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Circ Cardiovasc Genet. Author manuscript; available in PMC 2016 July 06. Published in final edited form as: Circ Cardiovasc Genet. 2015 December ; 8(6): 812–822. doi:10.1161/CIRCGENETICS.115.001145.

Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

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Sandra Guauque-Olarte, MSc, David Messika-Zeitoun, MD, Arnaud Droit, PhD, Maxime Lamontagne, MSc, Joël Tremblay-Marchand, BSc, Emilie Lavoie-Charland, MSc, Nathalie Gaudreault, BSc, Benoit J. Arsenault, PhD, Marie-Pierre Dubé, PhD, Jean-Claude Tardif, MD, Simon C. Body, MBChB, MPH, Jonathan G. Seidman, PhD, Catherine Boileau, PharmD, PhD, Patrick Mathieu, MD, Philippe Pibarot, DVM, PhD, and Yohan Bossé, PhD Centre de recherche Institut universitaire de cardiologie et de pneumologie de Québec, Quebec, Canada (S.G.-O., M.L., J.T.-M., E.L.-C., N.G., P.M., P.P., Y.B.); Departments of Molecular Medicine (A.D., Y.B.), Surgery (P.M.), and Medicine (P.P.), Laval University, Quebec, Canada; Cardiology Department, AP-HP, Bichat Hospital, Paris, France (D.M.-Z.); INSERM U698, Paris, France (D.M.Z.); Département de Génétique, Hôpital Bichat, 75018 Paris, France (C.B.); Centre de Recherche du CHUQ, Quebec, Canada (A.D.); Montreal Heart Institute, Department of Medicine (M.-P.D., J.C.T.), Université de Montréal, Montreal, Canada (B.J.A.); Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA (S.C.B.); and Department of Genetics, Harvard Medical School, Boston, MA (J.G.S.)

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Background—Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS.

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Methods and Results—A meta-analysis was performed combining the results of 2 genomewide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five singlenucleotide polymorphisms had P65 and 85 years, respectively.1 The actual increase of life expectancy will raise the incidence of AS and amplify this public health problem.2 The risk of death, valve replacement, or progression to heart failure is increased by 80% over a period of 5 years in patients with AS.3 No pharmacological treatments are available, and conventional cardiovascular drugs, such as, statin have failed to prevent the development of AS.4,5 Open heart surgery (aortic valve replacement) and more recently transcatheter aortic valve implantation are the only treatments available for symptomatic and severe cases. AS is the principal indication for valve replacement surgery in North America and Europe.6 In Canada and the United States, the cost of an aortic valve replacement surgery is estimated at $74 602 CAN7 and $190 194 USD,8 respectively. New treatment strategies for this growing disease are urgently needed. AS is a biologically active process involving pathways and cellular processes, such as inflammation, extracellular matrix remodeling, and calcification.6,9 A genetic component to AS is demonstrated,10,11 and candidate gene studies have identified polymorphisms associated with AS or AS-related phenotypes12 (Table I in the Data Supplement). Rare mutations in NOTCH1 have been associated with bicuspid aortic valve disease and severe valve calcification.13,14 A recent genome-wide association study (GWAS) identified the lipoprotein(a) (LPA) gene associated with aortic valve calcification.15

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We performed a GWAS meta-analysis with AS patients from Quebec and France to identify new susceptibility genes for this disease. Gene expression of susceptibility genes was evaluated by RNA sequencing (RNA-Seq) in human aortic valves with and without AS. Finally, the functional meanings of the newly identified single-nucleotide polymorphisms (SNPs) showing some association with AS and that are located in differentially regulated genes were extended by carrying out the first expression quantitative trait loci (eQTL) mapping study in human valve tissues. This integrative genomic study confirmed the role of runt-related transcription factor 2 (RUNX2) as a potential driver of AS development and identified a new AS susceptibility gene, namely CACNA1C, belonging to the calcium signaling pathway.

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Materials and Methods Figure 1 illustrates the overall study design. Populations

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The Quebec cohort consisted of 494 French Canadian patients who underwent aortic valve replacement for severe tricuspid nonrheumatic AS at the Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ). The France cohort consisted of 505 patients with degenerative AS from the (Aortic Stenosis in Elderly: Determinant of Progression; clinicalTrial.gov number NCT 00338676) and (Genetic of Aortic Valve Stenosis-Clinical and Therapeutic Implications; http://www.clinicaltrial.gov; unique identifier NCT00647088) studies. Inclusion criteria were at least mild AS defined as peak aortic jet velocity (Vpeak) > 2.0 m/s. Some patients from France had bicuspid valve based on echocardiographic examination (n=35+38 possible bicuspid). Exclusion criteria for individual cohorts are provided in Methods in the Data Supplement. A summary of the clinical characteristics of individuals from Quebec and Paris are shown in Table II in the Data Supplement. Written informed consent was obtained from all study participants after institutional review board approval.

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Control cohorts were obtained from the database of genotypes and phenotypes.16 Proper control groups were selected based on genetic ancestry, genotyping data available, and sample size (Data Supplement). For cases from Quebec, the Polycystic Ovary Syndrome Genetics-NUgene (PCOS; phs000368) cohort from United States was selected. This cohort is a control group of individuals recruited at the Northwestern University Medical Center for performing a GWAS on PCOS susceptibility. For cases from France, 1 European cohort was selected, namely the Genetic Epidemiology of Refractive Error in the Kooperative Gesundheitsforschung in der Region Augsburg Study (KORA; phs000303). KORA is a population-based study of adults randomly selected from 430 000 individuals in Augsburg and 16 surrounding counties in Germany. Table III in the Data Supplement presents the characteristics of the database of genotypes and phenotypes cohorts. Whole-Genome Genotyping, Quality Controls, and Imputation

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DNA was extracted from whole blood or buffy coat (Data Supplement). Whole-genome genotyping for the Quebec and France cohorts was performed at the McGill University and Genome Quebec Innovation Center (http://gqinnovationcenter.com) using the Illumina HumanOmniExpress-12v1.0 BeadChip testing 733 202 SNPs. Standard genotyping quality controls were performed independently in the Quebec, France, and database of genotypes and phenotypes cohorts. Details are provided and summarized in Tables IV and V in the Data Supplement. For the Quebec cohort, 639 052 SNPs and 474 patients remained after quality controls. For the France cohort, 654 363 SNPs and 486 samples remained. The PCOS cohort was also genotyped using the Illumina HumanOmniExpress-12v1.0 platform. The KORA cohort was genotyped using the Illumina HumanOmni2.5 platform testing 2 182 680 SNPs. For the PCOS cohort, 645 438 SNPs and 2988 individuals passed quality controls. For the KORA cohort, 1 492 728 SNPs and 1864 individuals passed quality controls. To increase whole-genome coverage, additional SNPs were inferred using the

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segmented haplotype estimation and imputation tool17/IMPUTE218 pipeline. Only SNPs that passed a certainty score cut off of 0.9 were retained (Data Supplement). Association Tests and Meta-Analysis

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To account for differences in clinical characteristics between AS cases from Quebec and France, genetic association tests were performed independently and then combined into a meta-analysis. Association tests were performed with additive logistic regression models. Seven significant principal components derived from EIGENSTRAT19 were used as covariates to adjust for potential population stratification. After adjustment, the genomic inflation factors for the Quebec-PCOS and the France-KORA comparisons were both 1.0. The meta-analysis was performed using the inverse variance method with fixed- and random-effect models. The meta-analysis included 6 635 034 SNPs after the exclusion of polymorphisms with alternate risk alleles (n=36 590). The significant P value cutoff was set to 5×10−8. Association tests, meta-analysis, and linkage disequilibrium calculation were performed with PLINK.20 Regional plots were created with LocusZoom.21 SNPs with GWAS P150 μmol/L) were selected. Exclusion criteria are provided in Methods in the Data Supplement. Normal noncalcified tricuspid valves (n=10) were obtained from patients undergoing heart transplantation. Control valves had no evidence of disease at postexplant examination. Stenotic and control groups were matched for age (±10 years). RNA was extracted from valve leaflets, and gene expression evaluated by the Illumina HiSeq 2000 platform (Data Supplement). TopHat25 was used to align reads, which were then assembled by Cufflinks.25 Gene expression between the 2 groups of valves were compared with Cuffdiff, DESeq,26 and edgeR.27 Genes with Benjamini–Hochberg adjusted P

Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease.

Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study ...
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