NKX2.5、GATA4、CITED2基因突变及环境因素与散发型先天性心脏病关系的初步探讨
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摘要
先天性心脏病(Congential heart disease,CHD)是常见的新生儿先天性畸形,近年来先心病发病呈上升趋势。约90%的先心病属于多基因病,多基因病的遗传因子和环境因子的性质相当复杂。某些高发家系仅仅是由单个基因座位与个别环境因子相互作用所致,而大多数散发型先心病是由多个基因座位的微小效应叠加在一起相互作用引起的,散发型非高发家系的先心病患者又占多基因遗传的90%以上,因此对散发型先心病发病机制研究更为重要。目前先心病的研究主要集中于从部分基因座位着手揭示多基因的分子病理机制。模式生物研究证实心脏转录因子功能异常可导致严重的先心病,其基因突变成为目前先天性心脏病发病机制研究的焦点。多基因疾病发生过程中参与的遗传因素存在明显的地区差异和种族差异。最早发现的心脏特异性同源盒转录因子NKX2.5(Homobox transcription factor,NKX2.5)基因国外人群共发现29种突变,突变率约4%。国内人群Li Tian在一个房间隔缺损家系中发现NKX2.5基因3个错义突变,而散发型先心病中至今仅孙淼发现了一个三碱基的缺失。国外人群研究GATA4基因突变率仅为约2%,而中国汉族人群唐朝晖在31例患者中却发现了两个新的错义突变Val267Met、Val380Met。提示中国汉族人群先天性心脏病患者转录因子基因突变检测率及突变位点可能与国外人群不同。CITED2基因(cAMP-responsive element-binding protein,CBP/p300)cAMP应答元件结合蛋白基因是转录激活因子家族中新发现的成员之一,目前发现的3种突变均集中于编码链的丝氨酸-甘氨酸富含区(serine-glycine-rich junction SGJ)序列。本课题拟研究散发型先心病患者与瓣膜缺损相关的NKX2.5、GATA4、CITED2基因,了解中国汉族人群中的先心病突变位点及分布情况,并进一步分析基因型与表型间的关系。
先心病由环境因素和遗传因素相互作用引起,即遗传易感性和环境致畸原的相互作用是先心病的发病原因。既往研究认为孕期母亲暴露于先心病高危因素会增加子代先心病风险,所以本研究还回顾性探讨孕期父母暴露因素与叶酸补充情况,了解叶酸补充与孕母自身叶酸代谢酶5,10亚甲基四氢叶酸还原酶基因(methylenetrahydrofolate reduclase gene,MTHFR)C677T基因型之间的交互作用,并比较病例组父代及对照组父代突变等位基因频率,分析其对子代发病的风险。
第一部分散发型先天性心脏病NKX2.5、GATA4、CITED2基因突变检测
第一节NKX2.5基因在散发型先天性心脏病中的突变检测
目的:研究中国人群散发型先天性心脏病NKX2.5基因编码链突变。
方法:收集135个散发型先天性心脏病患者和114例正常健康新生儿血液进行DNA抽提、PCR扩增NKX2.5基因的编码链,应用变性高效液相色谱仪(Denaturing high performance liquid chro matography DHPLC)检测并DNA测序验证突变。
结果:在室间隔缺损病人中发现一种新的错义突变,编码链第848位碱基C转换为A,导致283位密码子由CCG转换为CAG,编码的氨基酸由脯氨酸转换为谷胺酰胺(p.Pro283Gln),这种突变对照组未检测到。发现两种已报道单核苷酸多态性rs2277923和rs3729753,分别导致第21位密码子GAG转变为GAA和第303位密码子CTG转变为CTC,均为同义突变。基因型频率及等位基因频率与以往报道相同,两组没有差异。既往报道的33种突变位点本研究中未检测到。
结论:NKX2.5基因Pro283Gln突变位点可能是室间隔缺损患者的发病原因之一。
第二节GATA4基因在散发型先天性心脏病中的突变检测
目的:检测中国汉族人群散发型先天性心脏病患者GATA4基因编码链突变。
方法:应用PCR—DHPLC对135例散发型先天性心脏病患者和114例健康对照组进行GATA4基因编码序列的突变检测,对异常峰型PCR产物进行直接测序验证。
结果:发现两个新的错义突变和三个新的单核苷酸多态性位点。错义突变分别是在散发型VSD患者中发现的位于exonl的Pro163Ser,和散发型TOF患者位于exon6的Pro407Gln,对照组中均未检测到。位于编码链的新的核苷酸多态性位点Ala33Ala系同义突变。内含子区域检测到两个SNP改变。比较SNP基因型频率及等位基因频率病例组及对照组均没有显著性差异。
结论:GATA4基因错义突变可能是中国人群室间隔缺损和法洛氏四联症的发病原因之一。GATA4基因突变可能多存在于中国散发型先心病患者而且对应先心病表型呈多样化。
第三节GITED2基因在散发型先天性心脏病中的突变检测
目的分析先天性心脏病(congenital heart defects,CHD)患者的CITED2基因编码链基因突变的情况。
方法收集101例散发型CHD患者和104例正常健康新生儿血液进行DNA抽提、PCR扩增,应用DHPLC进行CITED2基因全部编码序列的突变检测,对有异常峰型的DNA进行直接测序,并与GeneBank进行比较。
结果首次在动脉导管未闭的患者发现CITED2基因的一种新的插入突变,在CITED2基因编码链碱基483位起始处插入一个重复9肽(c483_484ins27),导致蛋白的丝氨酸—甘氨酸富含区(SGJ)插入9个氨基酸p.Ser161-Gly162ins9。对照组中未检测到此突变。在CITED2基因的EP300结合基序未发现突变。
结论中国先心病患者中存在CITED2基因突变,新发现的CITED2基因的重复9肽插入突变c483_484ins27可能是导致动脉导管未闭发生的原因之一。
第二部分先心病患者父代NKX2.5、GATA4、CITED2基因突变检测
目的研究患者父代NKX2.5、GATA4、CITED2基因编码链的突变情况,了解患者父代基因突变或SNP对子代先心病发病风险的影响。
方法收集137例散发型CHD患者父亲或母亲和114例正常健康对照组父母血液进行DNA抽提、PCR扩增,应用DHPLC检测NKX2.5、GATA4、CITED2基因全部编码链的突变情况。对有异常峰型的DNA进行直接测序。
结果在法洛氏四联症患者(33)无先心病的父亲血液基因组中发现与患者相同的Pro163Ser突变。6个SNP位点患者父代中均存在,NKX2.5基因rs2277923多态性位点父代与子代基因型组合发现父代杂合型/子代野生型及父代野生型/子代杂合型较父代野生型/子代野生型后代有更大风险获得先天性心脏病(P＜0.05)
结论患者未患先心病的一级亲属中也可以存在GATA4基因Pro163Ser基因突变位点。
第三部分孕期环境高危因素与MTHFR基因C677T多态性对子代先心病发病风险研究
目的分析孕母孕早期环境高危因素,探讨叶酸摄入与MTHFR基因多态性间交互作用对先心病发病的影响。
方法通过病例对照研究回顾性分析98例先心病患者母亲和101例正常对照组母亲孕期危险因素暴露水平,及叶酸及复合维生素摄入情况,并进一步应用DHPLC检测MTHFRC677T多态性了解与叶酸摄入的交互作用。
结果Logistic回归分析有6项因素纳入先心病风险模型,孕母教育程度、没有定期产检、精神紧张、接触化学物质是子代先心病的高危因素,服用叶酸和复合维生素是先心病的保护因素。三种基因型对照组摄入叶酸情况均与病例组有显著差异(P＜0.05)。每天服用叶酸情况下,MTHFR基因各基因型比较病例组与对照组无统计学差异。仅间断服用叶酸组较未服用叶酸组各基因型比较差异无统计学意义,合并有时服用及未服用叶酸比较孕母基因型为CT与CC时发病相对风险,P值为0.087,OR值为2.018(0.95-4.285)。
结论提高孕母的自我保健意识是减少先心病危险因素暴露的重要措施。怀孕早期孕母叶酸的补充不足可能是导致子代先心病发病风险增高的独立因素,孕母MTHFR基因C677T基因型合并叶酸补充不足可能增加先心病风险。
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 mutations
Section 1.1 NKX2.5 mutations in patients with congenital heart disease
Objective: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 Diseases
Objective: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 disease
Objective 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.483_484ins27),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 offspring
Obsjective: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 Factors
Objective: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.
先心病由环境因素和遗传因素相互作用引起,即遗传易感性和环境致畸原的相互作用是先心病的发病原因。既往研究认为孕期母亲暴露于先心病高危因素会增加子代先心病风险,所以本研究还回顾性探讨孕期父母暴露因素与叶酸补充情况,了解叶酸补充与孕母自身叶酸代谢酶5,10亚甲基四氢叶酸还原酶基因(methylenetrahydrofolate reduclase gene,MTHFR)C677T基因型之间的交互作用,并比较病例组父代及对照组父代突变等位基因频率,分析其对子代发病的风险。
第一部分散发型先天性心脏病NKX2.5、GATA4、CITED2基因突变检测
第一节NKX2.5基因在散发型先天性心脏病中的突变检测
目的:研究中国人群散发型先天性心脏病NKX2.5基因编码链突变。
方法:收集135个散发型先天性心脏病患者和114例正常健康新生儿血液进行DNA抽提、PCR扩增NKX2.5基因的编码链,应用变性高效液相色谱仪(Denaturing high performance liquid chro matography DHPLC)检测并DNA测序验证突变。
结果:在室间隔缺损病人中发现一种新的错义突变,编码链第848位碱基C转换为A,导致283位密码子由CCG转换为CAG,编码的氨基酸由脯氨酸转换为谷胺酰胺(p.Pro283Gln),这种突变对照组未检测到。发现两种已报道单核苷酸多态性rs2277923和rs3729753,分别导致第21位密码子GAG转变为GAA和第303位密码子CTG转变为CTC,均为同义突变。基因型频率及等位基因频率与以往报道相同,两组没有差异。既往报道的33种突变位点本研究中未检测到。
结论:NKX2.5基因Pro283Gln突变位点可能是室间隔缺损患者的发病原因之一。
第二节GATA4基因在散发型先天性心脏病中的突变检测
目的:检测中国汉族人群散发型先天性心脏病患者GATA4基因编码链突变。
方法:应用PCR—DHPLC对135例散发型先天性心脏病患者和114例健康对照组进行GATA4基因编码序列的突变检测,对异常峰型PCR产物进行直接测序验证。
结果:发现两个新的错义突变和三个新的单核苷酸多态性位点。错义突变分别是在散发型VSD患者中发现的位于exonl的Pro163Ser,和散发型TOF患者位于exon6的Pro407Gln,对照组中均未检测到。位于编码链的新的核苷酸多态性位点Ala33Ala系同义突变。内含子区域检测到两个SNP改变。比较SNP基因型频率及等位基因频率病例组及对照组均没有显著性差异。
结论:GATA4基因错义突变可能是中国人群室间隔缺损和法洛氏四联症的发病原因之一。GATA4基因突变可能多存在于中国散发型先心病患者而且对应先心病表型呈多样化。
第三节GITED2基因在散发型先天性心脏病中的突变检测
目的分析先天性心脏病(congenital heart defects,CHD)患者的CITED2基因编码链基因突变的情况。
方法收集101例散发型CHD患者和104例正常健康新生儿血液进行DNA抽提、PCR扩增,应用DHPLC进行CITED2基因全部编码序列的突变检测,对有异常峰型的DNA进行直接测序,并与GeneBank进行比较。
结果首次在动脉导管未闭的患者发现CITED2基因的一种新的插入突变,在CITED2基因编码链碱基483位起始处插入一个重复9肽(c483_484ins27),导致蛋白的丝氨酸—甘氨酸富含区(SGJ)插入9个氨基酸p.Ser161-Gly162ins9。对照组中未检测到此突变。在CITED2基因的EP300结合基序未发现突变。
结论中国先心病患者中存在CITED2基因突变,新发现的CITED2基因的重复9肽插入突变c483_484ins27可能是导致动脉导管未闭发生的原因之一。
第二部分先心病患者父代NKX2.5、GATA4、CITED2基因突变检测
目的研究患者父代NKX2.5、GATA4、CITED2基因编码链的突变情况,了解患者父代基因突变或SNP对子代先心病发病风险的影响。
方法收集137例散发型CHD患者父亲或母亲和114例正常健康对照组父母血液进行DNA抽提、PCR扩增,应用DHPLC检测NKX2.5、GATA4、CITED2基因全部编码链的突变情况。对有异常峰型的DNA进行直接测序。
结果在法洛氏四联症患者(33)无先心病的父亲血液基因组中发现与患者相同的Pro163Ser突变。6个SNP位点患者父代中均存在,NKX2.5基因rs2277923多态性位点父代与子代基因型组合发现父代杂合型/子代野生型及父代野生型/子代杂合型较父代野生型/子代野生型后代有更大风险获得先天性心脏病(P＜0.05)
结论患者未患先心病的一级亲属中也可以存在GATA4基因Pro163Ser基因突变位点。
第三部分孕期环境高危因素与MTHFR基因C677T多态性对子代先心病发病风险研究
目的分析孕母孕早期环境高危因素,探讨叶酸摄入与MTHFR基因多态性间交互作用对先心病发病的影响。
方法通过病例对照研究回顾性分析98例先心病患者母亲和101例正常对照组母亲孕期危险因素暴露水平,及叶酸及复合维生素摄入情况,并进一步应用DHPLC检测MTHFRC677T多态性了解与叶酸摄入的交互作用。
结果Logistic回归分析有6项因素纳入先心病风险模型,孕母教育程度、没有定期产检、精神紧张、接触化学物质是子代先心病的高危因素,服用叶酸和复合维生素是先心病的保护因素。三种基因型对照组摄入叶酸情况均与病例组有显著差异(P＜0.05)。每天服用叶酸情况下,MTHFR基因各基因型比较病例组与对照组无统计学差异。仅间断服用叶酸组较未服用叶酸组各基因型比较差异无统计学意义,合并有时服用及未服用叶酸比较孕母基因型为CT与CC时发病相对风险,P值为0.087,OR值为2.018(0.95-4.285)。
结论提高孕母的自我保健意识是减少先心病危险因素暴露的重要措施。怀孕早期孕母叶酸的补充不足可能是导致子代先心病发病风险增高的独立因素,孕母MTHFR基因C677T基因型合并叶酸补充不足可能增加先心病风险。
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 mutations
Section 1.1 NKX2.5 mutations in patients with congenital heart disease
Objective: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 Diseases
Objective: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 disease
Objective 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.483_484ins27),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 offspring
Obsjective: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 Factors
Objective: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.
引文
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