ABCMdb

ABCB11 p.Met677Val

Reviews:	p.Met677Leu N p.Met677Val N
Predicted by SNAP2:	A: N (72%), C: N (72%), D: N (66%), E: D (63%), F: N (61%), G: N (61%), H: N (61%), I: N (87%), K: D (53%), L: D (63%), N: N (78%), P: N (66%), Q: N (72%), R: N (61%), S: N (82%), T: N (82%), V: N (87%), W: D (91%), Y: N (61%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Interindividual variability of canalicular ATP-bin... Hepatology. 2006 Jul;44(1):62-74. Meier Y, Pauli-Magnus C, Zanger UM, Klein K, Schaeffeler E, Nussler AK, Nussler N, Eichelbaum M, Meier PJ, Stieger B
Interindividual variability of canalicular ATP-binding-cassette (ABC)-transporter expression in human liver.
Hepatology. 2006 Jul;44(1):62-74., [PMID:16799996]

Abstract [show]
Interindividual variability in hepatic canalicular transporter expression might predispose to the development of hepatic disorders such as acquired forms of intrahepatic cholestasis. We therefore investigated expression patterns of bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4), multidrug resistance associated protein 2 (MRP2, ABCC2) and multidrug resistance protein 1 (MDR1, ABCB1) in healthy liver tissue of a white population. Protein expression levels were correlated with specific single nucleotide polymorphisms (SNPs) in the corresponding transporter genes. Hepatic protein expression levels from 110 individuals undergoing liver resection were assessed by Western blot analysis of liver plasma membranes enriched in canalicular marker enzymes. Each individual was genotyped for the following synonymous (s) and nonsynonymous (ns) SNPs: ABCB11: (ns:1457T>C and 2155A>G), ABCB4: (ns:3826A>G) and ABCC2 (ns:1286G>A,3600T>A and 4581G>A) and ABCB1 (ns:2677G>T/A and s:3435C>T). Transporter expression followed unimodal distribution. However, of all tested individuals 30% exhibited a high expression and 32% a low or very low expression phenotype for at least one of the four investigated transport proteins. Transporter expression levels did not correlate with age, sex, underlying liver disease, or presurgery medication. However, low BSEP expression was associated with the 1457C-allele in ABCB11 (P = .167) and high MRP2 expression was significantly correlated with the 3600A and 4581A ABCC2 variants (P = .006). In conclusion, the results demonstrate a considerable interindividual variability of canalicular transporter expression in normal liver. Furthermore, data suggest a polymorphic transporter expression pattern, which might constitute a risk factor for the development of acquired forms of cholestatic liver diseases.

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154 Box-plot analysis of (A) normalized BSEP-expression against genetic variants of 1457TϾC(V444A) and 2155A Ͼ G(M677V); (B) MDR3-expression against 3826A Ͼ G (R652G); (C) MRP2-expression against 1286G Ͼ A(V417I), 3600T Ͼ A(V1188E), and 4581G Ͼ A(C1515Y) and MDR1 against 3435C Ͼ T and 2677G Ͼ T/A(A893S/T). Login to comment
63 Primers and Probes of RealTime PCR for Allelic Discrimination of Single Nucleotide Polymorphisms (SNPs) in Whites Gene Exon cDNA PositionA SNPB GenBank Reference Amino Acid Exchange Sense-Antisense Primer Probesc ABCB11 13 1457 T Ͼ C rs2287617* V444A 5Ј-CTTTCTTCTCCAGATTCTAAATGACCTCA-3Ј/ VIC 5Ј-CCTGGTTTAATGACCATGT-3Ј 5Ј-GTCCTACCAGAGCTGTCATTTCC-3Ј FAM 5Ј-CTGGTTTAATGGCCATGT-3Ј ABCB11 17 2155 A Ͼ G Ref. 14,15** M677V 5Ј-TCATGCTGTGTTGAGTAGATGCA-3Ј/ VIC 5Ј- CTGAAGATGACATGCTT-3Ј 5Ј-GGTAGCTCCCTCTGCTAAAGGT-3Ј FAM 5Ј- ACTGAAGATGACGTGCTT-3Ј ABCB4 16 3826 A Ͼ G rs8187799* R652G 5Ј-TCCAGTCAGAAGAATTTGAACTAAATGATGAA-3Ј/ VIC 5Ј-CTGCCACTAGAATGG-3Ј 5Ј-GCCTAAATAGATTTCCAGCCATTTGG-3Ј FAM 5Ј-TGCCACTGGAATGG-3Ј ABCC2 10 1286 G Ͼ A rs2273697* V417I 5Ј-CCAACTTGGCCAGGAAGGA-3Ј/ VIC 5Ј-CTGTTTCTCCAACGGTGTA-3Ј 5Ј-GGCATCCACAGACATCAGGTT-3Ј FAM 5Ј-ACTGTTTCTCCAATGGTGTA-3Ј ABCC2 25 3600 T Ͼ A rs8187694* V1188E 5Ј-GCACCAGCAGCGATTTCTG-3Ј/ VIC 5Ј-ACACAATGAGGTGAGGAT-3Ј 5Ј-AGGTGATCCAGGAAAAGACACATTT-3Ј FAM 5Ј-ACAATGAGGAGAGGAT-3Ј ABCC2 32 4581 G Ͼ A rs8187710* C1515Y 5Ј-GTAATGGTCCTAGACAACGGGAAG-3Ј/ VIC 5Ј- AGAGTGCGGCAGCC -3Ј 5Ј-CCAGGGATTTGTAGCAGTTCTTCAG-3Ј FAM 5Ј-ATTATAGAGTACGGCAGCC-3Ј ABCB1 26 3435 CϾT rs1045642* synonym. Login to comment
141 Distribution of Genotypes and Allelic Frequencies of Investigated SNPs in Individuals With Low, Normal and High Transporter Expression Phenotypes SNP Study population Low expressorsA Normal expressorsB High expressionC 1) BSEP n ϭ 110 (100%) n ϭ 14 (100%) n ϭ 79 (100%) n ϭ 17 (100%) Alleles (2n) 220 (100%) 28 (100%) 158 (100%) 34 (100%) a) ABCB11 1457T>C (V444A): Genotypes: TT 19 (17%) 1 (7%) 14 (18%) 4 (24%) CC 29 (26%) 6 (43%) 19 (24%) 4 (24% TC 62 (56%) 7 (50%) 46 (58%) 9 (53%) Allelic frequency: C-allele 120 (55%) 19 (68%) 84 (53%) 17 (50%) b) ABCB11 2155A>G (M677V): Genotypes: AA 102 (93%) 14 (100%) 72 (91%) 16 (94%) AG 8 (7%) 7 (9%) 1 (6%) Allelic frequency: G-allele 8 (4%) 7 (7%) 1 (3%) 2) MDR3 n ϭ 110 (100%) n ϭ 13 (100%) n ϭ 86 (100%) n ϭ 11 (100%) Alleles (2n) 220 (100%) 26 (100%) 172 (100%) 22 (100%) ABCB4 3826A>G (R652G): Genotypes: AA 87 (89%) 8 (62%) 71 (83%) 8 (73%) AG 23 (21%) 5 (38%) 15 (17%) 3 (27%) Allelic frequency: G-allele 23 (10%) 5 (19%) 15 (9%) 3 (14%) 3) MRP2 n ϭ 110 (100%) n ϭ 11 (100%) n ϭ 90 (100%) n ϭ 9 (100%) Alleles (2n) 220 (100%) 22 (100%) 180 (100%) 18 (100%) a) ABCC2 1286G>A (V417I): Genotypes: GG 64 (58%) 7 (64%) 51 (57%) 6 (67%) AA 1 (1%) 1 (1%) GA 45 (41%) 4 (36%) 38 (42%) 3 (33%) Allelic frequency: A-allele 47 (26%) 4 (18%) 40 (22%) 3 (17%) b) ABCC2 3600T>A (V1188E): Genotypes: TT 95 (86%) 10 (91%) 80 (89%) 5 (56%) AA 1 (1%) 1 (11%) TA 14 (13%) 1 (9%) 10 (11%) 3 (33%) Allelic frequency: A-allele 16 (6%) 1 (5%) 10 (5%) 5 (28%) c) ABCC2 4581G>A (C1515Y): Genotypes: GG 95 (86%) 10 (91%) 80 (89%) 5 (56%) AA 1 (1%) 1 (11%) GA 14 (13%) 1 (9%) 10 (11%) 3 (33%) Allelic frequency: A-allele 16 (6%) 1 (5%) 10 (5%) 5 (28%) 4) MDR1 n ϭ 110 (100%) n ϭ 17 (100%) n ϭ 77 (100%) n ϭ 16 (100%) Alleles (2n) 220 (100%) 34 (100%) 154 (100%) 32 (100%) a) ABCB1 3435C>T: Genotypes: CC 23 (21%) 3 (18%) 16 (21%) 4 (25%) TT 28 (25%) 4 (24%) 20 (26%) 4 (25%) CT 59 (54%) 10 (58%) 41 (53%) 8 (50%) Allelic frequency: T-allele 115 (52%) 18 (53%) 81 (53%) 16 (50%) b) ABCB1 2677G>T/A (A893S/T): Genotypes: GG 31 (28%) 6 (35%) 20 (26%) 5 (31%) TT 21 (19%) 5 (29%) 15 (20%) 1 (6%) AA 1 (1%) 1 (6%) GT 48 (44%) 4 (24%) 35 (45%) 9 (56%) GA 4 (4%) 3 (4%) 1 (6%) TA 5 (5%) 1 (6%) 4 (5%) Allelic frequency: T/A-allele 95/11 (43%/5%) 15/3 (44%/9%) 69/7 (45%/5%) 11/1 (34%/3%) AIndividuals with phenotype low expressors (Ͻmean-1SD) and very low (Ͻmean-2SD) transporter expression levels. Login to comment
145 In contrast, no correlation between the second polymorphism 2155AϾG (M677V) and BSEP protein expression level was found, because the M677M variant was equally distributed between individuals with low and high BSEP protein expression phenotypes (Table 3). Login to comment

[hide] BSEP and MDR3 haplotype structure in healthy Cauca... Hepatology. 2004 Mar;39(3):779-91. Pauli-Magnus C, Kerb R, Fattinger K, Lang T, Anwald B, Kullak-Ublick GA, Beuers U, Meier PJ
BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis.
Hepatology. 2004 Mar;39(3):779-91., [PMID:14999697]

Abstract [show]
Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are characterized by a cholestatic pattern of liver damage, also observed in hereditary or acquired dysfunction of the canalicular membrane transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein type 3 (MDR3, ABCB4). Controversy exists whether a genetically determined dysfunction of BSEP and MDR3 plays a pathogenic role in PBC and PSC. Therefore, 149 healthy Caucasian control individuals (control group) were compared to 76 PBC and 46 PSC patients with respect to genetic variations in BSEP and MDR3. Sequencing spanned approximately 10,000 bp including promoter and coding regions as well as 50-350 bp of flanking intronic regions. In all, 46 and 45 variants were identified in BSEP and MDR3, respectively. No differences between the groups were detected either in the total number of variants (BSEP: control group: 37, PBC: 37, PSC: 31; and MDR3: control group: 35; PBC: 32, PSC: 30), or in the allele frequency of the common variable sites. Furthermore, there were no significant differences in haplotype distribution and linkage disequilibrium. In conclusion, this study provides an analysis of BSEP and MDR3 variant segregation and haplotype structure in a Caucasian population. Although an impact of rare variants on BSEP and MDR3 function cannot be ruled out, our data do not support a strong role of BSEP and MDR3 genetic variations in the pathogenesis of PBC and PSC.

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67 Five coding region changes were single nucleotide polymorphisms present in all of the studied populations at an allele frequency of Ͼ1% (synonymous: exon 5: T270C, exon 10: A957G, exon 24: G3084A; nonsynonymous: exon 13: T1331C 3 V444A and exon 17: A2029G 3 M677V). Login to comment
78 BSEP Genetic Variability Variant Number Amplicon DNA Position cDNA Position Nucleotide Reference Nucleotide Variant AA Change Control Group n ‫؍‬ 186 PBC n ‫؍‬ 140 PSC n ‫؍‬ 92 Pro 1 intron-1 (-2397) C T 0.005 0.000 0.000 Pro 2 intron-1 (-2080) CTCT delCTCT 0.102 0.049 0.048 Pro 3 intron-1 (-1994) T C 0.000 0.008 0.000 Pro 4 intron-1 (-1952) T C 0.480 0.425 0.405 Pro 5 intron-1 (-1820) G A 0.101 0.042 0.048 Pro 6 intron-1 (-1814) C T 0.000 0.008 0.000 Pro 7 intron-1 (-1746) G A 0.101 0.054 0.048 Pro 8 intron-1 (-1275) G A 0.025 0.034 0.057 Pro 9 intron-1 (-1239) G A 0.298 0.292 0.244 Pro 10 intron-1 (-1155) T C 0.616 0.565 0.595 Pro 11 intron-1 (-1118) C A 0.005 0.000 0.000 Pro 12 intron-1 (-1009) T C 0.021 0.033 0.060 Pro 13 intron-1 (-906) C T 0.026 0.016 0.036 Pro 14 intron-1 (-837) A G 0.015 0.008 0.012 Pro 15 intron-1 (-603) T C 0.015 0.009 0.000 2.1. amplicon 2 intron 1 (-50) G A 0.025 0.029 0.054 3.1. amplicon 3 intron 2 (-37) T G 0.000 0.000 0.022 4.1. amplicon 4 intron 3 (-20) T C 0.071 0.043 0.076 5.1. amplicon 5 exon 5 270 T C syn 0.027 0.036 0.064 5.2. amplicon 5 intron 5 (ϩ8) G A 0.049 0.019 0.026 7.1. amplicon 7 intron 6 (-77) A T 0.005 0.008 0.000 7.2. amplicon 7 exon 7 580 T C S194P_c 0.000 0.000 0.011 8.1. amplicon 8 intron 7 (-41) T C 0.005 0.000 0.000 8.2. amplicon 8 exon 8 779 G A G260D 0.000 0.008 0.000 9.1. amplicon 9 exon 9 851 T C V284A_c 0.005 0.000 0.000 10.1. amplicon 10 intron 9 (-69) C T 0.051 0.016 0.011 10.2. amplicon 10 intron 9 (-31) C T 0.046 0.015 0.022 10.3. amplicon 10 intron 9 (-24) G A 0.000 0.000 0.011 10.4. amplicon 10 intron 9 (-17) G A 0.046 0.015 0.022 10.5. amplicon 10 intron 9 (-15) A G 0.704 0.709 0.739 10.6. amplicon 10 exon 10 957 A G syn 0.046 0.015 0.022 12.1. amplicon 12 intron 12 (ϩ73) G T 0.010 0.000 0.000 13.1. amplicon 13 exon 13 1331 T C A444V_c 0.595 0.632 0.600 13.2. amplicon 13 intron 13 (ϩ70) C T 0.590 0.623 0.600 14.1. amplicon 14 exon 14 1530 C A syn 0.000 0.007 0.000 14.2. amplicon 14 intron 14 (ϩ32) T C 0.604 0.629 0.589 17.1. amplicon 17 exon 17 2029 A G M677V 0.042 0.016 0.014 18.1. amplicon 18 exon 18 2093 G A R698H_c 0.005 0.008 0.000 18.2. amplicon 18 exon 18 2134 T C syn 0.005 0.031 0.012 19.1. amplicon 19 intron 18 (-17) C A 0.432 0.418 0.565 20.1. amplicon 20 intron 19 (-17) T C 0.694 0.655 0.702 21.1. amplicon 21 intron 21 (ϩ18) A C 0.006 0.000 0.000 24.1. amplicon 24 exon 24 3084 G A syn 0.459 0.563 0.398 27.1. amplicon 27 Exon 27 3683 C T A1228V_c 0.000 0.008 0.000 28.1. amplicon 28 intron 27 (-34) G A 0.448 0.569 0.378 28.2. amplicon 28 3ЈUTR (ϩ82) G T 0.000 0.008 0.000 NOTE. Variants are numbered sequentially by exon. Login to comment

[hide] Sequence analysis of bile salt export pump (ABCB11... Pharmacogenetics. 2004 Feb;14(2):91-102. Pauli-Magnus C, Lang T, Meier Y, Zodan-Marin T, Jung D, Breymann C, Zimmermann R, Kenngott S, Beuers U, Reichel C, Kerb R, Penger A, Meier PJ, Kullak-Ublick GA
Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy.
Pharmacogenetics. 2004 Feb;14(2):91-102., [PMID:15077010]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) might be risk factors for ICP development. This study aimed to (i). describe the extent of genetic variability in BSEP and MDR3 in ICP and (ii). identify new disease-causing mutations. Twenty-one women with ICP and 40 women with uneventful pregnancies were recruited between April 2001 and April 2003. Sequencing of BSEP and MDR3 spanned 8-10 kb per gene and comprised the promoter region and 100-350 bp of the flanking intronic region around each exon. DNA sequencing of polymerase chain reaction fragments was performed on an ABI3700 capillary sequencer. MDR3 promoter activity of promoter constructs carrying different ICP-specific mutations was studied using reporter assays. A total of 37 and 51 variant sites were detected in BSEP and MDR3, respectively. Three non-synonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E). Furthermore, four ICP-specific splicing mutations were detected in MDR3 [intron 21, G(+1)A; intron 25, G(+5)C and C(-3)G; and intron 26, T(+2)A]. Activity of the mutated MDR3 promoter was similar to that observed for the wild-type promoter. Our data further support an involvement of MDR3 genetic variation in the pathogenesis of ICP, whereas analysis of BSEP sequence variation indicates that this gene is probably less important for the development of pregnancy-associated cholestasis.

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66 Unauthorized reproduction of this article is prohibited. Table 1 BSEP genetic variability Variant number Amplicon DNA position cDNA position Nucleotide reference Nucleotide variant AA change ICP (n ¼ 42) Control (n ¼ 80) Pro 1 Intron -1 (-2080) CTCT delCTCT 0.132 0.162 Pro 2 Intron -1 (-1952) T C 0.286 0.556 Pro 3 Intron -1 (-1820) G A 0.143 0.125 Pro 4 Intron -1 (-1746) G A 0.150 0.122 Pro 5 Intron -1 (-1275) G A 0.000 0.014 Pro 6 Intron -1 (-1239) G A 0.143 0.365 Pro 7 Intron -1 (-1155) T C 0.381 0.681 Pro 8 Intron -1 (-1009) T C 0.000 0.014 Pro 9 Intron -1 (-906) C T 0.000 0.026 Pro 10 Intron -1 (-603) T C 0.000 0.014 4.1. Amplicon 4 Intron 3 (-20) T C 0.000 0.075 5.1. Amplicon 5 Intron 5 (þ8) G A 0.000 0.032 6.1. Amplicon 6 Exon 6 402 C T Syn 0.000 0.038 6.2. Amplicon 6 Intron 6 (þ16) G A 0.000 0.038 10.1. Amplicon 10 Intron 9 (-69) C T 0.000 0.038 10.2. Amplicon 10 Intron 9 (-31) C T 0.000 0.038 10.3. Amplicon 10 Intron 9 (-17) G A 0.000 0.075 10.4. Amplicon 10 Intron 9 (-15) A G 0.857 0.650 10.5. Amplicon 10 Exon 10 957 A G Syn 0.000 0.075 10.6. Amplicon 10 Intron 10 (þ18) A T 0.000 0.038 12.1. Amplicon 12 Intron 11 (-91) C T 0.000 0.013 12.2. Amplicon 12 Exon 12 1244 G A R415Q_c 0.000 0.013 12.3. Amplicon 12 Intron 12 (þ73) G T 0.000 0.013 13.1. Amplicon 13 Exon 13 1331 T C A444V_c 0.833 0.513 13.2. Amplicon 13 Intron 13 (þ70) C T 0.833 0.514 14.1. Amplicon 14 Intron 13 (-45) C G 0.028 0.000 14.2. Amplicon 14 Intron 14 (þ32) T C 0.833 0.513 15.1. Amplicon 15 Exon 15 1772 A G N591S_c 0.026 0.000 15.2. Amplicon 15 Exon 15 1791 G T Syn 0.026 0.000 17.1. Amplicon 17 Exon 17 2029 A G M677V 0.000 0.056 18.1. Amplicon 18 Exon 18 2134 T C Syn 0.000 0.013 19.1. Amplicon 19 Intron 18 (-17) C A 0.619 0.583 20.1. Amplicon 20 Intron 19 (-17) T C 0.684 0.813 22.1. Amplicon 22 Intron 22 (-36) G A 0.000 0.013 24.1. Amplicon 24 Exon 24 3084 G A Syn 0.325 0.413 26.1. Amplicon 26 Exon 26 3561 A G Syn 0.000 0.016 28.1. Amplicon 28 Intron 27 (-34) G A 0.368 0.423 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_003742. Login to comment
81 P R415 Q V444A Cytoplasm N519S M677V K F K R M K Q I M C D F H G I S V G E L H T H I D D S F K I H D A I A D Q H E R Y F R R I H R Q R Y A L E Y D T A V R H S S I A F G E K R E L K E Y R E V F Q R H G I R K E V V A K G V V A D F K K T G H AA R K T L L E G L K F G F R A H R D F H A I D Q D R H S P G A L S Q V Q G A A G S S L G T R F G T L H R R S L A E H T T G I G K E R T E L A E I E K T A I Q K A H Q I R D A L E H V G T Q F A A S I K S G V F A L V F A C S I H G Y K I H A A R A I V Q T S S I S P G A K R S L D IV L A D T A L R T S K K S A I H V A L E P F K Q F H D R D T H D L L I K P L G V I E K G V K A H L E E Q Q D L L Q F S S K G C G G V S Q E P E P I E K T R E K Q R I S V L H G L R T I H GQ D A S T A E L I A V H A Q I V K Y Y A G L S P S G T T I H V S I P P F E L L Q I Q L R S H I K A A I V R S G S Q L S T P S R P D Y R F A QY V K E S E T T S I Q H A D T D K E G A T K Q D P D Y D K K V H V F H L Q A Q K E T H V G D V D C K F H G S D YF A D L A V Q K I I A H R L V G R E R C T S V H L E P I V H D H G Q H F G I K I T H G H E H T G R G Q A L H E A K I D E E S R A S I E G T A V Q I E H D LH Q K S R Q R S T E E D R K Y S R H A V S V K R E L L L T L Q S Q A D D E T S G S Y Q D A T I I G F L G V L RD E V L Y S L H A V D H K S V P P LR FY TT A TFRV I P H R I Q V L S K K E T H D L L L S E A H V A E S G LA I R A I A L V H G G Q K Q S Q Q P L D H Q E V L T D F G G E H A Y H A D V Q A A K I E H A I T T E D R G Y R I E A E E P V L D R Q I G I V D L G V T V H D H S R I D H L I R Y D P C F T L Q L I Q A S T E G P K H G P G V L A S A L D L H H H S P E V K I R P E K I D G T H H F E I V F GGHTHQKGYH LAIFSFGEIL K Y G D E S H C D I I P A P E V E E Q V P I D K D G A T T E F I S T A A A R P V R I L K F S A P E H S D S V I L R H H E E G F K K D G F Y H H D K K S S K D D G K K E D Q G V R L F R F S S S Q F F T D S I R L E S V G V F I D Y D D L E Q L E V Q I P H H T T V H V C A H L S S H T Q H H C G L L H I R T G G K T H I H E S E H F A T H T S R G H K T S G Y H P G T L V Q L V TL D E G E Y T G L I Q R S Q I H G F S QI P D K E E G G Y R Y F S Y V F R Y L HI S H E G L F S H K L I F L YG P G V Q G I A H A F L C G S L V L H FHI A G I Y V A V A I T G L I Q I Y F H V C A A A I T L V L I S V I P L I S I G A G T I G A S V S L F L S I I V G V L H L A H A S G C L E P F A T A C L L V V A H F C V S G F T Q L L Q G F A F A Y T S T H A L F H A F L C Y A L L I F C F V H G F F T G L F S F L Y A P T V T G A V H A S V G G T H V F G H I I A Q C F Y G F I S L L I S A V V L S V T A L A R A F G Y T P S S Y A C F A A H F I A H S A S I L V F F C F L P L S A A T G V H S T V A H I I A F S H L V YI V H G I Q R I C G F L L G F F R H H R V D Extracellular Fig. 1 Secondary structure of the bile salt export pump with non-synonymous coding region genetic variants. Login to comment

[hide] Genetic variability, haplotype structures, and eth... Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8. Lang T, Haberl M, Jung D, Drescher A, Schlagenhaufer R, Keil A, Mornhinweg E, Stieger B, Kullak-Ublick GA, Kerb R
Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11).
Drug Metab Dispos. 2006 Sep;34(9):1582-99. Epub 2006 Jun 8., [PMID:16763017]

Abstract [show]
Biliary excretion of bile salts and other bile constituents from hepatocytes is mediated by the apical (canalicular) transporters P-glycoprotein 3 (MDR3, ABCB4) and the bile salt export pump (ABCB11). Mutations in ABCB4 and ABCB11 contribute to cholestatic diseases [e.g., progressive familial intrahepatic cholestasis 2 (PFIC2), PFIC3, and intrahepatic cholestasis of pregnancy], and our objective was to establish genetic variability and haplotype structures of ABCB4 and ABCB11 in healthy populations of different ethnic backgrounds. All coding exons, 5 of 6 noncoding exons, 50 to 300 base pairs of the flanking intronic regions, and 2.5 to 2.8 kilobase pairs of the promoter regions of ABCB4 and ABCB11 were sequenced in 159 and 196 DNA samples of Caucasian, African-American, Japanese, and Korean origin. In total, 76 and 86 polymorphisms were identified in ABCB4 and ABCB11, respectively; among them, 14 and 28 exonic polymorphisms, and 8 and 10 protein-altering variants, of which 4 were predicted to have functional consequences. Both genes showed substantial ethnic differences with respect to allele number, frequency of common and population-specific sites, and patterns of linkage disequilibrium. Population genetic analysis suggested some selective pressure against changes in the protein, supporting the important endogenous role of these transporters. Haplotype variability was greater in ABCB11 than in ABCB4. An ABCB11 promoter haplotype was associated with significant decrease of activity compared with wild type. Our results contribute to a better understanding of the molecular basis and of ethnic differences in drug response, and provide a valuable tool for future research on the heredity of cholestatic liver injury.

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67 The numbers 1 to 53 in the variant ID column indicate all variants included in haplotype analysis and linkage disequilibrium estimation. Variant ID 5Ј Sequence Genetic Variation 3Ј Sequence Region Amino Acid Change CA AA JA Total n % n % n % n % 54 GTAGTCACA g.-15595CϾT TTTCAGAGC Promoter 186 0.5 92 0.0 88 0.0 366 0.3 1 ACACTCTCT g.-15281_-15278 delCTCT CACACAGCA Promoter 186 10.2 92 4.3 86 26.7 364 12.6 2 CCCCCTCCC g.-15150TϾC GCCCCCAGA Promoter 148 48.0 76 39.5 92 25.0 316 58.9 3 TGACTGTAG g.-15018GϾA GACCACAAC Promoter 158 10.1 78 0.0 92 29.3 328 13.1 4 ATTAAGCAC g.-14944GϾA ATCAACTCA Promoter 198 10.1 72 0.0 96 28.1 366 12.8 5 CTATTGGGA g.-14589AϾT TCTTTTCCC Promoter 198 0.0 90 2.2 88 0.0 376 0.5 55 TGAAGCAAA g.-14524AϾT TTTTTTTCC Promoter 198 0.0 90 1.1 88 0.0 376 0.3 6 TACATTTGC g.-14473GϾA TCAACTCAG Promoter 198 2.5 90 18.9 88 0.0 376 8.8 7 TTGCATAGA g.-14437GϾA GAAACATCT Promoter 198 29.8 94 22.3 96 14.6 388 24.2 8 ATTATATGT g.-14353TϾC ATAATTTTG Promoter 190 61.6 80 92.5 88 75.0 358 71.8 56 ATAAACCAT g.-14316CϾA TTATACATA Promoter 192 0.5 80 0.0 88 0.0 360 0.3 9 ACCATCTTA g.-14312TϾC ACATAAATT Promoter 192 0.0 80 0.0 88 3.4 360 0.8 57 ATAAATTCC g.-14300AϾT ATAGAGAAA Promoter 192 0.0 80 1.3 88 0.0 360 0.3 10 TTTAATTTC g.-14207TϾC GCAAATTAA Promoter 190 2.1 80 17.5 88 10.2 358 7.5 11 TTGTTACAC g.-14104CϾT TTAGGAGGA Promoter 196 2.6 92 0.0 96 0.0 384 1.3 12 CATGATAGC g.-14035AϾG CCCAACTCC Promoter 194 1.5 92 1.1 96 0.0 382 1.0 58 AAGGCTGGA g.-13910GϾA TGAGAGGCA Promoter 202 0.0 94 1.1 96 0.0 392 0.3 13 AGAGGAAGA g.-13814GϾA GCAGCACAA Promoter 194 0.0 94 6.4 88 0.0 376 1.6 14 GCACAAATA g.-13801TϾC ATTGGAGCT Promoter 194 1.5 94 0.0 88 0.0 376 0.8 15 CTCAGACTT g.-13662TϾC TGAGCAAGG Promoter 192 0.0 94 7.4 86 0.0 372 1.9 83 TTAAAGGTA g.-13523͓T͔9 GTCTTGTTA Promoter 200 10.0 90 11.1 96 28.1 386 14.8 84 TTAAAGGTA g.-13523͓T͔10 GTCTTGTTA Promoter 200 9.0 90 18.9 96 6.3 386 10.6 85 TTAAAGGTA g.-13523͓T͔11 GTCTTGTTA Promoter 200 65.0 90 53.3 96 45.8 386 57.5 86 TTAAAGGTA g.-13523͓T͔12 GTCTTGTTA Promoter 200 16.0 90 16.7 96 19.8 386 17.1 59 CTGGGCCAG g.-13595GϾA AGCATCTGG Promoter 198 0.0 94 1.1 96 0.0 388 0.3 16 CAAGCACAC g.-13333TϾC CTGTGTTTG Promoter 196 0.0 76 0.0 96 3.9 368 0.9 17 ATGTTTCTC g.-13297GϾA TATGTCACT Promoter 196 0.0 76 3.9 96 0.0 368 0.8 60 TCCACAGTG g.-13142GϾA AGTCCATTA Exon 1 194 0.0 76 0.0 92 1.1 362 0.3 18 TTGATTAAA g.-77GϾA AAGAAAGAA Intron 1 202 2.5 88 11.4 90 12.2 380 6.8 19 ATTTTTTTT g.1319delT CTGACAGAT Intron 2 198 0.0 88 3.4 92 0.0 378 0.8 20 TTTAAATCC g.3754TϾC TATGTTTTT Intron 3 198 7.1 62 32.3 88 12.5 348 12.9 21 GTTACAAGA g.3781TϾC GAGAAGAAA Exon 4 D36D 198 0.0 62 0.0 88 26.1 348 6.6 22 GAATCTAGT g.4542AϾT ACTAAATTA Intron 4 184 0.0 90 0.0 92 2.2 366 0.5 61 CAAGTTTCG g.4621GϾA TTTTCTTCA Exon 5 R52R 184 0.0 90 1.1 92 0.0 366 0.3 23 AGATGTTTT g.4735TϾC ATTGACTAC Exon 5 F90F 184 2.7 90 13.3 92 12.0 366 7.7 24 GGGTAGGTT g.4862GϾA TTTTTGTTT Intron 5 182 4.9 90 2.2 94 0.0 366 3.0 25 GCTGAACAT g.21416CϾT GAGAGCGAA Exon 6 I134I 192 0.0 86 18.6 88 0.0 366 4.4 26 AGCTCCTCC g.21507GϾA TATAATTTA Intron 6 196 0.0 92 18.5 92 0.0 380 4.5 27 ACAATGAGA g.21554TϾG GCAATGTGT Intron 6 196 0.0 92 0.0 92 4.3 380 1.1 62 TGTATTGAA g.22567AϾT GTACTTTCT Intron 6 198 0.5 94 0.0 94 0.0 386 0.3 63 TTTGAATGA g.24203TϾC CAAATTCAG Intron 7 192 0.5 90 0.0 94 0.0 376 0.3 64 TCTAGTGAT g.24248AϾG TTAATAAAA Exon 8 I206V 194 0.0 90 1.1 96 0.0 380 0.3 28 TACGGACTA g.27224TϾC GAGCTGAAG Exon 9 Y269Y 200 0.0 80 0.0 96 27.1 376 6.9 65 CTGATGAAG g.27268TϾC CATTTCATC Exon 9 V284A 200 0.5 80 0.0 96 0.0 376 0.3 66 GTGAGAAAA g.27313GϾA AGAGGTTGA Exon 9 R299K 200 0.0 80 0.0 96 1.0 376 0.3 29 ACTGCATCA g.31773CϾT GGCCTGTTT Intron 9 178 5.1 70 1.4 48 0.0 296 3.4 30 TGTTTCTGC g.31811CϾT GAAATTGAC Intron 9 196 4.6 72 4.2 86 0.0 354 3.4 31 TTGACTCAA g.31825GϾA CATTTTGTC Intron 9 196 4.6 72 26.4 86 0.0 354 7.9 32 GACTCAAGC g.31827AϾG TTTTGTCTT Intron 9 196 70.4 72 84.7 86 90.7 354 78.2 33 TAGAAAAGG g.31890AϾG ATAGTGATG Exon 10 G319G 196 4.6 72 26.4 86 0.0 354 7.9 34 GACTTATTG g.32034AϾT CCGAGACAT Intron 10 196 0.0 66 4.5 82 0.0 344 0.9 67 CCTCAGTGT g.38161CϾT ATAGTAGGA Exon 11 V366V 196 0.0 88 1.1 94 0.0 378 0.3 35 CATTTTTGA g.38248GϾA ACAATAGAC Exon 11 E395E 196 0.0 88 8.0 96 0.0 380 1.8 36 GCAGAGATA g.41348CϾT GCCAAAGAT Intron 11 198 0.0 72 2.8 80 0.0 350 0.6 37 CCACAAATT g.41622GϾT CTCATTTTC Intron 12 196 1.0 72 0.0 74 0.0 342 0.6 38 CAGTGACAA g.44255delT CTGAACTTT Intron 12 190 0.0 94 2.1 92 0.0 376 0.5 39 TCAACATGG g.44308TϾC CATTAAACC Exon 13 V444A 190 59.5 90 65.6 92 80.4 372 66.1 40 TTGATCAAA g.44481CϾT AGAAAGGTG Intron 13 188 59.0 90 65.6 92 80.4 370 65.9 68 CAAGGAGGC g.46246CϾT AATGCCTAC Exon 14 A535A 184 0.0 86 0.0 96 1.0 366 0.3 41 GGGAGAAAC g.46311TϾC AAGAGGTCG Intron 14 182 60.4 86 66.3 94 79.8 362 66.9 42 GTTGCTCAT g.48611CϾG GCTTGTCTA Exon 16 R616G 194 0.0 90 2.2 96 0.0 380 0.5 69 CGCTTGTCT g.48620AϾG CGGTCAGAG Exon 16 T619A 194 0.0 90 1.1 96 0.0 380 0.3 70 CAGAGCTGC g.48634AϾG GATACCATC Exon 16 A623A 194 0.0 90 1.1 96 0.0 380 0.3 43 GAAGATGAC g.49653AϾG TGCTTGCGA Exon 17 M677V 190 4.2 86 14.0 88 0.0 364 5.5 71 CCGGCAAC g.53835GϾA CTCCAAGTC Exon 18 R698H 196 0.5 82 0.0 94 0.0 372 0.3 72 GAACCTCCA g.53876TϾC TAGCTGTTG Exon 18 L712L 196 0.5 82 0.0 94 0.0 372 0.3 44 TTAATATAA g.59981CϾA CCTCTCTCT Intron 18 192 43.2 84 21.4 90 27.8 366 34.4 45 AATAGATTT g.73116_73119delATTT TTCTATTTA Intron 19 192 0.0 66 6.1 96 0.0 354 1.1 46 ATTTATAAT g.73132_73133insCAA AAAGTTACT Intron 19 194 0.0 66 6.1 96 0.0 356 1.1 47 ACTTTCTTG g.73148TϾC TTACTATCT Intron 19 196 69.4 66 93.9 96 0.0 358 82.1 qanal/courses/predoc97/blosum62.cmp), and Grantham values (Grantham, 1974). 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116 Two Caucasian-specific variants in exon 13 (c.1331TϾC; 59.4%) and exon 17 (c.2029AϾG; 4.2%) coded for amino acid substitutions p.V444A and p.M677V; one variant, detected in exon 16 (c.1846CϾG, 2.2%) in the African-American population sample, resulted in protein sequence alteration p.R616G; and the Japanese-specific exon 21 variant c.2594CϾT (2.4%) resulted in amino acid substitution p.A865V (Table 4). Login to comment
166 The most common ABCB11 protein-altering polymorphism was p.V444A, which was frequently observed in all groups [ABCB11 p.M677V was present in both the Caucasian (4.2%,) and the African-American (14%) population sample and p.A865V was only found in Japanese samples]. Login to comment
177 Amino Acid Change Scoring Systems for Nonsynonymous Variants Grantham SIFT PolyPhen Blosum62 EC/EU MDR3 D87E 45 1.00 0.48 2 EC P95S 74 0.48 0.87 -1 EC T175A 58 0.01 0.72 -1 EC I367V 29 0.23 0.96 3 EC E450G 98 0.01 0.13 -2 EC R590Q 43 0.01 2.51 1 EC R652G 125 0.36 1.47 -2 EU E1099G 98 0.04 1.58 -2 EC BSEP I206V 29 1.00 0.23 3 EU V284A 64 0.13 0.43 -2 EC R299K 26 1.00 0.38 2 EU V444A 64 0.63 0.78 -2 EC R616G 125 0.01 3.16 -2 EC T619A 58 0.00 1.78 -1 EC M677V 21 0.29 0.82 1 EU R698H 29 0.30 0.57 0 EC A865V 64 0.02 1.12 0 EC R958Q 43 0.04 0.24 1 EU neutral mutation model (Tajima, 1989). Login to comment

[hide] The bile salt export pump. Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19. Stieger B, Meier Y, Meier PJ
The bile salt export pump.
Pflugers Arch. 2007 Feb;453(5):611-20. Epub 2006 Oct 19., [PMID:17051391]

Abstract [show]
Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver disease.

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160 Their bile flow rate is slightly but not significantly lower in comparison to controls, but the total bile salt output into bile is massively reduced and their liver bile salt concen- S114R G238V V284L* C336S D482G R487H S593R E636G G982R G1004D R1153CD R1268Q E186G E297G R432T I498T I498T T923P A926P R1050C R1128H S194P G260D N519S A1228V V444A K461E M677V R698H PFIC2 BRIC2 acquired cholestasis SNP Fig. 2 Putative secondary structure of Bsep (NT-005403) generated with the TOPO program (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl). Login to comment

[hide] Prediction of drug-induced intrahepatic cholestasi... Expert Opin Drug Saf. 2007 Jan;6(1):71-86. Sakurai A, Kurata A, Onishi Y, Hirano H, Ishikawa T
Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump.
Expert Opin Drug Saf. 2007 Jan;6(1):71-86., [PMID:17181454]

Abstract [show]
Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

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120 H2N COOH S56L G238V G260D C336S L339V V444A K461E D482G T923P K930X G982R R1090X R1153C Outside Inside R1268Q A1228VE1186K R1128H R1057X R1050C A926P A865V R698H E636G M677V S593R E592Q N591S R575XA570T Q558H I498T R432T R415Q R299K E297G V284A I206V S194P E186G cholestasis Expert Opin. Drug Saf. (2007) 6(1) Table 1. Login to comment
124 [47] - 15 1907 A→G Glu636Gly PFIC2 [46] rs11568364 16 2029 A→G Met677Val - [39,41,44,102] - 16 2093 G→A Arg698His - [44] - 16 2098 A→del Frame shift at position 700 PFIC2? Login to comment

[hide] Mutations and polymorphisms in the bile salt expor... Pharmacogenet Genomics. 2007 Jan;17(1):47-60. Lang C, Meier Y, Stieger B, Beuers U, Lang T, Kerb R, Kullak-Ublick GA, Meier PJ, Pauli-Magnus C
Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury.
Pharmacogenet Genomics. 2007 Jan;17(1):47-60., [PMID:17264802]

Abstract [show]
OBJECTIVES: Increasing evidence suggests that a genetically determined functional impairment of the hepatocellular efflux transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) play a pathophysiological role in the development of drug-induced liver injury. The aim of this study was therefore to describe the extent of genetic variability in ABCB11 and ABCB4 in patients with drug-induced liver injury and to in vitro functionally characterize newly detected ABCB11 mutations and polymorphisms. METHODS: ABCB11 and ABCB4 were sequenced in 23 patients with drug-induced cholestasis and 13 patients with drug-induced hepatocellular injury. Ninety-five healthy Caucasians served as the control group. Reference and mutant BSEP were expressed in Sf9 cells and ATP-dependent transport of [H]-taurocholate was measured in a rapid filtration assay. RESULTS: Four highly conserved nonsynonymous mutations were specific for drug-induced liver injury [ABCB11: D676Y (drug-induced cholestasis) and G855R (drug-induced cholestasis); ABCB4: I764L (drug-induced cholestasis) and L1082Q (drug-induced hepatocellular injury)]. Furthermore, a polymorphism in exon 13 of ABCB11 (V444A), which is associated with decreased hepatic BSEP expression was significantly more frequent in drug-induced cholestasis patients than in drug-induced hepatocellular injury patients and healthy controls (76 versus 50 and 59% in drug-induced cholestasis patients, drug-induced hepatocellular injury patients and healthy controls, respectively; P<0.05). The in-vitro transport activity of the V444A and the D676Y BSEP constructs was similar, whereas the G855R mutation was nonfunctional. CONCLUSION: In summary, our data support a role of ABCB11 and ABCB4 mutations and polymorphisms in drug-induced cholestasis. Genotyping of selected patients with acquired cholestasis might help to identify individuals with a genetic predisposition.

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62 Amino acid exchanges were introduced by site-directed mutagenesis, using the Quickchange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, California, USA) with the following mutagenesis primer pairs: (i) replacement of alanine to valine at position 444: forward 50 -CTAAAT GACCTCAACATGGTCATTAAACCAGGGG-30 and reverse 50 -CCCCTGGTTTAATGACCATGTTGAGGTCAT TTAG-30 ; (ii) replacement of aspartic acid to tyrosine at position 676: forward 50 -GGATGCAACTGAAGATTA CATGCTTGCGAGG-30 and reverse 50 -CCTCGCAAG CATGTAATCTTCAGTTGCATCC-30 ; (iii) replacement of methionine to valine at position 677: forward 50 -GCAACTGAAGATGACGTGCTTGCGAGGACC-30 and reverse 50 -GGTCCTCGCAAGCACGTCATCTTCAGT TGC-30 . Login to comment
82 All calculations Table 3 ABCB11 (BSEP) variant sites in drug-induced liver injury Amplicon DNA position cDNA position Nucleotide reference Nucleotide variant AA change AF DC (n = 46) AF DH (n = 26) AF controls (n = 190) Amplicon - 1 Intron - 1 - 2080 CTCT delCTCT 0.02 0.00 0.10 Amplicon - 1 Intron - 1 - 1952 T C 0.43 0.54 0.48 Amplicon - 1 Intron - 1 - 1820 G A 0.09 0.04 0.10 Amplicon - 1 Intron - 1 - 1746 G A 0.09 0.04 0.10 Amplicon - 1 Intron - 1 - 1275 G A 0.07 0.04 0.03 Amplicon - 1 Intron - 1 - 1239 G A 0.24 0.35 0.30 Amplicon - 1 Intron - 1 - 1155 T C 0.57 0.65 0.62 Amplicon - 1 Intron - 1 - 1009 T C 0.09 0.04 0.02 Amplicon - 1 Intron - 1 - 837 A G 0.02 0.00 0.02 Amplicon 2 Intron 1 - 50 G A 0.09 0.04 0.02 Amplicon 4 Intron 3 - 20 T C 0.09 0.08 0.07 Amplicon 5 Exon 5 270 T C syn 0.09 0.04 0.03 Amplicon 5 Intron 5 8 G A 0.00 0.13 0.05 Amplicon 9 Exon 9 851 T C V284A_c 0.02 0.00 0.01 Amplicon 10 Intron 9 - 69 C T 0.00 0.04 0.05 Amplicon 10 Intron 9 - 31 C T 0.00 0.04 0.05 Amplicon 10 Intron 9 - 17 G A 0.00 0.04 0.05 Amplicon 10 Intron 9 - 15 A G 0.77 0.65 0.70 Amplicon 10 Exon 10 957 A G syn 0.00 0.04 0.05 Amplicon 13 Exon 13 1331 T C V444A_c 0.76 0.50 0.59 Amplicon 13 Intron 13 70 C T 0.76 0.50 0.59 Amplicon 14 Intron 14 32 T C 0.75 0.54 0.60 Amplicon 17 Exon 17 2026 G T D676Y 0.02 0.00 0.00 Amplicon 17 Exon 17 2029 A G M677V 0.00 0.04 0.04 Amplicon 18 Exon 18 2093 G A R698H_c 0.02 0.00 0.01 Amplicon 18 Exon 18 2134 T C syn 0.02 0.00 0.01 Amplicon 19 Intron 18 - 17 C A 0.52 0.62 0.43 Amplicon 20 Intron 19 - 17 T C 0.66 0.75 0.69 Amplicon 21 Exon 21 2563 G A G855R_c 0.02 0.00 0.00 Amplicon 24 Exon 24 3084 G A syn 0.50 0.62 0.46 Amplicon 28 Intron 27 - 34 G A 0.48 0.62 0.45 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_003742.2. Login to comment
97 Chemical classification of all causative drugs revealed that the most prominent structures were the b-lactam ring of antibacterials in Fig. 2 Extracellular V284A V444A G855R R698H D676Y M677V Cytoplasm Secondary structure of bile salt export pump (BSEP) with nonsynonymous coding region variants. Login to comment
118 Of the coding region changes, eight have been previously described in healthy Caucasians [23], including two frequent nonsynonymous polymorphisms (exon 13: 1331T > C-V444A and exon 17: 2029A > G- M677V). Login to comment
158 Comparable protein amounts were detected for the alanine and valine containing constructs in position 444 (Fig. 5a), while expression levels of the M677V, D676Y and G855R variants amounted to 40, 60 and 20% of expression levels of reference BSEP (Fig. 6a), respectively. Login to comment
161 Furthermore, adjusted taurocholate transport activities for M677V and D676Y were similar to reference BSEP, whereas hardly any transport activity was seen with the G855R construct (Fig. 6c). Login to comment
172 In contrast, no differences as to taurocholate transport activity or hepatic BSEP expression were observed for the second frequent ABCB11 polymorphism in exon 17 (M677V) [24]. Login to comment
194 Normalizedtransportactivity (in%ofref.BSEP) A 0 20 40 60 80 100 120 Ref. BSEP D676Y G855RM677V 140 Bile salt export pump (BSEP) expression and taurocholate transport in Sf9 membrane vesicles, expressing reference BSEP and the D676Y, M677V and the G855R constructs. Login to comment
195 (a) Expression levels of the D676Y, the M677V and the G855R constructs amounted to 60, 40 and 20% of reference BSEP, respectively. Login to comment

[hide] Missense mutations and single nucleotide polymorph... Hepatology. 2009 Feb;49(2):553-67. Byrne JA, Strautnieks SS, Ihrke G, Pagani F, Knisely AS, Linton KJ, Mieli-Vergani G, Thompson RJ
Missense mutations and single nucleotide polymorphisms in ABCB11 impair bile salt export pump processing and function or disrupt pre-messenger RNA splicing.
Hepatology. 2009 Feb;49(2):553-67., [PMID:19101985]

Abstract [show]
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.

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68 Continued Exon Nucleotide Change Predicted Protein Effect Location in Protein Associated Phenotype Prevalence or frequency* Any Defect(s) Identified Reference BRIC, 1 family (both hom) 15 c.1757CϾT T586I Adj WB BRIC 1 family (het) No splicing † 15 c.1763CϾT A588V Adj WB PFIC 2 families (both het) No protein 31, 32 15 c.1772AϾG N591S Adj WB SNP-ICP 2.6% 42 15 c.1779TϾA S593R NBF1 PFIC 1 family (het) 29 15 c.1791GϾT V597V NBF1 SNP 2.6% 42 16 c.1880TϾC I627T IC3 PFIC 1 family (het) ‡ 16 c.1964CϾT T655I IC3 BRIC / ICP / DC 1 family (het) Reduced levels of mature protein ‡ 17 c.2029AϾG M677V IC3 SNP 1.6-5.6% 39, 42-45 18 c.2093GϾA R698H IC3 SNP 0.3 - 0.8% 43, 45 18 c.2125GϾA E709K IC3 SNP-PFIC 1 family (het) ‡ 18 c.2130TϾC P710P IC3 SNP-PBC 0.5 - 3.1% 43 20-21 c.2412AϾC A804A TM8 SNP 1.1% 45 20-21 c.2453AϾT Y818F IC4 SNP-PFIC 2 families (hom) Reduced levels of mature protein ‡ 20-21 c.2494CϾT R832C IC4 PFIC 2 families (1 het, 1 consanguineous) Moderate differential splicing 31, 32 20-21 c.2576CϾG T859R IC4 PFIC 1 family (het) 31 22 c.2767AϾC T923P IC5 BRIC 1 family (het) 8 22 c.2776GϾC A926P IC5 BRIC 1 family (het) Mild exon skipping 8 23 c.2842CϾT R948C IC5 PFIC 2 families (both het) Immature protein 31 23 c.2935AϾG N979D TM11 PFIC 1 family (consanguineous) 31 23 c.2944GϾA G982R TM11 PFIC 4 families (1 hom, 1 consanguineous, 2 het) Immature protein 7, 29, 31 23 c.3011GϾA G1004D EC6 PFIC 1 family (hom) 28 24 c.3084AϾG A1028A TM12 SNP-PBC 39.86 - 56.3% Severe exon skipping 8, 43, 45 24 c.3148CϾT R1050C C term BRIC 2 familes (1 hom, 1 het) Immature protein 8 25 c.3329CϾA A1110E Adj WA PFIC 2 familes (both het) Mild exon skipping; immature protein 31 25 c.3346GϾC G1116R WA PFIC / BRIC 1 family (consanguineous) Mild exon skipping ‡ 25 c.3382CϾT R1128C NBF2 PFIC 1 family (consanguineous) Mild exon skipping; immature protein 31 25 c.3383GϾA R1128H NBF2 BRIC 1 family (hom) Mild exon skipping; greatly reduced levels of mature protein 8 26 c.3432CϾA S1144R NBF2 PFIC 1 family (het) Severe differential splicing 29 26 c.3457CϾT R1153C NBF2 PFIC 4 families (2 consanguineous, 2 het) Immature protein 7, 31, 36 26 c.3458GϾA R1153H NBF2 PFIC 4 families (2 consanguineous, 2 het) Severe differential splicing; immature protein 31 26 c.3460TϾC S1154P NBF2 PFIC 1 family (het) Severe differential splicing 31 26 c.3556GϾA E1186K NBF2 SNP 1%-10% Mild exon skipping ‡ 26 c.3589_3590 delCTinsGG L1197G NBF2 BRIC 1 family (het) † 27 c.3628AϾC T1210P Adj ABCm PFIC 1 family (hom) Immature protein 31 27 c.3631AϾG N1211D Adj ABCm SNP-PFIC 1 family (het) ‡ 27 c.3669GϾC E1223D ABCm Prolonged NNH 1 family (het) ‡ 27 c.3683CϾT A1228V Adj ABCm/WB SNP-PBC 0.8% 43 27 c.3691CϾT R1231W Adj ABCm/WB PFIC 1 family (het) Severe exon skipping; immature protein 30, 31 27 c.3692GϾA R1231Q Adj ABCm/WB PFIC 2 families (1 consanguineous, 1 het) No splicing; immature protein 31, 34 27 c.3724CϾA L1242I WB PFIC 1 family (het) 31 28 c.3892GϾA R1268Q¶ NBF2 PFIC 1 family (hom) Immature protein 7 *Prevalence or frequency is quoted depending on how data were presented in the original publication(s). Login to comment

[hide] Genetic variations of the ABC transporter gene ABC... Drug Metab Pharmacokinet. 2009;24(3):277-81. Kim SR, Saito Y, Itoda M, Maekawa K, Kawamoto M, Kamatani N, Ozawa S, Sawada J
Genetic variations of the ABC transporter gene ABCB11 encoding the human bile salt export pump (BSEP) in a Japanese population.
Drug Metab Pharmacokinet. 2009;24(3):277-81., [PMID:19571440]

Abstract [show]
The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3'-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361C>A (Gln121Lys), 667C>T (Arg223Cys), and 1460G>T (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations.

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53 Six variations previously reported in other ethnic groups were not detected: 616AÀG (Ile206Val; found with 0.011 frequency in African-Americans), 851TÀC (Val284Ala; 0.005 in Caucasians), 1846CÀG (Arg616Gly; 0.022 in African-Americans), 1855AÀG (Thr619Ala; 0.011 in African-Americans), 2029AÀG (Met677Val; 0.042 in Caucasians and 0.14 in African-Americans), and 2093GÀA (Arg698His; 0.005 in Caucasians).7) These variations might be ethnic-specific. Login to comment

[hide] Recent insights into the function and regulation o... Curr Opin Lipidol. 2009 Jun;20(3):176-81. Stieger B
Recent insights into the function and regulation of the bile salt export pump (ABCB11).
Curr Opin Lipidol. 2009 Jun;20(3):176-81., [PMID:19684528]

Abstract [show]
PURPOSE OF REVIEW: Generation of bile is an important function of the liver. Its impairment can be caused by inherited mutations or by acquired factors and leads to cholestasis. Bile salts are an important constituent of bile and are secreted by the bile salt export pump (BSEP) from hepatocytes. RECENT FINDINGS: Significant progress was made in the understanding of mechanisms and consequences of malfunctioning BSEP. This information was gained from extensive characterization of patients with inherited BSEP deficiency and the subsequent characterization of the identified mutations in heterologous expression systems. Furthermore and importantly, clinical evidence shows that patients with severe BSEP deficiency are at risk to develop hepatocellular carcinoma. Bile salts are now recognized to be important in the modulation of whole body energy homeostasis. Because BSEP is the rate-limiting step in hepatocellular bile salt transport, it controls the spill over of bile salts into the systemic circulation. Therefore, an indirect role of BSEP in energy homeostasis becomes more and more likely. SUMMARY: In summary, knowledge on the physiologic and pathophysiologic role of BSEP is rapidly progressing. It can be anticipated that the next major step in better understanding BSEP should come from information on structure-function relationship. However, given the difficulty in structure determination of mammalian transporters, this will require major efforts.

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52 To date, c.1331T>C (p.V444A) in exon 13 and c.2029A>G (p.M677V) in exon 17 of the BSEP gene are two nonsynonymous SNPs, which have consistently been observed with frequencies of higher than 0.5% [8,30-32] in different cohorts. Login to comment

[hide] Polymorphic variants in the human bile salt export... Pharmacogenet Genomics. 2010 Jan;20(1):45-57. Ho RH, Leake BF, Kilkenny DM, Meyer Zu Schwabedissen HE, Glaeser H, Kroetz DL, Kim RB
Polymorphic variants in the human bile salt export pump (BSEP; ABCB11): functional characterization and interindividual variability.
Pharmacogenet Genomics. 2010 Jan;20(1):45-57., [PMID:20010382]

Abstract [show]
OBJECTIVES: 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. METHODS: 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. RESULTS: For the most part, polymorphisms were rare and ethnic-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 to 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. CONCLUSION: Accordingly, our study indicates 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.

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146 Two common BSEP polymorphisms, 1331T > C (Val444Ala) and 2029A > G (Met677Val), were not associated with significant changes in taurocholate transport activity. Login to comment
156 Calnexin, an intracellular resident Table 1 Single-nucleotide polymorphisms in ABCB11 Allele frequencies (%) SNP rs number Amino acid change African-American European-American Asian-American Mexican-American Pacific Islanders 108T > C rs3815675 Synonymous 1.5 1.5 25 5.0 21.4 167C > T rs11568361 Ser56Leu 0.5 0 0 0 0 174C > T rs11568362 Synonymous 0.5 0 0 0 0 270T > C rs414877 Synonymous 3.0 3.5 5.0 0 7.1 402C > T rs11568377 Synonymous 3.5 0 0 0 0 585G > C rs11568365 Synonymous 0.5 0 0 0 0 616A > G rs11568357 Ile206Val 2.5 0 0 0 0 696G > T rs11568358 Synonymous 0 0.5 0 0 0 807T > C rs2287616 Synonymous 2.0 0.5 23.3 5.0 21.4 890A > G rs11568372 Glu297Gly 0 0.5 0 0 0 957A > G rs7563233 Synonymous 31.5 0.5 0 15.0 0 1281C > T rs11568360 Synonymous 0.5 0 0 0 0 1331T > C rs2287622 Val444Ala 53.0 57.1 66.7 50.0 92.9 1671C > T rs11568368 Synonymous 0 0.5 0 0 0 1674G > C rs11568369 Gln558His 0 0.5 0 0 0 1772A > G rs11568367 Asn591Ser 0 0.5 0 0 0 1774G > C rs11568370 Glu592Gln 0 0.5 0 0 0 1791G > T rs11568371 Synonymous 0 0.5 0 0 0 2029A > G rs11568364 Met677Val 15.0 5.5 1.7 5.0 0 2412A > G rs11568373 Synonymous 8.0 0 0 5.0 0 3084A > G rs97692 Synonymous 28.6 54.6 63.3 37.5 21.4 3258A > G rs11568359 Synonymous 7.0 0 0 0 0 3435A > G rs11568366 Synonymous 1.0 0 0 0 0 3556G > A rs1521808 Glu1186Lys 2.5 0 0 0 0 Allele frequencies for single-nucleotide polymorphisms (SNPs) in ABCB11 were determined from a DNA panel of ethnically defined healthy individuals - African-Americans (n = 100), European-Americans (n = 100), Asian-Americans (n = 30), Mexican-Americans (n = 10) and Pacific Islanders (n = 7). Login to comment

[hide] Role of the bile salt export pump, BSEP, in acquir... Drug Metab Rev. 2010 Aug;42(3):437-45. Stieger B
Role of the bile salt export pump, BSEP, in acquired forms of cholestasis.
Drug Metab Rev. 2010 Aug;42(3):437-45., [PMID:20028269]

Abstract [show]
Generation of bile is a key function of the liver. Its impairment leads to accumulation of cytotoxic bile salts in hepatocytes and, consequently, to liver disease. The bile salt export pump, BSEP, is critically involved in the secretion of bile salts into bile. Its function can be disturbed or abolished by inherited mutations. This will lead to progressive intrahepatic cholestais and severe liver disease. In addition to mutations, BSEP can be inhibited by acquired factors, such as xenobiotics or drugs, aberrant bile salt metabolites, or pregnancy. This inhibition will lead to acquired cholestasis. Some drugs are now known to be competitive inhibitors of Bsep. In addition, a polymorphism in the gene coding for BSEP has been identified as a potential susceptibility factor for acquired cholestasis.

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141 Until now, the nonsynonomous c.1331T>C (p.V444A) in exon 13 and c.2029A>G (p.M677V) in exon 17 have consistently been reported to have frequencies of higher than 0.5% (Saito etal., 2002; Pauli-Magnus etal., 2004; Lang etal., 2006). Login to comment

[hide] ATP8B1 and ABCB11 analysis in 62 children with nor... Hepatology. 2010 May;51(5):1645-55. Davit-Spraul A, Fabre M, Branchereau S, Baussan C, Gonzales E, Stieger B, Bernard O, Jacquemin E
ATP8B1 and ABCB11 analysis in 62 children with normal gamma-glutamyl transferase progressive familial intrahepatic cholestasis (PFIC): phenotypic differences between PFIC1 and PFIC2 and natural history.
Hepatology. 2010 May;51(5):1645-55., [PMID:20232290]

Abstract [show]
Progressive familial intrahepatic cholestasis (PFIC) types 1 and 2 are characterized by normal serum gamma-glutamyl transferase (GGT) activity and are due to mutations in ATP8B1 (encoding FIC1) and ABCB11 (encoding bile salt export pump [BSEP]), respectively. Our goal was to evaluate the features that may distinguish PFIC1 from PFIC2 and ease their diagnosis. We retrospectively reviewed charts of 62 children with normal-GGT PFIC in whom a search for ATP8B1 and/or ABCB11 mutation, liver BSEP immunostaining, and/or bile analysis were performed. Based on genetic testing, 13 patients were PFIC1 and 39 PFIC2. The PFIC origin remained unknown in 10 cases. PFIC2 patients had a higher tendency to develop neonatal cholestasis. High serum alanine aminotransferase and alphafetoprotein levels, severe lobular lesions with giant hepatocytes, early liver failure, cholelithiasis, hepatocellular carcinoma, very low biliary bile acid concentration, and negative BSEP canalicular staining suggest PFIC2, whereas an absence of these signs and/or presence of extrahepatic manifestations suggest PFIC1. The PFIC1 and PFIC2 phenotypes were not clearly correlated with mutation types, but we found tendencies for a better prognosis and response to ursodeoxycholic acid (UDCA) or biliary diversion (BD) in a few children with missense mutations. Combination of UDCA, BD, and liver transplantation allowed 87% of normal-GGT PFIC patients to be alive at a median age of 10.5 years (1-36), half of them without liver transplantation. CONCLUSION: PFIC1 and PFIC2 differ clinically, biochemically, and histologically at presentation and/or during the disease course. A small proportion of normal-GGT PFIC is likely not due to ATP8B1 or ABCB11 mutations.

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74 The 24 remaining mutations were single missense substitutions. In addition to the 41 disease-causing mutations, we found three other variants (p.V444A, p.G319G, p.M677V) considered SNPs.7,27 The p.V444A mutation was present on 44% of PFIC alleles Table 1. Login to comment
111 36†,k p.G1116F p.R387H na na Novel mutations described in this study are shown in bold. Login to comment
114 ‡Patient harboring the heterozygous ABCB11 mutation p.M677V. Login to comment

[hide] The bile salt export pump: clinical and experiment... Semin Liver Dis. 2010 May;30(2):125-33. Epub 2010 Apr 26. Lam P, Soroka CJ, Boyer JL
The bile salt export pump: clinical and experimental aspects of genetic and acquired cholestatic liver disease.
Semin Liver Dis. 2010 May;30(2):125-33. Epub 2010 Apr 26., [PMID:20422495]

Abstract [show]
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.

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40 Two nonsynonymous SNPs, c.1331T>C (p.V444A) in exon 13 and c.2029A>G (p.M677V), have been consistently observed and patients with at least one c.1331T allele tended to have lower levels of BSEP expression.49,51 The V444A variant is also associated with ICP and drug-induced cholestasis,46,49,51-53 but functional activity is not affected.51 It should be noted that these polymorphisms in BSEP that have been associated with ICP and drug cholestasis will require further validation and functional analyses in a larger group of patients. Login to comment
47 Two single nucleotide polymorphisms (SNPs; V444A and M677V, cross) are also represented and have been characterized by functional studies (gray star). Login to comment

[hide] The role of the sodium-taurocholate cotransporting... Handb Exp Pharmacol. 2011;(201):205-59. Stieger B
The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation.
Handb Exp Pharmacol. 2011;(201):205-59., [PMID:21103971]

Abstract [show]
Bile formation is an important function of the liver. Bile salts are a major constituent of bile and are secreted by hepatocytes into bile and delivered into the small intestine, where they assist in fat digestion. In the small intestine, bile salts are almost quantitatively reclaimed and transported back via the portal circulation to the liver. In the liver, hepatocytes take up bile salts and secrete them again into bile for ongoing enterohepatic circulation. Uptake of bile salts into hepatocytes occurs largely in a sodium-dependent manner by the sodium taurocholate cotransporting polypeptide NTCP. The transport properties of NTCP have been extensively characterized. It is an electrogenic member of the solute carrier family of transporters (SLC10A1) and transports predominantly bile salts and sulfated compounds, but is also able to mediate transport of additional substrates, such as thyroid hormones, drugs and toxins. It is highly regulated under physiologic and pathophysiologic conditions. Regulation of NTCP copes with changes of bile salt load to hepatocytes and prevents entry of cytotoxic bile salts during liver disease. Canalicular export of bile salts is mediated by the ATP-binding cassette transporter bile salt export pump BSEP (ABCB11). BSEP constitutes the rate limiting step of hepatocellular bile salt transport and drives enterohepatic circulation of bile salts. It is extensively regulated to keep intracellular bile salt levels low under normal and pathophysiologic situations. Mutations in the BSEP gene lead to severe progressive familial intrahepatic cholestasis. The substrates of BSEP are practically restricted to bile salts and their metabolites. It is, however, subject to inhibition by endogenous metabolites or by drugs. A sustained inhibition will lead to acquired cholestasis, which can end in liver injury.

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439 To date, c.1331T>C (p.V444A) in exon 13 and c.2029A>G (p.M677V) in exon 17 of the BSEP gene are two nonsynonymous SNPs, which consistently have been found with frequencies higher than 0.5% (Lang et al. 2006; Pauli-Magnus et al. 2004a; Saito et al. 2002a; Stieger 2009; Stieger et al. 2007) in different, unrelated cohorts. Login to comment

[hide] NR1H4 analysis in patients with progressive famili... Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):569-73. doi: 10.1016/j.clinre.2012.08.008. Epub 2012 Nov 9. Davit-Spraul A, Gonzales E, Jacquemin E
NR1H4 analysis in patients with progressive familial intrahepatic cholestasis, drug-induced cholestasis or intrahepatic cholestasis of pregnancy unrelated to ATP8B1, ABCB11 and ABCB4 mutations.
Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):569-73. doi: 10.1016/j.clinre.2012.08.008. Epub 2012 Nov 9., [PMID:23142591]

Abstract [show]
Farnesoid X receptor (FXR, NR1H4) controls bile acid homeostasis. NR1H4 variants may predispose to intrahepatic cholestasis of pregnancy (ICP). We report on NR1H4 analysis in eight patients with progressive familial intrahepatic cholestasis (PFIC) and in eight women with either ICP and/or drug-induced cholestasis (DIC) in whom no disease causing mutation in ATP8B1, ABCB11 and/or ABCB4 were found. No NR1H4 mutation was found in PFIC patients. In one woman with ICP/DIC, a NR1H4 heterozygous variant (c.-1G>T) was found. This suggests that a NR1H4 mutation is not or rarely involved in hepatocellular cholestasis of unknown cause.

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55 Table 1 Main clinical characteristics and ATP8B1, ABCB11 and NR1H4 genotypes in eight normal GGT PFIC patients of unknown cause. Patient Phenotype ATP8B1 genotype ABCB11 genotype NR1H4 genotype Liver histology and canalicular immunostaining Patient 1 Severe permanent cholestasis since age 5 mo, Serum BS = 208 òe;mol/L, deafness, UDCA and BD not tested, LT at 4.5 yr, alive at 25 yr No mutation p.V444A Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 2 Severe permanent cholestasis since age 1 mo, Serum BS = 161 òe;mol/L, Biliary BS = 5.7 mmol/L, Success of UDCA but persistent pruritus Alive at 5 yr p.R952Q Heterozygous p.V444A, p.M677V Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 3 Severe recurrent cholestasis since age 7 mo, Serum BS = 375 òe;mol/L, Biliary BS = 0.39 mmol/L, Success of UDCA, then of BD at 7.5 yr, Chronic diarrhea, and liver steatosis post BD, Alive at 19 yr p.R952Q Heterozygous No mutation No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 4 Severe permanent cholestasis since age 1 mo, Serum BS = 291 òe;mol/L, UDCA failure, BD not tested, LT at 3.2 yr, died post-LT p.R952Q Heterozygous p.V444A Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 5 Severe cholestasis since age 3 mo, Serum BS = 262 òe;mol/L, UDCA failure, BD success at 4.7 yr, Alive at 14 yr p.R952Q Heterozygous p.V444A Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 6 Severe permanent cholestasis since age 1 mo, Serum BS = 191 òe;mol/L, biliary BS = 0.09 mmol/L, TC = 7 mmol/L, TG = 4.5 mmol/L, UDCA and BD failure, LT at 6 yr, died post-LT No mutation No mutation No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 7 Cholestasis since age 2.5 yr, Serum BS = 42 òe;mol/L, Success of UDCA, Alive at 20 yr No mutation p.M677V, p.G319G Homozygous p.V444A Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis BSEP+ MDR3+ Patient 8 Severe cholestasis since age 1 week, Serum BS = 240 òe;mol/L, UDCA and BD not tested, Liver failure and LT at age 5 mo, Alive at 21 yr No mutation p.V444A Heterozygous No mutation Hepatocellular cholestasis, septal fibrosis Giant cells BSEP-, MDR3+ GGT: gammaglutamyl transferase; PFIC: progressive familial intrahepatic cholestasis; BS: bile salt concentration; UDCA: ursodeoxycholic acid; BD: biliary diversion; LT: liver transplantation; mo: month; yr: year; BSEP+: normal immunostaining; BSEP-: negative immunostaining; MDR3+: normal immunostaining; TC: serum fasting total cholesterol; TG: serum fasting triglycerides. Login to comment

[hide] The bile salt export pump (BSEP) in health and dis... Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12. Kubitz R, Droge C, Stindt J, Weissenberger K, Haussinger D
The bile salt export pump (BSEP) in health and disease.
Clin Res Hepatol Gastroenterol. 2012 Dec;36(6):536-53. doi: 10.1016/j.clinre.2012.06.006. Epub 2012 Jul 12., [PMID:22795478]

Abstract [show]
The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.

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185 PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Missense mutations M1V C336S D549V L1055P E135K E137K T87R V43I S701P G19R W342G G556R C1083Y E137K L198P M123T S56L L712L L50S A382G G562D A1110E E186G E297G S194P Q121K A865D M62K R387H A570T S1114R L198P R415Q L198P R128H A865G C68Y A390P L581F G1116E E297G V444A G260D I206V S874P C107R G410D A588V G1116F G374S D482G E297K V284A I939M I112T L413W S593R G1116R A390P N591S V444A G295C R958Q W114R I420T I627T S1120N R432T T655I T510T G295R F959C Y157C D440E E636G R1128C V444A T655I G295S F959V A167T G455E R698C S1144R I498T D676Y R299K T965S A167V K461E S699P R1153C A570T P710P R303K F971L I182K T463I E709K R1153H T586I L827I L339V F971Y M183T Q466K G758R S1154P G648V G855R H423R L1006F M183V R470Q G766R N1173D T655I E1186K V444A N1009H G188W Y472C Y818F T1210P T923P V444D K1145N M217R V481E R832C N1211D A926P V444G I1183T R223C D482G R832H V1212F R948C A459V S226L R487H T859R R1231Q G1004D I468I G238V R487P A865V R1231W R1050C R487L T242I N490D Q869P L1242I G1116R Q546K A257G I498T G877R D1243G R1128H Q558H V284L G499E S901R R1268Q L1197G E592Q E297G I512T R948C A1283V R1231Q V597M R303G N515T N979D G1292V R616G R303K R517H G982R G1298R T619A Q312H F540L G1004D M677L R313S I541L T1029K M677V G327E I541T G1032R R696Q W330R F548Y A1044P R698H Nonsense mutations (premature stop-codons) S25X Y472X Y772X R1090X E96X W493X Q791X V1147X W330X R520X R928X Q1215X Y354X I528X Y1041X R1235X R415X R575X R1057X E1302X R470X Q702X Q1058X Table 1 (Continued) PFIC BRIC/NFC ICP Other liver diseases Genetic variants without disease association Splice site mutations 76 + 3G > T 908 + 1delG 2178 + 1G > T 3057-2A > G Q159Q 77-1G > C 908 + 1G > T 2179-2A > G 3213 + 1delG Q361Q 99-1G > T 908 + 1G > A 2343 + 1G > T 3213 + 4A > G 150 + 3A > C 1435-13 -8del 2343 + 2T > C 3213 + 5G > A 390-1G > A 2012-8T > G 2611-2A > T 611 + 1G > A 2178 + 1G > A R1001R Deletions/insertions/frame shifts Q101Dfs8X L380Wfs18X G648Vfs5X Q1058Hfs38X F959Hfs1X T127Hfs6X A382 A388del K700Sfs12X I1061Vfs34X F959Gfs48X N199Ifs14X P456Pfs24X T919del L1165del L232Cfs9X H484Rfs5X K930Efs92X A1192Efs50X R303Sfs17X I528Sfs21X K930Efs79X T1256Tfs40X V368Rfs27X I610Qfs45X K969 K972del Synonymous variants without disease association R33R F90F L232L I416I G557G I876I A1028A K1145K D36D I134I Y269Y G418G V597V G937G K1070K R52R S136S Q312Q F427F A804A Y981Y T1086T D58D V195V G319G E395E A535A G817G G1004G A1110A The overview shows ࣈ 290 known variants of BSEP on the protein level, except splice site mutations, which are shown on cDNA level. Login to comment

[hide] Biosynthesis and trafficking of the bile salt expo... Mol Aspects Med. 2014 Jun;37:3-14. doi: 10.1016/j.mam.2013.05.001. Epub 2013 May 15. Soroka CJ, Boyer JL
Biosynthesis and trafficking of the bile salt export pump, BSEP: therapeutic implications of BSEP mutations.
Mol Aspects Med. 2014 Jun;37:3-14. doi: 10.1016/j.mam.2013.05.001. Epub 2013 May 15., [PMID:23685087]

Abstract [show]
The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter.

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168 The BSEP variants, V444A and M677V, have been reported to consistently occur with frequencies of greater that 50% (Lang et al., 2006; Saito et al., 2002). Login to comment

[hide] Liver transcript analysis reveals aberrant splicin... Mol Genet Metab. 2014 Nov;113(3):225-9. doi: 10.1016/j.ymgme.2014.07.006. Epub 2014 Jul 15. Davit-Spraul A, Oliveira C, Gonzales E, Gaignard P, Therond P, Jacquemin E
Liver transcript analysis reveals aberrant splicing due to silent and intronic variations in the ABCB11 gene.
Mol Genet Metab. 2014 Nov;113(3):225-9. doi: 10.1016/j.ymgme.2014.07.006. Epub 2014 Jul 15., [PMID:25085279]

Abstract [show]
BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is an autosomal recessive disease due to mutations in ABCB11. ABCB11 encodes the bile salt export pump (BSEP), the major transporter responsible for biliary bile acid secretion, which expression is restricted to hepatocytes. In some patients, molecular analysis of ABCB11 revealed either exonic or intronic variations - including common polymorphisms - predicted to affect splicing according to in silico analysis or in vitro minigene studies. Transcript analysis in liver tissue is the best way to determine whether the variations predicted to affect splicing are deleterious or not. METHODS AND RESULTS: We performed ABCB11 transcript analysis in liver tissue from five PFIC2 patients who had variations which were predicted to either affect splicing or not. Among eleven variants tested, only the silent c.3003A>G variant and the intronic c.3213+4A>G variant led to abnormal splicing as suggested by in silico analysis. CONCLUSION: ABCB11 liver transcript analysis is a useful tool to confirm or invalidate the predicted splicing effect of a silent or intronic ABCB11 variation.

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48 Byrne et al. [7] showed that the silent variants p.Gly319Gly and p.Ala1028Ala had respectively a mild and a severe exon skipping effect while p.Phe90Phe, p.Val444Ala, and p.Met677Val variants had no effect on splicing. Login to comment
72 Some variants were previously studied in minigene system [7] and predicted the following consequences: &#b0;no effect: p.Phe90Phe (rs4148777); p.Met677Val (rs11568364); p.Val444Ala (rs2287322); *mild exon skipping: p.Gly319Gly (rs7563233); p.Arg1128Cys (disease-causing mutation); **severe exon skipping: p.Ala1028Ala (rs497692). Login to comment
86 Table 3 ABCB11 exonic variations. Modification Location SSF NNSplice MaxEntScan HSF ESE Finder RESCUE-ESE Observed consequence on cDNA WT 3'ss N MUT 3'ss or WT 5'ss N MUT 5'ss WT N MUT c.3003ANG p.Arg1001Arg 54 nt from 5' ss 90.6 N 90.6 Creation of a cryptic donor 54 bp upstream scored 84.8 0.99 N 0.99 Creation of a cryptic donor 54 bp upstream scored 0.98 10.07 N 10.07 Creation of a cryptic donor 54 bp upstream scored 10 96.9 N 96.9 Creation of a cryptic donor 54 bp upstream scored 89.3 Loss: SRp55 Gain: 2 SF2/ ASF SRp40: 3.28 N 5.66 (72.5%) Loss 2 ESE Use of the cryptic 5'ss p.Tyr1002_Arg1019del c.3382CNT p.Arg1128Cys 30 nt from 5' ss 82.7 N 82.7 0.97 N 0.97 8.15 N 8.15 91.3 N 91.3 Loss: SC35 None found No change detected c.270TNC p.Phe90Phe In middle exon None found None found No change detected c.957ANG p.Gly319Gly 58 nt from 3' ss 95.9 N 95.9 0.98 N 0.98 8.61 N 8.61 94.4 N 94.4 SC35: 3.46 N 3.82 (+10%) Loss 2 ESE No change detected c.1331TNC p.Val444Ala 29 nt from 3' ss 92.9 N 92.9 0.99 N 0.99 12.39 N 12.39 94.7 N 94.7 SC35: 3.23 N 3.8 (+17%) Loss 1 ESE No change detected c.2029ANG p.Met677Val 46 nt from 5' ss 93.9 N 93.9 1.00 N 1.00 10.22 N 10.22 97.0 N 97.0 Loss: SRp40 Gain: SF2/ ASF None found No change detected c.3084ANG p.Ala1028Ala 27 nt from 3' ss 87.9 N 87.9 0.86 N 0.96 9.34 N 9.34 89.2 N 89.2 SRp55: 2.93 N 3.54 (+20%) SRp40: 2.86 N 4.30 (+50%) Loss 1 ESE No change detected c.3258ANG p.Thr1086Thr 44 nt from 3' ss 81.1 N 81.1 0.97 N 0.97 10.90 N 10.90 91.0 N 91.0 Gain SRp55 SRp55: 4.55 N 5.16 (-13%) None found No change detected In bold: disease-causing mutation. In italics: common variation. WT, wild type; MUT, mutant; ss, splice site; SRp, serine/arginine-rich protein, a family of conserved splicing factors. Login to comment
97 In accordance to in silico predictions and minigene system's results, no splice defect due to the p.Phe90Phe, p.Val444Ala and p.Met677Val polymorphic variants was observed. Login to comment
130 Patient 4 (P4) harbored heterozygous variants p.Gly319Gly on exon 10 (fragment C), p.Met677Val on exon 17 (fragment E) and p.Ala1028Ala on exon 24 (fragment G). Login to comment
134 Among those identified in the five PFIC2 patients, Byrne et al. [7] reported that some variants (p.Met677Val, p.Phe90Phe) had no effect on splicing while others affected splicing either mildly (p.Gly319Gly, p.Arg1128Cys), or severely (p.Ala1028Ala). Login to comment