DNA‐nuclear protein interactions around‐224 A/G Single Nucleotide Polymorphism in the Neuropeptide Receptor Y2 (NPY2R) Gene in Predisposition to Hypertension

Abstract

Hypertension (HTN), is an important risk factor for cardiovascular and kidney diseases. Multiple genes as well as environmental and lifestyle factors influence the risk of developing high blood pressure. Previous work in animal models and human populations identified Neuropeptide Y receptor 2 (NPY2R) as a candidate gene for HTN. In two independent Japanese populations, the GG genotype single nucleotide polymorphism (SNP) located 224 bp upstream of the transcription start site was associated with an increased risk of HTN. The aim of this study was to assess the DNA‐nuclear protein interactions surrounding the NPY2R‐224 A/G SNP. Two versions of double stranded oligonucleotide probes (50bp) corresponding to the sequence flanking NPY2R‐224A/G were synthesized and incubated with different amounts nuclear extracts for an electrophoretic shift assay (EMSA). In addition, we used CTCF antibody for a gel supershift assay. The DNA/protein reactions were loaded on a 4% non‐denaturing polyacrylamide gel. Interestingly, the EMSA showed an allele‐specific binding with the oligonucleotide containing an A nucleotide in ‐224 position. However, the oligonucleotide containing the G nucleotide in ‐224 position shows no DNA/nuclear protein interaction. Also, we were able to detect a supershift using the CTCF antibody. The data provides strong evidence for a functional role of NPY2R in genetic predisposition to HTN.