Atrial fibrillation (AF) is the most common cardiac arrhythmia. Despite its high frequency, current knowledge on the genetic bases of atrial fibrillation remains scant. Point-mutations in potassium channel encoding genes have been associated with familial AF, but account for only a small fraction of all AF cases ( < 10%). Recently, genome-wide association studies (GWAS) have unravelled common single nucleotide polymorphisms in three human chromosome loci (1q21, 16q22 and 4q25) highly associated to human atrial fibrillation (>40%). 1q21 is located intronic to KCNN3 potassium channel encoding gene whereas 16q22 is intronic to zing finger transcription factor ZFHX3. However, 4q25 SNP is located in a gene desert, being the closest gene the homeobox transcription factor PITX2. Evidence from our laboratory has demonstrated that impaired expression of PITX2 in the atrial myocardium is associated to AF. In addition, Pitx2 insufficiency in mice leads to increased atrial arrhythmogenesis, supporting the role of PITX2 in AF. However, it remains to be established if PITX2 controls ZFHX3 and/or KCNN3 expression, and whether epistatic relationship between these gene-associated SNPs occurs in the setting of AF. Zfhx3 and Kcnn3 expression is impaired in atrial and ventricular-specific Pitx2 conditional knock-outs . Over-expression of Pitx2 in HL1 cardiomyocytes reveals regulation of these genes by Pitx2. In addition, functional over-expression of Zfhx3 results in up-regulation of Kcnn3 but not of Pitx2. Overall these data illustrate that Pitx2 regulates Zfhx3, which in turn regulates Kcnn3. In order to unravel if epistatic relationships between AF associated SNPs were observed, a cohort of AF patients (n=65) and a control cohort (n=500) were re-sequenced for the following SNPs; rs2200733, rs13143308, rs6843082, rs10033464, rs3853445 at the 4q25 locus; rs1051614, rs13376333 at the 1q21 locus; and rs7193343, rs2106261 and rs74913256 at the 16q22 locus. All but rs3853445, rs1051614 and rs7193343 SNPs were significantly associated to AF patients as compared to controls, in line with previous reports. Epistatic associations were uncovered for several of the studies SNPs, including rs2200733, rs13143308 (4q25) and rs1051614 (1q21) SNPs, supporting thus a genetic link between PITX2 and KCNN3. Overall these data demonstrate that PITX2 is upstream of ZFHX3 and KCNN3.