The Research Of The Heart Development-related Gene NKX2.5 And GATA4 Mutations And The Congenital Heart Disease

Objective: In this experiment, congenital heart disease was studied to explore the mutations of the genes GATA4 and NKX2.5 in patients with congenital heart disease. We tried to research the pathogenesis of the congenital heart disease from the molecular level. Methods:Using PCR-DNA sequencing technology to detect all exons and their flanking sequences of the genes NKX2.5 and GATA4 in the peripheral blood from 26 cases of hospitalized children with sporadic congenital heart disease and their parents, to analyze sequencing and to detect whether there are mutations. We used the methods of microarray comparative genomic hybridization (array CGH) to carry out an analysis of copy number variation (CNV), and at the same time the PCR-DNA sequencing technology was used to check the sequence of all GATA4 exons.Results:1. In the sporadic patients with congenital heart disease, we analyzed the sequence of all exons of heart development-related gene NKX2.5.(1) In nine cases of patients with congenital heart disease, we found that encoding the 21st amino acid in the third base (T→C, C/T) had a mutation which located in exon 1.This is a synonymous mutation (E21E, E for Glutamate). Detection rate was 34.6%(9/26). Compared to SNP database of NCBI, it is proved to be a single nucleotide polymorphism (SNP) site, the A→G(c.63A>G,-172662024) polymorphism has been reported.(2) In one case of patients with congenital heart disease, we found that the 282nd base pair (G/T) had a synonymous mutation (P94P, P for proline), the detection rate was 0.04% (1/26), located in exon 1,c.282 A/C(-172661805) in the NCBI's SNP database was not found, it might be a new single nucleotide polymorphisms.(3) In the six cases of patients with congenital heart disease, we found that the 738th base pair (c.738A/T) existed a heterozygous non-synonymous mutation (N246K, by the asparagine mutation of lysine), detection rate was 19.2%(5/26) , located in exon 2 .(4) In one case of patients with congenital heart disease, we found two synonymous mutations c.769C/G(P257P, P for proline) and c.780C/A (G260G, G for glycine), detection rate was 0.04%(1/26), located in exon 2,the genome sequences were (-172659778) and (-172659767), the corresponding SNPs were not found in NCBI's SNP database. 2. In all exons of GATA4 detection, only in one case of patients with congenital heart disease we found that the 744th base sequence (c.744C>T) was bimodal, then by the reverse primer sequencing we confirmed that it belonged to synonymous mutation which located in exon 3 (N249N,N for asparagine), detection rate was 0.04%(1/26), and the genome sequence wa(s11606555). It may be a new single-nucleotide polymorphism (SNP) site, we need to further confirm that.3. The DNA samples which were provided by Suzhou (Fudan) Life Science Laboratory of Suzhou Municipal Hospital were selected. We used the methods of microarray comparative genomic hybridization (array CGH) to analyze the CNV of a family with congenital heart disease (ASD), and we found no co-segregation of the CNV and the disease. At the same time the GATA4 gene in a family with ASD and pulmonary stenosis was sequenced by the PCR-DNA sequencing technology, a common mutation in the 839th base C→T(c.839C>T)was identified in all four patients from three generations, but not in the 4 other normal healthy people in this family, nor in the 800 normal group, which are all 280T homozygote. This mutation was not found in the normal people. Therefore, this result implies that the mutation in GATA4 of this family with CHD is special to itself. Heterozygous c.839C>T(T280M, mutation of methionine by threonine) mutation identified in this study is predicted to be pathologic.Conclusion:In the gene sequencing of all exons of NKX2.5 and GATA4 from 26 cases of patients with congenital heart disease, non-synonymous mutations were not found in gene GATA4. We detected a non-synonymous mutation in the first part of NKX2.5 exon 2 and found 4 SNPs in the exons of NKX2.5 and 1 SNPs in the exon of GATA4.These gene mutations and gene polymorphisms may be related to congenital heart disease, but the exact correlation remains to be further studied. We detected a mutation of GATA4 from CHD pedigrees, this mutation in SNapShot typing from the other 800 cases of the control samples were not found, suggesting that this mutation was special to this family. Bioinformatics studies have shown that the locus mutation is related to the atrial septal defect.