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In vitro functional analysis of novel single nucleotide polymorphisms in OATP1B1
Author(s) -
Yin Peter,
Kim Richard B
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.663.4
Subject(s) - single nucleotide polymorphism , biology , nonsynonymous substitution , microbiology and biotechnology , in vitro , genetics , computational biology , gene , genotype , genome
The goal of our study is to functionally characterize six nonsynonymous single nucleotide polymorphisms (SNPs) in human organic anion transporting polypeptide 1B1 (OATP1B1) using in vitro transporter expression technologies. We hypothesize these SNPs will result in a decrease in transport activity compared to wild‐type. OATP1B1 cDNA packaged in pEF6/V5‐His TOPO was used as template, and 6 SNPs — 298G>;A (rs144508550), 419C>;T (rs147450830), 463C>;A (rs11045819), 1007C>;G (rs72559747), 1463G>;C (rs59502379), and 1738C>;T (rs71581941) — were introduced separately to wild‐type templates using QuikChange site‐directed mutagenesis. OATP1B1 variant cDNAs will then be transferred to pAd/CMV/V5‐DEST Gateway vector for further propagation as OATP1B1 variants expressed in adenovirus. We will determine the optimal titre of the OATP1B1‐ variant adenovirus for transporter expression using a monolayer cell lines such as HeLa. Subsequently, we will use prototypical substrates of OATP1B1 such as rosuvastatin to determine the relative affinity (K m ) and capacity (V max ) for uptake, followed by quantification of relative cell surface trafficking. Identification of new functional SNPs in OATP1B1 will give us further insights to genetic heterogeneity in OATP1B1 as a contributor to inter‐subject variation in response to OATP1B1 substrate drug therapy. Research is funded by the Canadian Institute for Health Research.