NKX2-6 related congenital heart disease: Biallelic homeodomain-disrupting variants and truncus arteriosus.

Congenital heart defects (CHD) are the most common birth defect and are both clinically and genetically heterogeneous. Truncus arteriosus (TA), characterized by a single arterial vessel arising from both ventricles giving rise to the coronary, pulmonary and systemic arteries, is rare and only responsible for 1% of all CHD. Two consanguineous families with TA were previously identified to have homozygous nonsense variants within the gene NKX2-6. NKX2-6 is a known downstream target of TBX1, an important transcriptional regulator implicated in the cardiac phenotype of 22q11.2 microdeletion syndrome. Herein, we report two siblings with TA presumably caused by compound heterozygous NKX2-6 variants without a history of consanguinity. Two in-house cohorts with conotruncal defects (CTD) were sequenced for variants in NKX2-6 and no additional cases of biallelic NKX2-6 variants were identified. The similar phenotype of these cases, and the clustering of variants that likely result in a truncated protein that disrupts the homeobox domain, suggest that biallelic loss of function for NKX2-6 is a rare genetic etiology for TA in particular, and possibly other types of CHD.

MESP1 Mutations in Patients with Congenital Heart Defects.

Identifying the genetic etiology of congenital heart disease (CHD) has been challenging despite being one of the most common congenital malformations in humans. We previously identified a microdeletion in a patient with a ventricular septal defect containing over 40 genes including MESP1 (mesoderm posterior basic helix-loop-helix transcription factor 1). Because of the importance of MESP1 as an early regulator of cardiac development in both in vivo and in vitro studies, we tested for MESP1 mutations in 647 patients with congenital conotruncal and related heart defects. We identified six rare, nonsynonymous variants not seen in ethnically matched controls and one likely race-specific nonsynonymous variant. Functional analyses revealed that three of these variants altered activation of transcription by MESP1. Two of the deleterious variants are located within the conserved HLH domain and thus impair the protein-protein interaction of MESP1 and E47. The third deleterious variant was a loss-of-function frameshift mutation. Our results suggest that pathologic variants in MESP1 may contribute to the development of CHD and that additional protein partners and downstream targets could likewise contribute to the wide range of causes for CHD.

Mutations in NTRK3 suggest a novel signaling pathway in human congenital heart disease.

Congenital heart defects (CHDs) are the most common major birth defects and the leading cause of death from congenital malformations. The etiology remains largely unknown, though genetic variants clearly contribute. In a previous study, we identified a large copy-number variant (CNV) that deleted 46 genes in a patient with a malalignment type ventricular septal defect (VSD). The CNV included the gene NTRK3 encoding neurotrophic tyrosine kinase receptor C (TrkC), which is essential for normal cardiogenesis in animal models. To evaluate the role of NTRK3 in human CHDs, we studied 467 patients with related heart defects for NTRK3 mutations. We identified four missense mutations in four patients with VSDs that were not found in ethnically matched controls and were predicted to be functionally deleterious. Functional analysis using neuroblastoma cell lines expressing mutant TrkC demonstrated that one of the mutations (c.278C>T, p.T93M) significantly reduced autophosphorylation of TrkC in response to ligand binding, subsequently decreasing phosphorylation of downstream target proteins. In addition, compared with wild type, three of the four cell lines expressing mutant TrkC showed altered cell growth in low-serum conditions without supplemental neurotrophin 3. These findings suggest a novel pathophysiological mechanism involving NTRK3 in the development of VSDs.

Analysis of chromosomal structural variation in patients with congenital left-sided cardiac lesions.

BACKGROUND: We sought to characterize the landscape of structural variation associated with the subset of congenital cardiac defects characterized by left-sided obstruction. METHODS: Cases with left-sided cardiac defects (LSCD) and pediatric controls were uniformly genotyped and assessed for copy number variant (CNV) calls. Significance testing was performed to ascertain differences in overall CNV incidence, and for CNV enrichment of specific genes and gene functions in LSCD cases relative to controls. RESULTS: A total of 257 cases of European descent and 962 ethnically matched, disease-free pediatric controls were included. Although there was no difference in CNV rate between cases and controls, a significant enrichment in rare LSCD CNVs was detected overall (p=7.30 × 10(-3) , case/control ratio=1.26) and when restricted either to deletions (p=7.58 × 10(-3) , case/control ratio=1.20) or duplications (3.02 × 10(-3) , case/control ratio=1.43). Neither gene-based, functional nor knowledge-based analyses identified genes, loci or pathways that were significantly enriched in cases as compared to controls when appropriate corrections for multiple tests were applied. However, several genes of interest were identified by virtue of their association with cardiac development, known human conditions, or reported disruption by CNVs in other patient cohorts. CONCLUSION: This study examines the largest cohort to date with LSCD for structural variation. These data suggest that CNVs play a role in disease risk and identify numerous genes disrupted by CNVs of potential disease relevance. These findings further highlight the genetic heterogeneity and complexity of these disorders.

The phenotypic spectrum of ZIC3 mutations includes isolated d-transposition of the great arteries and double outlet right ventricle.

Disease causing mutations for heterotaxy syndrome were first identified in the X-linked laterality gene, ZIC3. Mutations typically result in males with situs ambiguus and complex congenital heart disease; however affected females and one male with isolated d-transposition of the great arteries (d-TGA) have been reported. We hypothesized that a subset of patients with heart defects common to heterotaxy but without laterality defects would have ZIC3 mutations. We also sought to estimate the prevalence of ZIC3 mutations in sporadic heterotaxy. Patients with TGA (n = 169), double outlet right ventricle (DORV; n = 89), common atrioventricular canal (CAVC; n = 41), and heterotaxy (n = 54) underwent sequencing of ZIC3 exons. We tested 90 patients with tetralogy of Fallot (TOF) to correlate genotype with phenotype. Three potentially disease-related missense mutations were detected: c.49G > T (Gly17Cys) in a female with isolated DORV, c.98C > T (Ala33Val) in a male with isolated d-TGA, and c.841C > T (His281Tyr) in a female with sporadic heterotaxy. We also identified a novel insertion (CPFP333ins) in a family with heterotaxy. All were absent in 200 control patients and the 1000 Genomes Project (n = 629). No significant mutations were found in patients with TOF. Functional studies demonstrated reduced transcriptional activity of the ZIC3 His281Tyr mutant protein. ZIC3 mutations were rarely identified in isolated DORV and d-TGA suggesting that a subset of DORV and d-TGA may fall within the spectrum of laterality defects. ZIC3 mutations were found in 3.7% of patients with sporadic heterotaxy; therefore testing should be considered in patients with heterotaxy.