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Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in

Supplemental Digital Content is available in the text., Background: Most cases of congenital heart disease (CHD) are sporadic and nonsyndromic, with poorly understood etiology. Rare genetic variants have been found to affect the risk of sporadic, nonsyndromic CHD, but individual studies to date are of only moderate sizes, and none to date has incorporated the ohnolog status of candidate genes in the analysis. Ohnologs are genes retained from ancestral whole-genome duplications during evolution; multiple lines of evidence suggest ohnologs are overrepresented among dosage-sensitive genes. We integrated large-scale data on rare variants with evolutionary information on ohnolog status to identify novel genetic loci predisposing to CHD. Methods: We compared copy number variants present in 4634 nonsyndromic CHD cases derived from publicly available data resources and the literature, and >27 000 healthy individuals. We analyzed deletions and duplications independently and identified copy number variant regions exclusive to cases. These data were integrated with whole-exome sequencing data from 829 sporadic, nonsyndromic patients with Tetralogy of Fallot. We placed our findings in an evolutionary context by comparing the proportion of vertebrate ohnologs in CHD cases and controls. Results: Novel genetic loci in CHD cases were significantly enriched for ohnologs compared with the genome (χ2 test, P

Analysis of large scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in 8 individuals with large atrial septal defect (ASD) in a 5-generation pedigree. Mice with TPM1 mutation exhibited early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient induced pluripotent stem cell derived cardiomyocytes showed normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, was identified as a candidate suppressor. Mouse CRISRP knockin of both the TLN2/TPM1 variants rescued heart beating, with near term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolded with the suppression of its embryonic lethality by a TLN2 protective variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Introduction There is strong evidence from familial recurrence studies for a genetic predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF). TOF is the most common, cyanotic congenital heart disease (CHD) phenotype yet the cause for the majority of cases remains elusive. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Methods and Results 829 TOF patients underwent whole exome sequencing (WES), the largest cohort of non-syndromic TOF patients reported to date. The prevalence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database (gnomAD) and a scaled combined annotation-dependent depletion (CADD) score of ≥20. Clustering analysis of variants revealed that two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P Conclusion In summary, the NOTCH1 locus is the most frequent site of genetic variants predisposing to non-syndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients. Conflict of Interest None

Generalized lymphatic dysplasia (GLD) is a rare form of primary lymphoedema characterized by a uniform, widespread lymphoedema affecting all segments of the body, with systemic involvement such as intestinal and/or pulmonary lymphangiectasia, pleural effusions, chylothoraces and/or pericardial effusions. This may present prenatally as non-immune hydrops. Here we report homozygous and compound heterozygous mutations in PIEZO1, resulting in an autosomal recessive form of GLD with a high incidence of non-immune hydrops fetalis and childhood onset of facial and four limb lymphoedema. Mutations in PIEZO1, which encodes a mechanically activated ion channel, have been reported with autosomal dominant dehydrated hereditary stomatocytosis and non-immune hydrops of unknown aetiology. Besides its role in red blood cells, our findings indicate that PIEZO1 is also involved in the development of lymphatic structures.

Rationale: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, nonsyndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease phenotype. Rare genetic variants have been identified as important contributors to the risk of congenital heart disease, but relatively small numbers of TOF cases have been studied to date. Objective: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in the largest cohort of nonsyndromic TOF patients reported to date. Methods and Results: Eight hundred twenty-nine TOF patients underwent whole exome sequencing. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database and a scaled combined annotation-dependent depletion score of ≥20. The clustering of variants in 2 genes, NOTCH1 and FLT4 , surpassed thresholds for genome-wide significance (assigned as P −8 ) after correction for multiple comparisons. NOTCH1 was most frequently found to harbor unique, deleterious variants. Thirty-one changes were observed in 37 probands (4.5%; 95% CI, 3.2%–6.1%) and included 7 loss-of-function variants 22 missense variants and 2 in-frame indels. Sanger sequencing of the unaffected parents of 7 cases identified 5 de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y, and p.N1875S) were subjected to functional evaluation, and 2 showed a reduction in Jagged1-induced NOTCH signaling. FLT4 variants were found in 2.4% (95% CI, 1.6%–3.8%) of TOF patients, with 21 patients harboring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy disease. In addition to NOTCH1 , FLT4 and the well-established TOF gene, TBX1 , we identified potential association with variants in several other candidates, including RYR1 , ZFPM1 , CAMTA2 , DLX6 , and PCM1 . Conclusions: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4 . Together, variants in these genes are found in almost 7% of TOF patients.

Genetic factors predisposing to congenital heart disease (CHD) are characterized by extreme heterogeneity, comprising changes to the genome at all levels of variation from chromosomal aneuploidy to single nucleotide mutations. About 15–20% of patients with CHD have underlying genetic causes identifiable by currently standard clinical genetics laboratory testing, including patients with Down syndrome, Turner syndrome, 22q11 deletion syndrome, other chromosomal rearrangements, and multisystem conditions mediated by single nucleotide changes in particular genes (e.g. Noonan’s syndrome). Extracardiac malformations and/or neurodevelopmental abnormalities characteristic of these conditions are important diagnostic cues. Mendelian families with isolated non-syndromic CHD are very rare. In the remaining 80% of cases, CHD is apparently ‘sporadic’ and the empirical recurrence risk to a sibling of an index case in such families is approximately 3%. This low recurrence risk suggests that de novo events, that is, new mutations in affected offspring absent in the parents, are an important potential genetic cause of CHD. De novo copy number variations such as 1q21.1 duplication have been shown to contribute to aetiology in 5–10% of apparently sporadic patients. Recent studies have also shown that approximately 20% of patients without recognized syndromic presentations, but with CHD accompanied by extracardiac malformations and/or neurodevelopmental delay, may have pathogenic de novo single nucleotide changes discoverable by exome sequencing. Continuing advances in genomic technologies present the prospect of substantial progress in the understanding of the genetic predisposition to CHD, but further research in large cohort studies is required.

AimsFamilial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Here, we use whole exome sequencing to assess the prevalence of rare, potentially deleterious variants in candidate genes previously associated with both syndromic and non-syndromic TOF, in the largest cohort of non-syndromic TOF patients reported to date.Methods & Results829 non-syndromic TOF patients underwent whole exome sequencing. A systematic review of the literature was conducted which revealed 77 genes in which mutations had been reported in patients with TOF. The presence of rare, deleterious variants in the 77 candidate genes was determined, defined by a minor allele frequency of ≤ 0.001 and scaled combined annotation-dependent depletion (CADD) score of ≥ 20. We found a clustering of heterozygous rare, deleterious variants inNOTCH1(P=1.89E-15),DOCK6(P=2.93E-07),MYOM2(P= 7.35E-05),TTC37(P=0.016),MESP1(P=0.024) andTBX1(P=0.039), after correcting for multiple testing.NOTCH1was most frequently found to harbour deleterious variants. Changes were observed in 49 patients (6%; 95% confidence interval [CI]: 4.5% - 7.8%) and included six truncating/frameshift variants and forty missense variants. Sanger sequencing of the unaffected parents of thirteen cases identified fivede novovariants. Variants were not confined to a single functional domain of the NOTCH1 protein but significant clustering of variants was evident in the EGF-like repeats (P=0.018). ThreeNOTCH1missense variants (p.G200R, p.C607Y andde novop.N1875S) were subjected to functional evaluation and showed a reduction in Jagged1 ligand-induced NOTCH signalling. p.C607Y, which exhibited the most significant reduction in signalling, also perturbed S1 cleavage of the NOTCH1 receptor in the Golgi.ConclusionTheNOTCH1locus is a frequent site of genetic variants predisposing to non-syndromic TOF with 6% of patients exhibiting rare, deleterious variants. Our data supports the polygenic origin of TOF and suggests larger studies may identify additional loci.

Congenital heart disease (CHD) is the most common type of birth defect in humans. Most cases of CHD are sporadic with the specific interactions between genetic variants and environmental factors involved in their pathogenesis uncharacterised. Various whole exome sequencing studies have identified de novo mutations in different genes; however they have only explained a small percentage of CHD cases.Previous work from the group and others has identified chromosomal regions where rare copy number variants (CNVs) were significantly enriched in CHD cases compared to controls. We hypothesise that utilising available CNV data to prioritise candidate regions within which we will interrogate exome sequencing data in CHD cases will be a productive means of identifying causative genes. In this study we have undertaken various refinement steps to narrow down potentially causative candidate gene/s within deleted (DEL) and duplicated (DUP) CNV regions that have been previously shown to be highly associated with non-syndromic CHD patients. Firstly, we have generated case DEL and DUP CNV lists. This was done by updating a published meta-analysis study (our group have contributed to this study) though utilisation of DECIPHER, ISCA, ECARUCA databases and published studies, using different key terms to identify further non-syndromic CHD patients. We then utilised BedTools to compare these case lists with the corresponding control CNV lists generated by using controls from published literature, DECIPHER, the Database of Genomic Variants and the 1000Genome Phase 3 CNVs. The resulting unique cases CNV regions were annotated and compared against an in-house list of candidate genes (containing novel or rare variants) generated from an exome data analysis of 850 Tetralogy of Fallot (ToF) patients. Genes were further prioritised based on whether they have already an assigned human phenotype, on their ExAC CNV scores, probability of haploinsufficiency (pHI) and loss of function (LoF) intolerance scores. Initially, we have identified 10 586 genes for which 1986 genes are present in both DEL and DUP CNV regions, 2772 genes are unique in DUP and 3842 genes unique in DEL regions (Figure 1). Further analysis of genes in DEL regions and genes present in both types of CNVs revealed that 1,150/3,842 genes and 588/1,986 genes respectively have LoF variants in our ToF exome data. Additional filtering with pHI and pLI scores resulted in 57 genes collectively. This is an on-going work and our plan is to design a next-generation sequencing panel to screen our final candidate gene list in an additional 2000 CHD cases. We will focus on the most promising candidate gene emerging from the discovery experiment to perform functional work. Our experimental strategy will vary depending on what is known about the gene, and whether its involved or not in any well-known signalling pathway during heart development such as the Wnt and VEGF pathways.