Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations.
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SUBMITTER: Granados-Riveron JT
PROVIDER: S-EPMC3370385 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundSeveral genes encoding transcription factors are known to be the primary cause of congenital heart disease. NKX2-5 and GATA4 were the first congenital heart disease-causing genes identified by linkage analysis. This study designed to study the association of five single-nucleotide variants of NKX2-5, GATA4, and TBX5 genes with sporadic nonsyndromic cases of a congenital cardiac septal defect in Egyptian children.MethodsVenous blood samples from 150 congenital heart disease children (including a ventricular septal defect, atrial septal defect, tetralogy of Fallot, and patent ductus arteriosus) and 90 apparently healthy of matched age and sex were studied by polymerase chain reaction followed by direct sequencing in order to study two single-nucleotide variants of NKX2-5 (rs2277923, rs28936670), two single-nucleotide variants of GATA4 (rs368418329, rs56166237) and one single-nucleotide variant TBX5 (rs6489957). The distribution of genotype and allele frequency in the congenital heart diseases (CHD) group and control group were analyzed.ResultsWe found different genotype frequencies of the two variants of NKX2-5, as CT genotype of rs2277923 was present in 58% and 36% in cases and control respectively, and TT genotype present in 6% of the cases. Also regarding missense variant rs28936670, heterozygous AG presented in 82% of the cases. Also, we observed a five prime UTR variant rs368418329, GT (42% of the cases) and GG (46% of the cases) genotypes showed the most frequent presentation in cases. While regarding a synonymous variant rs56166237, GT and GG were the most presented in cases (41.4%, 56% respectively) in contrast to control group (20%, 1.7% respectively). Also, a synonymous variant in TBX5, the distribution of genotype frequency was significantly different between the CHD group and control group. CT genotype of TBX5 -rs6489957 was found in 12 ASD, 24 VSD, six PDA, three aortic coarctation and nine fallot that represent 42% of the cases.ConclusionsSignificantly higher frequency of different allelle of five variants was observed in cases when compared to the control group, with significant risky effect for the development of septal defect. In addition to two polymorphisms of NKX2-5 (rs2277923, rs28936670) variant in the cardiac septal defect, two variants in GATA4 (rs368418329, rs56166237) and one variant in TBX5 (rs6489957) seem to have a role in the pathogenesis of congenital heart disease.
| S-EPMC6503026 | biostudies-literature
Project description:BackgroundCongenital heart disease (CHD) is a common birth defect originating from both environmental and genetic factors. An overabundance of copy number variations (CNVs) affecting cardiac-related genes has previously been detected in individuals with CHD.ObjectiveTo evaluate if the presence of CNVs in the 22q11.2 region, and to determine whether GATA4, NKX2-5, TBX5, BMP, and CRELD1 genes contributed toward the pathogenesis of isolated incidences of CHDs in southwest China.MethodsIn total 167 patients from southwest China with sporadic CHD were studied, including 121 patients with ventricular septal defect (VSD), 24 with atrial septal defect (ASD), 12 with tetralogy of fallot (TOF), six VSD cases with TOF, two cases with patent ductus arteriosus (PDA), and two VSD cases with ASD. 22q11.2, GATA4, NKX2-5, TBX5, BMP4, and CRELD1 regions were screened using MLPA and copy number variation sequencing (CNV-Seq).ResultsA 2.5-2.8 Mb deletion in the 22q11.2 region was identified in 5 patients with CHD. Two of these patients were diagnosed with VSD, while two had VSD and ASD, and the other had TOF. 5 patients correspond to the same classical DiGeorge syndrome. A 0.86 Mb duplication in the 22q11.2 region was identified in a PDA patient, whom was without extracardiac symptoms.ConclusionThese data suggest that copy number variation in the 22q11.2 region is common in CHD patients in southwest China. Regardless of the presence or absence of extracardiac symptoms, results also indicate that it is necessary to perform prenatal screening for CHD.
| S-EPMC6818592 | biostudies-literature
Project description:Reports of somatic mutations found in hearts with cardiac septal defects have suggested that these mutations are aetiologic in pathologic cardiac development. However, the hearts in these reports had been fixed in formalin for over 22 years. Because of the profound implication of this finding, we attempted to replicate it using fresh frozen tissue obtained in the current era from 28 patients with septal defects who underwent cardiac surgery and who were enrolled in our congenital heart disease tissue bank.Our cohort included patients with atrial septal defects (ASD, n = 13), ventricular septal defects (VSD, n = 5), and atrioventricular canal defects (AVCD, n = 10). Cardiac tissue samples were collected both from diseased tissue located immediately adjacent to the defect and from anatomically normal tissue located at a site remote from the defect (right atrial appendage). Tissue samples were immediately frozen in liquid nitrogen and stored at -80 degrees C. Genomic DNA was isolated and amplified using the same methodology described in the previously published reports. 42 pathologic cardiac tissue samples were sequenced.One non-synonymous germline sequence variant was identified in one patient. Two synonymous germline sequence variants were identified in two separate patients. A common single nucleotide polymorphism (SNP) was identified in 16 patients. Based on the incidence of somatic mutations described in the previously published reports, our study was adequately powered to replicate the previous studies. No evidence of somatic mutations was found in this study.Somatic mutations in NKX2-5 do not represent an important aetiologic pathway in pathologic cardiac development in patients with cardiac septal defects.
| S-EPMC2628540 | biostudies-literature
Project description:Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood.A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors.The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.
| S-EPMC4671672 | biostudies-literature
Project description:Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene.To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR.Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression.In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.
| S-EPMC3252638 | biostudies-literature
Project description:Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.
| S-EPMC3349729 | biostudies-literature
Project description:Cardiac transcription factors are master regulators during heart development. Some were shown to transdifferentiate tail tip and cardiac fibroblasts into cardiomyocytes. However, recent studies have showed that controversies exist. Potential difference in tail tip and cardiac fibroblast isolation may possibly confound the observations. Moreover, due to the use of a cardiac reporter (Myh6) selection strategy for induced cardiomyocyte enrichment, and the lack of tracking signals for each transcription factors, individual roles of each transcription factors in activating cardiac gene expression in mammalian non-myoblastic cells have never been elucidated. Answers to these questions are an important step toward cardiomyocyte regeneration. Because mouse 10T1/2 fibroblasts are non-myoblastic in nature and can be induced to express genes of all three types of muscle cells, they are an ideal model for the analysis of cardiac and non-cardiac gene activation after induction. We constructed bi-cistronic lentiviral vectors, capable of expressing cardiac transcription factors along with different fluorescent tracking signals. By infecting 10T1/2 fibroblasts with Nkx2-5, Tbx5, Gata4 or Myocd cardiac transcription factor lentivirus alone or different combinations, we found that only Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression. Microarray-based gene ontology analysis revealed that Tbx5 alone activated genes involved in the Wnt receptor signaling pathway and inhibited genes involved in a number of cardiac-related processes. Myocd alone activated genes involved in a number of cardiac-related processes and inhibited genes involved in the Wnt receptor signaling pathway and non-cardiac processes. Gata4 alone inhibited genes involved in non-cardiac processes. Tbx5+Gata4+Myocd was the most effective activator of genes associated with cardiac-related processes. Unlike Tbx5, Gata4, Myocd alone or Tbx5+Myocd, Tbx5+Gata4+Myocd activated the fewest genes associated with non-cardiac processes. Conclusively, Tbx5, Gata4 and Myocd play different roles in cardiac gene activation in mammalian non-myoblastic cells. Tbx5+Gata4+Myocd activates the most cardiac and the least non-cardiac gene expression.
| S-EPMC3483304 | biostudies-literature
Project description:Congenital heart disease (CHD) is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.
| S-EPMC3633926 | biostudies-literature
Project description:Congenital heart disease (CHD) is the most common type of developmental defect, with high rates of morbidity in infants. The transcription factor GATA‑binding factor 4 (GATA4) has been reported to serve a critical role in embryogenesis and cardiac development. Our previous study reported a heterozygous GATA4 c.1306C>T (p.H436Y) mutation in four Chinese infants with congenital heart defects. In the present study, functional analysis of the GATA4 H436Y mutation was performed in vitro. The functional effect of GATA4 mutation was compared with GATA4 wild‑type using a dual‑luciferase reporter assay system and immunofluorescence. Electrophoretic mobility‑shift assays were performed to explore the binding affinity of the mutated GATA4 to the heart and neural crest derivatives expressed 2 (HAND2) gene. The results revealed that the mutation had no effect on normal nuclear localization, but resulted in diminished GATA‑binding affinity to HAND2 and significantly decreased gene transcriptional activation. These results indicated that this GATA4 mutation may not influence cellular localization in transfected cells, but may affect the affinity of the GATA‑binding site on HAND2 and decrease transcriptional activity, thus suggesting that the GATA4 mutation may be associated with the pathogenesis of CHD.
| S-EPMC6691264 | biostudies-literature
Project description:BackgroundCongenital heart disease (CHD) is the most prevalent type of birth defect in human, with high morbidity in infant. Several genes essential for heart development have been identified. GATA4 is a pivotal transcription factor that can regulate the cardiac development. Many GATA4 mutations have been identified in patients with different types of CHD.AimsIn this study, the NKX2-5, HAND1 and GATA4 coding regions were sequenced in a family spanning three generations in which seven patients had CHD. Disease-causing potential variation in this family was evaluated by bioinformatics programs and the transcriptional activity of mutant protein was analyzed by the dual luciferase reporter assay.ResultsA novel GATA4 mutation, c.C931T (p.R311W), was identified and co-segregated with the affected patients in this family. The bioinformatics programs predicted this heterozygous mutation to be deleterious and the cross-species alignment of GATA4 sequences showed that the mutation occurred within a highly conserved amino acid. Even though it resided in the nuclear localization signal domain, the mutant protein didn't alter its intracellular distribution. Nevertheless, further luciferase reporter assay demonstrated that the p.R311W mutation reduced the ability of GATA4 to activate its downstream target gene.ConclusionsOur study identified a novel mutation in GATA4 that likely contributed to the CHD in this family. This finding expanded the spectrum of GATA4 mutations and underscored the pathogenic correlation between GATA4 mutations and CHD.
| S-EPMC4938561 | biostudies-literature