Congenital heart disease risk loci identified by genome-wide association study in European patients

Abstract

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

Keywords: Cardiology; Cardiovascular disease; Genetics; Molecular genetics; iPS cells.

Figure 1. Identification of SNPs with genome-wide significance across the entire CHD study group.

(A) Manhattan plot of genome-wide P values for association with the entire CHD study group. (B) LocusZoom plot of the genomic region of rs185531658 on chromosome 5 (chr5). The index SNPs are indicated by a purple diamonds. The forest plot shows the significance of the SNP and the ORs of both collectives separately and together. Circles represent imputed SNPs and triangles represent genotyped SNPs. FE, fixed effects; RE, random effects. Patients with CHD, n = 2594; control participants, n = 8486. –log₁₀ P values were determined by association statistics from the GWAS (logistic regression).

Figure 2. SNPs associated with TGA.

(A) LocusZoom plot of the MACROD2 region on chromosome 20. (B) LocusZoom plot of the ZBTB10 region on chromosome 8. The index SNPs are indicated by purple diamonds, and the other SNPs are color coded depending on their degree of correlation (r²). Circles represent imputed SNPs and triangles genotyped SNPs. Patients with TGA, n = 399. –log₁₀ P values were determined by association statistics from the GWAS (logistic regression).

Figure 3. SNPs associated with anomalies of thoracic arteries and veins.

LocusZoom plot of the GOSR2 region on chromosome 17. The index SNPs are indicated by purple diamonds, and the other SNPs are color coded depending on their degree of correlation (r²). Circles represent imputed SNPs and triangles genotyped SNPs. Patients with ATAV, n = 486. –log₁₀ P values were determined by association statistics from the GWAS (logistic regression).

Figure 4. Role of SNP-carrying candidate genes in murine cardiac development.

(A) Schedule of differentiation of murine ESCs. (B) Relative gene expression of Macrod2, Gosr2, Wnt3, and Msx1 in GFP-negative cells and GFP-positive CPCs (n = 4 each). Data represent the mean ± SEM. (C) Schematic representation of the enrichment of murine CPCs and postnatal CMs. (D) Heatmap of genes differentially expressed in embryonic CPCs and adult CMs. (E) Expression of Macrod1, Wnt3, and Leprel1 in E9–E11 CPCs (n = 4), newborn CMs (n = 3), and adult CMs (n = 3). Data represent the mean ± SEM. ND, no expression detected. (F–H) Results of GSEA of 1649 genes overlapping between CHD-associated SNP-carrying genes and genes upregulated in CPCs according to (F) significance of GO terms, (G) coverage of GO terms, and (H) second-level GO terms showing molecular functions. *P < 0.05 and ***P < 0.001, by unpaired, 2-tailed Student’s t test or Mann-Whitney rank-sum test (B) and 1-way ANOVA or the Kruskal-Wallis test (E), correcting for multiple testing using the Holm-Sidak method.

Figure 5. Expression of SNP-candidate genes during murine embryonic cardiogenesis.

(A) UMAP plot of all mesodermal and neural crest cells of the cardiogenic region (n = 21,745). (B) Expression of marker genes in individual clusters. (C) Expression of Macrod2, Gosr2, Msx1, and Wnt3 in cardiogenic tissue at E7.75, E8.25, and E9.25.

Figure 6. Expression of SNP-carrying candidate genes during differentiation of human iPSCs and in pediatric and adult aortic tissue.

(A) Schedule of directed cardiac differentiation of human iPSCs. (B) Expression of MACROD2, GOSR2, WNT3, and MSX1 during directed cardiac differentiation of human iPSCs. Data represent the mean ± SEM of at least 2 independent experiments, each run in duplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 versus day 0 (D0), by unpaired, 2-tailed Student’s t test. (C) Expression of MACROD2, GOSR2, WNT3, and MSX1 in aortic tissues of pediatric patients (n = 35, 24, 23, and 6, respectively) and adult surgical patients (n = 15, 9, 10, and 20, respectively). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by Mann-Whitney rank-sum test.

Figure 7. Role of SNP-carrying candidate genes in human cardiac development.

(A) Unbiased clustering of embryonic cells (n = 669) into biological entities. Cells are labeled on the basis of age as well as anatomical localization for purposes of visualization. (B) Relative expression of MACROD2, GOSR2, WNT3, and MSX1 in embryonic heart cells. (C) Schedule of scRNA-Seq analysis of cells from atria and ventricles (n = 17,782). (D) Clustering of embryonic and adult cells and identification of cell types. (E) Expression of candidate genes in the integrated data set split by embryonic cells (upper panel) and adult cells (lower panel). (F) Expression of candidate genes associated with TGA (turquoise), ATAV (orange), or septal defects (red) in vitro and in vivo during different stages of the developing murine and human heart. hiPS, human iPSCs; miPS, murine iPSCs; SMC, smooth muscle cells.