisms at pre-mRNA splicing and protein processing/functional levels. We show that aberrant pre-mRNA splicing occurs in a considerable number of cases, leading to reduced levels of normal mRNA. Thus, primary defects at either the protein or the mRNA level (or both) contribute significantly to BSEP deficiency. These results will help to develop mutation-specific therapies for children and adults suffering from intrahepatic cholestasis due to BSEP deficiency.

Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families.

BACKGROUND & AIMS: Patients with severe bile salt export pump (BSEP) deficiency present as infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy. METHODS: Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome. RESULTS: Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7 families, only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS: With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those carrying 2 null mutations, is essential.

ATP8B1 gene expression is driven by a housekeeping-like promoter independent of bile acids and farnesoid X receptor.

BACKGROUND: Mutations in ATP8B1 gene were identified as a cause of low γ-glutamyltranspeptidase cholestasis with variable phenotype, ranging from Progressive Familial Intrahepatic Cholestasis to Benign Recurrent Intrahepatic Cholestasis. However, only the coding region of ATP8B1 has been described. The aim of this research was to explore the regulatory regions, promoter and 5'untranslated region, of the ATP8B1 gene. METHODOLOGY/PRINCIPAL FINDINGS: 5'Rapid Amplification of cDNA Ends using human liver and intestinal tissue was performed to identify the presence of 5' untranslated exons. Expression levels of ATP8B1 transcripts were determined by quantitative reverse-transcription PCR and compared with the non-variable part of ATP8B1. Three putative promoters were examined in vitro using a reporter gene assay and the main promoter was stimulated with chenodeoxycholic acid. Four novel untranslated exons located up to 71 kb upstream of the previously published exon 1 and twelve different splicing variants were found both in the liver and the intestine. Multiple transcription start sites were identified within exon -3 and the proximal promoter upstream of this transcription start site cluster was proven to be an essential regulatory element responsible for 70% of total ATP8B1 transcriptional activity. In vitro analysis demonstrated that the main promoter drives constitutive ATP8B1 gene expression independent of bile acids. CONCLUSIONS/SIGNIFICANCE: The structure of the ATP8B1 gene is complex and the previously published transcription start site is not significant. The basal expression of ATP8B1 is driven by a housekeeping-like promoter located 71 kb upstream of the first protein coding exon.

VPS33B mutation with ichthyosis, cholestasis, and renal dysfunction but without arthrogryposis: incomplete ARC syndrome phenotype.

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare multisystem disorder first described in 1979 and recently ascribed to mutation in VPS33B, whose product acts in intracellular trafficking. Arthrogryposis, spillage of various substances in the urine, and conjugated hyperbilirubinemia define an ARC core phenotype, in some patients associated with ichthyosis, central nervous system malformation, deafness, and platelet abnormalities. We describe a patient with cholestasis, aminoaciduria, ichthyosis, partial callosal agenesis, and sensorineural deafness who, although homozygous for the novel VPS33B mutation 971delA/K324fs, predicted to abolish VPS33B function, did not exhibit arthrogryposis. The phenotypes associated with VPS33B mutation may include incomplete ARC.

Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency.

Hepatocellular carcinoma (HCC) is rare in young children. We attempted to see if immunohistochemical and mutational-analysis studies could demonstrate that deficiency of the canalicular bile acid transporter bile salt export pump (BSEP) and mutation in ABCB11, encoding BSEP, underlay progressive familial intrahepatic cholestasis (PFIC)--or "neonatal hepatitis" suggesting PFIC--that was associated with HCC in young children. We studied 11 cases of pediatric HCC in the setting of PFIC or "neonatal hepatitis" suggesting PFIC. Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11 was performed in leukocyte DNA from available patients and parents. Among the 11 nonrelated children studied aged 13-52 months at diagnosis of HCC, 9 (and a full sibling, with neonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) had immunohistochemical evidence of BSEP deficiency; the eleventh child did not. Mutations in ABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNA could be studied (n = 7). These mutations were confirmed in the parents (n = 14). With respect to the other 3 children with BSEP deficiency, mutations in ABCB11 were demonstrated in all 5 parents in whom leukocyte DNA could be studied. Thirteen different mutations were found. In conclusion, PFIC associated with BSEP deficiency represents a previously unrecognized risk for HCC in young children. Immunohistochemical evidence of BSEP deficiency correlates well with demonstrable mutation in ABCB11.

The human bile salt export pump: characterization of substrate specificity and identification of inhibitors.

BACKGROUND & AIMS: The bile salt export pump (BSEP) is the major bile salt transporter in the liver canalicular membrane. Our aim was to determine the affinity of the human BSEP for bile salts and identify inhibitors. METHODS: Human BSEP was expressed in insect cells. Adenosine triphosphatase (ATPase) assays were performed, and bile salt transport studies were undertaken. RESULTS: The BSEP gene, ABCB11, was cloned and a recombinant baculovirus was generated. Infected insect cells expressed a 140-kilodalton protein that was absent in uninfected and in mock-infected cells. An ATPase assay showed BSEP to have a high basal ATPase activity. Transport assays were used to determine the Michaelis constant for taurocholate as 4.25 micromol/L, with a maximum velocity of 200 pmol x min(-1) x mg(-1) protein. Inhibition constant values for other bile salts were 11 micromol/L for glycocholate, 7 micromol/L for glycochenodeoxycholate, and 28 micromol/L for taurochenodeoxycholate. Cyclosporin A, rifampicin, and glibenclamide were proved to be competitive inhibitors of BSEP taurocholate transport, with inhibition constant values of 9.5 micromol/L, 31 micromol/L, and 27.5 micromol/L, respectively. Progesterone and tamoxifen did not inhibit BSEP. CONCLUSIONS: The human BSEP is a high-affinity bile salt transporter. The relative affinities for the major bile salts differ from those seen in rodents and reflect the different bile salt pools. BSEP is competitively inhibited by therapeutic drugs. This is a potentially significant mechanism for drug-induced cholestasis.

Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity.

BACKGROUND & AIMS: The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler's disease (progressive familial intrahepatic cholestasis type 1) are unknown. METHODS: Interactions among the apical sodium-dependent bile acid transporter, the farnesoid X receptor (FXR), and familial intrahepatic cholestasis-1 were studied in the ileum of children with progressive familial intrahepatic cholestasis type 1 and in Caco-2 cells. RESULTS: Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with progressive familial intrahepatic cholestasis type 1. Paradoxically, ileal lipid-binding protein mRNA expression was repressed, suggesting a central defect in bile acid response. Ileal FXR and short heterodimer partner mRNA levels were reduced in the same 3 patients. In Caco-2 cells, antisense-mediated knock-down of endogenous familial intrahepatic cholestasis-1 led to up-regulation of apical sodium-dependent bile acid transporter and down-regulation of FXR, ileal lipid-binding protein, and short heterodimer partner mRNA. In familial intrahepatic cholestasis-1-negative Caco-2 cells, the activity of the human apical sodium-dependent bile acid transporter promoter was enhanced, whereas the human FXR and bile salt excretory pump promoters' activities were reduced. Overexpression of short heterodimer partner but not of the FXR abrogated the effect of familial intrahepatic cholestasis-1 antisense oligonucleotides. FXR cis-element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from familial intrahepatic cholestasis-1-negative Caco-2 cells. CONCLUSIONS: Loss of familial intrahepatic cholestasis-1 leads to diminished nuclear translocation of the FXR, with the subsequent potential for pathologic alterations in intestinal and hepatic bile acid transporter expression. Marked hypercholanemia and cholestasis are predicted to develop, presumably because of both enhanced ileal uptake of bile salts via up-regulation of the apical sodium-dependent bile acid transporter and diminished canalicular secretion of bile salts secondary to down-regulation of the bile salt excretory pump.

Characterization of mutations in ATP8B1 associated with hereditary cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1-3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened.