Family Based Whole Exome Sequencing Reveals the Multifaceted Role of Notch Signaling in Congenital Heart Disease

<div><p>Left-ventricular outflow tract obstructions (LVOTO) encompass a wide spectrum of phenotypically heterogeneous heart malformations which frequently cluster in families. We performed family based whole-exome and targeted re-sequencing on 182 individuals from 51 families with multiple affected members. Central to our approach is the family unit which serves as a reference to identify causal genotype-phenotype correlations. Screening a multitude of 10 overlapping phenotypes revealed disease associated and co-segregating variants in 12 families. These rare or novel protein altering mutations cluster predominantly in genes (<i>NOTCH1</i>, <i>ARHGAP31</i>, <i>MAML1</i>, <i>SMARCA4</i>, <i>JARID2</i>, <i>JAG1</i>) along the Notch signaling cascade. This is in line with a significant enrichment (Wilcoxon, p< 0.05) of variants with a higher pathogenicity in the Notch signaling pathway in patients compared to controls. The significant enrichment of novel protein truncating and missense mutations in <i>NOTCH1</i> highlights the allelic and phenotypic heterogeneity in our pediatric cohort. We identified novel co-segregating pathogenic mutations in <i>NOTCH1</i> associated with left and right-sided cardiac malformations in three independent families with a total of 15 affected individuals. In summary, our results suggest that a small but highly pathogenic fraction of family specific mutations along the Notch cascade are a common cause of LVOTO.</p></div

Design and rationale of a genetic cohort study on congenital cardiac disease: experiences from a multi-institutional platform in Quebec

Background Congenital cardiac disease is the most common malformation, and a substantial source of mortality and morbidity in children and young adults. A role for genetic factors is recognised for these malformations, but overall few predisposing loci have been identified. Here we report the rationale, design, and first results of a multi-institutional congenital cardiac disease cohort, assembled mainly from the French-Canadian population of the province of Quebec and centred on families with multiple affected members afflicted by cardiac malformations.[...

Clinical features of three families carrying pathogenic <i>NOTCH1</i> mutations.

<p>Clinical features of three families carrying pathogenic <i>NOTCH1</i> mutations.</p

Variant filtering criteria and candidate gene prioritization.

<p>Variants were called using the GATK pipeline as outlined in the method section. Several stringent quality and allele filtering thresholds were applied prior to filtering for allele frequency and co-segregation among family members. Annotation and filtering of coding SNVs was performed using the ANNOVAR pipeline for RefSeq gene annotations [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006335#pgen.1006335.ref065" target="_blank">65</a>].</p

Overview of the study cohort.

<p><b>(A)</b> Prevalence and phenotypes associated with CHD in the general population and in the presented cohort. The numbers highlighted in grey indicate the prevalence of CHD phenotypes in 1 million live births according to Fahed et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006335#pgen.1006335.ref003" target="_blank">3</a>]. The graph highlighted in blue represents the distribution of overlapping CHD phenotypes in our cohort, which show a similar distribution of what is expected from the general CHD population. <b>(B)</b> Principal component analysis of individuals from the French Canadian pediatric cohort presented in this study with 2504 individuals from the 1000 Genomes phase 3 release for the five human super populations (AFR: purple, AMR: red, EUR: blue, EAS:green, SAS:yellow). Study subjects (black dots) and controls (grey dots) predominantly cluster with individuals of European descent (blue dots).</p

<i>NOTCH1</i> mutations in three independent families.

<p><b>(A)</b> Pedigrees of families harboring high impact <i>NOTCH1</i> mutations. Colors represent the different phenotype associations. The (+/-) symbols indicate mutation carrier status. <b>(B)</b> Schematic representation of the <i>NOTCH1</i> locus. Clustering of protein truncating mutations is observed in the extracellular part of the protein in the EGF domains 1–36.</p

Association between variant pathogenicity and allele frequencies.

<p><b>(A)</b> Variant pathogenicity based on scaled CADD scores is significantly higher in familial cases compared to unaffected family members (p < 0.05, Wilcoxon-rank sum test) and controls from the Mendel dataset (p < 0.01, Wilcoxon-rank sum test). <b>(B)</b> This enrichment of variant pathogenicity is due to a small fraction of variants exclusively observed in cases exceeding CADD scores >20 (p < 0.05, chi-square test). In contrast, there was no significant enrichment observed for rare (ExAC MAF < 0.1%) or novel deleterious variants in cases compared to unaffected family members or population controls.</p

Candidate gene prioritization in family based whole exome sequencing.

<p><b>(A)</b> Cumulative sum of unique and rare (ExAC <0.1%) deleterious variants co-segregating with disease for each gene in the whole exome dataset against the genome-wide RVI score percentiles. The red line indicates a transformed z-score thresold cutoff (p-value < 0.01), highlighting genes which have acquired more than 10 rare deleterious variants in the whole-exome dataset (z-score > 2). Candidate genes <i>NOTCH1</i> and <i>KMT2D</i> in the top 10% of intolerant genes as well as likely false positive <i>MUC16</i> and <i>MUC6</i> in the top 10% of tolerant genes, are highlighted in red. <b>(B)</b> Pathway enrichment of genes among the 10^% RVI percentile which harbor two or more rdSNVs in 106 whole-exome sequenced probands. Ranked Gene Set Enrichment analysis was performed on z-score based gene ranking with the weighted parameter and 1000 permutations for 50 BROAD derived hallmark gene sets.</p

Candidate genes in familial LVOTO cluster predominantly along the Notch signaling axis.

<p>Candidate genes in familial LVOTO cluster predominantly along the Notch signaling axis.</p