Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11).

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.

Genetic polymorphisms in the multidrug resistance-associated protein 3 (ABCC3, MRP3) gene and relationship to its mRNA and protein expression in human liver.

AIMS: To determine the genetic variability of multidrug resistance protein 3 (MRP3). METHODS: Genomic DNA samples from 103 Caucasians were systematically screened for genetic variations to find a potential relationship with hepatic MRP3 expression. Sequencing comprised all 31 exons, approximately 100 bp of the flanking intronic regions and 2 kb of the 5' UTR. RESULTS: In total, 51 mutations were identified. Fifteen SNPs were located in the coding exons of MRP3, six of which are nonsynonymous mutations. SNPs 39G>C (allele frequency: 0.5%, located in exon 1), 202C>T (1.6%, exon 2), 1037C>T (0.5%, exon 9), 1537C>A (0.5%, exon 12), 3890G>A (5.2%, exon 27) and 4267G>A (0.6%, exon 29) resulted in Lys13Asn, His68Tyr, Ser346Phe, Gln513Lys, Arg1297His and Gly1423Arg amino acid substitutions, respectively. A splice site mutation (1339-1G>T) was found at the intron 10-exon 11 boundary. To evaluate, whether mutations in the MRP3 gene correlate with human hepatic MRP3 expression, we analyzed the genetic variants in Caucasian liver samples, whose MRP3 mRNA (n = 84) and protein (n = 50) expression has been determined by real time quantitative PCR and Western Blot, respectively. We found a significant correlation of a polymorphism in the 5' promoter region (-211C>T) of MRP3 with mRNA expression. Individuals homozygous and heterozygous for the -211C>T promoter polymorphism had significantly lower MRP3 transcript levels compared to wild-type individuals (P < 0.05). Accordingly, electrophoretic mobility shift assay demonstrated that -211C>T polymorphism affected the binding of nuclear factors. CONCLUSIONS: Multiple genetic polymorphisms of MRP3 exist in Caucasians. The -211C>T promoter polymorphism appears to be associated with altered hepatic MRP3 mRNA expression.

Identification of genetic variations of the human organic cation transporter hOCT1 and their functional consequences.

By systematic mutation screening of the polyspecific organic cation transporter hOCT1 (SLC22A1) in 57 Caucasians, 25 genetic variations were identified and further analysed for population frequency. Five mutations resulting in the amino acid changes Arg61Cys, Cys88Arg, Phe160Leu, Gly401Ser, and Met420del, with respective allele frequencies of 9.1, 0.6, 22, 3.2, and 16%, were functionally characterized upon expression in Xenopus oocytes. Phe160Leu and Met420del exhibited substrate affinities and selectivites identical to hOCT1 wild-type. In contrast, uptake of 0.1 microm [3H]1-methyl-4-phenylpyridinium ([3H]MPP) by Arg61Cys, Cys88Arg and Gly401Ser were reduced to 30, 1.4 and 0.9% compared to wild-type, respectively. Since transport of 1 microm [3H]serotonin by Cys88Arg and Gly401Ser was reduced to only 13 and 12% of wild-type, these mutants exhibit a changed substrate selectivity. The data show that the mutants Arg61Cys, Cys88Arg and Gly401Ser could affect the disposition of OCT1 substrates and as a consequence may alter the duration and intensity of effects of drugs and neurotransmitters which are substrates for hOCT1.