| ObjectivesNKX2-5, GATA4 and TBX5 are the major cardiac transcription factor studied widely currently which related to congenital heart disease (CHD). Many transcription factors, such as NKX2-5, GATA4, TBX5 regulate the formation of the heart singly or interact with each other. Studies have found that, the missing or anomaly of transcription factor Nkx2-5, GATA4 and TBX5 will affect the expression of pathway related and myocardial development, resulting in congenital heart disease such as atrial septal defects (ASD) and ventricular septal defect (VSD). This study was conducted to research the association between Nkx2-5, GATA4 and TBX5 gene single nucleotide polymorphism (SNPs) and the hereditary susceptibility to CHD, and verify the functional SNP related to CHD.MethodsA hospital-based of case-control study was conducted.158 Chinese Han and Zhuang of Guangxi who with VSD and 68 ASD were collected and 234 patients without heart related diseases matched in age, sex and nation were selected as controls. The basic situation and the medical record information were obtained by epidemiological survey method, we collected peripheral blood and extracted genomic DNA. The National Institute of Environmental Health Sciences (NIEHS) database was used to screened the SNPs of Nkx2-5, GATA4 and TBX5 gene, the eligible SNPs includers6882776 of NKX2-5,rs11067101、rs883079、rs3782467 of TBX5 and rs867858、rs904018、rs804287、rs4841588 of GATA4 were selected. TaqMan MGB real-time fluorescent quantitative polymerase chain reaction(RT-PCR)technology was applied on genotyping for the screened SNPs. t test, chi-square test and the multi-factor Logistic regression model in SPSS 18.0 software were used to analyze the relationship between SNPs and the susceptibility of CHD. Hardy-weinberg equilibrium (HWE) test was performed on a control group of each locus through the hardy-weinberg equilibrium software. The function of locus which were related to the susceptibility of CHD was predicted by microRNA.org database (http://www.microrna.org/). Screened on the website of http://www.microrna.org/and SNPInFO software for miRNA was further conducted if the locus was the potential targeted sites of a miRNA. The dual luciferase reporter gene assays were carried out to verify the relationship then.Results(1) Information about the study subjects:There were no statistical differences in the distribution of age and nation between the cases and controls, it’s significantly differences in the distribution of gender. But the distributions of age, gender, nation between the controls and the VSD cases has no statistical significance (P>0.05).(2) HWE test:The genotype distributions of rs904018 in the control is not meet the HWE (χ2=126.39, P=0.000), the others are all in the HWE. All were tested by HaploView4.2 software.(3) Relationship between genotype and the risk of CHD:The statistical analysis reveal that genotype CT/TT of rs4841588 and AC/CC of rs867858 can increase risk of CHD, also can increase the risk of ASD alone. Genotype CT/TT of rs883079 might reduce risk of CHD, also can reduce the risk of ASD and VSD separately. The other SNPs are not relevant to CHD.(4) Stratification analysis of nation and gender:The result showed that genotype frequency of rs4841588, rs867858 and rs883079 are statistically associated with the risk of CHD.(5) Interaction analyze:There is a gene-gene interaction between rs804287 and rs11067101(OR=0.877,95% CI:0.784-0.981,P=0.021),rs4841588 and rs867858 (OR=1.082,95% CI:1.005～1.163,P=0.035)(6) Dual luciferase detection assays:The result of function assays indicated that the relative luciferase activities of wild-type plasmid with miR375 mimic is 21.35±1.47, the group without miR375 mimic is 22.95±0.96, the group mut-type plasmid with miR375 mimic is 24.19±0.52. The latter two have no statistical difference to the former one, P>0.05.ConclusionMutation in rs883079 might reduce the risk of CHD, mutation of rs867858 and rs4841588 might increase the risk of CHD. The onset of CHD may have ethnic and gender differences. Locus rs883079 is not the binding site of miR-375 with the target genes TBX5. |
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