ABCMdb

PMID: 18987030

Dixon PH, van Mil SW, Chambers J, Strautnieks S, Thompson RJ, Lammert F, Kubitz R, Keitel V, Glantz A, Mattsson LA, Marschall HU, Molokhia M, Moore GE, Linton KJ, Williamson C
Contribution of variant alleles of ABCB11 to susceptibility to intrahepatic cholestasis of pregnancy.
Gut. 2009 Apr;58(4):537-44. Epub 2008 Nov 5., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Val444Ala ABCB11 p.Val444Ala ABCB11 p.Asn591Ser ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Methods: ABCB11 variation in ICP was investigated by screening for five mutant alleles (E297G, D482G, N591S, D676Y and G855R) and the V444A polymorphism (c.1331T.C, rs2287622) in two ICP cohorts (n = 333 UK, n = 158 continental Europe), and controls (n = 261) for V444A. Login to comment 5 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Results: E297G was observed four times and D482G once. Login to comment 6 ABCB11 p.Asn591Ser ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr N591S was present in two patients; D676Y and G855R were not observed. Login to comment 7 ABCB11 p.Val444Ala The V444A polymorphism was associated with ICP (allelic analysis for C vs T: OR 1.7 (95% CI 1.4 to 2.1, p,0.001)). Login to comment 9 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Structural analyses suggest that E297G and D482G destabilise the protein fold of BSEP. Login to comment 10 ABCB11 p.Val444Ala ABCB11 p.Asn591Ser The molecular basis of V444A and N591S was not apparent from the Sav1866 structure. Login to comment 12 ABCB11 p.Asn591Ser N591S is a recurrent mutation; however, the mechanism may be independent of protein stability or function. Login to comment 13 ABCB11 p.Val444Ala The V444A polymorphism is a significant risk factor for ICP in this population. Login to comment 18 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCC2 (encoding MRP2) variation has also been implicated in ICP in a South American cohort,23 but this has not been replicated to date in European populations.24 BSEP is a high affinity liver-specific transporter, which is responsible for the export of conjugated bile acids into the bile canaliculus.25-27 It is a member of the ABC transporter family of proteins, of which there are 48 members in the human genome and whose functions include the transport of a range of substances including lipids, drugs, cholesterol and bile salts.28 Two mutant alleles of ABCB11, namely E297G and D482G, have been found frequently in European families and one or both are present in 58%.29 The function of BSEP and the role of ABCB11 mutations in PFIC and BRIC indicate that variation in this gene could be involved in the aetiology of ICP. Login to comment 19 ABCB11 p.Val444Ala ABCB11 p.Val444Ala ABCB11 p.Asn591Ser ABCB11 p.Asn591Ser ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr This was initially proposed in the Finnish population following analysis of a two SNP (single nucleotide polymorphism) haplotype in 57 cases.30 However, a subsequent study in a larger cohort of 142 ICP cases failed to replicate the initial findings, leaving the role of BSEP open to question.31 A recent study of the entire coding region of the BSEP gene identified a single potential mutation (N591S) in a cohort of 21 women with ICP.32 In addition, a polymorphism (c.1331C.T, V444A, rs2287622) possibly affecting BSEP expression has been suggested to play a role in ICP32 33 and has recently been reported to be associated with drug-induced cholestasis (DIC).34 This association has been replicated recently in a slightly larger cohort.24 We sought to clarify the role of ABCB11 mutations in predisposition to ICP by screening a large cohort of patients for the two common mutant alleles, together with the N591S mutation found previously in a case of ICP32 and the two recently described mutations (D676Y and G855R) associated with DIC.34 We also genotyped the V444A polymorphism in the largest case/control study to date in ICP. Login to comment 20 ABCB11 p.Val444Ala A control group of 261 European women with normal pregnancies were also genotyped for the V444A polymorphism. Login to comment 21 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Val444Ala ABCB11 p.Asn591Ser BSEP is homologous with the bacterial multidrug resistance protein (Sav1866) for which two structures have recently been determined at high resolution by x ray crystallography.35 36 We therefore used this structure to consider the possible effects of E297G, D482G, N591S and V444A on BSEP, all of which were identified in patients with ICP, to provide insights into potential mutational mechanisms. Login to comment 36 ABCB11 p.Val444Ala Genotyping of the c.1331C.T polymorphism (rs2287622, which results in V444A) was also performed by DNA sequencing of ABCB11 exon 13. Login to comment 48 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr RESULTS Sequencing DNA sequence was generated for the UK ICP cohort (333 patients) for exons 9 and 14 which contain the common European mutations E297G and D482G, together with exons 15, 17 and 21 containing the previously described ICP-linked mutation (N591S) and the two DIC-linked mutations (D676Y and G855R), respectively. Login to comment 49 ABCB11 p.Glu297Gly Analysis of this sequence revealed that three patients were heterozygous for E297G. Login to comment 51 ABCB11 p.Glu297Gly One carrier (patient number 1, table 1) with a family history of ICP had a maternal sample available which was sequenced and demonstrated to be heterozygous for the E297G mutation. Login to comment 52 ABCB11 p.Asp482Gly Analysis of exon 14 did not identify any carriers of D482G, and no carriers of the DIC mutations were identified. Login to comment 54 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser In this cohort, a single occurrence each of E297G and D482G was identified together with two occurrences of N591S. Login to comment 57 ABCB11 p.Val444Ala Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 Genotyping and statistics Genotyping of the V444A polymorphism in exon 13 in both cohorts resulted in the allele frequencies shown in table 2 and fig 1. Login to comment 62 ABCB11 p.Val444Ala Taken together, these results indicate that individuals with the C allele of the V444A polymorphism have increased risk for the development of ICP in this population, with homozygotes for this allele (CC, alanine) being at highest risk, with test for trend p,0.001. Login to comment 75 ABCB11 p.Asp482Gly Replacing this conserved aspartic acid with a glycine (as in BSEP D482G) would probably destabilise this region of the protein and may prevent correct folding of the entire b-roll. Login to comment 76 ABCB11 p.Val444Ala A putative functional or structural significance for the V444A variant of BSEP is less evident because the residue is not highly conserved in the NBDs of ABC transporters, and because replacing the valine with a similarly small and non-polar alanine is unlikely to be very detrimental. Login to comment 77 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser S363 occupies the position of V444 in Sav1866. S363 hydrogen-bonds with D341 in an adjacent antiparallel b-strand within the Sav1866 NBD (fig 2C), but as it is only one of many bonds between the Table 1 Clinical features of ICP patients with heterozygous ABCB11 mutations Patient No Cohort Biochemistry (highest level measured) Other maternal clinical findings Fetal and labour complications Family history of ICPBile acids* ALT* GGT* E297G 1 UK 32 133 27 - - Yes 2{ UK 41 166 16 - M, H No 3{ UK 118 229 NP G, C Pr No 4 CE 41 82 NP - - No D482G 5 CE 74 98 15 J, P, BR - No N591S 6 CE 270 195 29 - - - 7 CE 32 60 11 - - - *Normal ranges: bile acids, ,14 mmol/l; alanine aminotransferase (ALT), (31 IU/l; c-glutamyl transferase (GGT), ,30 IU/l; bilirubin (BR), ,17 mmol/l. Login to comment 83 ABCB11 p.Val444Ala Table 2 Frequency counts for genotypes and alleles of the V444A polymorphism Genotype TotalCC CT TT Cases 222 (45%) 212 (43%) 57 (12%) 491 Controls 75 (29%) 133 (51%) 53 (20%) 261 Total 297 345 110 752 C (%) T (%) Total Cases 656 (67) 326 (33) 982 Controls 283 (54) 239 (46) 522 Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 two strands (and because the aliphatic side chain of V444 is unable to form a similar bond) it is unlikely to be critical for the function or folding of the domain. Login to comment 84 ABCB11 p.Asn591Ser Likewise, the functional or structural significance of the N591S variant of BSEP is not apparent from the crystal structure of Sav1866 (fig 2D). Login to comment 88 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser Indeed, in the sequence of MJ0796 from Methanococcus jannaschii this position is occupied by a serine, suggesting that the serine side chain is likely to be tolerated as a replacement for N591 in BSEP.38 DISCUSSION We report here the identification of seven heterozygous carriers of BSEP (ABCB11) mutations (four E297G, one D482G and two N591S) in a large cohort of patients with ICP. Login to comment 90 ABCB11 p.Val444Ala Our work has also confirmed the role of the V444A polymorphism as a susceptibility locus for ICP, as suggested by prior studies in smaller cohorts.24 32 A previous study analysed the ABCB11 gene in ICP cases but sequenced the entire gene in a small number of patients.32 Haplotype analysis of the ABCB11 locus in a European Swedish population failed to identify a difference in the distribution of common haplotypes across this region.19 We chose to focus on frequent mutant PFIC/BRIC alleles together with ICP and DIC- associated mutations in order to analyse a much larger population of ICP cases than has been performed previously. Login to comment 91 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly To understand the effects of PFIC and BRIC mutations, several groups have studied the effects of the E297G and D482G mutations on BSEP expression, transport, localisation, function and stability, in vitro. Login to comment 92 ABCB11 p.Asp482Gly The D482G mutation has been studied in mouse,39 rat40-42 and human43 BSEP and, when expressed in mammalian cell lines at physiological temperature, all of the studies report reduced plasma membrane expression of the recombinant protein (quantified at 5%42 and 26%41 of the level of the wild-type protein). Login to comment 94 ABCB11 p.Asp482Gly These data can be explained if the D482G mutant has a protein folding defect that slows progression through the trafficking pathway, resulting in much of the protein being degraded via the proteasome. Login to comment 95 ABCB11 p.Asp482Gly Our structural analysis is entirely consistent with this interpretation and provides a molecular explanation for the observed phenotype, as the D482G change should destabilise the b-roll of NBD1. Login to comment 96 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly Similar data, implicating primarily a defect in protein folding, have also been reported for the E297G mutation40 41 43 44 However, one study42 observed that while E297G in rat Bsep is expressed at the plasma membrane of HEK293 cells at roughly 40% of the level of the wild-type protein, its ATPase activity (measured in membrane vesicles prepared from insect cells) was uncoupled from the transport of taurocholate. Login to comment 97 ABCB11 p.Glu297Gly Other studies report low transport activity for the E297G mutant of rat40 41 and human44 BSEP. Login to comment 98 ABCB11 p.Glu297Gly However, one study concluded, after a large correction for the low level of expression, that the E297G mutant in human BSEP could drive wild-type levels of taurocholate transport activity.43 The reason for this discrepancy is not clear but may be related to the protein background, expression system or experimental methodology used by the different groups. Login to comment 100 ABCB11 p.Val444Ala The fold Figure 1 Genotype and allele frequencies for the V444A variant in cases (n = 491) and controls (n = 261). Login to comment 102 ABCB11 p.Val444Ala (B) Genotype frequencies in cases and controls for the V444A variant; The y-axis indicates frequency of the genotype expressed as a percentage. ORs for CC vs TT and CC vs CT comparisons together with 95% CIs are shown. Login to comment 103 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly ABCB11 p.Val444Ala ABCB11 p.Val444Ala Table 3 Allelic analysis for the V444A polymorphism associated with cholestasis Allele OR (95%CI) p Value C vs T 1.70 (1.4 to 2.1) ,0.001 Table 4 Genotypic analysis for the V444A polymorphism associated with cholestasis OR (95% CI) p Value CC vs CT 1.9 (1.3 to 2.6) ,0.001 CC vs TT 2.8 (1.7 to 4.4) ,0.001 CC and CT vs TT 1.9 (1.3 to 2.9) 0.001 Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 Figure 2 The structure of Sav1866 suggests a molecular mechanism for the bile salt export pump (BSEP) mutations E297G and D482G (A) Schematic representation of the drug exporter Sav1866 with two bound ADPs shown as spheres (pdb 2HYD). Login to comment 115 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The N-terminal Biliary tract Gut 2009;58:537-544. doi:10.1136/gut.2008.159541 of the E297G mutant is likely to be less stable, resulting in reduced expression at the plasma membrane, but, because of the position of this residue at the interface between the domains, mutant protein at the cell surface may also be deficient in communication between the bile acid-binding sites and the ATP catalytic sites, offering a molecular explanation for the uncoupling observed in one of the studies.42 However, the underlying codon change in the D482G mutant may also impair RNA splicing,45 raising the possibility that the phenotypic effect of the 482G codon may be mediated earlier in the gene expression pathway. Login to comment 118 ABCB11 p.Asn591Ser The N591S mutation has been previously described in a case of ICP in a screen of 21 cases.32 This residue is not highly conserved across ABC transporter NBDs, suggesting that folding and/or function may not be affected by this change. Login to comment 122 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Our results suggest that the common E297G and D482G mutations of this gene do not play a major role in ICP predisposition in our population. Login to comment 123 ABCB11 p.Glu297Gly ABCB11 p.Asn591Ser Examination of clinical information for the E297G and N591S carriers failed to identify any factor to distinguish them from the general ICP population. Login to comment 124 ABCB11 p.Asp482Gly However, the single carrier of D482G was a primigravid woman who had an extended phenotype with markedly elevated liver enzymes and hyperbilirubinaemia in the postnatal period. Login to comment 127 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly The 444A allele may contribute to susceptibility in a considerably higher number of cases of ICP than the E297G and D482G mutations. Login to comment 128 ABCB11 p.Val444Ala ABCB11 p.Val444Ala A study of 21 ICP cases suggested a role for the V444A polymorphism in the disease,32 which was also supported by a further, slightly extended study.24 In addition, a second cholestatic phenotype, DIC, has been associated with this polymorphism.34 This polymorphism has biological plausibility, as indicated by functional evidence: there is a trend for low BSEP activity in liver specimens from individuals who harbour this polymorphism.33 V444A has been described as a mutation in three recent case reports of ICP46 47 and BRIC.48 Functional assays of BSEP activity in Sf9 cells failed to demonstrate a difference in taurocholate transport activity between constructs carrying valine or alanine at position 444.34 However, carriers of the alanine allele exhibit lower hepatic BSEP protein expression.33 Analysis of the equivalent residue (S363) in Sav1866 provided no compelling argument for a possible effect on BSEP folding or function. Login to comment 129 ABCB11 p.Val444Ala Comprehensive studies are warranted to quantify definitively the effect of V444A on BSEP expression and function, and an effect at the level of mRNA stability should also be examined to explain the reported associations with susceptibility to cholestasis. Login to comment 157 ABCB11 p.Val444Ala However, our study also indicates that V444A confers susceptibility to ICP in the largest cohort to be studied to date, indicating that genetic variation of ABCB11 may play a more central role in the aetiology of ICP than previously suspected. Login to comment