ABCMdb

ABCB4 p.Arg652Gly

Predicted by SNAP2:	A: N (53%), C: D (59%), D: D (85%), E: N (53%), F: D (80%), G: D (80%), H: D (75%), I: N (57%), K: N (72%), L: D (53%), M: N (57%), N: N (66%), P: D (85%), Q: N (61%), S: N (78%), T: N (72%), V: D (80%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: 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
146 For the ABCB4 3826AϾG(R652G) polymorphism, there was a trend of lower MDR3 expression levels for the 3826AG(R652G) variant (1.03 Ϯ 0.43) compared with 3826AA (R652R) (0.90 Ϯ 0.56) (P ϭ .086) (Fig. 5B), but low and high MDR3 expression phenotypes were similarly distributed between the two variants (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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110 Five coding region changes were single nucleotide polymorphisms (synonymous: exon 4: C175T, exon 6: C504T, exon 8: A711T; nonsynonymous: exon 6: A523G 3 T175A and exon 16: A1954G 3 R652G). Login to comment
98 MDR3 Genetic Variability Variant Number Amplicon DNA Position cDNA Position Nucleotide Reference Nucleotide Variant AA Change Control Group n ‫؍‬ 112 PBC n ‫؍‬ 152 PSC n ‫؍‬ 92 Pro1 amplicon -3 intron -4 (-394) T G 0.048 0.052 0.058 Pro2 amplicon -3 exon -3 (-410) T C non-coding 0.049 0.051 0.071 Pro3 amplicon -3 exon -3 (-387) C A non-coding 0.008 0.000 0.000 Pro4 amplicon -2 exon -2 (-229) C T non-coding 0.114 0.103 0.152 Pro5 amplicon -1 intron -2 (-221) C T 0.111 0.109 0.152 Pro6 amplicon -1 intron -2 (-204) A G 0.135 0.101 0.152 Pro7 amplicon -1 exon -1 (-44) A C non-coding 0.016 0.000 0.000 Pro8 amplicon 1 intron -1 (-301) C G 0.175 0.147 0.217 Pro9 amplicon 1 intron -1 (-220) C T 0.048 0.051 0.065 Pro10 amplicon 1 intron -1 (-201) C G 0.135 0.110 0.163 Pro11 amplicon 1 intron -1 (-184) T C 0.262 0.353 0.233 Pro12 amplicon 1 intron -1 (-86) C A 0.008 0.000 0.000 4.1. amplicon 4 exon 4 175 C T syn 0.127 0.123 0.156 4.2. amplicon 4 exon 4 217 C G L73V_c 0.000 0.007 0.000 5.1. amplicon 5 intron 4 (-92) C T 0.034 0.007 0.033 5.2. amplicon 5 intron 5 (ϩ113) A G 0.164 0.167 0.217 6.1. amplicon 6 intron 5 (-66t to -62) AGAAA delAGAAA 0.079 0.021 0.014 6.2. amplicon 6 exon 6 504 C T syn 0.571 0.493 0.524 6.3. amplicon 6 exon 6 523 A G T175A_c 0.032 0.014 0.012 7.1. amplicon 7 intron 6 (-56) T C 0.000 0.007 0.000 7.2. amplicon 7 intron 6 (-44) C A 0.000 0.000 0.012 8.1. amplicon 8 intron 7 (-61) C G 0.008 0.000 0.000 8.2. amplicon 8 exon 8 711 A T syn 0.151 0.169 0.239 8.3. amplicon 8 exon 8 728 A C D243A_c 0.000 0.007 0.000 9.1. amplicon 9 exon 9 927 T C syn 0.008 0.000 0.000 10.1. amplicon 10 exon 10 1099 A G I367V_c 0.008 0.000 0.000 12.1. amplicon 12 intron 11 (-88) T delT 0.070 0.081 0.116 12.2. amplicon 12 exon 12 1304 A C K435T_c 0.000 0.007 0.000 12.3. amplicon 12 intron 12 (ϩ130) G T 0.065 0.083 0.100 13.1. amplicon 13 intron 12 (-40) A G 0.952 0.944 0.930 14.1. amplicon 14 exon 14 1633 C T R545C_c 0.000 0.000 0.011 15.1. amplicon 15 intron 14 (-39) A G 0.008 0.007 0.011 15.2. amplicon 15 exon 15 1769 G A R590Q_c 0.008 0.000 0.000 15.3. amplicon 15 intron 15 (ϩ6) T C splicing 0.000 0.007 0.000 16.1. amplicon 16 exon 16 1954 A G R652G 0.073 0.086 0.128 16.2. amplicon 16 intron 16 (ϩ55) A G 0.053 0.071 0.128 17.1. amplicon 17 intron 17 (ϩ16) C T 0.951 0.913 0.955 20.1. amplicon 20 intron 20 (ϩ40) A G 0.063 0.092 0.102 23.1. amplicon 23 intron 22 (-38) T delT 0.008 0.000 0.000 23.2. amplicon 23 intron 23 (ϩ92) G delG 0.000 0.000 0.032 26.1. amplicon 26 exon 26 3296 A G E1099G_c 0.018 0.000 0.000 26.2. amplicon 26 intron 26 (ϩ11) insA 0.000 0.000 0.024 27.1. amplicon 27 intron 26 (-16) T C 0.918 0.911 0.939 28.1. amplicon 28 intron 27 (-72) T C 0.073 0.090 0.065 28.2. amplicon 28 exon 28 3751 A C G1251Q_c 0.000 0.007 0.000 NOTE. Variants are numbered sequentially by exon. Login to comment
112 Alignment of all mammalian MDR3 sequences indicated that with the exception of R652G all nonsynonymous coding variants were in codons for an evolutionarily conserved amino acid. 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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75 Unauthorized reproduction of this article is prohibited. Table 2 MDR3 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 amplicon -3 Intron -4 (-394) T G 0.050 0.066 Pro 2 Amplicon -3 Intron -4 (-394) insA 0.025 0.000 Pro 3 Amplicon -3 Intron -4 (-10) C T 0.028 0.000 Pro 4 Amplicon -3 Exon -3 (-410) T C Non-coding 0.053 0.066 Pro 5 Amplicon -3 Exon -3 (-358) A G Non-coding 0.026 0.000 Pro 6 Amplicon -2 Intron -3 (-31) C T 0.024 0.000 Pro 7 Amplicon -2 Exon -2 (-229) C T Non-coding 0.095 0.154 Pro 8 Amplicon -2 Intron -2 (þ234) C T 0.024 0.000 Pro 9 Amplicon -1 Intron -2 (-221) C T 0.095 0.150 Pro 10 Amplicon -1 Intron -2 (-204) A G 0.095 0.150 Pro 11 Amplicon 1 Intron -1 (-301) C G 0.167 0.225 Pro 12 Amplicon 1 Intron -1 (-292) G A 0.000 0.013 Pro 13 Amplicon 1 Intron -1 (-285) T C 0.000 0.013 Pro 14 Amplicon 1 Intron -1 (-220) C T 0.071 0.063 Pro 15 Amplicon 1 Intron -1 (-201) C G 0.167 0.138 Pro 16 Amplicon 1 Intron -1 (-184) T C 0.333 0.275 Pro 17 Amplicon 1 Exon 1 (-10) C A Non-coding 0.024 0.000 4.1. Amplicon 4 Exon 4 175 C T Syn 0.095 0.154 5.1. Amplicon 5 Intron 4 (-61) C T 0.000 0.025 5.2. Amplicon 5 Intron 5 (þ113) A G 0.167 0.213 6.1. Amplicon 6 Intron 5 (-62) AGAAA delAGAAA 0.026 0.027 6.2. Amplicon 6 Exon 6 459 T C Syn 0.026 0.000 6.3. Amplicon 6 Exon 6 504 C T Syn 0.525 0.500 6.4. Amplicon 6 Exon 6 523 A G T175A_c 0.025 0.000 8.1. Amplicon 8 Exon 8 711 A T Syn 0.167 0.213 9.1. Amplicon 9 Intron 8 (-36) T G 0.000 0.016 9.2. Amplicon 9 Exon 9 959 C T S320F_c 0.050 0.000 12.1. Amplicon 12 Intron 11 (-88) T delT 0.125 0.103 12.2. Amplicon 12 Intron 11 (-70) A G 0.000 0.015 12.3. Amplicon 12 Intron 12 (þ86) G A 0.024 0.000 12.4. Amplicon 12 Intron 12 (þ130) G T 0.067 0.227 13.1. Amplicon 13 Intron 12 (-40) G A 0.950 0.919 14.1. Amplicon 14 Intron 13 (-198) A C 0.000 0.013 14.2. Amplicon 14 Exon 14 1584 G C E528D 0.000 0.013 16.1. Amplicon 16 Exon 16 1954 A G R652G 0.100 0.163 16.2. Amplicon 16 Intron 16 (þ55) A G 0.100 0.145 17.1. Amplicon 17 Intron 17 (þ16) T C 0.975 0.879 18.1. Amplicon 18 Intron17 (-139) A G 0.024 0.000 18.2. Amplicon 18 Exon 18 2285 G A G762E_c 0.024 0.000 19.1 Amplicon 19 Exon 19 2324 C T T775M_c 0.000 0.013 20.1. Amplicon 20 Intron 20 (þ40) A G 0.100 0.176 21.1. Amplicon 21 Intron 21 (þ1) G A Splicing 0.025 0.000 21.2. Amplicon 21 Intron 21 (þ98) insTGT 0.000 0.013 21.3. Amplicon 21 Intron 21 (þ158) T C 0.150 0.064 23.1. Amplicon 23 Intron 23 (þ65) T A 0.000 0.013 25.1. Amplicon 25 Intron 25 (þ5) G C Splicing 0.029 0.000 26.1. Amplicon 26 Intron 25 (-3) C G Splicing 0.028 0.000 26.2. Amplicon 26 Intron 26 (þ2) T A Splicing 0.028 0.000 26.3. Amplicon 26 Intron 26 (þ53) A G 0.000 0.013 27.1. Amplicon 27 Intron 26 (-16) T C 0.921 0.871 28.1. Amplicon 28 Intron 27 (-72) T C 0.125 0.186 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_000443. Login to comment
110 Alignment of all mammalian MDR3 sequences indicated that, with the exception of E528D and R652G, all non-synonymous coding variants were in codons for an evolutionarily conserved amino acid. Login to comment
142 Q R I K R I Q I D F H G I E H D T T E L H S I D D T L K I S E G I G D K R Q R K F F H A I L R G A V A G V T R I G G Q H L E K A Q K H L L G A E I F E YA R R T L I E G L S H A F K A H R D F H S H D Q D K H S T G A L A Q V Q G A T G T R L R S R H G A L L K R E I A E T A T S L T Q E R V Y H S E F L P Y R H S V Q K K I K D E L E A A G H E L A K A Y D A T Q G K K V Q E P I L I E A I S C Q Q R I A I E V V Q G L S L P A K L S H KL H A D T A L R T A K K A I H V V K E Y G K K F D A R V K Q Q R I L A P V F Q G G A V Q H T G H E Q H L K A A S C S S G V L L A Q L G I R H D G Y A G Q L S G G P R A H V P F G R S TT A D L L L I E I H A D L I Q I K Q A L L G V V H S F Y S L Q R E F L Q V T S K G K L H V Q H I D Q S V V E R V G D K H V V F H Y P V K E TE E D L A S T T I H R L S T A E S Y S D I D F K E L L L D G Q E A A K A A A V E L P K K Y F G H I T F E Y L A TE E V V K E S Q E R T C I V G D A L K R A H H I P I F A P L G V T K G L P K D Y F H D L E R H A T G H K P T S E L G I S S K F D G V T K T K K R K H I F R Y S D H Q L T L D K A A Q K A E G V F V D K G A T H F S S L S F H V H L L P K K H T R E E E F P I H A Y Y Y Y H T D S R G H K A I I E S K Q Q K C H I F VG P G D D A F R F C G H R F R D V G A HY L I V H G Y G H Q V F G I GE L H H P S G L G I A H A T T H G H S LFL G A G L A Y I A T L V L S P I I I G HT T V F H S I L F G A F I V G Q S A P C A F S LI L F L F I I S G F T F F L Q G F T F G F A T F V G H F V Y G S H L A F A A S Y Y L L I F I FV A F S P G L Q G I A H A T V C G A H L I A L I L S I S F H I Y A T L L L V F I A IS F G A V A LV H AG S S F G I T Q A F H Y F S Y A L L L V V A I I P V S A I V G A Q H G T G I I I S F I F V V YG I A H F Q Q F A G F I V G F I A K A Y E K D L S F A S L A E A R E A H E K G I K I K A ISAH I E E T D L D H R V T E H K K E S V K F Y V G D K A A T V A H R L S A L L EI K H P A H G H Q K S L D C L A Q V E A D F G A I V V Q G S H S S T Q H H S E F E L H R G R T T I A E E G EE H F L R S K R K L K H S Q H S T QQ QG ID A IVK K G G S L L E A T S Q D R I A I R Q P K I L L A H A V L A V E R G A G P K F D T L Q L E V A K K R I E Y A H A F H H T K D E V H C Y G R G I E E S V V G Q T S F L V P T I H L R E I Y D R H F H V I Q P Y L R Q D I I T G E D H D S T V Q L T G S G C G K S V K H L G K L V Q G S Q V T A R H V K I A V F S Y P S H D P H G E K L G K I S D H E KAGLHF QIIILS F S D I K P H H D I I D V P P V H A E L G D T E V F V I I A A G R A H A F A D P K V L K F H K T E H L R R S L Q T G A Q H IAL V I T S H A I G L L A A S H A V L V V V S Q H T F A A L S K E Y I K T175A E528D T775M R652G S Cytoplasm R T G762E S320F Extracellular K Fig. 2 Secondary structure of multidrug resistance protein 3 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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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
63 The numbers 1 to 42 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 KO JA Total n % n % n % n % 43 TCAATGCAC g.-7676AϾT GTCTCACAA PromA 110 0.9 96 0.0 88 0.0 294 0.3 44 CTACCCTCT g.-7554TϾC CAATGCCTC PromA 110 0.9 96 0.0 88 0.0 294 0.3 45 GAGTGAAGT g.-7253GϾA TAGAAATCT PromA 106 0.0 96 1.0 94 0.0 296 0.3 46 AATTTAGAA g.-7114AϾT ACTCAATAG PromA 108 0.0 96 1.0 94 0.0 298 0.3 1 AAGAGGAAA g.-7094GϾC TTTCTTGTA PromA 108 13.0 96 30.2 94 24.5 298 22.1 47 CAAGAATTT g.-6816CϾT ATTAGGCAA PromA 102 0.0 96 1.0 92 0.0 290 0.3 48 GAGAGAGAG g.-6639AϾC GAGCTGAAT PromA 110 0.9 90 0.0 92 0.0 292 0.3 49 GAGAGAGAG g.-6637_-6636 delAG CTGAATCAG PromA 110 0.0 90 1.1 92 0.0 292 0.3 2 GTGCCTTTG g.-6588GϾT GTGTGCTGG PromA 110 2.7 88 0.0 92 2.2 290 1.7 3 AAAGAAGAA g.-6540CϾT GAAACCAAA PromA 108 14.8 86 26.7 90 27.8 284 22.5 4 TTAGTGACC g.-6325AϾG AAAGTTTGG PromA 108 17.6 90 31.1 92 26.1 290 24.5 5 ATTCTTTTT g.-6014GϾT TACAAACCC PromA 108 16.7 94 28.7 88 26.1 290 23.4 50 ACTGGTGCT g.-5941GϾA TGGGCACTA PromA 108 0.0 94 0.0 88 1.1 290 0.3 6 TGAAGTCAC g.-5859GϾA TGGCCAGAG PromA 108 16.7 94 28.7 88 26.1 290 23.4 7 ATGAGATGA g.-5717TϾC ATATATGTG PromA 110 0.0 92 1.1 86 3.5 288 1.4 8 CCTTCTTTA g.-5610TϾC ATGCCTAAA PromA 110 100.0 96 100.0 94 97.9 300 99.3 9 TAAGTGTGG g.-5570GϾC CAGCAATTA PromA 110 12.7 96 29.2 94 24.5 300 21.7 51 TACTCTCAC g.-5509GϾA GCTCTTATG PromA 110 0.0 96 1.0 94 0.0 300 0.3 10 CTCTCTTGT g.-5236CϾT TGAGTAATA PromA 108 15.7 90 27.8 94 26.6 292 22.9 52 GAGGATAAA g.-2551AϾT AAGAAAGAT PromB 126 0.0 88 0.0 90 1.1 304 0.3 11 AGCCTTACA g.-2478TϾG CAATGCATA PromB 126 4.8 88 0.0 90 2.2 304 2.6 12 GAAGGAATT g.-1921TϾC GGGTTGATT PromB/Exon -3 122 4.9 92 0.0 82 1.2 296 2.4 53 GAAGAGAAT g.-1899CϾA CTCATGGTC PromB/Exon -3 122 0.8 92 0.0 82 0.0 296 0.3 13 ATCCTAATA g.-1603AϾT CACCCTTAT PromB 128 0.0 94 2.1 86 1.2 308 1.0 14 TTTATAGAT g.-1584CϾT CAATGACTG PromB/Exon -2 118 11.0 94 29.8 74 23.0 286 20.3 54 ACACCAGGG g.-1510TϾG CCACCCAGC PromB 126 0.0 94 1.1 68 0.0 288 0.3 15 CTTATACCA g.-1484TϾC GCTCTGCTT PromB 126 0.0 94 1.1 68 5.9 288 1.7 55 TTTGAAAGT g.-1146CϾT TCCGGTTTC PromB 126 0.0 92 1.1 82 0.0 300 0.3 16 TGGTAGGAG g.-1031CϾT AGAGACAAT PromB 126 11.1 92 30.4 82 24.4 300 20.7 56 GAGACAATT g.-1020CϾG AATACAGAC PromB 126 0.0 92 1.1 82 0.0 300 0.3 17 ATTCAATAC g.-1014AϾG GACAGAAGT PromB 126 13.5 92 30.4 82 24.4 300 21.7 18 GAACTGGGG g.-682AϾC TGCGGAAGC PromB/Exon -1 124 1.6 70 0.0 64 0.0 258 0.8 19 AGGCTCCAG g.-495CϾG CTGATCTCG PromB 126 17.5 86 29.1 84 28.6 296 24.0 20 GCGCCCCGG g.-414CϾT GGCAAGAGC PromB 126 4.8 86 3.5 84 6.0 296 4.7 21 GGCAGGCTG g.-395CϾG GCCCCTGGC PromB 126 13.5 86 3.5 84 6.0 296 8.4 22 GCCCGCGCC g.-378TϾC AGCCTGGGG PromB 126 26.2 86 27.9 84 20.2 296 25.0 57 GCGTTTCCC g.-292GϾT GGCCGGACG PromB 128 0.0 96 0.0 92 1.1 316 0.3 58 CCGGACGCG g.-280CϾA GTGGGGGGC PromB 126 0.8 86 0.0 84 0.0 296 0.3 59 CCCTGCCAG g.-186AϾG CACGCGCGA PromB/Exon 1 128 0.0 96 1.0 92 0.0 316 0.3 23 TGCCCCCGG g.-75GϾT CCCCGCGAC PromB 128 0.0 96 0.0 92 1.1 316 0.3 24 TTTATGTCG g.12597CϾT TGGGTACCA Exon 4 L59L 126 12.7 96 25.0 94 21.3 316 19.0 60 CAAATTTGT g.12680TϾG GATACTGCA Exon 4 V86V 126 0.0 96 0.0 94 1.1 316 0.3 61 ATTTGTTGA g.12683TϾG ACTGCAGGA Exon 4 D87E 126 0.0 96 0.0 94 1.1 316 0.3 62 TTCTCCTTT g.12705CϾT CAGGTAAGC Exon 4 P95S 126 0.0 96 0.0 94 1.1 316 0.3 63 TAGCTTTCA g.20782TϾG ACATTTAAA Intron 4 114 0.0 88 1.1 70 0.0 272 0.4 25 TTTTAAAAA g.20813CϾT CTGGCAATG Intron 4 116 3.4 90 1.1 70 0.0 276 1.8 26 TCACCTATT g.21044AϾG TTATCATTT Intron 5 122 16.4 52 15.4 46 32.6 220 19.5 27 AAAAGAAAA g.22281_22284delGAAAA AAGAAAAGA Intron 5 126 7.9 88 0.0 84 0.0 298 3.4 28 TGACATCAA g.22490CϾT GACACCACT Exon 6 N168N 126 42.9 88 37.5 90 44.4 304 47.7 29 GAACTCAAT g.22509AϾG CGCGGCTAA Exon 6 T175A 126 3.2 88 0.0 90 0.0 304 1.3 64 CTCTGCAGC g.23831CϾT GTTTGGGCA Exon 7 A232A 128 0.0 94 0.0 90 1.1 312 0.3 65 AAGGGTTGA g.25313CϾG CAGAGTGCC Intron 7 124 0.8 96 0.0 90 0.0 310 0.3 30 TGTCCAGAT g.25376AϾT CTCTCGGCA Exon 8 I237l 126 15.1 96 27.1 90 27.8 312 22.4 66 GTTAATATA g.28354TϾC GCATCATAT Exon 9 Y309Y 128 0.8 96 0.0 92 0.0 316 0.3 67 GCATATGTG g.30584AϾG TCTTTGATA Exon 10 I367V 118 0.8 92 0.0 92 0.0 302 0.3 68 TAATATTTA g.31823TϾG AGGAATTCC Intron 11 128 0.0 96 0.0 92 1.1 316 0.3 31 ACTTTTTTT g.31941delT CAAATTTCA Intron 11 128 7.0 96 3.1 94 1.1 318 4.1 69 ACCCTGATG g.32140AϾG GGGCACAGT Exon 12 E450G 128 0.0 96 1.0 94 0.0 318 0.3 70 ACAAATTTG g.32232CϾT GTGTGAATC Intron 12 128 0.0 96 1.0 94 0.0 318 0.3 32 GGCAATGCC g.32277GϾT ATGGATAAT Intron 12 124 6.5 96 3.1 94 3.2 314 4.5 33 CAGCTATTA g.35024AϾG ATGGTTAAA Intron 12 124 95.2 94 70.2 94 72.3 312 80.8 71 ACAGGTAAA g.35273GϾA CCTCTGATA Intron 13 126 0.0 96 0.0 94 1.1 316 0.3 72 AGTGTGCCA g.43862AϾG TACTGTAAC Intron 14 122 0.8 88 0.0 72 0.0 282 0.4 34 TGTGCCAAT g.43864AϾG CTGTAACCC Intron 14 124 0.0 88 1.1 72 2.8 284 1.1 73 TAGCACACC g.43938GϾA ACTGTCTAC Exon 15 R590Q 124 0.8 88 0.0 74 0.0 286 0.3 35 GCTGCCACT g.48606AϾG GAATGGCCC Exon 16 R652G 124 7.3 86 2.3 74 1.4 284 4.2 36 GCTACAATT g.48771AϾG TTGAAATTC Intron 16 114 5.3 86 2.3 70 1.4 270 3.3 37 TTGCAAACA g.51576CϾT CACATAACA Intron 17 122 95.1 70 74.3 80 71.3 272 82.7 38 TTACATAAC g.56969AϾG TGGTTTTAG Intron 20 126 6.3 60 3.3 66 1.5 252 4.4 74 ATATTTTAC g.58304TϾC GTATTAATG Intron 21 124 0.0 96 0.0 92 1.1 212 0.3 39 CTGTATTAA g.58312TϾC GTCTAGAAC Intron 21 122 4.1 96 1.0 92 1.1 310 2.3 75 AAAGGAGGC g.63395delT GAAGAGATG Intron 22 124 0.8 92 0.0 94 0.0 310 0.3 76 AATATTAAG g.63598AϾT TTATTCTAT Intron 23 124 0.0 92 1.1 94 0.0 310 0.3 40 ATGGTCAAG g.68988AϾG AGCAAAGAA Exon 26 E1099G 112 1.8 92 0.0 84 0.0 288 0.7 41 TATTTATAA g.72169TϾC TTGGTTAAC Intron 26 122 91.8 90 65.6 92 75.0 304 78.9 42 GTAACATTT g.73092TϾC CAAATTTAC Intron 27 124 7.3 94 1.1 86 1.2 304 3.6 n, number of alleles analyzed (number of subjects times 2). 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110 These included three Caucasian variants in exon 6 (c.523AϾG; allele frequency 3.2%), exon 16 (c.1954AϾG; 7.3%), and exon 26 (c.3296AϾG; 1.8%), resulting in amino acid substitutions p.T175A, p.R652G, and p.E1099G, respectively. Login to comment
145 In contrast, ABCB4 p.R652G is a more common variant, which changes an evolutionary conserved amino acid to an evolutionary less acceptable one with probably altered physicochemical properties. Login to comment
146 On the other hand, SIFT and PSIC scores of this variant do not confirm functional consequences of ABCB4 p.R652G. Login to comment
163 ABCB4 p.R652G was the only protein-altering variant with high allele frequency in all groups (7.2% in Caucasian, 1.4% in Japanese, and 2.3% in Korean). Login to comment
271 Only two variants from our study had been related to cholestatic disease earlier, ABCB4 c.523AϾG (p.T175A), the second most prevalent nonsynonymous change in Caucasians (Rosmorduc et al., 2001), and the most prevalent nonsynonymous variant, ABCB4 c.1954GϾA (p.R652G), which was present in all of our population samples (Jacquemin et al., 2001). Login to comment
283 For instance, the MDR3 variant p.R652G was present in PFIC3 patients and healthy subjects and hence cannot, alone, be sufficient to cause cholestasis. Login to comment
284 It was suggested that p.R652G had no or mild consequences under normal conditions, but resulted in cholestasis during pregnancy (Jacquemin et al., 2001). 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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142 With the exception of the I764L, which was located in the eighth transmembrane domain of MDR3, all other nonsynonymous variants were Table 6 ABCB4 (MDR3)1 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 = 112) Amplicon - 3 Intron - 4 - 394 T G 0.02 0.00 0.05 Amplicon - 3 Intron - 4 - 197 G T 0.00 0.04 0.00 Amplicon - 3 Exon - 3 - 410 T C Noncoding 0.02 0.00 0.05 Amplicon - 2 Exon - 2 - 228 C T Noncoding 0.04 0.08 0.11 Amplicon - 1 Intron - 2 - 221 C T 0.04 0.08 0.11 Amplicon - 1 Intron - 2 - 204 A G 0.04 0.08 0.13 Amplicon 1 Intron - 1 - 301 C G 0.07 0.08 0.17 Amplicon 1 Intron - 1 - 220 C T 0.02 0.00 0.05 Amplicon 1 Intron - 1 - 201 C G 0.04 0.08 0.13 Amplicon 1 Intron - 1 - 184 T C 0.33 0.46 0.26 Amplicon 4 Exon 4 175 C T syn 0.05 0.08 0.13 Amplicon 5 Intron 4 - 61 C T 0.11 0.08 0.03 Amplicon 5 Intron 5 113 A G 0.07 0.08 0.16 Amplicon 6 Intron 5 - 62 AGAAA delAGAAA 0.05 0.04 0.08 Amplicon 6 Intron 5 - 50 GAAA delGAAA 0.02 0.00 0.00 Amplicon 6 Exon 6 504 C T syn 0.62 0.46 0.57 Amplicon 6 Exon 6 523 A G T175A_c 0.02 0.00 0.03 Amplicon 8 Exon 8 711 A T syn 0.07 0.08 0.15 Amplicon 11 Intron10 - 43 T C 0.02 0.00 0.00 Amplicon 12 Intron 11 - 88 T delT 0.05 0.00 0.07 Amplicon 12 Intron 11 - 81 T delT 0.04 0.04 0.00 Amplicon 12 Exon 12 1314 G A syn 0.00 0.04 0.00 Amplicon 12 Intron 12 130 G T 0.08 0.04 0.06 Amplicon 13 Intron 12 - 40 G A 0.02 0.00 0.05 Amplicon 15 Intron 14 - 39 A G 0.02 0.08 0.01 Amplicon 15 Exon 15 1769 G A R590Q_c 0.02 0.00 0.01 Amplicon 16 Exon 16 1954 A G R652G 0.09 0.04 0.07 Amplicon 16 Intron 16 55 A G 0.09 0.04 0.05 Amplicon 17 Intron 17 16 T C 0.03 0.00 0.05 Amplicon 18 Exon 18 2290 A C I764L_c 0.02 0.00 0.00 Amplicon 20 Intron 20 40 A G 0.11 0.00 0.06 Amplicon 23 Intron 23 70 G T 0.00 0.04 0.00 Amplicon 25 Exon 25 3245 T A L1082Q_c 0.00 0.04 0.00 Amplicon 27 Intron 26 - 16 T C 0.35 0.58 0.92 Amplicon 28 Intron 27 - 72 T C 0.11 0.00 0.07 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_000443.2. Login to comment
149 Of the coding region changes, eight have previously been described in healthy Caucasians, including three synonymous and two nonsynonymous changes (synonymous: exon 4: 175C > T, exon 6: 504C > T, exon 8: 711A > T; nonsynonymous: exon 6: 523A > G-T175A, exon 15: 1769G > A-R590Q and exon 16: 1954A > G-R652G). Login to comment
163 The estimated IC50 was 50 and 60 mmol/l for reference BSEP Fig. 4 Extracellular I764L T175A R652G R590Q Cytoplasm L1082Q Secondary structure of multidrug resistance protein 3 (MDR3) with nonsynonymous coding region variants. Login to comment

[hide] Clinical features and genotype-phenotype correlati... J Pediatr Gastroenterol Nutr. 2011 Jan;52(1):73-83. Colombo C, Vajro P, Degiorgio D, Coviello DA, Costantino L, Tornillo L, Motta V, Consonni D, Maggiore G
Clinical features and genotype-phenotype correlations in children with progressive familial intrahepatic cholestasis type 3 related to ABCB4 mutations.
J Pediatr Gastroenterol Nutr. 2011 Jan;52(1):73-83., [PMID:21119540]

Abstract [show]
OBJECTIVES: The aim of the study was to estimate the frequency of ABCB4 mutations among children with chronic intrahepatic cholestasis with elevated gamma-glutamyl-transpeptidase (gamma-GT) activity and to characterize the genotypes with respect to severity of symptoms, response to ursodeoxycholic acid therapy, and outcome. PATIENTS AND METHODS: Molecular analysis of ABCB4 in 133 Italian children was performed, and ABCB4 mutations were classified as disease-causing mutations or benign substitutions according to the prediction algorithm PolyPhen. RESULTS: : Twenty-eight patients were identified carrying 31 mutations (20 disease causing). Twenty patients carried 2 mutated alleles and 8 only 1. At presentation (1-204 months), 20 children were symptomatic with jaundice and/or pruritus, whereas in 8 biochemical cholestasis was a fortuitous finding. Cirrhosis developed in 15 and 6 progressed to terminal liver failure. Disease-causing mutations on both alleles were found to be associated with reduced liver expression of ABCB4 protein, lack of response to ursodeoxycholic acid therapy, and progression to cirrhosis and end-stage liver disease, whereas mild genotypes, including single heterozygous mutations, were generally associated with less severe disease and, often, absence of symptoms. CONCLUSIONS: ABCB4 mutations are responsible for a chronic liver disease in more than one-third of patients with chronic intrahepatic cholestasis and elevated gamma-GT activity. In patients with severe ABCB4 genotype, the disease is often progressive with risk of developing cirrhosis and liver failure during the first 2 decades of life. Patients with mild genotypes, including single heterozygous mutations, have variable expressions of liver disease that may be influenced by comorbidity factors and modulated by still unknown genetic modifiers.

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131 Distribution of ABCB4 SNPs in 68 patients with chronic intrahepatic cholestasis without ABCB4 mutations and 112 healthy subjects ABCB4 genotypes ABCB4 alleles SNPsÃ NCBI annotation Location Patients Controls Fisher P Patients Controls Fisher Pwt het homo wt het homo Allele 1 Allele 2 Allele 1 Allele 2 c.175 C>T (p.L59L) rs2302387 Exon 4 48 19 1 57 26 3 0.72 115 21 140 32 0.54 c.287-61 C>T rs45626341 Intron 4 64 4 0 97 5 0 1.00 132 4 199 5 1.00 c.504 T>C (p.N168N) rs1202283 Exon 6 13 36 19 28 56 20 0.29 62 74 112 96 0.15 c.711 A>T (p.I237I) rs2109505 Exon 8 48 17 3 66 37 3 0.34 113 23 169 43 0.48 c.834-66 G>T rs1014283 Intron 8 46 19 3 63 37 3 0.50 111 25 163 43 0.68 c.1231-81 delT rs3834727 Intron 11 58 8 1 94 6 0 0.15 124 10 194 6 0.07 c.1357-40 G>A Not annotated Intron 12 54 13 1 92 18 0 0.45 121 15 202 18 0.45 c.1954 A>G (p.R652G) rs2230028 Exon 16 59 8 1 94 15 0 0.47 126 10 203 15 1.00 c.2064þ55 A>G rs1017054 Intron 16 59 8 1 94 15 0 0.47 126 10 203 15 1.00 c.2211þ16 T>C rs31668 Intron 17 54 13 1 86 17 0 0.45 121 15 189 17 0.45 c.2478þ40 A>G rs788404 Intron 20 59 7 2 87 11 0 0.30 125 11 185 11 0.38 c.2479-66 delG rs5885584 Intron 20 59 7 2 91 17 0 0.11 125 11 199 17 1.00 c.2683-197 T>C rs17149547 Intron 21 59 7 2 85 11 0 0.34 125 11 181 11 0.50 c.3508-16 C>T rs31653 Intron 26 54 13 1 88 18 1 0.85 121 15 194 20 0.71 c.3655-72 T>C rs45549641 Intron 27 60 6 2 81 11 0 0.24 126 10 173 11 0.65 Ã Positions are based on translation start site of ATG (NM_018849). Login to comment

[hide] Multidrug resistance protein 3 R652G may reduce su... World J Gastroenterol. 2009 Dec 14;15(46):5855-8. Chen XQ, Wang LL, Shan QW, Tang Q, Lian SJ
Multidrug resistance protein 3 R652G may reduce susceptibility to idiopathic infant cholestasis.
World J Gastroenterol. 2009 Dec 14;15(46):5855-8., [PMID:19998509]

Abstract [show]
AIM: To evaluate the role of genetic factors in the pathogenesis of idiopathic infant cholestasis. METHODS: We performed a case-control study, including 78 infants with idiopathic infant cholestasis and 113 healthy infants as controls. Genomic DNA was extracted from peripheral venous blood leukocytes using phenol chloroform methodology. Polymerase chain reaction was used to amplify the multidrug resistance protein 3 (MDR3) R652G fragment, and products were sequenced using the ABI 3100 Sequencer. RESULTS: The R652G single nucleotide polymorphism (SNP) was significantly more frequent in healthy infants (allele frequency 8.0%) than in patients (allele frequency 2.60%) (P < 0.05), odds ratio, 0.29; 95% confidence interval, 0.12-0.84. The conjugated bilirubin in patients with the AG genotype was significantly lower than in those with the AA genotype (44.70 +/- 6.15 micromol/L vs 95.52 +/- 5.93 micromol/L, P < 0.05). CONCLUSION: MDR3 R652G is negatively correlated with idiopathic infant cholestasis. Children with the R652G SNP in Guangxi of China may have reduced susceptibility to infant intrahepatic cholestasis.

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7 Polymerase chain reaction was used to amplify the multidrug resistance protein 3 (MDR3) R652G fragment, and products were sequenced using the ABI 3100 Sequencer. Login to comment
8 RESULTS: The R652G single nucleotide polymorphism (SNP) was significantly more frequent in healthy infants (allele frequency 8.0%) than in patients (allele frequency 2.60%) (P < 0.05), odds ratio, 0.29; 95% confidence interval, 0.12-0.84. Login to comment
10 CONCLUSION: MDR3 R652G is negatively correlated with idiopathic infant cholestasis. Login to comment
11 Children with the R652G SNP in Guangxi of China may have reduced susceptibility to infant intrahepatic cholestasis. Login to comment
13 Key words: Multidrug resistance protein 3; Single nucleotide polymorphisms; R652G; Infant; Cholestasis Peer reviewer: Tamara Alempijevic, MD, PhD, Assistant Professor, Clinic for Gastroenterology and Hepatology, Clinical Centre of Serbia, 2 Dr Koste Todorovica St., Belgrade 11000, Serbia Chen XQ, Wang LL, Shan QW, Tang Q, Lian SJ. Login to comment
14 Multidrug resistance protein 3 R652G may reduce susceptibility to idiopathic infant cholestasis. World J Gastroenterol 2009; 15(46): 5855-5858 Available from: URL: http://www.wjgnet.com/1007-9327/15/ 5855.asp DOI: http://dx.doi.org/10.3748/wjg.15.5855 INTRODUCTION Idiopathic infant cholestasis is of unknown etiology and can occur in either a sporadic or a familial form. Login to comment
18 Studies have investigated the involvement of MDR3 variants in the development of disease, and we were interested to note that the MDR3 R652G single nucleotide polymorphism (SNP) has a high allele frequency in generally healthy people[7] . Login to comment
19 Our aim was to study the MDR3 R652G SNP distribution frequency in idiopathic infant cholestasis and healthy infants and to determine whether there were any differences between them and, if so, their relevance. Login to comment
24 Polymerase chain reaction (PCR) amplified the MDR3 R652G and sequence analysis Genomic DNA was extracted from peripheral venous blood leukocytes using standard phenol chloroform procedures. Login to comment
26 Primers for MDR3 R652G were forward (5'-CATCCATTTGGAGACACACACAC-3') and reverse (5'-GTAGCAGTCATCTGTGCCTGAA A-3')[7] . Login to comment
27 The primary PCR product fragments were 348 bp. PCRs for generating the R652G fragments were generally performed in a reaction volume of 50 μL with 100 ng of genomic DNA, 1.5 U of Taq polymerase (Fermentas), 10 × PCR buffer (Fermentas), 1.5 mmol/L of MgCl2 (Fermentas), 200 μmol/L deoxynucleoside- 5-triphosphate (Takara) and 20 μmol of each primer. Login to comment
32 Comparison of the frequency of the R652G SNP was made between patients and controls, and the analysis of association with the phenotype was performed by χ 2 test or Fisher`s exact test when appropriate. Login to comment
44 PCR products sequence analysis showed a heterozygous substitution A>G (Figure 1) in codon 652 which creates an amino acid substitution in codon R652G in exon 16. Login to comment
46 Test results showed that the R652G genotype distribution in the 2 groups was in line with the Hardy-Weinberg equilibrium, indicating that the selected sample was representative of the population. Login to comment
51 Table 2 MDR3 R652G genotypes and allele frequencies in patients and controls n (%) Groups n Genotypes Allele P-value A/A A/G G/G frequency (%) Patients 78 74 (94.9) 4 (5.1) 0 2.6 < 0.051 Controls 113 95 (84.1) 18 (15.9) 0 8.0 1 Fisher`s exact test between patients and controls for genotype frequency. Login to comment
53 A A G G C T G C C A C T N G A A T G G C C C C A A A Figure 1 Sequence of the multidrug resistance protein-3 (MDR3) single nucleotide polymorphism R652G (A>G). Login to comment
57 DISCUSSION Our study is the first report of the MDR3 R652G SNP in children in China. Login to comment
58 Our data showed that the R652G SNP had a significantly higher proportional distribution in normal infants than in idiopathic infant cholestasis patients. Login to comment
61 For this reason, we can infer that the R652G variant has a specific protective effect in the normal population. Login to comment
62 In previous studies, in the analysis of MDR3 gene sequence variants in different countries, the R652G SNP was the only protein-altering variant with high allele frequency in all groups. Login to comment
65 R652G is a MDR3 gene mutation that results in non-synonymous amino acid substitutions but is not associated with disease. Login to comment
66 In Switzerland, a study showed that the MDR3 R652G variant was 7% in healthy Caucasians, 9% in drug-induced cholestatic patients and 4% in hepatocellular/mixed liver injury[9] . Login to comment
67 Pauli-Magnus et al[10] reported the R652G variant in 10% of intrahepatic cholestasis pregnancy cases and 16.3% in healthy controls. Login to comment
68 The previous studies indicated that the R652G variant was prevalent in the general population. Login to comment
69 Furthermore, studies of MDR3 R652G in a variety of diseases showed no evidence that the R652G variant was related to disease. Login to comment
70 In our study, the R652G variant was 15.9% in healthy children, with a higher frequency than in infant cholestasis patients. Login to comment
73 There is also another phenomenon whereby R652G may have a protective effect and reduce the risk of suffering from disease. Login to comment
74 Jacquemin et al[11] reported one patient with intrahepatic cholestasis of pregnancy carrying a R652G substitution, and whose biliary phospholipids were lower than other patients. Login to comment
78 A recent study of gallstones in sibling pairs and controls showed that R652G variant frequency was 18% in patients and 25% in controls. Login to comment
88 In conclusion, the MDR3 R652G SNP is negatively correlated with cholestasis (OR, 0.29, 95% CI, 0.12-0.84). Login to comment
89 Children in Guangxi of China with the R652G variant may have reduced susceptibility to infant intrahepatic cholestasis. Login to comment
94 However, an interesting phenomenon is that there was no positive evidence for the MDR3 R652G variant being involved in the pathogenesis of intrahepatic cholestasis. Login to comment
95 This study investigated the MDR3 R652G distributed allele frequency in idiopathic infant cholestasis and healthy infants to determine whether there were any difference and, if so, their relevance. Login to comment
98 In order to evaluate the role of the MDR3 R652G variant in the pathogenesis of idiopathic infant cholestasis, the authors analyzed the MDR3 R652G polymorphism in a case-control study in Guangxi Chinese infants. Login to comment
99 The authors found that the R652G variant was significantly more frequent in healthy infants than in patients. Login to comment
100 The results showed that the R652G variant has a protective effect in healthy infants and reduces the possibility of suffering from idiopathic infant cholestasis. Login to comment
104 MDR3 R652G and infant intrahepatic cholestasis was not the same as a previous study. Login to comment
106 Applications The study results suggest that the MDR3 R652G variant has a protective effect in healthy infants. Login to comment
107 This will give further information for comparing geographical regions, and it is very important to establish particular characteristics of MDR3 R652G that can be useful in the differential diagnosis of idiopathic infant cholestasis, and furthermore, may establish the influence of such an single nucleotide polymorphism (SNP) in prognosis. Login to comment
108 Terminology MDR3 R652G: MDR3 R652G is a SNP of the MDR3 in the 652 coding site. Login to comment
110 The R652G is a gene mutation where the adenine (A) mutates into guanine (G) which causes AGA>GGA resulting in arginine substitution by glycine (R652G). Login to comment

[hide] ABCB4 gene mutations and single-nucleotide polymor... J Med Genet. 2009 Oct;46(10):711-5. Epub 2009 Jul 6. Bacq Y, Gendrot C, Perrotin F, Lefrou L, Chretien S, Vie-Buret V, Brechot MC, Andres CR
ABCB4 gene mutations and single-nucleotide polymorphisms in women with intrahepatic cholestasis of pregnancy.
J Med Genet. 2009 Oct;46(10):711-5. Epub 2009 Jul 6., [PMID:19584064]

Abstract [show]
AIM: To evaluate the nature and frequency of ATP-binding cassette subfamily B member 4 (ABCB4) gene variants in a series of French patients with intrahepatic cholestasis of pregnancy (ICP). METHODS: In this prospective study, the entire ABCB4 gene coding sequence was analysed by DNA sequencing in 50 unrelated women with ICP defined by pruritus and raised serum alanine aminotransferase activity or bile acid concentration, with recovery after delivery. Genomic variants detected in patients with ICP were sought in 107 control pregnant women. Patients with ICP and controls were of Caucasian origin. RESULTS: Eight genomic variants were observed. One nonsense mutation (p.Arg144Stop) and two missense mutations (p.Ser320Phe and p.Thr775Met) were revealed each in one heterozygous patient. A third missense mutation (p.Arg590Gln) was detected in three heterozygous patients and in two homozygous patients also homozygous for a particular haplotype of three single-nucleotide polymorphisms (c.175C>T, c.504T>C, c.711A>T). The chromosomal frequency of the p.Arg590Gln variant was significantly different between the ICP and control group (7.0% vs 0.5%; p = 0.0017; OR 16.03, 95% CI 1.94 to 132.16). An association was also found between allele T of the c.504T>C silent nucleotide polymorphism and ICP (68.0% vs 53.7%; p = 0.017; OR 1.83, 95% CI 1.08 to 3.11). The chromosomal frequency of the p.Arg652Gly variant did not differ between the ICP and control group (p = 0.40). CONCLUSIONS: This study shows that 16% of Caucasian patients with ICP bear ABCB4 gene mutations, and confirms the significant involvement of this gene in the pathogenesis of this complex disorder.

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8 The chromosomal frequency of the p.Arg652Gly variant did not differ between the ICP and control group (p = 0.40). Login to comment
55 The sequence variant c.1954A.G, p.Arg652Gly was detected in three heterozygous patients with ICP, and in nine heterozygous and one homozygous control women. Login to comment
56 The distribution of the p.Arg652Gly alleles was not significantly different between the ICP and control group. Login to comment
74 Furthermore, Arg590 is well conserved during evolution (fig 1), and the missense mutation p.Arg590Gln is localised in the first ATP-binding domain of the ABCB4 protein, where it substitutes a basic amino acid for a Table 3 Allele frequencies of ABCB4 genomic variants in 50 patients with intrahepatic cholestasis of pregnancy (100 chromosomes) and 107 controls (214 chromosomes) Genomic variant Exon Allele identification ICP (n (%)) Controls (n (%)) p Value Deduced effect Protein domain c.175 C.T 4 C 86 (86) 180 (84.1) 0.66 Silent T 14 (14) 34 (15.9) c.462 C.T 6 C 99 (99) 214 (100) 0.14 p.Arg144Stop (nonsense) Intracytoplasmic first loopT 1 (1) 0 (0) c.504 T.C 6 T 68 (68) 115 (53.7) 0.017 Silent C 32 (32) 99 (46.3) c.711 A.T 8 A 86 (86) 176 (82.2) 0.40 Silent T 14 (14) 38 (17.8) c.959 C.T 9 C 99 (99) 214 (100) 0.14 p.Ser320Phe (missense) TM5 T 1 (1) 0 (0) c.1769 G.A 15 G 93 (93) 213 (99.5) 0.0017 p.Arg590Gln (missense) NBD1 A 7 (7) 1 (0.5) c.1954 A.G 16 A 97 (97) 202 (94.4) 0.40 p.Arg652Gly (missense) NBD1 G 3 (3) 12 (5.6) c.2324 C.T 19 C 99 (99) 214 (100) 0.32 p.Thr775Met (missense) TM8 T 1 (1) 0 (0) ICP, intrahepatic cholestasis of pregnancy; NBD, nucleotide-binding domain; TM, transmembrane domain; n, number of chromosomes. Login to comment
79 The chromosomal frequency of the p.Arg652Gly variant was not significantly different between patients with ICP and controls. Login to comment
90 No ABCB4 mutation was found in these three patients, except the p.Arg652Gly variant, considered to be a neutral polymorphism, which was found in one of them. Login to comment

[hide] A new splicing site mutation of the ABCB4 gene in ... Dig Liver Dis. 2009 Sep;41(9):671-5. Epub 2009 Mar 3. Tavian D, Degiorgio D, Roncaglia N, Vergani P, Cameroni I, Colombo R, Coviello DA
A new splicing site mutation of the ABCB4 gene in intrahepatic cholestasis of pregnancy with raised serum gamma-GT.
Dig Liver Dis. 2009 Sep;41(9):671-5. Epub 2009 Mar 3., [PMID:19261551]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy is a liver disorder with a multifactorial etiology characterized by maternal pruritus, abnormal liver function tests and increased fetal risk. The main biochemical finding is the increase in total serum bile acid concentrations. In a subgroup of women, the serum gamma-glutamyl transpeptidase level is also increased. There is evidence that dysfunction of the ABCB4 gene might play a role in intrahepatic cholestasis of pregnancy development. AIM: To investigate the role of the ABCB4 gene in Italian women with intrahepatic cholestasis of pregnancy and raised gamma-glutamyl transpeptidase by, analyzing the complete coding sequence and mRNA splicing products. METHODS: Among 299 women with intrahepatic cholestasis of pregnancy, 10 showing raised gamma-glutamyl transpeptidase were enrolled in this study. DNA and RNA were extracted from peripheral blood mononuclear cells using standard procedures. The 27 coding exons and the promoter region were amplified by polymerase chain reaction and analyzed by sequencing. Reverse transcript-polymerase chain reaction analysis of ABCB4 mRNA and cDNA analysis were also performed. RESULTS: A novel splicing mutation that causes a truncated protein of 249 amino acid was identified in a woman who had the highest serum levels of gamma-glutamyl transpeptidase, alkaline phosphatase, bile acids, and the highest pruritus score. We identified also one already described p.R590Q mutation in a woman who had significantly higher serum levels of alkaline phosphatase, aspartate, and alanine aminotransferase. CONCLUSIONS: Our study demonstrates that splicing mutations in the ABCB4 gene can cause ICP in women with high gamma-glutamyl transpeptidase and thus a complete analysis of coding sequence and cDNA products is required.

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67 Patients Involved regions Nucleotide positiona Reference nucleotide Nucleotide variant Effect on proteinb GenBank accession numberc 9d Promoter c.-495 C G 3, 9 Promoter c.-378 T C 2 Exon 4 c.175 C T p.L59L 2, 3, 4, 5d , 7, 9, 10 Exon 6 c.504 T C p.N168N 9 Intron 7 c.709-2 A G p.I237VfsX250 EU142941 2, 7 Exon 8 c.711 A T p.I237I 2, 7 Intron 8 c.834-66 G T 7 Intron 11 c.1231-81 T delT 2 Exon 15 c.1769 G A p.R590Q 7 Exon 16 c.1954 A G p.R652G 7 Intron 16 c.2064+55 A G 2 Intron 17 c.2211+16 T C 7 Intron 20 c.2478+40 A G 7 Intron 20 c.2479-67 G delG 7 Intron 21 c.2682+22 T C EU142942 7 Intron 21 c.2683-197 T C 7 Intron 27 c.3655-72 T C In bold the two mutations which were most likely to cause an impairment of about 50% of the ABCB4 floppase activity in ICP patients n. 2 and n. 9. a Nucleotide A of the ATG of the initiator Met codon is position +1 of cDNA sequence (reference sequence GenBank NM 018849); b The protein GenBank reference number is NP 061337. c GenBank accession number of the new sequences identified in this study. Login to comment

[hide] Common variants of ABCB4 and ABCB11 and plasma lip... Lipids. 2009 Jun;44(6):521-6. Epub 2009 Apr 30. Acalovschi M, Tirziu S, Chiorean E, Krawczyk M, Grunhage F, Lammert F
Common variants of ABCB4 and ABCB11 and plasma lipid levels: a study in sib pairs with gallstones, and controls.
Lipids. 2009 Jun;44(6):521-6. Epub 2009 Apr 30., [PMID:19408031]

Abstract [show]
Most epidemiological surveys have confirmed the association of low HDL-cholesterol and high triglyceride levels with cholesterol gallstones. Our objective was to analyze the relationship between plasma lipid levels and common polymorphisms of ABCB11 (encoding the bile salt export pump, BSEP) and ABCB4 (encoding the phospholipid transporter into bile, MDR3) genes. Plasma lipids were measured in 108 index patients of sib pairs with gallstones and in 260 controls. Using PCR-based assays with 5'-nuclease and fluorescence detection (TaqMan), the ABCB11 coding SNP p.A444V and four haplotype-tagging SNPs covering the ABCB4 gene (c.504C > T, c.711T > A, p.R652G, rs31653 in intron 26) were genotyped. Plasma lipids were compared in carriers of the common versus rare allele of these polymorphisms using Student's t test and Pearson's correlation. BMI and triglyceride levels were higher and HDL-cholesterol levels were lower in affected siblings than in controls. Among cases, triglyceride and cholesterol levels were higher in carriers of the common versus rare (hetero/homozygous carriers) allele of the SNPs p.A444V of ABCB11 and C.504C > T of ABCB4. HDL-cholesterol was lower in carriers of the common allele of rs31653. In controls, significant differences of cholesterol and HDL-cholesterol levels were found in carriers of ABCB4 polymorphisms. Our results do not support the hypothesis of a link between ABCB4 and ABCB11 polymorphisms, lithogenic dyslipidemia, and gallstone risk.

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3 Using PCR-based assays with 50 -nuclease and fluorescence detection (TaqMan), the ABCB11 coding SNP p.A444V and four haplotype-tagging SNPs covering the ABCB4 gene (c.504C [ T, c.711T [ A, p.R652G, rs31653 in intron 26) were genotyped. Login to comment
31 Employing PCR-based assays with 50 -nuclease and fluorescence detection (TaqMan), the following SNPs were genotyped: the ABCB11 coding SNP p.A444V (rs2287622), which is known to affect transporter expression (rsXX), and four haplotype-tagging SNPs covering the ABCB4 gene (c.504C [ T - rs1202283; c.711T [ A - rs2109505; p.R652G - rs8187799; rs31653 in intron 26), which were identified in our previous study [13]. Login to comment
61 patients Age (years) BMI (kg/m2 ) Triglycerides (mg/dl) Cholesterol (mg/dl) HDL cholesterol (mg/dl) ABCB11 p.A444 V rs2287622 Common 28 55.14 ± 12.45 27.82 ± 4.54 198.03 ± 118.71* 228.96 ± 56.5 52.13 ± 17.74 Rare 80 54.28 ± 10.8 29.36 ± 5.39 162.53 ± 85.86 207.78 ± 44.44 48.61 ± 14.8 ABCB4 c.504C [ T rs1202283 Common 34 56.26 ± 10.14 29.86 ± 5.85 204.67 ± 122.54* 230.55 ± 48.56§ 51.17 ± 15.86 Rare 74 54.0 ± 11.53 28.94 ± 5.36 159.02 ± 77.04 204.42 ± 46.44 48.36 ± 15.02 ABCB4 c.711T [ A rs2109505 Common 75 53.77 ± 10.24 29.06 ± 5.56 173.77 ± 97.35 213.36 ± 49.01 49.23 ± 14.56 Rare 33 56.9 ± 13.22 28.7 ± 4.37 170.87 ± 92.49 214.21 ± 48.78 49.5 ± 16.75 ABCB4 p.R652G rs8187799 Common 83 54.36 ± 11.97 29.45 ± 5.27 181.31 ± 102.99 213.55 ± 48.34 49.39 ± 14.52 Rare 21 55.95 ± 7.61 28.86 ± 5.7 159.61 ± 64.21 209.38 ± 54.05 46.31 ± 16.16 ABCB4 rs31653 Common 89 54.5 ± 11.03 29.12 ± 5.4 172.95 ± 96.19 210.61 ± 48.64 48.77 ± 15.75* Rare 19 55.0 ± 12.18 27.66 ± 4.2 170.94 ± 94.85 230.78 ± 46.74 54.53 ± 13.07 Values given in bold are significantly different from controls-carriers of the same allele (Table 4) * Significantly different than in carriers of the rare allele Table 4 Age, BMI, and plasma lipid levels of 260 controls in relation to the common and rare (homozygous and heterozygous carriers) alleles of the ABCB4 and ABCB11 polymorphisms Allele No. Login to comment
62 controls Age (years) BMI (kg/m2 ) Triglycerides (mg/dl) Cholesterol (mg/dl) HDL cholesterol (mg/dl) ABCB11 p.A444 V rs2287622 Common 79 53.99 ± 10.55 26.28 ± 4.11 128.11 ± 59.43 228.4 ± 37.15 57.11 ± 15.11 Rare* 181 52.76 ± 11.18 25.78 ± 5.04 140.33 ± 91.75 229.21 ± 53.62 55.01 ± 13.41 ABCB4 c.504C [ T rs1202283 Common 69 52.41 ± 10.25 26.26 ± 5.54 152.52 ± 108.06 231.01 ± 53.21 55.06 ± 13.26 Rare 191 53.15 ± 11.25 25.81 ± 4.46 130.92 ± 71.95 227.47 ± 47.69 55.84 ± 14.12 ABCB4 c.711T [ A rs2109505 Common 185 52.46 ± 9.87 26.32 ± 4.92 142.27 ± 92.03 233.89 ± 50.93* 56.34 ± 14.48 Rare 75 54.17 ± 13.32 24.98 ± 4.26 122.15 ± 53.0 214.45 ± 41.29 53.85 ± 12.42 ABCB4 p.R652G rs8187799 Common 192 52.37 ± 10.96 26.21 ± 4.62 142.06 ± 90.91 234.19 ± 51.2* 51.47 ± 14.22 Rare 68 54.58 ± 10.95 25.13 ± 5.13 121.25 ± 54.36 212.14 ± 39.65 56.09 ± 13.30 ABCB4 rs31653 Common 219 52.93 ± 10.70 25.43 ± 4.77 133.73 ± 84.53 228.92 ± 50.42 56.32 ± 14.21* Rare 38 52.79 ± 12.63 26.10 ± 4.81 148.08 ± 67.14 223.0 ± 40.30 51.61 ± 11.66 * Significantly different from carriers of the rare allele hydroxylase gene (CYP7A1) [17] or the gene encoding the CCK receptor A [18], no variants have been linked to cholesterol gallstones. Login to comment

[hide] ABCB4 sequence variations in young adults with cho... Liver Int. 2009 May;29(5):743-7. Epub 2008 Oct 24. Nakken KE, Labori KJ, Rodningen OK, Nakken S, Berge KE, Eiklid K, Raeder MG
ABCB4 sequence variations in young adults with cholesterol gallstone disease.
Liver Int. 2009 May;29(5):743-7. Epub 2008 Oct 24., [PMID:19018976]

Abstract [show]
BACKGROUND AND AIMS: Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)-binding cassette, sub-family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid-associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged <40 years. PATIENTS AND METHODS: Hundred and four patients (mean age 30.6 years, range 12-39) were included in the study and the ABCB4 gene was sequenced. The frequency of missense mutations found in the patient material was measured in 95 healthy controls. The potential functional implications of the ABCB4 missense variations were assessed by computerized analysis (BLOSUM62 and Grantham substitution matrices, polymorphism phenotyping and sorting intolerant from tolerant). RESULTS: One patient was heterozygous for a frameshift mutation (c.1399_1400ins10/p.Y467F fsX25). Another patient was heterozygous for a nonsense mutation (c.3136C>T/p.R1046X). These two mutations are considered detrimental to ABCB4 protein function. In addition, six missense mutations were found in the ABCB4 gene, and three of these were only present in patients. CONCLUSION: In our study, <2% of young gallstone patients were found to be heterozygous for detrimental ABCB4 mutations. The functional implication of several missense mutations remains to be clarified. Thus, mutations in the ABCB4 gene are a rare cause of gallstone disease.

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62 These were c.337A4G/p.M113L, c.523A4G/p.T175A, c.1584G4 C/ p.E528D, c.1769G4 A/p.R590Q, c.1954A4G/p.R652G and c.3318G4 C/p.Q1106H). Login to comment
80 The nucleotide change c.1954A4G replaces residue 652 arginine to glycine (p.R652G) and was found in 16 patients, and one patient was homozygous. Login to comment
88 Table 1. Summary of patient characteristics having ABCB4 gene variations with possible effects on the ABCB4 protein and the occurrence of these variations in healthy controls Patient (ID) (n = 104) Gender/ethnicity Age Indication for surgery Gallstone Location Nucleotide change Peptide change Status Controls (n = 95) 1-16 Female Exon 16 c.1954A 4 G p.R652G 9 17-22 Female/Norwegian 21 Choledocholithiasis Multiple, 5 mm Exon 6 c.523A 4 G p.T175A heterozygous 3 Female/Iraq 25 Cholecystolithiasis Multiple, 5-10 mm Female/Norwegian 28 Cholecystolithiasis Two, 15 mm Female/African 31 Cholecystitis Multiple, 5 mm1solitary, 20 mm Female/Norwegian 32 Cholecystolithiasis Multiple, 5 mm Female/Norwegian 34 Cholecystolithiasis Multiple, 5-10 mm 23 Female/Norwegian 32 Cholecystolithiasis Two, 20 mm Exon 14 c.1584G 4 C p.E528D heterozygous 0 24-25 Female/Norwegian 23 Cholecystolithiasis Multiple, 5 mm Exon 15 c.1769G 4 A p.R590Q heterozygous 1 Female/Norwegian 37 Cholecystolithiasis Multiple, o 5 mm 26 Female/Norwegian 40 Cholecystitis Solitary, 30 mm Exon 25 c.3136C 4 T p.R1046X heterozygous - 27 Female/Pakistani 30 Cholecystolithiasis Three, 10 mm Exon 13 c.1399_1400 ins10 p.Y467F fsX25 heterozygous - 28 Ã Female/Pakistani 32 Cholecystolithiasis Multiple, o 5 mm Exon 5 c.337A 4 G p.M113L heterozygous 0 Exon 26 c.3318G 4 C p.Q1106H 0 The variation p.R652G, considered to be without functional significance for the ABCB4 product, is shown without patient characteristics (16 patients). Login to comment
94 Grantham values range from 5 to 215; low values ( o 50) indicate chemical similarity and high values ( 4 50) indicate more radical differences) Scoring Systems for Nonsynonymous Variants Amino acid change Grantham Blosum62 SIFT PolyPhen EC/EU p.M113L 15 2 0.17 1.211 EC p.T175A 58 0 0 0.845 EC p.E528D 45 2 0.25 0.617 EC p.R590Q 43 1 0 1.951 EC p.R652G 125 À 2 0.42 1.237 EU p.Q1106H 24 0 0.03 0.185 EC Blosum62 values range from À 4 to13, with negative values indicating less acceptable and positive values indicating more acceptable substitutions. Login to comment
119 The c.1954A 4G (p.R652G) variation was found in one patient, which was homozygous for the variation in addition to 15 patients with heterozygous variations. Login to comment

[hide] Hepatobiliary phospholipid transporter ABCB4, MDR3... Dig Liver Dis. 2008 May;40(5):366-70. Epub 2007 Dec 20. Floreani A, Carderi I, Paternoster D, Soardo G, Azzaroli F, Esposito W, Montagnani M, Marchesoni D, Variola A, Rosa Rizzotto E, Braghin C, Mazzella G
Hepatobiliary phospholipid transporter ABCB4, MDR3 gene variants in a large cohort of Italian women with intrahepatic cholestasis of pregnancy.
Dig Liver Dis. 2008 May;40(5):366-70. Epub 2007 Dec 20., [PMID:18083082]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy is a multifactorial disorder of pregnancy associated with a genetic background. AIM: To evaluate the genetic contribution of ABCB4, MDR3 gene in the development of intrahepatic cholestasis of pregnancy in a large cohort of Italian subjects. METHODS: This study represents an extension of a previous multicentre-prospective study including three Italian referral centres. In all, we enrolled 96 women at the 3rd trimester of pregnancy. Genomic DNA was extracted from peripheral venous blood leucocytes by standard procedures. Polymerase chain reaction was used to amplify exon 14, 15 and 16 of MDR3 gene. RESULTS: We found 3 non-synonymous heterozygous mutations in exon 14 (E528D, R549H, G536A), 1 in exon 15 (R590Q) and 2 in exon 16 (R652G, T6671). MDR3 gene variants in exons 14, 15 and 16 occurred in 7/96 of pregnant mothers with intrahepatic cholestasis of pregnancy (7.2%), and in none of 96 pregnant controls matched for age and parity. All seven patients had normal gamma-glutamyl transpeptidase, normal bilirubin, but high levels of both alanine transferase and serum bile acids. One had cholesterol biliary lithiasis. The outcome of pregnancy was normal in four cases (with vaginal delivery), while there was one fetal distress. CONCLUSIONS: MDR3 mutations are responsible for the development of intrahepatic cholestasis of pregnancy in only a small percentage of Italian women. Further genetic studies are warranted, however, to clarify the role of different mutations in intrahepatic cholestasis of pregnancy.

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10 We found 3 non-synonymous heterozygous mutations in exon 14 (E528D, R549H, G536A), 1 in exon 15 (R590Q) and 2 in exon 16 (R652G, T6671). Login to comment
54 The median peak serum transaminases were 114.6 U/L (range 42-1117) and 164.5 U/L (range 47-1186) for AST Table 2 Clinical details of the patients with ICP N = 96 (%) Median age (range) 34 years (19-47) Parity 0001 68 70.8 0002 22 22.9 0003 6 6.3 Family history of ICP 8 8.3 Median AST (U/L; range) 114.6 (42-1117) Median ALT (U/L; range) 164.5 (47-1186) 10.4 Median total bile salts (␮M; range) 14.35 (3-115) Total bile salts >40 ␮M 10 10.4 Median total bilirubin (mg/dL) 0.61 (0.23-2.35) Abnormal bilirubin 10 11.4 Median serum GGT (U/L; range) 20.5 (4-147) Abnormal serum GGT 11 Normal values: AST, aspartate aminotransferase <40 U/L; ALT, alanine transferase <45 U/L; total bile salts <2 ␮M. Table 3 Polymorphisms of exons 14, 15 and 16 found in the study population cDNA (exon) N refer N var AA change 1584 (14) G C E528D 1606 (14) G A G536R 1646 (14) G A R549H 1769 (15) G A R590Q 1954 (16) A G R652G 2000 (16) C T T667I andALTrespectively.Eighty-sixpatientshadbilirubinwithin the normal range, while 10 (10.4%) had a slight rise in total bilirubin (2-3 mg/dL). Login to comment
58 Sequence analysis of exon 16 showed 2 mutations: (1) AGA-GGA mutation in codon 652, resulting in the substitution of the wild-type arginine with a mutant glycine (R652G); (2) ACT-ATT mutation in codon 667 with a substitution of the wild-type threonine with a mutant isoleucine (T667I). Login to comment
59 R652G variant in exon 16 occurred in two patients. Login to comment
80 There is a difference, however, in the phenotypic expression of this condition by comparison Table 4 Clinical details of ICP patients with MDRR3 mutations Patient #1 E528D Patient #2 R549H Patient #3 G536R Patient #4 R590Q Patient #5 R652G Patient #6 T667I Patient #7 R652G Onset of pruritus 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester Parity 0002 0001 0001 0001 0001 0001 0001 Previous ICP No Yes Yes No No No No Peak of AST (U/L) 163 88 129 62 789 119 60 Peak of ALT (U/L) 98 121 137 88 1186 125 78 Bilirubin (mg/dL) 0.60 0.61 0.89 1.2 0.57 0.3 0.78 Peak of GGT (U/L) 8 15 19 10 25 35 5 Cholesterol lithiasis No No Yes No No No No Total bile salts (␮M/L) 3.3 6.3 10.1 60 76.3 25.3 4.0 Delivery Vaginal Vaginal Caesarean Vaginal Caesarean Caesarean Vaginal ICP, intrahepatic cholestasis of pregnancy; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Login to comment
55 Table 3 Polymorphisms of exons 14, 15 and 16 found in the study population cDNA (exon) N refer N var AA change 1584 (14) G C E528D 1606 (14) G A G536R 1646 (14) G A R549H 1769 (15) G A R590Q 1954 (16) A G R652G 2000 (16) C T T667I andALTrespectively.Eighty-sixpatientshadbilirubinwithin the normal range, while 10 (10.4%) had a slight rise in total bilirubin (2-3 mg/dL). Login to comment
60 R652G variant in exon 16 occurred in two patients. Login to comment
81 There is a difference, however, in the phenotypic expression of this condition by comparison Table 4 Clinical details of ICP patients with MDRR3 mutations Patient #1 E528D Patient #2 R549H Patient #3 G536R Patient #4 R590Q Patient #5 R652G Patient #6 T667I Patient #7 R652G Onset of pruritus 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester 3rd trimester Parity 0002 0001 0001 0001 0001 0001 0001 Previous ICP No Yes Yes No No No No Peak of AST (U/L) 163 88 129 62 789 119 60 Peak of ALT (U/L) 98 121 137 88 1186 125 78 Bilirubin (mg/dL) 0.60 0.61 0.89 1.2 0.57 0.3 0.78 Peak of GGT (U/L) 8 15 19 10 25 35 5 Cholesterol lithiasis No No Yes No No No No Total bile salts (òe;M/L) 3.3 6.3 10.1 60 76.3 25.3 4.0 Delivery Vaginal Vaginal Caesarean Vaginal Caesarean Caesarean Vaginal ICP, intrahepatic cholestasis of pregnancy; AST, aspartate aminotransferase; ALT, alanine aminotransferase. Login to comment

[hide] Prenatal molecular diagnosis of inherited cholesta... J Pediatr Gastroenterol Nutr. 2007 Apr;44(4):453-8. Jung C, Driancourt C, Baussan C, Zater M, Hadchouel M, Meunier-Rotival M, Guiochon-Mantel A, Jacquemin E
Prenatal molecular diagnosis of inherited cholestatic diseases.
J Pediatr Gastroenterol Nutr. 2007 Apr;44(4):453-8., [PMID:17414143]

Abstract [show]
OBJECTIVES: Progressive familial intrahepatic cholestasis (PFIC) and to a lesser extent, Alagille syndrome, often lead to end-stage liver disease during childhood. We report our experience of DNA-based prenatal diagnosis of PFIC1-3 and Alagille syndrome. PATIENTS AND METHODS: Four molecular antenatal diagnoses were performed in 3 PFIC families and 17 in 11 Alagille syndrome families. DNA was isolated from chorionic villus or cultured amniocyte samples from women, without pregnancy complications. RESULTS: All four foetuses with a family history of PFIC1, 2, or 3 were heterozygous for an ATP8B1, ABCB11, or ABCB4 mutation and pregnancies were continued. Three of the infants were healthy after birth, and 1 premature infant, who had an ABCB4 mutation, experienced transient neonatal cholestasis. Among the families with a history of de novo JAG1 mutation, none of the foetuses was mutated, versus 40% of those with a history of familial mutation. Of 4 pregnant women with a JAG1-mutated foetus, 3 cut short their pregnancy and 1 gave birth to a child with overt Alagille syndrome. CONCLUSIONS: Molecular antenatal diagnosis of PFIC1-3 and Alagille syndrome is reliable because clinical outcome after birth corresponded to molecular foetal data.

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69 Despite this favourable evolution, the entire ABCB4 gene downstream of the deletion was sequenced to exclude the possible presence of another mutationonthe second allele.We identified ahomozygous missense mutation, 1986A>G (R652G), believed to constitute a polymorphism (7). Login to comment
77 Mutational status and results of prenatal molecular diagnosis in PFIC families Disease and family Propositus: phenotype and mutation Foetal genetic status Evolution of pregnancy and after birth PFIC1: family 1 CC, ATP8B1 gene: compound heterozygous IVS9-1G>A, 1336G>A (G446R) Foetus: heterozygous IVS9-1G>A Continued pregnancy: 12 mo of follow-up: healthy, no cholestasis PFIC2: family 2 Unknown CC status, vitamin K deficiency, cerebral haemorrhage, deceased, ABCB11 gene: compound heterozygous IVS24þ5G>A, 3701delC (E1192fs1242X) Foetus: heterozygous 3701delC (E1192fs1242X) Continued pregnancy: 3 mo of follow-up: healthy, no cholestasis PFIC3: family 3 CC, LT, ABCB4 gene: homozygous 1744delT (571fs586X) Foetus 1: heterozygous 1744delT (571fs586X) Continued pregnancy: neonatal cholestasis; favourable evolution under UDCA treatment with normalization of liver tests; UDCA was stopped and clinical status and liver tests were normal at 7 y Complete sequencing of ABCB4 in neonate: homozygous 1986A>G (R652G) Foetus 2: absence of 1744delT (571fs586X) mutation Continued pregnancy: 4 y of follow-up: healthy, no cholestasis Heterozygous 1986A>G (R652G) CC, indicates chronic neonatal cholestasis; LT, liver transplantation; UDCA, ursodeoxycholic acid. Login to comment
109 In addition, the presence of the R652G polymorphism on the foetus`s second allele may have favoured the expression of transient neonatal cholestasis because it has been shown that when this polymorphism is homozygous, it may be associated with a trend towards a low MDR3 protein level in normal human liver (7,8,33). Login to comment

[hide] Intrahepatic cholestasis of pregnancy: the severe ... Gut. 2007 Feb;56(2):265-70. Epub 2006 Aug 4. Wasmuth HE, Glantz A, Keppeler H, Simon E, Bartz C, Rath W, Mattsson LA, Marschall HU, Lammert F
Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene.
Gut. 2007 Feb;56(2):265-70. Epub 2006 Aug 4., [PMID:16891356]

Abstract [show]
BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterised by troublesome maternal pruritus, raised serum bile acid levels and increased fetal risk. Mutations of the ABCB4 gene encoding the hepatobiliary phospholipid transporter have been identified in a small proportion of patients with cholestasis of pregnancy. In a recent prospective study on 693 patients with cholestasis of pregnancy, a cut-off level for serum bile acid (> or =40 micromol/l) was determined for increased risk of fetal complications. OBJECTIVES: To investigate whether common combinations of polymorphic alleles (haplotypes) of the genes encoding the hepatobiliary ATP-binding cassette (ABC) transporters for phospholipids (ABCB4) and bile acids (ABCB11) were associated with this severe form of cholestasis of pregnancy. METHODS: For genetic analysis, 52 women with bile acid levels > or =40 micromol/l (called cases) and 52 unaffected women (called controls) matched for age, parity and geographical residence were studied. Gene variants tagging common ABCB4 and ABCB11 haplotypes were genotyped and haplotype distributions were compared between cases and controls by permutation testing. RESULTS: In contrast with ABCB11 haplotypes, ABCB4 haplotypes differed between the two groups (p = 0.019), showing that the severe form of cholestasis of pregnancy is associated with the ABCB4 gene variants. Specifically, haplotype ABCB4_5 occurred more often in cases, whereas haplotypes ABCB4_3 and ABCB4_7 were more common in controls. These associations were reflected by different frequencies of at-risk alleles of the two tagging polymorphisms (c.711A: odds ratio (OR) 2.27, p = 0.04; deletion intron 5: OR 14.68, p = 0.012). CONCLUSION: Variants of ABCB4 represent genetic risk factors for the severe form of ICP in Sweden.

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285 In the study by Pauli-Magnus et al,13 the intron 5 deletion was detected in one patient with ICP and two controls, indicating that this polymorphism is not ICP specific, but study participants were not matched for age, parity or geographical residence.13 Although we observed markedly different allele and genotype frequencies for the SNP c.1954ARG in our ICP cases and controls, it has been assumed that the resulting missense mutation (R652G) is a neutral polymorphism that occurs with similar frequencies in cases and controls.12 17 29 39 However, in contrast with a French study,12 in our Swedish patients and in patients from Switzerland,13 ICP seemed to be more prevalent among carriers of arginine at codon 652, and sequence comparisons of ABCB4 genes identifies that the glycine residue is conserved in other species such as rat and hamster. Login to comment

[hide] Novel resequencing chip customized to diagnose mut... Gastroenterology. 2007 Jan;132(1):119-26. Epub 2006 Oct 21. Liu C, Aronow BJ, Jegga AG, Wang N, Miethke A, Mourya R, Bezerra JA
Novel resequencing chip customized to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis.
Gastroenterology. 2007 Jan;132(1):119-26. Epub 2006 Oct 21., [PMID:17241866]

Abstract [show]
BACKGROUND & AIMS: Inherited syndromes of intrahepatic cholestasis commonly result from mutations in the genes SERPINA1 (alpha(1)-antitrypsin deficiency), JAG1 (Alagille syndrome), ATP8B1 (progressive familial intrahepatic cholestasis type 1 [PFIC1]), ABCB11 (PFIC2), and ABCB4 (PFIC3). However, the large gene sizes and lack of mutational hotspots make it difficult to survey for disease-causing mutations in clinical practice. Here, we aimed to develop a technological tool that reads out the nucleotide sequence of these genes rapidly and accurately. METHODS: 25-mer nucleotide probes were designed to identify each base for all exons, 10 bases of intronic sequence bordering exons, 280-500 bases upstream from the first exon for each gene, and 350 bases of the second intron of the JAG1 gene and tiled using the Affymetrix resequencing platform. We then developed high-fidelity polymerase chain reactions to produce amplicons using 1 mL of blood from each subject; amplicons were hybridized to the chip, and nucleotide calls were validated by standard capillary sequencing methods. RESULTS: Hybridization of amplicons with the chip produced a high nucleotide sequence readout for all 5 genes in a single assay, with an automated call rate of 93.5% (range, 90.3%-95.7%). The accuracy of nucleotide calls was 99.99% when compared with capillary sequencing. Testing the chip on subjects with cholestatic syndromes identified disease-causing mutations in SERPINA1, JAG1, ATP8B1, ABCB11, or ABCB4. CONCLUSIONS: The resequencing chip efficiently reads SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 with a high call rate and accuracy in one assay and identifies disease-causing mutations.

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70 Diagnosisa Mutation 1 ␣1AT deficiency SERPINA1 T638C (Val213Ala, homozygous) and G1024A (Glu342Lys, homozygous)45,46 2 ␣1AT deficiency SERPINA1 T638C (Val213Ala, homozygous) and G1024A (Glu342Lys, homozygous)45,46 3 ␣1AT deficiency SERPINA1 T638C (Val213Ala, homozygous) and G1024A (Glu342Lys, homozygous)45,46 4 Alagille syndrome JAG1 C2230T (Arg744stop, heterozygous)47 5 Alagille syndrome JAG1 IVS19 ϩ1 G to A, heterozygous48 6 Alagille syndrome JAG1 C2650T (Glu884Stop, heterozygous)b 7 Alagille syndrome JAG1 C2650T (Glu884Stop, heterozygous)b 8 PFIC1 ATP8B1 C2788T (Arg930stop, heterozygous)28 9 PFIC1 ATP8B1 T1982C (Ile661Thr, heterozygous)15 10 PFIC1 ATP8B1 569-base pair deletion (including first 17 base pairs in exon 23, homozygous)b 11 PFIC2 ABCB11 C3457T (Arg1153Cys, heterozygous)17 12 PFIC2 ABCB11 C2782T (Arg928Stop, heterozygous)b 13 PFIC3 ABCB4 A874T (Lys292Stop, homozygous) and A1954G (Arg652Gly, homozygous)49 14 Biliary atresia ATP8B1 IVS 26 ϩ8 G to T, heterozygousb 15 Biliary atresia No nonsynonymous polymorphism 16 Biliary atresia No nonsynonymous polymorphism 17 Biliary atresia JAG1 C2612G (Pro871Arg, heterozygous)50 18 Biliary atresia SERPINA1 G302A (Arg101His, heterozygous)51 NOTE. Login to comment

[hide] Linkage between a new splicing site mutation in th... Hepatology. 2007 Jan;45(1):150-8. Schneider G, Paus TC, Kullak-Ublick GA, Meier PJ, Wienker TF, Lang T, van de Vondel P, Sauerbruch T, Reichel C
Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy.
Hepatology. 2007 Jan;45(1):150-8., [PMID:17187437]

Abstract [show]
Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus and elevated bile acid serum concentrations in late pregnancy. Splicing mutations have been described in the multidrug resistance p-glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women. Pedigrees studied were not large enough for linkage analysis. Ninety-seven family members of a woman with proven ICP were asked about pruritus in earlier pregnancies, birth complications and symptomatic gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1) gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were analyzed in 55 relatives. We identified a dominant mode of inheritance with female restricted expression and a new intronic MDR3 mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe deletion via cryptic splicing site activation. Linkage analysis of the ICP trait versus this intragenic MDR3 variant yielded a LOD score of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an unknown locus gave a posterior probability of >0.9966 in favor of MDR3 as causative ICP locus. During the episode of ICP the median gamma-glutamyl transpeptidase (gamma-GT) activity was 10 U/l (95% CI, 6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported in seven heterozygous women (22 pregnancies) and none in five women (14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease was more prevalent in heterozygous relatives (7/21) than in relatives without the mutation (1/34), (P = 0.00341). CONCLUSION: This study demonstrates that splicing mutations in the MDR3 gene can cause ICP with normal gamma-GT and may be associated with stillbirths and gallstone disease.

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68 MDR3 Genetic Variability in the Index Patient Variant DNA Position Nucleotide Reference Nucleotide Variant Effect c.-3335_-3336insAG* Prom A5 insAG n.a. c.-316A>T* Prom B6 A T n.a. c.345-50_-46 delGAAAA Intron 5 GAAAA del GAAAA n.a. c.504C>T Exon6 C T syn c.1231-81delT Intron11 del T n.a. c.1954A>G Exon 16 A G R652G c.2064؉55A>G Intron 16 A G n.a. c.2478؉40A>G* Intron 20 A G n.a. c.3486؉5G>A* Intron 26 G A splicing c.3634-72T>C Intron 27 T C n.a. NOTE. Login to comment

[hide] ABCB4 gene mutations and primary sclerosing cholan... Gastroenterology. 2004 Apr;126(4):1220-2; author reply 1222-3. Rosmorduc O, Hermelin B, Boelle PY, Poupon RE, Poupon R, Chazouilleres O
ABCB4 gene mutations and primary sclerosing cholangitis.
Gastroenterology. 2004 Apr;126(4):1220-2; author reply 1222-3., [PMID:15057773]

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57 The previously described Arg652Gly polymorphism was detected with a similar frequency in all groups (about 6%). Login to comment

[hide] ABCB4 gene mutation-associated cholelithiasis in a... Gastroenterology. 2003 Aug;125(2):452-9. Rosmorduc O, Hermelin B, Boelle PY, Parc R, Taboury J, Poupon R
ABCB4 gene mutation-associated cholelithiasis in adults.
Gastroenterology. 2003 Aug;125(2):452-9., [PMID:12891548]

Abstract [show]
BACKGROUND & AIMS: We recently put forward arguments in favor of ABCB4 gene (adenosine triphosphate-binding cassette, subfamily B, member 4) defects as a risk factor for symptomatic cholelithiasis in adults. In this study, we characterized ABCB4 gene mutations in a series of patients with symptomatic cholelithiasis to determine the genetic basis and the clinical phenotype of ABCB4 gene mutation-associated cholelithiasis. METHODS: We analyzed the entire ABCB4 gene coding sequences in a first group of 32 patients who had a clinical history compatible with the syndrome previously described, in a second group of 28 patients who presented with a classic gallstone disease that justified a cholecystectomy, and in a third group of 33 patients without a history of cholelithiasis. RESULTS: We identified both heterozygous and homozygous ABCB4 gene point mutations in 18 of 32 (56%) patients who presented with clinical criteria of the syndrome, whereas no mutation was detected in the 2 other groups of patients (P < 0.001). Three independent clinical features were strongly associated with point mutations: recurrence of symptoms after cholecystectomy (odds ratio, 8.5); intrahepatic hyperechoic foci, intrahepatic sludge, or microlithiasis (odds ratio, 6.1); and age <40 years at the onset of symptoms (odds ratio, 3.0). ABCB4 gene point mutations were detected exclusively in the patients who showed 2 or 3 of these clinical features. CONCLUSIONS: Our results show that ABCB4 gene mutations represent a major genetic risk factor in a symptomatic and recurring form of cholelithiasis in young adults.

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67 A previously described Arg652Gly SNP was detected at a similar frequency (approximately 5%) in patients with and Table 3. Login to comment
90 Characterization of ABCB4 Gene SNPs and Determination of the Main Allele Frequency in Patients With and Without LPAC Syndrome and in Control Subjects SNP1 SNP2 SNP3 SNP4 SNP5 SNP characteristics SNP localization Exon 4 Exon 5 Exon 6 Exon 8 Exon 16 Nucleotide change 175C3T 342T3C 504C3T 711A3T 1954A3G Amino acid Leu59 Thr114 Asn168 Ile237 Arg652Gly Most frequent allele CTG ACT AAC ATA AGG Group of patients and controls Mutation, LPAC phenotype (score Ն2) 21/24 (87.5%) 24/24 (100%) 16/30 (53.3%) 21/24 (87.5%) 23/24 (95.8%) No mutation, LPAC phenotype (score Ն2) 27/28 (96.4%) 27/28 (96.4%) 3/28 (10.7%) 27/28 (96.4%) 27/28 (96.4%) No mutation, no LPAC phenotype (score Ͻ2) 52/56 (92.8%) 56/56 (100%) 29/56 (51.8%) 51/56 (91.1%) 51/56 (91.1%) Control subjects 54/66 (81.8%) 65/66 (98.5%) 36/66 (54.5%) 50/66 (89.3%) 61/66 (92.4%) Bonferroni-adjusted P value (5 comparisons) 0.05 0.99 0.001 0.14 0.99 NOTE. The table indicates the most frequent allele to total number of alleles ratio for each tested SNP in the different groups of patients. Login to comment

[hide] The wide spectrum of multidrug resistance 3 defici... Gastroenterology. 2001 May;120(6):1448-58. Jacquemin E, De Vree JM, Cresteil D, Sokal EM, Sturm E, Dumont M, Scheffer GL, Paul M, Burdelski M, Bosma PJ, Bernard O, Hadchouel M, Elferink RP
The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood.
Gastroenterology. 2001 May;120(6):1448-58., [PMID:11313315]

Abstract [show]
BACKGROUND & AIMS: We have specified the features of progressive familial intrahepatic cholestasis type 3 and investigated in 31 patients whether a defect of the multidrug resistance 3 gene (MDR3) underlies this phenotype. METHODS: MDR3 sequencing, liver MDR3 immunohistochemistry, and biliary phospholipid dosage were performed. RESULTS: Liver histology showed a pattern of biliary cirrhosis with patency of the biliary tree. Age at presentation ranged from the neonatal period to early adulthood. Sequence analysis revealed 16 different mutations in 17 patients. Mutations were identified on both alleles in 12 patients and only on 1 allele in 5. Four mutations lead to a frame shift, 2 are nonsense, and 10 are missense. An additional missense mutation probably representing a polymorphism was found in 5 patients. MDR3 mutations were associated with abnormal MDR3 canalicular staining and a low proportion of biliary phospholipids. Gallstones or episodes of cholestasis of pregnancy were found in patients or parents. Children with missense mutations had a less severe disease and more often a beneficial effect of ursodeoxycholic acid therapy. CONCLUSIONS: At least one third of the patients with a progressive familial intrahepatic cholestasis type 3 phenotype have a proven defect of MDR3. This gene defect should also be considered in adult liver diseases.

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57 To detect a polymorphism, a total of 100 control chromosomes were screened by restriction enzyme analysis for the missense mutation R652G, which was found in 5 of the 31 patients. Login to comment
113 R652G Mutation and Polymorphism The nonconservative missense mutation R652G located in the linker region, was identified in 5 affected patients (patients LM, GaM, LH, LF, and VHC), but was found heterozygous with the same frequency in 50 unaffected unrelated healthy individuals (data not shown). Login to comment
121 Among heterozygous parents, 3 mothers had experienced episodes of cholestasis of pregnancy.23,26 These parents were heterozygous for a mutation leading to a premature truncation of the protein in 2 cases and for the R652G missense mutation in 1 case. Login to comment
126 MDR3 Mutations, Immunohistochemistry, and Biliary Phospholipids in Patients With High GGT PFIC (PFIC3) Patients Exon Nucleotide mutation Amino acid change Protein consequence Family MDR3 liver Phospholipids (%) bile Homozygous Deletion/Insertion M Y M I (sister of M Y) 2 111 A Ͼ G, splice donor site, exon/intron boundary 2/3 27 Frame shift in NH2 terminus, then truncation ND Absent ND 1.6 B Ka 6 426-432 del 132 Frame shift in TMD 2, 29 novel amino acids then truncation Mother ϩ/- Father ϩ/- 1 sibling ϩ/ϩ Absent ND B Sa 14 1744 del T 571 Frame shift, 15 novel amino acids then truncation Mother ϩ/- (ICP) Father ϩ/- 3 siblings ϩ/ϩ Absent ND P G 24 2975-2984 del Compound heterozygous 981 Frame shift in TMD 12, 3 novel amino acids then truncation Mother ϩ/ϩ Father ϩ/- ND 14.9 Bo S Bo N (sister of Bo S) 16 Nonsense 1938 C Ͼ T Q636X Linker region Truncation Mother ϩ/- Father ϩ/- 2 siblings ϩ/- Absent 2 ND B Aa 23 2901 C Ͼ T R957X TMD 11 Truncation Mother ϩ/- (ICP) Father ϩ/- 4 siblings ϩ/- or ϩ/ϩ Absent ND S F 10 Missense 1069 G Ͼ T S346I Serine to isoleucine in TMD6 Mother ϩ/- Father ϩ/- Faint 1 B H 11 1216 A Ͼ G E395G Glutamate to glycine between TMD 6 and first Walker A motif ND ND ND N I 14 1653 A Ͼ T I541F Isoleucine to phenylalanine in first Walker B motif Mother ϩ/- Father ϩ/- Absent ND M M 14 1699 T Ͼ G L556R Leucine to arginine in first Walker B motif ND ND ND P G 24 2979 G Ͼ A Compound heterozygous G983S Glycine to serine in TMD 12 Mother ϩ/- Father ϩ/ϩ ND 14.9 P A 6 Heterozygous Missense 444 T Ͼ C W138R Trytophan to arginine in TMD 2 Mother ϩ/- Father ϩ/ϩ ND 6.7 M Aa 12 1302 A Ͼ G T424A Threonine to alanine in first Walker A motif ND gallbladder lithiasis in father Faint 6.3 P K 12 1307 G Ͼ A V425M Valine to methionine in first Walker A motif ND Normal ND G A 14 1723 A Ͼ G D564G Aspartate to glycine in first Walker B motif ND ND ND G M 17 2132 T Ͼ C F711S Phenylalanine to serine in TMD 7 ND ND ND L M 16 Polymorphism 1986 A Ͼ G Homozygous R652G Arginine to glycine in linker region Mother ϩ/- (suspicion of ICP) Father ϩ/- ND ND Ga M " Heterozygous " " Mother ϩ/- Father ϩ/ϩ 1 sibling ϩ/- ND 9.7 L H L F (sister of L H) " Heterozygous " " ND ND ND VH C " Heterozygous " " Mother ϩ/ϩ Father ϩ/- ND ND NOTE. Login to comment
152 plantation so far. Among these 7 patients, 5 have the R652G polymorphism. Login to comment
171 An additional missense mutation (R652G) was located in the linker region. Login to comment
172 Although it is a nonconservative mutation, it was found heterozygous in control chromosomes with the same frequency, and this excludes the possibility that R652G, by itself, can cause clinical symptoms. Login to comment
174 One possibility is that the R652G mutation has mild consequences, explaining the favorable outcome of patients with this mutation under UDCA therapy, but combined with another mutation or in circumstances such as pregnancy leads to clinical symptoms. Login to comment

[hide] The spectrum of liver diseases related to ABCB4 ge... Semin Liver Dis. 2010 May;30(2):134-46. Epub 2010 Apr 26. Davit-Spraul A, Gonzales E, Baussan C, Jacquemin E
The spectrum of liver diseases related to ABCB4 gene mutations: pathophysiology and clinical aspects.
Semin Liver Dis. 2010 May;30(2):134-46. Epub 2010 Apr 26., [PMID:20422496]

Abstract [show]
Class III multidrug resistance P-glycoproteins, Mdr2 in mice and MDR3 in human, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion. The role of an ABCB4 gene defect in liver disease has been initially proven in a subtype of progressive familial intrahepatic cholestasis called PFIC3, a severe pediatric liver disease that may require liver transplantation. Several ABCB4 mutations have been identified in children with PFIC3 and are associated with low level of phospholipids in bile leading to a high biliary cholesterol saturation index. ABCB4 mutations are associated with loss of canalicular MDR3 protein and /or loss of protein function. There is evidence that a biallelic or monoallelic ABCB4 defect causes or predisposes to several human liver diseases (PFIC3, low phospholipid associated cholelithiasis syndrome, intrahepatic cholestasis of pregnancy, drug-induced liver injury, transient neonatal cholestasis, adult biliary fibrosis, or cirrhosis). Most patients with MDR3 deficiency have a favorable outcome with ursodeoxycholic acid (UDCA) therapy, but some PFIC3 patients who do not respond to UDCA treatment still require liver transplantation. The latter should be good candidates for a targeted pharmacologic approach and/or to cell therapy in the future.

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144 Site-directed mutagenetic analysis of P-glycoprotein has shown that mutations in transmembrane domains are important for substrate specificity and that mutations localized near Walker motifs may disrupt the function of the transporter.88,91-93 Alternatively, missense mutations might result in intracellular misprocessing of MDR3 as shown for other ABC transporters.44,46,74,94 Indeed, such missense mutations were associated with a decreased level of MDR3 canalicular protein.4 Whatever the mechanism, a low level of biliary phospholipids found in patients with missense mutations demonstrates a functional MDR3 deficiency.4 Interestingly, some affected children had missense mutations that probably represented a polymorphism (p.R652G).4 It may be that this polymorphism has mild clinical consequences, explaining the favorable outcome of patients with this mutation following UDCA therapy, or that in certain circumstances such as pregnancy, may lead to clinical symptoms.6,22,63,82,95 In human liver, this polymorphism is associated with low expression of MDR3.95 It has been shown for MDR1 P-glycoprotein that amino acid polymorphisms may affect the protein function.96 Ideally, it would be desirable to have a functional means to distinguish disease mutations from normal variants.32,46,97,98 In our experience, in silico tools are useful to predict if an intronic mutation should lead to abnormal splicing.99 Compared with children who have ABCB4 mutations that lead to a truncated protein, children with ABCB4 missense mutations have a less severe disease, with a later onset in life and a slower progression, which can be favorably modified by chronic administration of UDCA in $50% of cases33 and be compatible with life until adolescence or early adulthood. Login to comment
148 In our experience, the threshold percentage of biliary phospholipids that predicts a positive answer to UDCA Table 1 Genotype-Phenotype Correlations in PFIC3 Children with ABCB4 Mutations Patients Homozygous for a Mutation Leading to a Truncated Protein Patients Homozygous or Heterozygous for a mutation Leading to a Missense Protein N 12 34(4*) MDR3 canalicular staining 5 Patients tested: 8 Patients tested: negative in 5 negative in 3, faint in 3, positive in 2 Mean age at first symptoms (range) 9 Months 4 years (1-28) (1 month-20 years) Biliary phospholipid concentration 3 Patients tested: 6 Patients tested(1*): Mean % of total biliary lipid concentration (range) 0.05-0.13 mM 0.4-3 mM <2% 7.6% (3.3-14) Response to UDCA 7 Patients tested: 29 Patients tested: negative in 7 negative in 4, unknown in 8 (1*), positive in 183* Liver transplantation No: 1, alive at 3 years No: 23 (3*), alive at a mean age of 13 years (range: 2-33) Mean age at liver transplantation (range) Yes: 11 Yes: 10 (1*) 5.5 years Lost to follow-up: 1 (2.5-9) 14 years (5-30) (n*) Including n patients with the p.R652G amino acid polymorphism. Login to comment

[hide] Role of multidrug resistance 3 deficiency in pedia... Semin Liver Dis. 2001 Nov;21(4):551-62. Jacquemin E
Role of multidrug resistance 3 deficiency in pediatric and adult liver disease: one gene for three diseases.
Semin Liver Dis. 2001 Nov;21(4):551-62., [PMID:11745043]

Abstract [show]
Class III multidrug resistance P-glycoproteins, mdr2 in mice and MDR3 in humans, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion. The role of an MDR3 gene defect in liver disease was initially suspected in a subtype of progressive familial intrahepatic cholestasis called PFIC3. Several MDR3 mutations have been identified in children with PFIC3 and are associated with a low level of phospholipids in bile, leading to a high biliary cholesterol saturation index. Mutations leading to a truncated protein are associated with an absence of canalicular MDR3 protein. The phenotypic spectrum of PFIC3 ranges from neonatal cholestasis to cirrhosis in young adults. There is now strong evidence that in addition to PFIC3, an MDR3 defect can be involved in intrahepatic cholestasis of pregnancy and in cholesterol gallstone disease. Therefore, at least three human liver diseases are due to a single gene deficiency. Patients with PFIC3 due to MDR3 deficiency may benefit from ursodeoxycholic acid therapy and could be good candidates for cell therapy in the future.

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110 TMD11 Patient 2 PFIC3 571 Truncation 41 ICP Frameshift Patient 3 PFIC3 R652G Polymorphism ? Login to comment
112 Patient 4* No R652G Polymorphism > N progesterone Linker ? Login to comment

[hide] ABCB4 and ABCB11 mutations in intrahepatic cholest... Dig Liver Dis. 2013 Mar;45(3):226-32. doi: 10.1016/j.dld.2012.08.011. Epub 2012 Sep 27. Anzivino C, Odoardi MR, Meschiari E, Baldelli E, Facchinetti F, Neri I, Ruggiero G, Zampino R, Bertolotti M, Loria P, Carulli L
ABCB4 and ABCB11 mutations in intrahepatic cholestasis of pregnancy in an Italian population.
Dig Liver Dis. 2013 Mar;45(3):226-32. doi: 10.1016/j.dld.2012.08.011. Epub 2012 Sep 27., [PMID:23022423]

Abstract [show]
BACKGROUND: Genetic alterations in the ATP-binding cassette subfamily B member 4 (ABCB4) and ATP-binding cassette subfamily B member 11 (ABCB11) have been associated to the onset of intrahepatic cholestasis of pregnancy (ICP) in predisposed women. AIMS: To identify new and/or frequent ABCB4 and ABCB11 genes variants in a cohort of Italian patients with ICP and to evaluate the possible pathogenetic role for the novel mutations identified. METHODS: DNA of 33 unrelated Italian women with obstetric cholestasis were screened for mutations in the entire coding sequence of ABCB4 and ABCB11 genes. Polymerase chain reaction and automated sequencing was performed on the 27 coding exons of both genes. RESULTS: Genotyping revealed 11 mutations, 5 of whom were novel variants: 2 localized on ABCB4 (p.I587DfsX603, p.I738LfsX744) and 3 on ABCB11 (p.V284D, p.Q558H, p.P731S). The most severe phenotypes were associated with the variants p.I587DfsX603, p.I738LfsX744 and p.V284D. Moreover, the already described mutation p.N510S found in ABCB4 seems to be strictly involved in the onset of ICP in that particular patient. CONCLUSIONS: Our data support the hypothesis of a significant involvement of ABCB4 mutations in the onset of ICP, but also confirm an important role for ABCB11 mutations in increasing the susceptibility to cholestasis of pregnancy.

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163 However, the absence of any disease-causing mutation in 22/33 patients, apart from the presence of the p.V444A and p.R652G polymorphisms (data not shown), suggest that other genes or other factors (comorbility) may play a role in the aetiology of ICP. Login to comment
164 However, the absence of any disease-causing mutation in 22/33 patients, apart from the presence of the p.V444A and p.R652G polymorphisms (data not shown), suggest that other genes or other factors (comorbility) may play a role in the aetiology of ICP. Login to comment

[hide] Selected ABCB1, ABCB4 and ABCC2 polymorphisms do n... PLoS One. 2014 Apr 14;9(4):e94675. doi: 10.1371/journal.pone.0094675. eCollection 2014. Ulzurrun E, Stephens C, Ruiz-Cabello F, Robles-Diaz M, Saenz-Lopez P, Hallal H, Soriano G, Roman E, Fernandez MC, Lucena MI, Andrade RJ
Selected ABCB1, ABCB4 and ABCC2 polymorphisms do not enhance the risk of drug-induced hepatotoxicity in a Spanish cohort.
PLoS One. 2014 Apr 14;9(4):e94675. doi: 10.1371/journal.pone.0094675. eCollection 2014., [PMID:24732756]

Abstract [show]
BACKGROUND AND AIMS: Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS: A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS: None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS: Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

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63 Samples and controls were genotyped for the ABCB1 c.1236T.C (p.Gly412Gly, rs1128503), c.3435T.C (p.Ile1145Ile, rs1045642), ABCB4 c.1954A.G (p.Arg652Gly, rs2230028) and ABCC2 c.-1549A.G (rs1885301), c.-24C.T (rs717620), c.1249G.A (p.Val417Ile, rs2273697), c.3972C.T (p.Ile1324Ile, rs3740066) and c.4544G.A (p.Cys1515Tyr, rs8187710) using a validated 59-nuclease PCR based assay with allele specific fluorescent probes (TaqMan SNP Genotyping Assays, Applied Biosystems, Foster City, CA, USA) as previously described [23]. Login to comment

[hide] ABCB4 mutations underlie hormonal cholestasis but ... World J Gastroenterol. 2014 May 21;20(19):5867-74. doi: 10.3748/wjg.v20.i19.5867. Jirsa M, Bronsky J, Dvorakova L, Sperl J, Smajstrla V, Horak J, Nevoral J, Hrebicek M
ABCB4 mutations underlie hormonal cholestasis but not pediatric idiopathic gallstones.
World J Gastroenterol. 2014 May 21;20(19):5867-74. doi: 10.3748/wjg.v20.i19.5867., [PMID:24914347]

Abstract [show]
AIM: To investigate the contribution of ABCB4 mutations to pediatric idiopathic gallstone disease and the potential of hormonal contraceptives to prompt clinical manifestations of multidrug resistance protein 3 deficiency. METHODS: Mutational analysis of ABCB4, screening for copy number variations by multiplex ligation-dependent probe amplification, genotyping for low expression allele c.1331T>C of ABCB11 and genotyping for variation c.55G>C in ABCG8 previously associated with cholesterol gallstones in adults was performed in 35 pediatric subjects with idiopathic gallstones who fulfilled the clinical criteria for low phospholipid-associated cholelithiasis syndrome (LPAC, OMIM #600803) and in 5 young females with suspected LPAC and their families (5 probands, 15 additional family members). The probands came to medical attention for contraceptive-associated intrahepatic cholestasis. RESULTS: A possibly pathogenic variant of ABCB4 was found only in one of the 35 pediatric subjects with idiopathic cholesterol gallstones whereas 15 members of the studied 5 LPAC kindreds were confirmed and another one was highly suspected to carry predictably pathogenic mutations in ABCB4. Among these 16, however, none developed gallstones in childhood. In 5 index patients, all young females carrying at least one pathogenic mutation in one allele of ABCB4, manifestation of LPAC as intrahepatic cholestasis with elevated serum activity of gamma-glutamyltransferase was induced by hormonal contraceptives. Variants ABCB11 c.1331T>C and ABCG8 c.55G>C were not significantly overrepresented in the 35 examined patients with suspect LPAC. CONCLUSION: Clinical criteria for LPAC syndrome caused by mutations in ABCB4 cannot be applied to pediatric patients with idiopathic gallstones. Sexual immaturity even prevents manifestation of LPAC.

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26 In our previous study[10] we focused on the role of the common variants c.523A>G (p.Thr175Ala) and c.1954A>G (p.Arg652Gly) in ABCB4, c.1331T>C (p.Val444Ala) in ABCB11 and c.55 G>C (p.Asp19His) in ABCG8 in pediatric gallstone disease. Login to comment
78 None of these changes is reportedly associated with hepatobiliary disease, with the possible exception of c.1954A>G (p.Arg652Gly), found previously in a heterozygous state in subjects 4, 8 and 26[10] . Login to comment
94 Table 2 Known variations in ABCB4, ABCB11 and ABCG8 found in 35 pediatric subjects with idiopathic gallstones in pediatric patients with idiopathic gallstones who met clinical criteria for the diagnosis of LPAC was the variation c.2318G>T (p.Gly773Val ) found in a heterozygous state in only one affected subject. Login to comment
95 The nucleotide change c.1954A>G found in 3 other pediatric gallstone subjects is common in the European, Caucasian, and African general population, but it has also been found in a patient with LPAC and low biliary phospholipid in whom its predicted consequence p.Arg652Gly was hypothesized to be conditionally penetrant, leading to clinical symptoms only under certain circumstances, such as pregnancy, or when combined with another mutation[18] . Login to comment
99 Interestingly, the ABCB4 variation c.523A>G (p. Thr175Ala), found in three index patients with LPAC, Patient. Login to comment
100 ID Variations in the coding sequence of ABCB4 ABCB11 low expression allele ABCG8 variation c.147C>T c.175C>T c.459T>C c.504C>T c.711A>T c.1954A>G c.1331T>C c.55G>C Ser49Ser Leu59Leu Phe153Phe Asn168Asn Ile237Ile Arg652Gly Val444Ala Asp19His rs8187789 rs2302387 rs2230027 rs1202283 rs2109505 rs2230028 rs2287622 rs11887534 1 CC CC TT TT AA AA CC GG 2 CC CC TT CT AA AA TC GG 3 CC CC TT CT AA AA TC GG 4 CC CC TT CT AT AG TC GG 5 CC CC TT TT AA AA CC GG 6 CC CC TT CC AA AA TC GG 7 CC CC TT CT AA AA TC GG 8 CC CC TT CC AA AG TC GG 9 CC CC TT TT AA AA TT GG 10 CC CC TT TT AA AA CC GG 11 CC CC TT TT AA AA TC GG 12 CC CC TT TT AA AA CC GG 13 CC CC TT CT AA AA TC GG 14 CC CC TT CC AA AA CC GG 15 CC CC TT TT AA AA TC GC 16 CC CC TT CC AA AA TC GC 17 CC CC TT CC AA AA TC GG 18 CC CC TT TT AA AA TC GC 19 CC CC TT CT AA AA TC GC 20 CC CC TT CT AA AA TT GG 21 CC CC TT TT AA AA TC GG 22 CC CC TT CT AA AA TT GG 23 CC CC TT CT AA AA TT GG 24 CC CT TT CC AT AA CC GG 25 CC CC TT CC AA AA TC GG 26 CT CT TC CC AA AG TC GG 27 CC CC TT TT AA AA TC GG 28 CC CC TT TT AA AA TT GG 29 CC CC TT CT AA AA TT GG 30 CC CC TT TT AA AA CC GC 31 CC CC TT CT AA AA TT GC 32 CC CC TT CT AA AA TT GG 33 CC CC TT TT AA AA TC GG 34 CC CC TT CC AA AA TT GG 35 CC CC TT TT AA AA CC GG Allelic frequency of variant alleles in patients with gallstones, HapMap populations and Czech population controls Allele T T C T T G C G Gallstone patients 0.014 0.029 0.014 0.571 0.029 0.043 0.471 0.086 HapMap CEU 0 0.112 01 0.664 0.175 0.075 0.408 0.085 HapMap HCB 0 0.167 01 0.344 0.222 0.023 0.333 0.022 HapMap JPT 0 0.273 01 0.442 0.300 0.023 0.261 0.011 HapMap YRI 0.042 0.525 0.11 0 0.362 0.392 0.425 0.042 Czech controls (n = 150) n.d. n.d. n.d. n.d. n.d. 0.090 0.400 0.0672 1 Results from corresponding populations studied in Environmental Genome Project (NIEHS ES15478 project). Login to comment
25 In our previous study[10] we focused on the role of the common variants c.523A>G (p.Thr175Ala) and c.1954A>G (p.Arg652Gly) in ABCB4, c.1331T>C (p.Val444Ala) in ABCB11 and c.55 G>C (p.Asp19His) in ABCG8 in pediatric gallstone disease. Login to comment
77 None of these changes is reportedly associated with hepatobiliary disease, with the possible exception of c.1954A>G (p.Arg652Gly), found previously in a heterozygous state in subjects 4, 8 and 26[10] . Login to comment