52 Genetic Loci Influencing Myocardial Mass

Abstract

Background: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death.

Objectives: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass.

Methods: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment.

Results: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo.

Conclusions: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets.

Keywords: QRS; electrocardiogram; genetic association study; heart failure; left ventricular hypertrophy.

FIGURE 1. Genome-Wide Associations and Candidate Genes

This overlay Manhattan plot shows the results for the genome-wide associations with QRS traits among Europeans. Single nucleotide polymorphisms (SNPs) reaching genome-wide significance (p < 1 × 10⁻⁸) are colored red (novel loci) or blue (previously reported loci). Candidate genes have been identified by 1 or multiple strategies: n = nearest; c = coding nonsynonymous variant; g = GRAIL (Gene Relationships Across Implicated Loci) tool; e = expression quantitative trait loci (eQTL); and d = DEPICT (Data-Driven Expression-Prioritized Integration for Complex Traits) tool. The presence of associated eQTL, coding SNPs, deoxyribonuclease (DNAse) hypersensitivity sites, chromatin states, or transcription factor binding sites are indicated for lead SNPs (light blue) or those in high (r² > 0.8) linkage disequilibrium (dark blue). 12LS = 12-lead sum product; Cor = Cornell voltage product; Dur = QRS duration; MAF = minor allele frequency; Sok = Sokolow-Lyon product.

FIGURE 2. Functional Annotations

(A) The 52 sentinel SNPs are significantly enriched in deoxyribonuclease hypersensitivity sites (DHS) of the human fetal heart compared with the matched random distribution of HapMap SNPs. (B) The effect of physical distance between SNPs that meet genome-wide significance (p < 1 × 10⁻⁸) on enrichment of fetal heart relative to all other tissues at DHS. The enrichment is strongest at the SNP’s location and decreases after 100 base pairs from the SNP sites. (C) SNPs associated with QRS traits are enriched for the activating histone modifications H3K27ac, H3K4me3, H3K4me1, and H3K36me3 in the human left ventricle, which increased at more stringent genome-wide association study (GWAS) p value thresholds. The repressive mark H3K27me3 is not enriched, whereas H3K9me3 is significantly reduced, suggesting that QRS-trait loci are predominantly expressed in the left ventricle. (D) To capture greater complexity, we performed an integrative analysis in an 18-state “expanded” ChromHMM model representative of different functional regions of the genome. The 52 loci for the 18-state model were enriched using the 6 core histone marks (left); the total number of the 52 loci overlapped by each feature is shown (right). (E) SNPs (p < 1 × 10⁻⁸) were also significantly enriched for various factors in the human heart, mouse heart, and the HL-1 cell line. CI = confidence interval; TSS = transcription start site; other abbreviations as in Figure 1.

FIGURE 3. Functional Follow-Up of rs6781009 in the SCN5A Locus

(A) In vivo activity of 4 exemplar human cardiac enhancers in embryonic transgenic mice stained for LacZ enhancer reporter activity (dark blue) are shown. (For additional examples of previously described enhancers near lead SNPs, see Online Figure 13.) (B) Position of the regulatory element containing rs6781009 on the SCN5A-SCN10A locus. GWAS signals are plotted on a −log(p) scale in dark blue. The regulatory element is bound by TBX3, TBX5, and P300 (lower black traces) in mice, and the contact profile of the SCN5A promoter obtained by 4C-seq human cardiac ventricular tissue revealed an interaction between this regulatory element and the SCN5A promoter (upper black trace and contact profile). Normalized contact intensities (gray dots) and their running median trends (black line) are depicted for the SCN5A promoter viewpoint. Medians are computed for 4-kb windows, and the gray band displays the 20% to 80% percentiles for these windows. Below the profile, statistical enrichment across differently scaled window sizes (from 2 kb [top row] to 50 kb [bottom rom]) is depicted of the observed number of sequenced ligation products over the expected total coverage of captured products, with the latter being estimated on the basis of a probabilistic background model. Local changes in color codes indicate regions that are statistically enriched for captured sequences. The lowest box shows the linkage disequilibrium pattern for the HapMap CEU population. (C) Luciferase assay performed in H10 cells showing a high constitutive activity for the enhancer core element (0.6 kb) containing the major allele for rs6781009, which is reduced for the minor allele in both a large enhancer construct (1.5 kb), as well as in the core enhancer element (0.6 kb). *p < 0.01. (D) Dorsal views of hearts containing the human regulatory element with the major versus minor allele for rs6781009 in a LacZ reporter vector, showing specific expression of the enhancer in the interventricular septum (ivs) for the major allele, which is absent for the minor allele. *p < 0.05. la = left atrium; lv = left ventricle; ra = right atrium; rv = right ventricle; other abbreviations as in Figure 1.

FIGURE 4. Heart-Specific RNAi Knockdown in Drosophila

Cardiac defects upon heart-specific ribonucleic acid interference (RNAi) knockdown are seen in Drosophila. (A) Wild-type dorsal heart tube stained with the F-actin stain phalloidin. The magnified region (right) is highlighted. Arrowheads point to ostia (inflow tracks), and the arrow shows the circumferential orientation of myofibrils. (B) Cka/Striatin RNAi induces myofibrillar disarrangement. Myofibrils are oriented in a disorganized, mainly anterior-posterior orientation with gaps in between (arrow). (C) Knockdown of NACα/NACA causes severe cardiac tissue disintegration. Adult cardiomyocyte tissue may be completely absent (asterisk), whereas some heart-associated longitudinal muscles are still present (arrowheads). At larval stages, the heart is much less affected, suggesting a maturation or remodeling defect. (D) Knockdown of EcR/NR1H blocks cardiac remodeling and causes myofibrillar disarray (arrow). Ventral longitudinal muscles are also abnormal (arrowhead).

FIGURE 5. Functional Connections of Gene Expression Networks

In the DEPICT (Data-Driven Expression-Prioritized Integration for Complex Traits) analysis, (A) plots show the enrichment of loci associated with QRS traits in specific physiological systems. (B) In a graphic display of DEPICT gene set enrichment analysis, meta-gene sets are represented by nodes colored according to statistical significance, and similarities between them are indicated by edges scaled according to their correlation (only correlations with r > 0.3 are shown).

CENTRAL ILLUSTRATION. Gene-Related Cardiac Conditions

In this study, a number of gene-containing loci were identified that influence a variety of abnormalities in the dysfunctional heart, myocardium, and cardiac myocytes. Analyzing the QRS genome-wide association study results using the DEPICT (Data-Driven Expression-Prioritized Integration for Complex Traits) tool identified 43 meta-gene sets (Figure 5B); 1 of these is the “dilated heart left ventricle,” of which the effects of the individual gene sets are visualized in the illustration. The correlation substructure and the p values of this gene set are also displayed in Online Figure 12.