1715-P: Identifying Potentially Functional Variants in Regulatory Regions in KCNQ1 that Associate with Type 2 Diabetes in American Indians

A prior genome-wide association study in 7701 Pima Indians utilized a custom Axiom array (Affymetrix) which contained 474,196 SNPs. These SNPs tagged 92% of common variants (R²≥0.85, minor allele frequency ≥0.05) identified in the Pima Indian genome. SNP rs79788804, which tags 38 additional SNPs in intron 15 of KCNQ1, achieved genome-wide significance in its association with type 2 diabetes (T2D; OR=1.31, p=2 x10^-8). A 3 SNP haplotype (rs79788804, rs2237892 and rs2299620) with >1 maternally-derived alleles strongly associated with T2D (OR_M=2.05, p=2x10^-12). ENCODE and Islet regulome databases mapped putative strong enhancers to 4 regions in intron 15 with multiple epigenetic marks and transcription factor binding sites. To validate these regulatory regions, we tested gene activation from the putative enhancers using CRISPR-CAS9-based acetyltransferase (dCas9^p300) in human pancreatic beta cells (EndoC-βH3). Guide RNA specific acetylation by dCas9^p300 ata 341 bp region spanning rs2299620, rs2237896, rs2237897 and rs74046911 resulted in a 1.5 fold gene activation for KCNQ1 and nearby gene TRPM5 (both p=0.001). A chromatin immunoprecipitation-qPCR assay confirmed that dCas9^P300 acetylated chromatin at this targeted region. We further assessed the effects of 5 transcription factors (NKX2.2, MAFB, NKX6.1, FOXA2, PDX1) on transcriptional activity under control of these 4 enhancer regions. Mouse pancreatic β-cells (NIT1) were co-transfected with a vector expressing each transcription factor and a luciferase reporter construct containing the KCNQ1 SNPs. NKX2.2 had a 5.5 fold activation for the construct containing the same 341 bp KCNQ1 region, as compared to a 4.1 fold activation for the construct containing KCNQ1 promoter alone (p=0.02). Our data suggested that a 341 bp region containing rs2299620, rs2237896, rs2237897 and rs74046911 may harbor functional variant(s) which affect expression of KCNQ1 and TRPM5.