Candidate gene resequencing in a large bicuspid aortic valve-associated thoracic aortic aneurysm cohort: SMAD6 as an important contributor

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter = 4.0 cm in adults, or a Z-score = 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells.

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects

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

Copy number variation analysis in bicuspid aortic valve-related aortopathy identifies TBX20 as a contributing gene

International audienceBicuspid aortic valve (BAV) is the most common congenital heart defect (CHD), affecting 1-2% of the population. BAV is associated with thoracic aortic aneurysms (TAAs). Deleterious copy number variations (CNVs) were found previously in up to 10% of CHD cases. This study aimed at unravelling the contribution of deleterious deletions or duplications in 95 unrelated BAV/TAA patients. Seven unique or rare CNVs were validated, harbouring protein-coding genes with a role in the cardiovascular system. Based on the presence of overlapping CNVs in patients with cardiovascular phenotypes in the DECIPHER database, the identification of similar CNVs in whole-exome sequencing data of 67 BAV/TAA patients and suggested topological domain involvement from Hi-C data, supportive evidence was obtained for two genes (DGCR6 and TBX20) of the seven initially validated CNVs. A rare variant burden analysis using next-generation sequencing data from 637 BAV/TAA patients was performed for these two candidate genes. This revealed a suggestive genetic role for TBX20 in BAV/TAA aetiology, further reinforced by segregation of a rare TBX20 variant with the phenotype within a BAV/TAA family. To conclude, our results do not confirm a significant contribution for deleterious CNVs in BAV/TAA as only one potentially pathogenic CNV (1.05%) was identified. We cannot exclude the possibility that BAV/TAA is occasionally attributed to causal CNVs though, or that certain CNVs act as genetic risk factors by creating a sensitised background for BAV/TAA. Finally, accumulative evidence for TBX20 involvement in BAV/TAA aetiology underlines the importance of this transcription factor in cardiovascular disease

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

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

<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

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

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