Background Genome wide association studies have identified 23 loci for atrial fibrillation (AF), but the mechanisms responsible for these associations, as well as the causal genes and genetic variants, remain undefined. Methods and Results To identify the effect of common genetic variants on gene expression that might explain the mechanisms linking genome wide association loci with AF risk, we performed RNA sequencing of left atrial appendages from a biracial cohort of 265 subjects. Combining gene expression data with genome-wide single nucleotide polymorphism (SNP) data, we found that approximately two-thirds of the expressed genes were regulated in cis by common genetic variants at a false discovery rate of <0.05, defined as cis-expression quantitative trait loci (cis-eQTLs). 12 of 23 reported AF genome wide association loci displayed genome-wide significant cis-eQTLs, at PRRX1 (chromosome 1q24), SNRNP27 (1q24), CEP68 (2p14), FKBP7 (2q31), KCNN2 (5q22), FAM13B (5q31), CAV1 (7q31), ASAH1 (8p22), MYOZ1 (10q22), C11ORF45 (11q24), TBX5 (12q24), and SYNE2 (14q23), suggesting that altered expression of these genes plays a role in AF susceptibility. Allelic expression imbalance was employed as an independent method to characterize the cis-control of gene expression. 1248 of 5153 queried genes had cis-SNPs that significantly regulated allelic expression at a false discovery rate of <0.05. Conclusions We provide a genome wide catalog of the genetic control of gene expression in human left atrial appendage. These data can be used to confirm the relevance of genome wide association loci and to direct future functional studies to identify the genes and genetic variants responsible for complex diseases such as AF.

Background Genome-wide studies reveal that genetic variants at chromosome 4q25 constitute the strongest locus associated with atrial fibrillation (AF), the most frequent arrhythmia. However, the mechanisms underlying this association are unknown. Our goal is to find and characterize left atrial expressed transcripts in the chromosome 4q25 AF risk locus that may play a role in AF pathogenesis. Methods and Results RNA sequencing performed on human left/right pairs identified an intergenic long noncoding RNA (lncRNA) adjacent to the PITX2 gene, which we have named PANCR (PITX2 adjacent noncoding RNA). In a human tissue screen, PANCR was expressed specifically in the left atria and eye, and in no other chambers of the heart. The levels of PANCR and PITX2c RNAs were highly correlated in 233 human left atrial appendage samples. PANCR levels were not associated with either atrial rhythm status or the genotypes of the chromosome 4q25 AF risk variants. Both PANCR and PITX2c RNAs were induced early during differentiation of human embryonic stem cells into cardiomyocytes. Since lncRNAs often control gene expression, we performed siRNA-mediated knockdown of PANCR; and, this treatment repressed PITX2c expression and mimicked the effects of PITX2c knockdown on global mRNA and miRNA expression. Cell fractionation studies demonstrate that PANCR is primarily localized in the cytoplasm. Conclusions PANCR and PITX2c are coordinately expressed early during cardiomyocyte differentiation from stem cells. PANCR knockdown decreased PITX2c expression in differentiated cardiomyocytes, altering the transcriptome in a manner similar to PITX2c knockdown.

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. 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 distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic.