Figure 7

Functional characterization of SNPs in the PIGB gene. (A) Patterns of linkage disequilibrium (LD) within ~200 Kb surrounding the rs3797418 SNP among three different ethnic groups using HapMap3 R2 data as a reference. The top 7 SNPs in PIGB are arranged in order from 5′ to 3′, as shown in the gene structure above each plot. Black indicates combinations where R² = 1 and linkage of disequilibrium (LOD) ≥ 2; light grey, combinations where 0 < R² < 1 and LOD ≥ 2; white, where R² = 0 and LOD < 2. (B) Association between SNPs and PIGB expression measured by QRT-PCR assay and microarray using 37 randomly selected LCLs. (C) Effect of the nonsynonymous coding SNP (rs2290344, M161T) on PIGB mRNA expression, protein expression and response to gemcitabine. PIGB mRNA and protein levels were determined in SU86 and MDA-MB-231 cells transfected with either PIGB wild-type or variant constructs with GST-tags. Antibody against GST (Antibody #2622, Cell Signaling Inc.) was used for detection of PIGB expression, and Antibody against MUC1 (VU4H5 Mouse mAb #4538, Cell Signaling Inc.) served as a loading control in Western Blot assay. Gemcitabine cytotoxicity performed with the MTS assay was performed in cells transfected with WT and variant constructs. No differences were observed between WT and variant SNP for any of the phenotypes tested. (D) EMSAs were performed for two regulatory SNPs in PIGB gene. The arrows indicate different binding pattern between WT and variant sequences for rs11636687 and rs28668016.