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Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

ABSTRACT: Background. The coronary heart disease (CHD) risk locus on 21q22 (lead SNP rs9982601) lies within a "gene desert." The aim of this study was to assess if this locus is associated with CHD risk factors and to identify the functional variant(s) and gene(s) involved. Methods. A phenome scan was performed with UCLEB Consortium data. Allele-specific protein binding was studied using electrophoretic mobility shift assays. Dual-reporter luciferase assays were used to assess the impact of genetic variation on expression. Expression quantitative trait analysis was performed with Advanced Study of Aortic Pathology (ASAP) and Genotype-Tissue Expression (GTEx) consortium data. Results. A suggestive association between QT interval and the locus was observed (rs9982601??p = 0.04). One variant at the locus, rs28451064, showed allele-specific protein binding and its minor allele showed 12% higher luciferase expression (p = 4.82 × 10-3) compared to the common allele. The minor allele of rs9982601 was associated with higher expression of the closest upstream genes (SLC5A3 1.30-fold increase p = 3.98 × 10-5; MRPS6 1.15-fold increase p = 9.60 × 10-4) in aortic intima media in ASAP. Both rs9982601 and rs28451064 showed a suggestive association with MRPS6 expression in relevant tissues in the GTEx data. Conclusions. A candidate functional variant, rs28451064, was identified. Future work should focus on identifying the pathway(s) involved.

SUBMITTER: Beaney KE

PROVIDER: S-EPMC5387827 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

Beaney Katherine E KE Smith Andrew J P AJP Folkersen Lasse L Palmen Jutta J Wannamethee S Goya SG Jefferis Barbara J BJ Whincup Peter P Gaunt Tom R TR Casas Juan P JP Ben-Shlomo Yoav Y Price Jacqueline F JF Kumari Meena M Wong Andrew A Ong Ken K Hardy Rebecca R Kuh Diana D Wareham Nicholas N Kivimaki Mika M Eriksson Per P Humphries Steve E SE Consortium Ucleb U

Disease markers 20170328

<i>Background</i>. The coronary heart disease (CHD) risk locus on 21q22 (lead SNP rs9982601) lies within a "gene desert." The aim of this study was to assess if this locus is associated with CHD risk factors and to identify the functional variant(s) and gene(s) involved. <i>Methods</i>. A phenome scan was performed with UCLEB Consortium data. Allele-specific protein binding was studied using electrophoretic mobility shift assays. Dual-reporter luciferase assays were used to assess the impact of ...[more]

PMID: 28458444

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Project description:A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the ?1 subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide/cGMP signaling pathway. The mechanism linking common variants in this region with coronary risk is not known.Gene expression and protein expression were analyzed with quantitative polymerase chain reaction and immunoblotting, respectively. Putative allele-specific transcription factors were identified with in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed with reporter gene assays. To assess the effect of zinc finger E box-binding homeobox 1 transcription factor (ZEB1), siRNA-mediated knockdown and overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes was analyzed with vascular smooth muscle cell migration assays and platelet aggregation analyses.Whole-blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the nonrisk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of nonrisk allele promoter activity and a significant reduction of endogenous GUCY1A3 expression. Ex vivo-studied platelets from homozygous nonrisk allele carriers displayed enhanced inhibition of ADP-induced platelet aggregation by the nitric oxide donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil compared with homozygous risk allele carriers. Moreover, pharmacological stimulation of sGC led to reduced migration only in vascular smooth muscle cells homozygous for the nonrisk allele. In the Hybrid Mouse Diversity Panel, higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta.Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the nonrisk allele, leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.

Dataset Information

Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

Publications

Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

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