Abstract 18159: A Functional Variant for Atrial Fibrillation Affects PITX2c Expression by Interacting With TFAP2a

Abstract

Background: To date, 17 loci associated with atrial fibrillation (AF) have been identified, and there are 4 regions at 4q25 around PITX2 gene strongly associated with AF. However, the functional variants at all known AF loci remain elusive. Methods and Results: Among the 4 AF loci at 4q25, we focused on one associated region of ~84 kb extending from Chr4: 111,520,926 to 111,604,727 that includes the PITX2 gene itself (defined as all SNPs with r 2 > 0.6 from the sentinel SNP rs1448818). To identify potential functional variants , we analyzed DNase hypersensitivity, histone modification and mammalian conservation from ENCODE and Roadmap Epigenomics Project. Additionally, cap analysis of gene expression (CAGE) data was also investigated. We identified 13 candidate regions, 5 of which showed enhancer activity in zebrafish skeletal muscle and heart. Using luciferase assays in a mouse atrial cell line (HL-1), we then identified one SNP (rs2595104) in which the T risk allele was associated with 31% decreased activity compared to the G non-risk allele (p = 0.007). rs2595104 lies ~10 kb upstream of PITX2c transcriptional start site and is in high linkage disequilibrium (LD) (r 2 = 0.67) with rs1448818. In silico investigation of rs2595104 using UniPROBE and HaploReg database revealed differential binding of each allele to activating enhancer binding protein 2 alpha (TFAP2a). Electrophoretic mobility shift assays showed that TFAP2a bound robustly to the non-risk G allele at rs2595104 but not to the risk T allele. Chromatin immunopricipitation followed by allele-specific qPCR in human embryonic stem cell-derived cardiomyocytes (hESC-CM) further suggested that TFAP2a was preferentially recruited to the non-risk G allele at rs2595104 in an AF-relevant cell type. hESC-CMs with homozygous deletion of rs2595104-containing enhancer using CRSPR-Cas9 expressed less PITX2c (35 ± 6%, p = 0.036), compared to the wild-type hESC-CMs. Finally, mutating the G allele at rs2595104 to T allele by CRSPR-Cas9 led to 21% reduction in PITX2c level in hESC-CMs (p = 0.027). Conclusion: We have found that the AF-associated SNP rs2595104 alters PITX2c expression via interaction with TFAP2a. Such a pathway may ultimately contribute to AF susceptibility at the PITX2 locus.