Genetic and Functional Studies of LociAssociated with Atrial Fibrillation

Atrial Fibrillation (AF) is the most common sustained arrhythmia, and is associated with an increased risk of mortality, morbidity and stroke. Genome wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region that is closest to the PITX2 gene. The PITX2c isoform expressed specifically in the left atria plays a role in cardiac left/right asymmetry during development and hemizygous knockout mice are susceptible to pacing induced arrhythmia. The Cleveland Clinic Lone AF GWAS identified four independent AF risk SNPs at the chromosome 4q25 locus, with the most significant SNP identified located ~171kb distal to PITX2c. In addition to PITX2c, our group identified an uncharacterized PITX2 Adjacent long intergenic Noncoding RNA (PANCR), ~2 kb proximal to PITX2. We hypothesized that the AF associated SNPs located on chromosome 4q25 might directly affect expression of PITX2c and/or PANCR. We determined that PITX2c and PANCR levels were positively correlated with each other in 223 left atrial appendages. Expression of both genes was examined in a panel of 33 human tissues, and both were highly expressed in left atria and eye. Using left atrial appendages surgically obtained from subjects of European ancestry, we obtained SNP genotypes via microarray and measured PITX2c and PANCR expression via quantitative RT-PCR (qRT-PCR). We found that the AF risk SNPs were not associated with PITX2c or PANCR expression. During differentiation of H9 human embryonic stem cells into cardiomyocytes, both transcripts were induced early prior to expression of cardiac troponin, suggesting they are coordinately expressed. Knock-down of PANCR in differentiated H9 cardiomyocytes led to decreased expression of itself and of PITX2c. RNAseq after knock-down of PITX2 and PANCR independently and simultaneously revealed a large number of genes that were differentially altered by the different conditions. We conclude that expression of PITX2c and PANCR in human adult left atrial appendages is not associated with the 4q25 AF risk SNPs. We speculate that these risk SNPs may alter expression of these genes in a different location or during cardiac development, and we plan to study this using differentiating H9 cells.