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

Abstract

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.

Methods and results: A meta-analysis was performed combining the results of 2 genome-wide 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 single-nucleotide polymorphisms had P<5×10(-6) in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10(-5)). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene.

Conclusions: This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway.

Trial registration: ClinicalTrials.gov NCT00647088.

Keywords: RNA-Seq; aortic valve calcification; aortic valve stenosis; eQTL; gene expression; genome-wide association study; genomics.

© 2015 American Heart Association, Inc.

Figures

Figure 1

Overview of the study design. The flow chart shows the steps of the genome-wide association studies (GWAS) meta-analysis, pathway analysis, gene expression study of aortic valves, and valve expression quantitative trait loci (eQTL) mapping study. DE indicates differentially expressed genes; FC, fold change; GSA-SNP, gene set analysis-single nucleotide polymorphisms; KORA, Kooperative Gesundheitsforschung in der Region Augsburg Study; and PCOS, Polycystic Ovary Syndrome Genetics-NUgene.

Figure 2

Manhattan plot showing the results of the genome-wide association studies (GWAS) meta-analysis. The y axis represents P in −log10 scale combining the results of the 2 independent GWAS. The horizontal red line indicates the genome-wide significance cutoff (P=5×10−8). The horizontal blue line indicates the P cutoff of 5×10−6.

Figure 3

Boxplots of genome-wide association studies (GWAS) P values for significant gene sets. Corrected P values for each pathway are shown in parentheses. The vertical dashed line indicates P=0.01. Each point represents a gene. Please see Data Supplement for more details about gene set analysis single-nucleotide polymorphisms. ECM indicates extracellular matrix; and GnRH, gonadotropin-releasing hormone.

Figure 4

Integration of the genome-wide association studies (GWAS) and RNA-Seq results with known aortic valve stenosis (AS)–related biological processes. Integration of genes differentially expressed between calcified and normal valves and with/near single-nucleotide polymorphisms with P<1×10−4 in biological processes known to be implicated in AS development and progression. Cyan indicates down-regulated gene; Red, upregulated genes. Some genes can belong to >1 biological process. This figure was modified from Bossé et al. Name of all genes are provided in Table XI in the Data Supplement.

Figure 5

Alterations of the calcium signaling pathway in aortic valve stenosis (AS). A, Heat map of genes differentially expressed in the RNA-Seq experiment that belong to the calcium signaling pathway. Genes in yellow are upregulated and genes in blue are downregulated in calcified valves (TAVc) compared to normal valves (TAVn). The vertical white line separates the 2 groups. Each row represents a different gene indicated at the right. The calcium signaling pathway genes are ADRB1 (adrenoceptor beta 1), AGTR1 (angiotensin II receptor, type 1), BDKRB2 (bradykinin receptor B2), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1C subunit), CACNA1H (calcium channel, voltage-dependent, T type, alpha 1H subunit), EDNRA (endothelin receptor type A), ERBB3 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3), HRH1 (histamine receptor H1), HTR4 (5-hydroxytryptamine [serotonin] receptor 4, G protein-coupled), P2RX6 (purinergic receptor P2X, ligand-gated ion channel, 6), and TBXA2R (thromboxane A2 receptor). B, Ingenuity Pathway Analysis system (IPA) of the calcium pathway showing only genes that are altered (single-nucleotide polymorphisms with P<0.01 or genes differentially expressed) or regulated as predicted by the molecule activity predictor (MAP). Symbol indicates gene function (see legend). Genes in yellow are upregulated and genes in blue are downregulated in TAVc. Predicted activation and inhibition of genes because of the differential expression in TAVc are shown in orange and violet, respectively. TAVc indicates calcified tricuspid aortic valve; and TAVn, normal tricuspid valve.

Figure 6

RUNX2 is a susceptibility gene of aortic valve stenosis (AS) upregulated in calcified aortic valve and associated with expression quantitative trait loci (eQTL)-single-nucleotide polymorphisms (SNPs). A, Regional plots showing SNP rs114193529 located in RUNX2. The y axis shows the P values in −log10 scale for SNPs 1 Mb up- and downstream of the sentinel SNP (purple dot). The extent of linkage disequilibrium (LD; r2 values) for all SNPs with the sentinel SNP is indicated by colors. The location of genes is shown at the bottom. RUNX2 is framed in red. SNPs are plotted based on their chromosomal position on build hg19. B, Forest plot of overall effect size for rs114193529 in the 2 independent genome-wide association studies (GWAS) and meta-analysis. The black filled squares represent the odds ratio (OR) for each cohort and are proportional to the study size. The horizontal lines represent the 95% confidence intervals of the OR. The dashed grey and the dotted red vertical lines represent an OR of 1.0 and the OR of the meta-analysis, respectively. C, Gene expression levels of RUNX2 in human aortic valves with and without AS measured by RNA-Seq and expressed as mean FPKM (fragments per kilobase of exon per million fragments mapped). Cuffdiff adjusted P value is indicated. The number of valves assessed per group is indicated in parenthesis. Error bars represent the standard error. D, E, and F, Boxplots of mRNA expression levels for RUNX2 in human aortic valves according to genotyping groups for SNPs rs1200428, rs12209785, and rs35565233, respectively. The left y axis presents the z-score of log2-tranformed mRNA expression levels. The grey bar on the right y axis presents the percent variance in expression levels explained by the genotype. The x axis represents the 3 genotyping groups for each SNP, with the number of subjects in parenthesis. TAVc indicates calcified tricuspid aortic valve; and TAVn, normal tricuspid valve.

Figure 7

CACNA1C a new susceptibility gene of aortic valve stenosis (AS). A, Regional plot showing single-nucleotide polymorphism (SNP) rs2239118 located in CACNA1C. The y axis shows the P values in −log10 scale for SNPs 1 Mb up- and downstream of the sentinel SNP (purple dot). The extent of linkage disequilibrium (LD; r2 values) for all SNPs with the sentinel SNP is indicated by colors. The location of genes is shown at the bottom. CACNA1C is framed in red. SNPs are plotted based on their chromosomal position on build hg19. B, Forest plot of overall effect size for rs2239118 in the 2 independent genome-wide association studies (GWAS) and meta-analysis. The black filled squares represent the odds ratio (OR) for each cohort and are proportional to the study size. The horizontal lines represent the 95% confidence intervals of the OR. The dashed grey and the dotted red vertical lines represent an OR of 1.0 and the OR of the meta-analysis, respectively. C, Boxplot of gene expression levels in human aortic valves for CACNA1C according to genotyping groups of rs2239118. The left y axis presents the z-score of log2-tranformed mRNA expression levels. The grey bar on the right y axis presents the percent variance in gene expression explained by the genotype. Number of patients per genotype is presented in parenthesis. D, Gene expression levels of CACNA1C in human aortic valves measured by RNA-Seq and expressed as mean FPKM (fragments per kilobase of exon per million fragments mapped). Cuffdiff adjusted P value is indicated. The number of valves assessed per group is indicated in parenthesis. Error bars represent the standard error. TAVc indicates calcified tricuspid aortic valve; and TAVn, normal tricuspid aortic valve.