Integrative analysis of genomic variants reveals new associations of candidate haploinsufficient genes with congenital heart disease

Abstract

Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.

Fig 1. CNV burden test on known gene sets.

The forest plot shows the odds ratio (dots), the 95% confidence intervals indicating the certainty about the OR (interrupted line) and the P-value in the indicated gene sets.

Fig 2. CNV burden test on constraint LOF genes at different observed/expected LOF ratio thresholds.

The forest plot shows the odds ratio (dots), the 95% confidence intervals indicating the certainty about the OR (interrupted line) and the P-value in the indicated gene sets.

Fig 3. CNV deletion distribution across the 22 autosomes.

The plot shows the distribution of rare CNV deletions (green track) in CHD cases, the differences between the overlapping CNV deletions in cases and controls (black track) and highlight the location of the 63 significant loci discovered (in magenta).

Fig 4. Comparison of the distribution of LOEUF metric at different level of significance of nsDNV-enriched genes.

X-axis denotes the P-values from the DNV analysis (binned). Y-axis denotes the o/e LOF ratio upper bound fraction (LOEUF). All groups were compared against the LOEUF distribution of all protein-coding genes (purple). Differences between the distributions were tested using a two-sided Wilcoxon rank sum test. ****: P<0.0001, ns: non-significant.

Fig 5. Comparison of the mean expression (heart) distribution at different metaP cut-offs.

Panels show three different heart development stages: early development, maturation and infant/adult. X-axis denotes the combined p-value from DNV and CNV analysis (metaP, at different cut-offs). Y-axis denotes the genes’ mean expression in the heart (log scale). The 21 significant candidate CHD genes (Table 1) are contained in the fraction with the higher expression (red box). Differences between the distributions were tested using a two-sided Wilcoxon rank sum test (reference group: all genes). ****: P<0.0001, ***: P<0.001, **: P<0.01, *: P<0.05, ns: non-significant.

Fig 6. Identification of functional networks enriched for proteins encoded by genes affected by CNVs and/or DNVs associated with CHD.

The protein-protein interaction networks (a-d, for clusters 1, 3, 8 and 9 respectively) were identified using GeNets (S4 Fig). Proteins are shown as nodes, interactions as edges. Enrichment for CNVs (blue) and DNVs (green) are highlighted. Proteins with no specific enrichment for CNV and/or DNVs but with B-H adjusted metaP < 0.05 are highlighted in red. The size of the circles denotes if the genes was found significantly highly and/or differentially expressed in the heart (large circles: significant expression; small circles: non-significant). The distribution of CHD case-CNVs and control-CNVs are shown for each cluster. Significant difference in the CNV distribution was calculated using a Wilcox rank sum test. The horizontal bar plots show the top ten GO enriched terms for each cluster (output from Enrichr tool). X-axis in the horizontal bar plot denotes the combined score from Enrichr, which is computed by multiplying the log-transformed p-value and the z-score. Bar color encoded the GO biological process significant level (dark blue: FDR < 5%, light blue: FDR 5–10%, grey: FDR > 10%).