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Functional Characterization of the GUCY1A3 Coronary Artery Disease Risk Locus.
- Source :
-
Circulation [Circulation] 2017 Aug 01; Vol. 136 (5), pp. 476-489. Date of Electronic Publication: 2017 May 09. - Publication Year :
- 2017
-
Abstract
- 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 α <subscript>1</subscript> 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.<br />Methods: 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.<br />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 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.<br />Conclusions: 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.<br /> (© 2017 American Heart Association, Inc.)
- Subjects :
- Alleles
Blood Platelets metabolism
Cell Line
Cell Movement drug effects
Coronary Artery Disease pathology
Cyclic GMP metabolism
Genetic Loci
Genotype
HEK293 Cells
Homozygote
Humans
Muscle, Smooth, Vascular cytology
Muscle, Smooth, Vascular drug effects
Muscle, Smooth, Vascular metabolism
Nitric Oxide metabolism
Nitroprusside pharmacology
Platelet Aggregation drug effects
Polymorphism, Single Nucleotide
Promoter Regions, Genetic
RNA Interference
RNA, Messenger metabolism
RNA, Small Interfering metabolism
Risk
Sildenafil Citrate pharmacology
Soluble Guanylyl Cyclase metabolism
Transcription Factors genetics
Transcription Factors metabolism
Zinc Finger E-box-Binding Homeobox 1 antagonists & inhibitors
Zinc Finger E-box-Binding Homeobox 1 genetics
Zinc Finger E-box-Binding Homeobox 1 metabolism
Coronary Artery Disease genetics
Soluble Guanylyl Cyclase genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1524-4539
- Volume :
- 136
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Circulation
- Publication Type :
- Academic Journal
- Accession number :
- 28487391
- Full Text :
- https://doi.org/10.1161/CIRCULATIONAHA.116.024152