Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus

Thorsten Kessler, MD; Jana Wobst, MS; Bernhard Wolf, MD; Juliane Eckhold, BS; Baiba Vilne, PhD; Ronja Hollstein, MS; Simon von Ameln, PhD; Tan An Dang, MS; Hendrik B. Sager, MD; Philipp Moritz Rumpf, MD; Redouane Aherrahrou, PhD; Adnan Kastrati, MD; Johan L.M. Bjoerkegren, MD, PhD; Jeanette Erdmann, PhD; Aldons J. Lusis, PhD; Mete Civelek, PhD; Frank J. Kaiser, PhD; Heribert Schunkert, MD

Circulation 136. 5 (9 May 2017): 476-489. 

Background—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 (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. The mechanism linking common variants in this region with coronary risk is not known.

Methods—Gene and protein expression were analyzed using quantitative polymerase chain reaction (qPCR) and immunoblotting, respectively. Putative allele-specific transcription factors were identified using in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed using reporter gene assays. To assess the effect of ZEB1, siRNA-mediated knockdown as well as overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes were analyzed using vascular smooth muscle cell (VSMC) migration assays and platelet aggregation analyses.

Results—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 non-risk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of non-risk allele promoter activity, as well as a significant reduction of endogenous GUCY1A3 expression. Ex vivo studied platelets from homozygous non-risk allele carriers displayed enhanced inhibition of adenosine diphosphate-induced platelet aggregation by the NO donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil as compared to homozygous risk allele carriers. Moreover, pharmacologic stimulation of sGC led to reduced migration only in VSMC homozygous for the non-risk allele. In the Hybrid Mouse Diversity Panel higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta.

Conclusions—Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the non-risk 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.