Cause Of Congenital Heart Disease Research Articles

Specification for genetic diagnosis of congenital heart disease

Congenital heart disease (CHD) is one of the most common congenital malformations and a major cause of mortality among neonates and children. Conventional methods for the diagnosis of CHD have relied on clinical features and imaging findings. With the rapid development of genetic techniques, to identify the cause of CHD through genetic diagnosis has gained great significance for the early diagnosis, treatment, and prevention of CHD. However, currently there is still a lack of norms and standards for the genetic diagnosis of CHD. In view of this, experts from the relevant fields have formulated the present norm by integrating the latest research advances on CHD-related genes with the current clinical practice on the diagnosis and treatment of CHD and status quo of genetic diagnosis in China. The norm has been recommended by the Cardiology Section of the Chinese Medical Education Association, the Medical Genetics Branch and the Heart Group of Pediatric Surgery Branch of the Chinese Medical Association, which has formulated the procedures and norms of genetic testing, prenatal diagnosis, and genetic counseling for CHD, with an aim to provide reference for clinicians as the standards for the integrated diagnosis, early treatment, and prevention of CHD.

Proteome profiling of early gestational plasma reveals novel biomarkers of congenital heart disease.

Prenatal diagnosis of congenital heart disease (CHD) relies primarily on fetal echocardiography conducted at mid-gestational age-the sensitivity of which varies among centers and practitioners. Anobjective method for early diagnosis is needed. Here, we conducted a case-control study recruiting 103 pregnant women with healthy offspring and 104 cases with CHD offspring, including VSD (42/104), ASD (20/104), and other CHD phenotypes. Plasma was collected during the first trimester and proteomic analysis was performed. Principal component analysis revealed considerable differences between the controls and the CHDs. Among the significantly altered proteins, 25 upregulated proteins in CHDs were enriched in amino acid metabolism, extracellular matrix receptor, and actin skeleton regulation, whereas 49 downregulated proteins were enriched in carbohydrate metabolism, cardiac muscle contraction, and cardiomyopathy. The machine learning model reached an area under the curve of 0.964 and was highly accurate in recognizing CHDs. This study provides a highly valuable proteomics resource to better recognize the cause of CHD and has developed a reliable objective method for the early recognition of CHD, facilitating early intervention and better prognosis.

Triple repeated fetal congenital heart disease linked to PLD1 mutation: a case report

BackgroundCongenital heart disease occurs in approximately 1 in 100 cases. Although sibling occurrence is high (3–9%), the causative genes for this disease are still being elucidated. PLD1 (Phospholipase D1) is a recently discovered gene; however, few case reports have been published on it. In this report, we describe a case of triplicate fetal congenital heart disease that was diagnosed as a PDL1 mutation. Our objective is to explore the clinical manifestations of PLD1 mutations in this particular case.Case presentationA 32-year-old Japanese woman (gravida, para 0) was introduced since fetus four chamber view was not clear and was diagnosed with ductus arteriosus-dependent left ventricular single ventricle and pulmonary atresia at 21 weeks and 1 day of gestation during her first pregnancy. Artificial abortion using Gemeprost was performed at 21 weeks and 5 days of gestation. The second pregnancy was diagnosed as pulmonary atresia with intact ventricular septum with cardiomegaly, a cardiothoracic area ratio of more than 35%, and a circulatory shunt at 13 weeks and 3 days of gestation. Subsequently, intrauterine fetal death was confirmed at 14 weeks and 3 days of gestation. Regarding the third pregnancy, fetal ultrasonography at 11 weeks and 5 days of gestation showed mild fetal hydrops and moderate tricuspid valve regurgitation. At 16 weeks and 5 days of gestation, the fetus was suspected to have a left ventricular-type single ventricle, trace right ventricle, pulmonary atresia with intact ventricular septum, or cardiomyopathy. Cardiac function gradually declined at 26 weeks of gestation, and intrauterine fetal death was confirmed at 27 weeks and 5 days of gestation. The fourth pregnancy resulted in a normal heart with good progression and no abnormal baby. We submitted the first and second fetuses’ umbilical cord, third fetus’ placenta, and the fourth fetus’ blood to genetic testing using whole exome analysis with next generation sequencing. Genetic analysis identified hemizygous PLD1 mutations in the first, second, and third fetuses. The fourth fetus was heterozygous. In addition, the parents were heterozygous for PLD1. This case is based on three consecutive cases of homozygosity for the PLD1 gene in the sibling cases and the fetuses with recurrent right ventricular valve dysplasia. This will elucidate the cause of recurrent congenital heart disease and intrauterine fetal death and may serve as an indicator for screening the next fetus. To date, homozygous mutations in PLD1 that repeat three times in a row are not reported, only up to two times. The novelty of this report is that it was repeated three times, followed by a heterozygous live birth.ConclusionsThis report is consistent with previous reports that mutations in PLD1 cause right ventricular valve dysplasia. However, there have been few case reports of PLD1 mutations, and we hope that this report will contribute to elucidate the causes of congenital heart disease, especially right ventricular valve dysplasia, and that the accumulation of such information will provide more detailed information on PLD1 mutations in heart disease.

Abstract P3200: SH3BGR Affects Energy Metabolism In Neonatal Rat Cardiomyocytes

Background: The SH3 domain-binding glutamic acid-rich (SH3BGR) gene maps to the genetic fragment associated with Down's syndrome (DS), one of the predominant causes of congenital heart diseases (CHD). We have previously shown that SH3BGR augments cardiomyocyte apoptosis, however, its possible role in cardiomyocyte metabolism is not known yet. Methods: Functional effects of SH3BGR knockdown or overexpression were studied in neonatal rat ventricular cardiomyocytes (NRVCMs) using seahorse metabolic assays, quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. Results: Since we found significantly altered cardiac levels of SH3BGR in human patients suffering from cardiac hypertrophy, which is reportedly associated with mitochondrial dysfunction, we determined if loss- or gain-of-function of SH3BGR impacts mitochondrial metabolism. We observed significant reduction in oxidative phosphorylation complexes and associated genes. Interestingly, seahorse metabolic activity assays revealed impairment in mitochondrial bioenergetics by both knockdown and overexpression of SH3BGR suggesting the importance of its stead-state levels in cardiomyocyte mitochondrial metabolism. NRVCMs showed inability to meet high energy demand on altered SH3BGR expression indicated by higher proton leak and impeded ATP production. These observations suggest altered mitochondrial respiration, which was further supported by alterations in the expression levels of respective metabolic genes. Combining these results with our previous findings suggests that SH3BGR plays an important role in cardiomyocyte oxidative phosphorylation, cell viability and apoptosis. Conclusion: Our data provide novel insights into potential role of SH3BGR in cardiomyocyte mitochondrial bioenergetics. These findings will help us understand how genetic deletion or SH3BGR mutations are associated with CHD e.g., using CRISPR/CAS mediated genetic manipulations using human patient derived induced pluripotent stem cells.

Abstract P3188: Establishing The Mitochondrial Citrate Transporter As A Regulator Of Cardiac Morphogenesis

Congenital heart disease (CHD) results from structural and functional defects of the heart that arise during embryonic development. CHD represents the most common type of birth defect and in the United States alone, affects ~1% of births per year. One common genetic cause of CHD is 22q11.2 deletion syndrome (22q11.2DS), where ~75% of 22q11.2DS patients present with CHD. SLC25A1 , which is found within the 22q11.2DS critical deletion region, encodes for the mitochondrial citrate carrier, a solute transporter localized to the inner mitochondrial membrane. In developing a knockout mouse model to study the in vivo functions of SLC25A1, we uncovered an unexpected perinatal-lethal phenotype that suggests roles for SLC25A1 in heart development. Hearts from Slc25a1 knockout embryos display a striking array of cardiac malformations including persistence of an expanded zone of trabeculated myocardium, reduced compact myocardium, and ventricular septal defects. Analysis of mitochondrial structure and function reveal that loss of Slc25a1 causes mitochondrial ultrastructural and functional defects. Lastly, transcriptomics analyses of metabolism-related genes revealed that Slc25a1 deletion causes widespread alterations in metabolic gene expression in a dosage-dependent manner. Our results highlight a novel role for SLC25A1 in cardiac morphogenesis and the transcriptional control of metabolic networks in the developing heart and point to a new role for SLC25A1 in the metabolic maturation of the developing myocardium.

Novel missense mutation in NKX2.6 gene (c.389 G > C, Arg130Pro) as a potentially pathogenic variant in pediatric patients with congenital heart disease

Congenital heart diseases (CHD) are the most common anomalies that occur during heart embryogenesis and are associated with significant mortality and morbidity in infants. It has been found that genomic alterations in cardiac-specific transcription factor genes play an important role in the pathogenesis of CHD. However, the prevalence of point mutations in these genes has not yet been determined. The aim of this research was to investigate the relationship of NKX2.6 gene mutations as a candidate gene in patients with congenital heart diseases. In the present study, two coding exons and flanking introns of the NKX2.6 gene, encoding a homeodomain-containing transcription factor, were sequenced in a cohort of patients. Five heterozygous NKX2.6 missense mutations, Ser37Cys, Glu54Val, Ala129Glu, Arg130Pro, Ser232Thr, and a synonymous variation, Asp68Asp, were identified in patients. They were not observed in controls, and the altered amino acids were evolutionarily highly conserved across species. The structural and functional consequences of missense mutations were assessed using various prediction servers. The consensus results showed that the Arg130Pro mutation is pathogenic/deleterious and likely to significantly affect protein function. To our knowledge, this is the first study on the association of NKX2.6 missense mutations with susceptibility to CHD in Iranian patients; our results present the spectrum of NKX2.6 mutations associated with patients and suggest that these missense mutations could be important as an uncommon cause of CHD.

Contribution of Previously Unrecognized RNA Splice-Altering Variants to Congenital Heart Disease.

Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing. We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10-6) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach. We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including KMT2D and RBFOX2. In 1 case, we confirmed the variant's predicted impact on RNA splicing in RNA transcripts from the proband's cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes HECTD1 and DYNC2H1. In addition, SpliceAI-a predictive algorithm for altered RNA splicing-has a positive predictive value of ≈93% in our cohort. Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD. URL: https://clinicaltrials.gov; Unique identifier: NCT01196182.

Increased gestational palmitic acid predisposes offspring to congenital heart disease

Identification of the causes for congenital heart disease (CHD) is a prerequisite for precise prevention and personalized treatment of CHD. Zhao etal.1 show increased that gestational serum palmitic acid (PA) predisposes offspring to CHD by perturbating the MARS/K-Hcy/GATA4 signaling pathway.

The genetic etiology of critical congenital heart disease

Congenital Heart Disease (CHD) is the most common kind of birth defect. Congenital heart disease is the most common birth defect and the leading cause of death in newborns. The causes of CHD are complicated and involve both genes and the environment. Congenital heart disease includes problems with the septum, the valves, and the outflow tract. Correctional heart surgery and new strategies for managing CHD have massively enhanced life expectancy. 490 percent of CHD newborns who live through their first year will become adults. Studies of the molecular genetics of humans and animal models of development are enhancing our understanding of normal heart development and cardiac diseases. A recent study demonstrates that microRNAs are implicated in congenital heart diseases. Epigenetic variables were eventually revealed to influence heart development. Several genes are responsible for congenital cardiac abnormalities as well as genetic disorders. This paper describes the categorization, environmental, and genetic causes of Coronary Heart Disease (CHD), the role of key CHD-causing genes, and potential options for preventing CHD.

Association of social determinants with the severity of congenital heart disease.

Congenital heart diseases are the most prevalent congenital malformations and cause greater morbi-mortality in newborns and infants. The aim of this study was to analyze the social determinants in families with children with the severity of congenital heart disease. Analytical cross-sectional study in 140 families of children with congenital heart disease to whom a structured survey was applied addressing topics related to family structure, health, economic conditions, exposure factors, and other social conditions relevant to the study, during 1 year. In all, 53.7% of the studied population belonged to low socioeconomic levels. No association was found between the severity of the heart disease and the presence of pathological antecedents in the parents. The families resided in urban areas. Also, 28.3% of the mothers had four or fewer prenatal controls during pregnancy. Only 22% of heart diseases were diagnosed during pregnancy. It was found that exposure to cigarette and wood smoke during pregnancy, in addition to low socioeconomic status, was associated with greater severity of heart disease (RACHS-1 and STS-Score), when evaluated by pathophysiological groups (cyanotic/non-cyanotic/single ventricle). Exposure to cigarette smoke, wood smoke during pregnancy, and low socioeconomic status turned out to be social determinants associated with the severity of heart disease analyzed by pathophysiological groups. The social component has not been well characterized as a cause of congenital heart disease, especially in countries like ours, where the existence of gaps and social inequities have a high impact. The findings of this study could have an impact on public health to the extent that policies are implemented to reduce exposure to cigarettes, especially during pregnancy. Knowledge of these changes and their measurement in this type of pathology could open the door to the creation of policies aimed at their prevention, focusing on the local risk factors found, which can impact the disease.

Infracardiac Total Anomalous Pulmonary Venous Drainage: A Case Report

Anomalous pulmonary venous drainage is a rare cause of congenital heart disease with varied clinical presentation. Symptoms can differ depending on the degree of venous obstruction. An 8-day-old term baby presented with tachypnea and was subsequently found to have an unobstructed infracardiac total anomalous pulmonary venous drainage. She underwent an uneventful surgical repair. The diagnosis of total anomalous pulmonary venous drainage can be delayed or missed, and a high index of suspicion is necessary.

A novel KLF13 mutation underlying congenital patent ductus arteriosus and ventricular septal defect, as well as bicuspid aortic valve.

Recently, mutations in the Kruppel-like factor 13 (KLF13) gene encoding a Kruppel-like transcription factor have been reported to cause congenital heart disease (CHD). However, due to pronounced genetic heterogeneity, the mutational spectrum of KLF13 in other cohorts of cases suffering from distinct types of CHD remain to be ascertained. In the present investigation, by Sanger sequencing of KLF13 in 316 unrelated cases affected by different forms of CHD, a new mutation in heterozygous status, NM_015995.3: c.430G>T; p.(Glu144*), was detected in an index patient affected with patent ductus arteriosus (PDA) and ventricular septal defect (VSD), as well as bicuspid aortic valve (BAV), with a mutation frequency of ~0.32%. Genetic investigation of the available family members of the proband demonstrated that the truncating mutation co-segregated with CHD. The nonsense mutation was not observed in 400 unrelated volunteers without CHD who were enrolled as control subjects. Quantitative biological measurements with dual luciferase reporters revealed that Glu144*-mutant KLF13 did not transactivate the downstream genes vascular endothelial growth factor A and natriuretic peptide A. In addition, the mutation abrogated the synergistic transcriptional activation between KLF13 and T-box transcription factor 5, a well-established CHD-causing gene. In conclusion, the present study indicates that genetically defective KLF13 contributes to familial PDA and VSD, as well as BAV, which expands the phenotypic spectrum linked to KLF13, and reveals a novel molecular pathogenesis of the disease, providing a new molecular target for the early prophylaxis and individualized treatment of CHD.

The TBX1/miR-193a-3p/TGF-β2 Axis Mediates CHD by Promoting Ferroptosis

Congenital heart disease (CHD) is the most common noninfectious cause of death during the neonatal stage. T-box transcription factor 1 (TBX1) is the main genetic determinant of 22q11.2 deletion syndrome (22q11.2DS), which is a common cause of CHD. Moreover, ferroptosis is a newly discovered kind of programmed cell death. In this study, the interaction among TBX1, miR-193a-3p, and TGF-β2 was tested using quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and dual-luciferase reporter assays. TBX1 silencing was found to promote TGF-β2 messenger ribonucleic acid (mRNA) and protein expression by downregulating the miR-193a-3p levels in H9c2 cells. In addition, the TBX1/miR-193a-3p/TGF-β2 axis was found to promote ferroptosis based on assessments of lipid reactive oxygen species (ROS) levels, Fe2+ concentrations, mitochondrial ROS levels, and malondialdehyde (MDA) contents; Cell Counting Kit-8 (CCK-8) assays and transmission electron microscopy; and Western blotting analysis of glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), NADPH oxidase 4 (NOX4), and acyl-CoA synthase long-chain family member 4 (ACSL4) protein expression. The protein expression of NRF2, GPX4, HO-1, NOX4, and ACSL4 and the level of MDA in human CHD specimens were also detected. In addition, TBX1 and miR-193a-3p expression was significantly downregulated and TGF-β2 levels were high in human embryonic CHD tissues, as indicated by the H9c2 cell experiments. In summary, the TBX1/miR-193a-3p/TGF-β2 axis mediates CHD by inducing ferroptosis in cardiomyocytes. TGF-β2 may be a target gene for CHD diagnosis and treatment in children.

The genetics of congenital heart disease: The role of advanced genomic approaches

As the leading non-infectious cause of paediatric morbidity and mortality worldwide, congenital heart disease (CHD) is a significant social and healthcare burden, especially in low- and lower-middle-income countries, including South Africa. The aetiology of CHD is poorly understood, though heritable genetic factors have been shown to contribute to the risk of CHD in individuals of European ancestry. In this review, we highlight the impact that advanced genomic approaches have had on the understanding of the role of genetics in CHD. We also summarise current knowledge of the genetics of CHD in Africa, and the challenges and opportunities for conducting genomic research in these populations. Chromosomal microarrays and next-generation sequencing platforms allow for high-throughput screening of patients for genetic mutations and show great potential for the identification of genetic causes of CHD. Advancing our understanding of the genetic architecture and risk factors associated with CHD in patients of African descent is the fi rst step towards improving CHD diagnosis and management. Therefore, exploring the genetics of CHD has the potential to improve the quality of life for children born with CHD in Africa, by enabling clinicians to identify familial inheritance patterns and predict recurrent risks and prognostic outcomes pre- and post-surgical intervention.

Taiwanese Clinical Experience with Noninvasive Prenatal Testing for DiGeorge Syndrome

Objective: DiGeorge syndrome (DGS) is associated with microdeletions of chromosome 22q11. It is the second most common cause of congenital heart disease and is an important consideration whenever a conotruncal cardiac anomaly is identified. The availability of noninvasive prenatal testing (NIPT) is altering the practice of prenatal genetics and maternal-fetal medicine, resulting in a decline in invasive testing. Antenatal ultrasound and other biomarkers have their own limitation. NIPT was proposed to screen DGS with cell-free DNA in Taiwan. Here, we present our experience of prenatal diagnosis of DGS in our center. Methods: This was a retrospective study between November 1, 2019, and August 31, 2020, in Taiwan. Data were collected from 7,826 pregnant women self-referred for DGS screening with massive parallel shotgun sequencing-based NIPT. High-risk cases subsequently received amniocentesis for array comparative genomic hybridization (aCGH) to confirm the diagnosis. Characteristics of pregnancies were documented when participants received the test. Report of NIPT was completed 2 weeks after the test. Follow-up on high-risk cases was completed by telephone interview on January 30, 2021. Results: Thirteen cases showed high risk by NIPT, and 7 cases were confirmed by aCGH. The sensitivity and specificity were 100% (95% confidence interval [CI] 64.57–100.00%) and 99.92% (95% CI 99.83–99.96%). The prevalence of DGS was 1 in 1,118 pregnancies. The positive predictive rate was 53.85% (95% CI 29.14–76.79%). One true positive (TP) showed US anomaly, and 5 TPs selected termination. Discussion/Conclusion: NIPT demonstrated good performance in DGS screening. Detection of 22q11.2 deletion could be combined with routine screening to facilitate proper intervention.

Impact of maternal hyperglycemia on cardiac development: Insights from animal models.

Congenital heart disease (CHD) is the leading cause of birth defect-related death in infants and is a global pediatric health concern. While the genetic causes of CHD have become increasingly recognized with advances in genome sequencing technologies, the etiology for the majority of cases of CHD is unknown. The maternal environment during embryogenesis has a profound impact on cardiac development, and numerous environmental factors are associated with an elevated risk of CHD. Maternal diabetes mellitus (matDM) is associated with up to a fivefold increased risk of having an infant with CHD. The rising prevalence of diabetes mellitus has led to a growing interest in the use of experimental diabetic models to elucidate mechanisms underlying this associated risk for CHD. The purpose of this review is to provide a comprehensive summary of rodent models that are being used to investigate alterations in cardiac developmental pathways when exposed to a maternal diabetic setting and to summarize the key findings from these models. The majority of studies in the field have utilized the chemically induced model of matDM, but recent advances have also been made using diet based and genetic models. Each model provides an opportunity to investigate unique aspects of matDM and is invaluable for a comprehensive understanding of the molecular and cellular mechanisms underlying matDM-associated CHD.

Abstract P331: Upregulated TBX1 by genetic variants are associated with human congenital heart disease

Congenital heart disease (CHD) is the most common human birth defect worldwide. The cause of CHD is so far not well understood. Uncovering genetic factors leading to CHD is still a pressing task to be solved. TBX1 is one of the transcription factors early expressed in embryonic cardiac progenitors. In animal models, imbalanced TBX1 activity leads to cardiac defects. Given the dosage effect of TBX1, it is possible that genetic variant altering TBX1 function or expression level would affect heart development and contribute to CHD. In order to study the association of genetic variants of TBX1 and CHD susceptibility, we performed genetic screening in 409 CHD patients and 213 healthy controls. Bioinformatic and in vitro functional studies were performed to evaluate the impact of genetic variants. One single nucleotide polymorphism (SNP), rs41260844, in TBX1 promoter region was identified to be associated with CHD. The minor allele of rs41260844 is associated with higher CHD risk and shows increased TBX1 promoter activity (Fig A). Further study showed the minor allele attenuates TBX1 promoter binding affinity with nuclear protein(s) (Fig B). In addition, a novel case-specific missense rare mutation, p.P164L, in T-box domain was identified and predicted as a deleterious mutation. Functional analysis showed a trend of increased TBX1 function with the rare mutation. In summary, we concluded that a higher TBX1 expression level or activity is associated with CHD susceptibility, which could affect TBX1 downstream targets and thus disrupt the balance of the complex regulation network during cardiogenesis.

Cardiovascular Development and Congenital Heart Disease Modeling in the Pig

BackgroundModeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high‐efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects.Methods and ResultsPig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3‐dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late‐stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality.ConclusionsThe major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.

Functional analysis of novel genetic variants of NKX2-5 associated with nonsyndromic congenital heart disease.

NKX2-5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N-terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine-rich domain (p.P247A). Variant-p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N-terminal variants-p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT-PCR. Nonetheless, variant p.R95L affected the synergy of NKX2-5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes-Irx4, Mef2c, Bmp10, Myh6, Myh7, and Myocd is also observed in response to NKX2-5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N-terminal variants supports the emerging role of this region during cardiac-developmental pathways.

Molecular Genetics and Complex Inheritance of Congenital Heart Disease.

Congenital heart disease (CHD) is the most common congenital malformation and the leading cause of mortality therein. Genetic etiologies contribute to an estimated 90% of CHD cases, but so far, a molecular diagnosis remains unsolved in up to 55% of patients. Copy number variations and aneuploidy account for ~23% of cases overall, and high-throughput genomic technologies have revealed additional types of genetic variation in CHD. The first CHD risk genotypes identified through high-throughput sequencing were de novo mutations, many of which occur in chromatin modifying genes. Murine models of cardiogenesis further support the damaging nature of chromatin modifying CHD mutations. Transmitted mutations have also been identified through sequencing of population scale CHD cohorts, and many transmitted mutations are enriched in cilia genes and Notch or VEGF pathway genes. While we have come a long way in identifying the causes of CHD, more work is required to end the diagnostic odyssey for all CHD families. Complex genetic explanations of CHD are emerging but will require increasingly sophisticated analysis strategies applied to very large CHD cohorts before they can come to fruition in providing molecular diagnoses to genetically unsolved patients. In this review, we discuss the genetic architecture of CHD and biological pathways involved in its pathogenesis.