Rapid identification of three functionally relevant polymorphisms in the OATP1B1 transporter gene using Pyrosequencing.
BACKGROUND
Clinical evidence suggests there are three single nucleotide polymorphisms (SNPs) of the solute carrier organic anion transporter family member B1 (SLCO1B1) gene for which in vivo evidence for a functional relevance for organic anion transporter polypeptides subgroup C (OATP1B1, formerly OATP-C) has been provided. These genetic variants have been shown to lead to altered pharmacokinetics of OATP1B1 substrates, mainly pravastatin, but also the irinotecan metabolite SN-38, estrone-3-sulfate, and estradiol-17beta-glucuronide. The authors therefore developed reliable and quick screening assays to identify the SLCO1B1 SNPs -11187G>A, 388A>G and 521T>C, in order to facilitate the judgment of their clinical role and to identify allelic frequencies of SNPs and haplotypes in a Caucasian random sample.


METHODS
Three simplex Pyrosequencing assays were developed and the three selected SLCO1B1 SNPs were screened for in 250 DNA samples from healthy young female and male unrelated volunteers of Caucasian ethnicity. SLCO1B1 haplotypes involving DNA positions -11187, 388 and 521 were identified by in silico haplotyping.


RESULTS
A clear identification of the three single nucleotide polymorphisms in the 250 DNA samples was possible and was verified by routine implementation of 40 control samples obtained by conventional sequencing. The frequencies of the variant alleles -11187A, 388G and 521C were 0.09, 0.47 and 0.12, respectively. All observed frequencies of heterozygous of homozygous carriers of SLCO1B1 alleles were in agreement with the Hardy-Weinberg equilibrium. SLCO1B1 haplotypes reportedly associated with altered substrate pharmacokinetics, i.e., SLCO1B1*15B (-11187G/388G/521C) and *17 (-11187A/388G/521C), were found at allelic frequencies of 0.09 and 0.02, respectively.


CONCLUSIONS
The presently developed Pyrosequencing assays allowed for quick and reliable identification of those SLCO1B1 SNPs that had been proposed to cause functional alternations in OATP1B1 with shown consequences for the pharmacokinetics of drugs that are OATP1B1 substrates.
Rohrbacher
Maren
M
pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor Stern Kai 7, D-60590 Frankfurt, Germany.
Kirchhof
Anja
A
Skarke
Carsten
C
Geisslinger
Gerd
G
Lötsch
Jörn
J
D000328:Adult; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D005006:Ethnicity; D005260:Female; D005820:Genetic Testing; D006801:Humans; D027381:Liver-Specific Organic Anion Transporter 1; D008297:Male; D027361:Organic Anion Transporters / Q000235:genetics*; D010599:Pharmacokinetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014481:United States
D017931:DNA Primers; D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; C503999:SLCO1B1 protein, human; D004247:DNA