EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis.

Hydrops fetalis describes fluid accumulation in at least 2 fetal compartments, including abdominal cavities, pleura, and pericardium, or in body tissue. The majority of hydrops fetalis cases are nonimmune conditions that present with generalized edema of the fetus, and approximately 15% of these nonimmune cases result from a lymphatic abnormality. Here, we have identified an autosomal dominant, inherited form of lymphatic-related (nonimmune) hydrops fetalis (LRHF). Independent exome sequencing projects on 2 families with a history of in utero and neonatal deaths associated with nonimmune hydrops fetalis uncovered 2 heterozygous missense variants in the gene encoding Eph receptor B4 (EPHB4). Biochemical analysis determined that the mutant EPHB4 proteins are devoid of tyrosine kinase activity, indicating that loss of EPHB4 signaling contributes to LRHF pathogenesis. Further, inactivation of Ephb4 in lymphatic endothelial cells of developing mouse embryos led to defective lymphovenous valve formation and consequent subcutaneous edema. Together, these findings identify EPHB4 as a critical regulator of early lymphatic vascular development and demonstrate that mutations in the gene can cause an autosomal dominant form of LRHF that is associated with a high mortality rate

Whole Exome Sequencing Reveals the Major Genetic Contributors to Nonsyndromic Tetralogy of Fallot

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<5×10−8) 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

Scientific Business Abstracts of the 113th Annual Meeting of the Association of Physicians of Great Britain and Ireland:Whole exome sequencing reveals the major genetic contributors to non-syndromic Tetralogy of Fallot

Objectives: 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 (CHD) phenotype. Rare genetic variants are important contributors to CHD risk, but relatively small numbers of TOF cases have been studied to date.Methods/Results: We used whole exome sequencing to assess the prevalence of unique, deleterious variants in 829 patients. The clustering of variants in two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P &lt; 5 108 ) after correction for multiple comparisons. NOTCH1 was most frequently found to harbour 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 seven cases identified five de novo variants. Three NOTCH1 variants were subjected to functional evaluation and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI: 1.6–3.8%) of TOF patients, with 21 patients harbouring 22 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 (exomewide corrected P &lt; 0.05) with variants in several other candidates including RYR1, ZFPM1, CAMTA2, DLX6 and PCM1.Conclusion: 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

Integration of Large-Scale Genomic Data Sources With Evolutionary History Reveals Novel Genetic Loci for Congenital Heart Disease

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 &gt;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&lt;0.0001, OR =1.253 [95% CI, 1.199–1.309]). We identified 54 novel candidate protein-coding genes supported by both: (1) copy number variant and whole-exome sequencing data; and (2) ohnolog status. Conclusions: We have identified new CHD candidate loci, and show for the first time that ohnologs are overrepresented among CHD genes. Incorporation of evolutionary metrics may be useful in refining candidate genes emerging from large-scale genetic evaluations of CHD

Predicting congenital renal tract malformation genes using machine learning

Abstract Congenital renal tract malformations (RTMs) are the major cause of severe kidney failure in children. Studies to date have identified defined genetic causes for only a minority of human RTMs. While some RTMs may be caused by poorly defined environmental perturbations affecting organogenesis, it is likely that numerous causative genetic variants have yet to be identified. Unfortunately, the speed of discovering further genetic causes for RTMs is limited by challenges in prioritising candidate genes harbouring sequence variants. Here, we exploited the computer-based artificial intelligence methodology of supervised machine learning to identify genes with a high probability of being involved in renal development. These genes, when mutated, are promising candidates for causing RTMs. With this methodology, the machine learning classifier determines which attributes are common to renal development genes and identifies genes possessing these attributes. Here we report the validation of an RTM gene classifier and provide predictions of the RTM association status for all protein-coding genes in the mouse genome. Overall, our predictions, whilst not definitive, can inform the prioritisation of genes when evaluating patient sequence data for genetic diagnosis. This knowledge of renal developmental genes will accelerate the processes of reaching a genetic diagnosis for patients born with RTMs

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability

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 eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation