Preliminary Exploration On The Relationship Of NKX2.5 Gene, GATA4 Gene, CITED2 Gene And Environmental Factors With The Sporadic Congenital Heart Disease

Recently incidence of congenital heart disease(CHD),malformation of the cardiovascular system that is present at or near the time of birth,ascends.Classical studies have proposed the multifactorial inheritance hypothesis,genes interacting with encironment factors,for the etiology of 85 to 90 percent of CHDs.An autosomal dominant mode of inheritance with reduced penetrance is likely for the familial CHDs; whenas nearly 90 percent of sporadic CHDs are caused by more than one alleles at a number of loci interacting with environmental factors.Then it is more important to identify genetic causes of sporadic CHD.Presently the etiologic investigation of CHD is focused on partial gene locus.Gene targeting exeriments of transcription factor in mice result in cardiac malformation,mutations in the gene encoding the transcription factor were found to cause sporadic human congenital heart disease.Many reports have implicated that there are obvious area- and race- specifics in the inherited factors during the development of polygenic disease.NKX2.5,indenpendently described as a cardic-specific homebox gene,has been identified approximately 29 missense and nonsense mutaions in foreign population.The incidence rate of mutation is 4%. Whenas in Chinese population only three novel mutions were described in a familial ASD and a three bases insertion in a sporadic CHD until now.GATA4,a conserved zinc finger transcription factor,which mutation rate just 2%in foreign population, was found two novel mution Val267Met,Val380Met in 31 CHD cases.These give a hint that the mutation loci and detection rate of transcription factors in Chinese population may be differ from foreign population.CITED2 gene,cAMP-responsive element-binding protein,is a new member of transcription activefactor family.The discovered mutations cluster mainly in the serine-glycine rich junction.In the present study we performed a mutation screening in patients with sporadic CHD to further investigate the extent of the NKX2.5,GATA4 and CITED2 gene and explore if there are different distributing compared with foreign population.Besides inheritance,the occurrence of CHD are also attributing to environment. Where a combination of genes from both parents,in addition to unknown environmental factors,produce the trait or condition.Some types of congenital heart defects are known to occur more often when the mother comes in contact with certain substances during the first few weeks of pregnancy,while the baby's heart is developing.In this study we explored the possible environmental risk factors and folic acid supplement.We also analyzed the gene-environment interaction between maternal methylenetrahydrofolate reduclase gene(MTHFR) C677T genotype with periconceptional folate supplementation and compared the frequencies of alleles and gentypes to investigate the occurrence of CHD in their offspring.Section 1 Screening of Chinese sporadic patients with Congenital heart disease for NKX2.5,GATA4 and CITED2 mutationsSection 1.1 NKX2.5 mutations in patients with congenital heart diseaseObjective:The purpose of this study was to estimate the frequency of NKX2.5 mutations in specific cardiovascular anomalies.Methods:We tested genomic deoxyribonucleic acid from 135 prospectively recruited patients with various CHD for NKX2.5 mutation by polymerase chain reaction(PCR) - denaturing high-performance liquid chromatography(DHPLC) and direct sequencing.Results:One novel mutation C848A(CCG-CAG) in the NKX2.5 coding region, was identified in sporadic patient with VSD.The mutation was missense nucleotide substitutions in exon2,resulting in the substitution of amino acid from proline acid to glutamine(Pro283Gln).None of the controls found this mutation.Two previous reported Single Nuclear Polymorphism(SNP) were also detected.Both rs2277923 and rs3729753 were synonymous mutation.There were no differences between two groups of their alleles and genotypes frequencies.Conclusion:Pro283Gln mutation in NKX2.5 gene may be one of the VSD etiologic causes.Section 1.2 GATA4 Mutations in sporadic Chinese Patients with Congenital Heart DiseasesObjective:To identify the mutations in GATA4 gene in patients with CHD of Chinese Han population.Method:By DHPLC and direct sequencing,we analyzed the coding of GATA4 in DNA isolated from blood samples of 135 patients with diverse congenital heart disease and 114 unrelated healthy individuals. Results:Two different genomic missense mutations and three novel SNP were identified in the region of GATA4.In patient with Tetralogy of Fallot Pro163Ser was located in the transcriptional activation domain 2(TAD2) and in patient with outlet membranous ventricular septal defect Pro407Gln was found in the C-terminal of GATA4.None of these mutations were detected in 114 healthy Chinese control.One SNP(p.Ala33Ala) was synonymous mutation.Another two were in the intron.There were no differences between two groups of their alleles and genotypes frequencies.Conclusions:This study had shown that genomic GATA4 missense mutation may relative commonly occur in sporadic Chinese patients with diverse phenotypes of CHD.Section 1.3 Insertion mutation in the serine-glycine rich junction of CITED2 as potential molecular cause for congenital heart diseaseObjective To detect coding regions mutation of CITED2 gene in patients with congenital heart disease(CHD).Methods The blood cell genomic DNA of 101 patients with CHD and 104 normal neonatal was isolated.Whole coding regions of CITED2 were amplified by PCR.The PCR products were detected by DHPLC and samples with different melting profile shapes were sequenced,compared to GeneBank sequence databases.Results A novel insertion mutation was first identified in patients with patent ductus arteriosus(PDA) compared with 104 healthy controls.Mutation was located between 483C and 484G(c.483₄84ins27),which resulted a 9-mer peptides repeat insertion in the serine-glycine rich junction(SGJ) of CITED2 amino acid sequence. Other patients and healthy individuals were normal.Conclusion There was mutation in CITED2 gene of Chinese CHD.The 9-mer peptides insertional mutation newly found may be one of the causes for PDA.Section 2 Effect of NKX2.5,GATA4,CITED2 genes mutations in parents on the risk of CHD in offspringObsjective:To explore the role of NKX2.5,GATA4,CITED2 genes mutations in Chinese parents whose offsprings with CHD.Methods:The blood cell genomic DNA of 137 individual parents of CHD patients and 114 individual parents of controls were collected.The whole coding regions of NKX2.5,GATA4,CITED2 gene were deterimined by PCR-DHPLC.Results:In the coding region of GATA4,the same mutation Pro163Ser was also detected in the father of TOF patients.Six SNPs were examined in parents.Compared two groups found that in rs2277923 of NKX2.5 parentGA/offspringGG and parentGG/offspringGA genotype combination have increased risk for CHD of offspring(p＜0.05).Conclusion:Pro163Ser mutation in GATA4 gene can occur in healthy first degree relative of CHD.Section 3 congenital heart diseases in offspring analysis on methylenetetrahydrofolate reduetase gene C677T Polymorphism and Environmental Risk FactorsObjective:To explore congenital heart diseases(CHD) in their offsprings in association with parental MTHFR gene C677T genotype,Periconceptional folate supplementation and environmental factors.Methods:Retrospective case-control study was carried out to investigate periconceptional folate supplementation and environmental factors in the 98 parents with CHD offsprings and 101 parents with normal.The mother'MTHFR gene 677C-T mutation was also identified.The possible risk factors were analysed by simple and multiple factors logistic regression methods.Results:Results revealed that 6 factors were related to the the occurrence of CHD in the offsprings:education degree of gestation mother,not received prenatal examination,under depressed or nerous condition during pregnancy and maternal exposures to harmful substance was the risk factors;periconceptional folate and Compound Vitamin supplementation were protection factors.there were significant difference between case and control group in folate supplement(P＜0.05).The maternal MTHFR 677CT and TT genotypes in combination with every day use of periconceptional folate supplements were associated with no increased risk for CHD in offspring,however in combination with not use of folate a two-fold(OR 2.018 95%CI 0.95-4.285) increased risk.Conclusion:To improve self health care of gestational mother was the most important protection measures to avoid the risk factors exposure.Periconceptional folate supplement deficiency may be the independence risk factor for CHD.Mother carrying MTHFR heterozygotes(CT) genotype in combination with folate deficiency may increase risk for CHD.