Introduction:
Genome-wide association studies have identified 14 independent loci for atrial fibrillation (AF). The 4q25 locus upstream of the left-right asymmetry gene
PITX2
is, by far, the strongest association signal for AF. However, as with most GWAS loci, the functional variants are noncoding, presumed to be regulatory, and remain unknown. We therefore sought to rapidly identify the functional variants at an AF locus by combining high throughput sequencing and massively parallel reporter assays.
Methods and Results:
We sequenced a ~750kb region encompassing the
PITX2
locus in 462 individuals with early-onset AF from the MGH AF Study and 464 referents from the Framingham Heart Study. The SNP most significantly associated with AF in our sequenced sample was rs2129983, which is 140kb from
PITX2
(OR=2.43,
P
=8.9X10
-16
). rs2129983 is approximately 1.7kb from the most significantly associated SNP in a prior AF GWAS, rs6817105 (r
2
=0.52). From the targeted sequencing analysis, we identified 262 SNVs with a MAF >0.5% within a genomic region bounded by SNPs with an r2 greater than 0.4 with the top variant. To identify functional variants, we then utilized a massively parallel reporter assay (MPRA) in order to measure enhancer activity at each SNP across the entire AF locus. In both HL-1 and C2C12 myoblasts, MPRA identified many distinct SNP regions with differential enhancer activity. Using AF-association status as a standard, we were able to identify a series of variants that have both differential activity in either cell line tested and also a high level of association (rs17042076, rs4469143). Mechanistically, these functional SNPs are predicted to alter transcription factor binding.
Conclusions:
We have comprehensively identified the AF-associated variation at 4q25 and determined which of these variants are functional through differential enhancer activity. Here, in addition to identifying the causative variation for AF at 4q25, we provide a generalizable pathway for translating this work to other loci, a method that could expedite the identification of causative genetic variants at other disease loci.
A massively parallel 3′ UTR reporter assay reveals relationships between nucleotide content, sequence conservation, and mRNA destabilization
