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Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations.

ABSTRACT: 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.

SUBMITTER: Granados-Riveron JT

PROVIDER: S-EPMC3370385 | biostudies-literature | 2012 Mar-Apr

REPOSITORIES: biostudies-literature

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Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations.

Granados-Riveron Javier T JT Pope Mark M Bu'lock Frances A FA Thornborough Christopher C Eason Jacqueline J Setchfield Kerry K Ketley Ami A Kirk Edwin P EP Fatkin Diane D Feneley Michael P MP Harvey Richard P RP Brook J David JD

Congenital heart disease 20111020 2

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 mu ...[more]

PMID: 22011241

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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.

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.

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.

Dataset Information

Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations.

Publications

Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations.

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