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Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO., Competing Interests: MM, EK, RP, DA, DH, GD, PC, VJ, TD, SD, EW, CM, IM, PT, RH No competing interests declared, (© 2023, Moradi Marjaneh, Kirk, Patrick et al.)

Background: The most common cyanotic congenital heart disease (CHD) requiring management as a neonate is transposition of great arteries (TGA). Clinically, up to 50% of TGA patients develop some form of neurodevelopmental disability (NDD), thought to have a significant genetic component. A "ciliopathy" and links with laterality disorders have been proposed. This first report of whole genome sequencing in TGA, sought to identify clinically relevant variants contributing to heart, brain and laterality defects., Methods: Initial whole genome sequencing analyses on 100 TGA patients focussed on established disease genes related to CHD (n = 107), NDD (n = 659) and heterotaxy (n = 74). Single variant as well as copy number variant analyses were conducted. Variant pathogenicity was assessed using the American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines., Results: Fifty-five putatively damaging variants were identified in established disease genes associated with CHD, NDD and heterotaxy; however, no clinically relevant variants could be attributed to disease. Notably, case-control analyses identified significantly more predicted-damaging, silent and total variants in TGA cases than healthy controls in established CHD genes (P < .001), NDD genes (P < .001) as well as across the three gene panels (P < .001)., Conclusion: We present compelling evidence that the majority of TGA is not caused by monogenic rare variants and is most likely oligogenic and/or polygenic in nature, highlighting the complex genetic architecture and multifactorial influences on this CHD sub-type and its long-term sequelae. Assessment of variant burden in key heart, brain and/or laterality genes may be required to unravel the genetic contributions to TGA and related disabilities., Competing Interests: Conflict of interest None., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Congenital heart disease (CHD) affects up to 1% of live births. However, a genetic diagnosis is not made in most cases. The purpose of this study was to assess the outcomes of genome sequencing (GS) of a heterogeneous cohort of CHD patients., Methods: Ninety-seven families with probands born with CHD requiring surgical correction were recruited for genome sequencing. At minimum, a proband-parents trio was sequenced per family. GS data were analyzed via a two-tiered method: application of a high-confidence gene screen (hcCHD), and comprehensive analysis. Identified variants were assessed for pathogenicity using the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines., Results: Clinically relevant genetic variants in known and emerging CHD genes were identified. The hcCHD screen identified a clinically actionable variant in 22% of families. Subsequent comprehensive analysis identified a clinically actionable variant in an additional 9% of families in genes with recent disease associations. Overall, this two-tiered approach provided a clinically relevant variant for 31% of families., Conclusions: Interrogating GS data using our two-tiered method allowed identification of variants with high clinical utility in a third of our heterogeneous cohort. However, association of emerging genes with CHD etiology, and development of novel technologies for variant assessment and interpretation, will increase diagnostic yield during future reassessment of our GS data.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5 +/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Up to 20% of children with congenital heart disease (CHD) undergoing cardiac surgery develop neurodevelopmental disabilities (NDD), with some studies reporting persistent impairment. Recent large-scale studies have demonstrated shared genetic mechanisms contributing to CHD and NDD. In this study, a targeted approach was applied to assess direct clinical applicability of this information., Methods: A gene panel comprising 148 known CHD and/or NDD genes was used to sequence 15 patients with CHD + NDD, 15 patients with CHD, and 15 healthy controls. The number and types of variants between the 3 groups were compared using Poisson log-linear regression, and the SNP-set (Sequence) Kernel Association Test-Optimized was used to conduct single-gene and gene-pathway burden analyses., Results: A significant increase in rare (minor allele frequency < 0.01) and novel variants was identified between the CHD + NDD cohort and controls, P < .001 and P = .001, respectively. There was also a significant increase in rare variants in the CHD cohort compared with controls (P = .04). Rare variant burden analyses implicated pathways associated with "neurotransmitters," "axon guidance," and those incorporating "RASopathy" genes in the development of NDD in CHD patients., Conclusions: These findings suggest that an increase in novel and rare variants in known CHD and/or NDD genes is associated with the development of NDD in patients with CHD. Furthermore, burden analyses point toward rare variant burden specifically in pathways related to brain development and function as contributors to NDD. Although promising variants and pathways were identified, further research, utilizing whole-genome approaches, is required prior to demonstrating clinical utility in this patient group., (Copyright © 2018 Elsevier, Inc. All rights reserved.)

Mutations in the Nkx2-5 gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the Nkx2-5 gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new Nkx2-5 point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of Nkx2-5 -driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by Nkx2-5 in the heart and show that Nkx2-5 -dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how Nkx2-5 regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by NKX2-5 mutations in patients., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

Background: Exome sequencing is an established strategy to identify causal variants in families with two or more members affected by congenital heart disease (CHD). This unbiased approach, in which both rare and common variants are identified, makes it suitable to research complex, heterogeneous diseases such as CHD., Methods and Results: Exome sequencing was performed on two affected members of a three generation family with atrial septal defects (ASD), suggesting a dominant inheritance pattern. Variants were filtered using two bioinformatics pipelines and prioritised according to in silico prediction programs. Segregation studies and functional analyses were used to assess co-segregation with disease and effects on protein function, respectively. Following the data and in silico analyses, ten candidate variants were prioritised. Of these, SRPK2 (c.2044C>T[p.Arg682Trp]) and NOTCH1 (c.3835C>T[p.Arg1279Cys]), co-segregated with disease in the family; however, previous functional analyses on SRPK2 make this an unlikely candidate. Functional analyses in the variant (c.3835C>T[p.Arg1279Cys]) of the known CHD gene NOTCH1 demonstrated a non-significant decrease in signalling activity., Conclusion: This study demonstrates both the potential, as well as the challenges, of applying exome sequencing to complex diseases such as CHD. While in silico evidence and segregation analyses in the NOTCH1 p.Arg1279Cys variant are highly suggestive of pathogenicity, the minimal change in signalling capacity suggests that other variants may be required for CHD development. This study highlights the difficulties of applying exome sequencing in familial, non-syndromic CHD in the clinical environment and a cautionary note in the interpretation of apparently causal abnormalities in silico without supportive functional data., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Our understanding of the genetics of congenital heart disease (CHD) is rapidly expanding; however, many questions, particularly those relating to sporadic forms of disease, remain unanswered. Massively parallel sequencing technology has made significant contributions to the field, both from a diagnostic perspective for patients and, importantly, also from the perspective of disease mechanism. The importance of de novo variation in sporadic disease is a recent highlight, and the genetic link between heart and brain development has been established. Furthermore, evidence of an underlying burden of genetic variation contributing to sporadic and familial forms of CHD has been identified. Although we are still unable to identify the cause of CHD for most patients, recent findings have provided us with a much clearer understanding of the types of variants and their individual contributions and collectively mark an important milestone in our understanding of both familial and sporadic forms of disease., (Copyright © 2017 American College of Cardiology Foundation. All rights reserved.)

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Background: Many genes have been implicated in the development of congenital heart disease (CHD). Next-generation sequencing offers opportunities for genetic testing but is often complicated by logistic and interpretative hurdles., Objectives: This study sought to apply next-generation sequencing technology to CHD families with multiple affected members using a purpose-designed gene panel to assess diagnostic potential for future clinical applications., Methods: We designed a targeted next-generation sequencing gene panel for 57 genes previously implicated in CHD. Probands were screened in 16 families with strong CHD histories and in 15 control subjects. Variants affecting protein-coding regions were classified in silico using prediction programs and filtered according to predicted mode of inheritance, minor allele frequencies, and presence in databases such as dbSNP (Single Nucleotide Polymorphism Database) and ESP (Exome Sequencing Project). Disease segregation studies were conducted in variants identified in CHD cases predicted to be deleterious and with minor allele frequencies <0.1%., Results: Thirteen potential disease-causing variants were identified in 9 families. Of these, 5 variants segregated with disease phenotype, revealing a likely molecular diagnosis in 31% of this cohort. Significant increases in the number of "indels, nonsense, and splice" variants, as well as variants classified as "probably damaging" were identified in CHD cases but not in control subjects. Also, there was a significant increase in the total number of "rare" and "low" frequency variants (minor allele frequencies <0.05) in the CHD cases., Conclusions: When multiple relatives are affected by CHD, a gene panel-based approach may identify its cause in up to 31% of families. Identifying causal variants has implications for clinical care and future family planning., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown., Methods and Results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein., Conclusions: Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.

About 80% of congenital heart disease (CHD) is multifactorial and arises through various combinations of genetic and environmental contributors. About 20% of cases can be attributed to chromosomal anomalies, Mendelian syndromes, non-syndromal single gene disorders or teratogens. Down syndrome and velocardiofacial syndrome are the most commonly seen syndromes in patients with CHD. To date, more than 30 genes have been linked to non-syndromal forms of CHD. Their contribution to CHD remains unknown but is presumed to be relatively small. There is limited evidence for the contribution of specific environmental factors to CHD causation. However, folic acid supplementation in the pre- and peri-conception period, ensuring rubella vaccination has been completed before pregnancy, and maintaining good glycaemic control in mothers with diabetes may reduce the risk of CHD in infants. Recurrence risks vary between different types of non-syndromal CHD with multifactorial inheritance, and can be as high as 10% when two or more siblings are affected. Generally, the recurrence risk increases if a parent rather than a sibling is affected, particularly when the affected parent is the mother. Individualised recurrence risks can be generated for members of families affected by CHD after obtaining a detailed family history, including accurate cardiac diagnoses for all affected members. High-throughput genetic techniques can accelerate gene discovery and improve our ability to provide individualised genetic counselling.

Background.  Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt-Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design.  In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results.  Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions.  In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts., (© 2011 Wiley Periodicals, Inc.)

Cited2 is a transcriptional coactivator that is required for normal development of the embryo and placenta. Cited2-null mice die during gestation with fully penetrant heart defects and partially penetrant laterality defects. The laterality defects occur due to the loss of Nodal expression in the left lateral plate mesoderm (LPM). The cause of the heart defects that arise independently of laterality defects is unknown; they might occur due to an intrinsic requirement for Cited2 in the developing heart, or to disturbances in left-right patterning of the early embryo. Herein it is established that deletion of Cited2 from the heart progenitors does not alter development, and that heart defects in Cited2-null embryos arise due to an extra-cardiac requirement for Cited2 in establishing the left-right body axis. In addition, we provide evidence supporting a role for Cited2 in tissues of the embryo vital for left-right patterning (the node and LPM). Molecular and genetic analysis reveals that Cited2 is required for the initiation, but not propagation of, the left-sided determinant Nodal in the LPM. Moreover, a new role for Cited2 is identified as a potentiator of bone morphogenetic protein (BMP) signalling, counteracting the initiation of Nodal expression in the LPM. These data define Cited2 as a key regulator of left-right patterning in the mammalian embryo, and reveal that the role of Cited2 in cardiac development lies in its extra-cardiac functions. The clinical relevance of these findings lies in the fact that heterozygous mutation of human CITED2 is associated with congenital heart disease and laterality defects.

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2-5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

Congenital heart disease (CHD) represents one of the most common birth defects, but the genetic causes remain largely unknown. Mutations in GATA4, encoding a zinc finger transcription factor with a pivotal role in heart development, have been associated with CHD in several familial cases and a small subset of sporadic patients. To estimate the pathogenetic role of GATA4 in CHD, we screened for mutations in 357 unrelated patients with different congenital heart malformations. In addition to nine synonymous changes, we identified two known (A411V and D425N) and two novel putative mutations (G69D and P163R) in five patients with atrial or ventricular septal defects that were not seen in control subjects. The four mutations did not show altered GATA4 transcriptional activity in synergy with the transcription factors NKX2-5 and TBX20. Our data expand the spectrum of mutations associated with cardiac septal defects but do not support GATA4 mutations as a common cause of CHD.

Congenital heart defects (CHD) are collectively the most common form of congenital malformation. Studies of human cases and animal models have revealed that mutations in several genes are responsible for both familial and sporadic forms of CHD. We have previously shown that a mutation in MYH6 can cause an autosomal dominant form of atrial septal defect (ASD), whereas others have identified mutations of the same gene in patients with hypertrophic and dilated cardiomyopathy. In the present study, we report a mutation analysis of MYH6 in patients with a wide spectrum of sporadic CHD. The mutation analysis of MYH6 was performed in DNA samples from 470 cases of isolated CHD using denaturing high-performance liquid chromatography and sequence analysis to detect point mutations and small deletions or insertions, and multiplex amplifiable probe hybridization to detect partial or complete copy number variations. One non-sense mutation, one splicing site mutation and seven non-synonymous coding mutations were identified. Transfection of plasmids encoding mutant and non-mutant green fluorescent protein-MYH6 fusion proteins in mouse myoblasts revealed that the mutations A230P and A1366D significantly disrupt myofibril formation, whereas the H252Q mutation significantly enhances myofibril assembly in comparison with the non-mutant protein. Our data indicate that functional variants of MYH6 are associated with cardiac malformations in addition to ASD and provide a novel potential mechanism. Such phenotypic heterogeneity has been observed in other genes mutated in CHD.

Rationale: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors., Objective: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger-type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor., Methods and Results: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or alphaMHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls., Conclusions: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network.

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Objectives: We sought to examine the importance of mutations in the cardiac transcription factor gene NKX2-5 in patients with an atrial septal defect (ASD), patent foramen ovale (PFO), or hypoplastic left heart syndrome (HLHS)., Background: Mutations in NKX2-5 have been found in families showing secundum ASD and atrioventricular (AV) conduction block and in some individuals with tetralogy of Fallot. The prevalence of NKX2-5 mutations in sporadic cases of ASD/PFO and other forms of congenital heart disease is unknown., Methods: A cohort of 146 individuals with secundum ASD, PFO complicated by paradoxical embolism, or HLHS were evaluated. Patients with ASD or PFO were ascertained irrespective of family history or associated cardiac abnormalities. The coding region of the NKX2-5 locus was amplified by polymerase chain reaction and sequenced., Results: Among 102 ASD and 25 PFO patients screened, 13 patients (10%) had a positive family history and 5 patients (4%) had AV conduction block. We found one previously documented NKX2-5 missense mutation, T178M, in members of a family with ASD without AV conduction block. One NKX2-5 mutation-positive child from this family had HLHS, although no mutations were subsequently found in 18 patients with sporadic or familial HLHS. In a second ASD family without AV conduction block, we found a missense change, E21Q, previously reported as pathogenic. Because this change did not segregate with disease status, we propose that it is a non-disease-causing polymorphism., Conclusions: Our findings suggest that NKX2-5 mutations are a relatively infrequent cause of sporadic ASD and HLHS. Screening for NKX2-5 mutations may be warranted in individuals with ASD and a positive family history, irrespective of the presence or absence of AV conduction block.

Congenital heart disease is a significant cause of morbidity and mortality in humans, and gene mutations that underlie some of these anomalies are now being described. The NKX2.5 gene, which encodes a homeobox transcription factor, was initially discovered in mice through its similarity to the tinman gene of the fruitfly Drosophila. Tinman is required for formation of the dorsal pulsatile vessel or 'heart' of the fly. Tinman and NKX2.5 share structural and functional features, and in mice the gene is required for normal cardiac looping and differentiation of chamber myocardium. Humans with heterozygous mutations in the NKX2.5 gene generally have a disorder involving progressive atrio-ventricular conduction block and atrial septal defect, although sometimes other abnormalities including tetralogy of Fallot. The TBX5 gene, which encodes another cardiac transcription factor that collaborates with NKX2.5, is also an important cardiac disease gene, with heterozygous mutations responsible for Holt-Oram (hand/heart) syndrome. These contributions to human pathology underscore the relevance of studying biological phenomena in lower organisms, and examination of other genes acting in this and associated pathways will expand our knowledge of congenital abnormalities and disease predisposition, and improve genetic counseling.