A Functional Variant Associated with Atrial Fibrillation Regulates PITX2c Expression through TFAP2a

Area of Science:

• Genetics
• Cardiology
• Molecular Biology

Background:

• The genetic locus 4q25 is strongly associated with atrial fibrillation (AF).
• Altered PITX2c expression is a suspected mechanism, but the functional impact of specific AF-associated variants remains unclear.
• Four independent association signals exist at this locus, necessitating detailed variant analysis.

Purpose of the Study:

• To investigate the functional role of AF-associated variants in the 4q25 region, specifically their effect on PITX2c gene expression.
• To identify the molecular mechanisms by which genetic risk variants contribute to AF susceptibility.
• To pinpoint the specific variant and its regulatory impact on PITX2c.

Main Methods:

• Bioinformatic analysis of regulatory elements using ENCODE and Roadmap Epigenomics data.
• In vivo enhancer activity assays in zebrafish embryos.
• CRISPR-Cas9 gene editing in human stem-cell-derived cardiomyocytes to assess variant effects.
• Electrophoretic mobility shift assays (EMSA) to study transcription factor binding.

Main Results:

• A single nucleotide polymorphism (SNP), rs2595104, was identified as a candidate regulatory variant.
• The AF risk allele of rs2595104 significantly reduced enhancer activity.
• CRISPR-Cas9 editing confirmed that the rs2595104 risk allele diminishes PITX2c expression in cardiomyocytes.
• Activating enhancer binding protein 2 alpha (TFAP2a) binding was reduced at the risk allele compared to the non-risk allele.

Conclusions:

• The AF-associated SNP rs2595104 directly impacts PITX2c expression.
• This effect is mediated through altered binding of TFAP2a to the non-risk versus risk allele.
• This mechanism provides a potential pathway linking genetic variation at 4q25 to atrial fibrillation susceptibility.