【作者】 仇小强；

【导师】 聂绍发；

【作者基本信息】 华中科技大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 研究目的1、探讨父母亚甲基四氢叶酸还原酶(MTHFR)基因C677T、胱硫醚β-合成酶(CBS)基因T833C、环境因素与子代先天性心脏病(CHD)发生的相关性;了解CHD及室间隔缺损(VSD)类型的危险因素;分析危险因素交互作用对VSD发生的影响。2、筛选单纯性VSD血清标志蛋白并构建其血清诊断模型,为进一步从蛋白质分子水平研究单纯性VSD的致病机理及发展其血清学诊断方法奠定基础。3、了解广西神经管畸形(NTDs)的基本分布情况,分析环境及行为危险因素。4、了解广西健康育龄人群MTHFR基因位点突变的分布及其与妊娠结局的联系。5、了解育龄妇女对NTDs的认知度、增补叶酸的依从性和评价宣教效果,为预防NTDs寻求更有效的干预模式。研究方法1、采用1∶1配对病例对照研究方法,对来自广西的115对CHD患儿与对照儿父母进行访谈式调查并抽取父母静脉血3ml作基因型分析,应用PCR限制性片段长度多态性(PCR-RFLP)方法检测MTHFR基因C677T,PCR扩增阻滞突变体系(PCR-ARMS)方法检测CBS基因T833C。对CHD、VSD可能的危险因素进行Logistic回归分析,对筛选出VSD的危险因素之间作交互作用分析,其他数据分析作x~2检验。2、用CM10芯片检测56例单纯性VSD、85例儿科常见病患儿及23例其它CHD患儿血清,用Biomark Wizard软件分析数据、筛选VSD血清差异蛋白,用BiomarkerPatterns软件构建诊断模型。3、以入户或电话采访的方式对NTDs和对照的双亲进行调查,获取他们是否暴露于相关环境危险因素的资料,对暴露因素进行Logistic回归分析。4、抽取早孕妇女静脉血,提取全血DNA,用PCR-RFLP方法检测MTHFR基因位点多态性,再比较其妊娠结局。5、对参加NTDs知识学习和未参加学习的新婚育龄妇女按调查表内容展开调查,比较两组调查结果;并收集孕妇服用叶酸情况,追踪其妊娠结局。结果用卡方检验和方差分析进行统计分析。研究结果1、115例CHD各类型组成VSD 46例、PDA 14例、F4 13例、ASD 6例、其他单纯类型8例、复杂型CHD 28例。广西健康人群的MTHFR基因C/C、C/T、T/T三种基因型构成比分别为64.8%、29.6%、5.7%,C等位基因频率为79.6%,T等位基因频率为20.4%,CBS基因T/T、T/C、C/C三种基因型构成比分别为42.6%、47.8%、9.6%,T等位基因频率为66.5%,C等位基因频率为33.5%。母亲MTHFR基因的C与T等位基因频率比较差异具有统计学意义(P=0.036,OR=1.595,95%CI:1.029～2.471);对于VSD类型,父母组合的MTHFR基因C与T等位基因频率比较差异具有统计学意义(P=0.032,OR=1.724,95%CI:1.043～2.849)。Logistic回归分析表明,母亲孕早期接触农药、妊娠合并症、孕早期感冒、孕期情绪状况、母MTHFR基因型、母CBS基因型为子代发生CHD的危险因素;妊娠合并症、孕早期服中成药、孕期情绪状况、母MTHFR基因型为子代发生VSD的危险因素。交互作用分析表明妊娠合并症与孕期情绪状况、母MTHFR基因型与妊娠合并症、孕早期服中成药、孕期情绪状况间对VSD的发生存在交互作用。2、25例壮族单纯性VSD与36例壮族儿科常见病儿血清蛋白质谱比较,有7个蛋白成分在VSD血清中高表达,5个蛋白下调;21例汉族单纯性VSD与33例汉族儿科常见病儿血清蛋白质谱比较,有7个蛋白成分高表达,1个蛋白下调;不分民族56例单纯性VSD与85例儿科常见病儿血清蛋白质谱比较,有10个蛋白成分高表达,11个蛋白下调;不分民族56例单纯性VSD与23例其它CHD血清蛋白质谱比较,有9个蛋白成分高表达,16个蛋白下调;不分民族56例单纯性VSD、85例儿科常见病儿与23例其它CHD血清蛋白质谱比较,有25个蛋白成分在三组之间有统计学差异;3、在单纯性VSD与儿科常见病的血清诊断模型中,完整模型ROC曲线下面积为0.984,预测准确率为96.51%,灵敏度为97.06%,特异度为96.15%,阳性预测值为94.29%,阴性预测值为98.04%,简化模型分别为0.913、88.37%、97.06%、82.69%、78.57%和97.73%,两模型各主要诊断指标比较,除特异度为完整模型优于简化模型外,其它均无统计学差异;在单纯性VSD与非VSD的血清诊断模型中,完整模型ROC曲线下面积为0.988,预测准确率为95.00%,灵敏度为100.00%,特异度为92.42%,阳性预测值为87.18%,阴性预测值为100.00%,简化模型分别为0.948、94.00%、88.24%、96.97%、93.75%和94.12%,两模型主要诊断指标比较无统计学意义。4、广西七城市NTDs发生率为9.87/万,无脑儿、脑积水占的比例较高,居住地在农村占的比例比城市略高。母亲营养缺乏、患有妊娠并发症、孕期发烧和父亲饮酒是NTDs发生的危险因素。5、MTHFR基因多态性检测结果为野生基因型59.5%,杂合突变型34%,纯合突变型6.5%,三组基因型围产儿的出生体重和新生儿评分异常率的差别无统计学意义。6、554名妊娠妇女中,在孕前和孕后3个月每天服用叶酸的83名,占15.0%,怀孕后开始服用的122名,占22.2%,断续服用的27名,占4.9%,未服用叶酸的322名,占57.9%;文化程度初中和初中以下的妇女服用叶酸率较低,分别为33.3%和33.1%,大专的是48.996,大学及以上是65.096。7、育龄妇女知道叶酸可以预防NTDs的有48%,知道服用叶酸预防NTDs从孕前开始的只有13.4%,计划服用叶酸的只有23%,知道NTDs类型的有15%,知道NTDs发生的可能原因有26.2%,经过宣传讲解后,对NTDs和叶酸的认知率都提高到78%以上;文化程度为初中及以下的知道叶酸可以预防NTDs的只有14.28%,计划孕前开始服用叶酸的只有7.12%,而大学及以上的分别是86.96%,73.91%。8、服用叶酸组出生缺陷检出率为8.96‰,平均出生体重为3237.0克,早产儿发生率为6.43‰;未服用叶酸组分别为13.33‰、3119.3克和8.50‰,其中平均出生体重的差别有统计学意义。研究结论1、母亲MTHFR基因677TT型与子代CHD发生有关,父母双方MTHFR基因677C→T突变与子代VSD发生有关,父母CBS基因833T→C突变与子代CHD无相关性。母亲孕早期接触农药、妊娠合并症、孕早期感冒、孕期情绪状况、母MTHFR基因型为子代发生CHD的危险因素;妊娠合并症、孕早期服中成药、孕期情绪状况、母MTHFR基因型为子代发生VSD的危险因素。不分型CHD总的危险因素与VSD的危险因素不尽相同,交互作用分析提示危险因素共同引起子代VSD发生的过程中,存在着主要由某两个危险因素交互作用影响的可能,亦提示各类型CHD的发生存在相同的情况。2、单纯性VSD、儿科常见病儿与其它CHD血清蛋白质存在差异,质荷比6441、6640、8570及8700的蛋白质可能是VSD的血清蛋白标志物。3、两种VSD血清简化诊断模型具有较高的准确性,有较好的应用前景。4、表面增强激光解吸/电离飞行时间质谱技术是寻找VSD标志蛋白和发展VSD血清学诊断技术的有效工具。5、为了减少NTDs的发生,育龄妇女除了增补叶酸以外,还应避免暴露于某些环境危险因素。6、不同地区、不同人群MTHFR基因分布各有特点,存在明显的群体差异,检测得到广西健康人群的MTHFR基因C677位点突变分布可为今后优生保健的健康筛查工作提供依据。7、育龄妇女对叶酸和NTDs的知晓率较低,叶酸预防NTDs知识尚未得到很好的普及,叶酸利用率较低,通过健康干预使育龄妇女对NTDs的知晓率和叶酸的利用率均显著提高。更多还原

【Abstract】 Objective1.To explore congenital heart diseases(CHD) in offspring in association with parental MTHFR gene C677T and CBS gene T833C;To identify the risk factors of CHD and VSD type with Logistic regression;To explore the interaction among the risk factors of VSD type.2.Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) technology to screen serum protein markers,and to construct serum diagnostic model of isolated ventricular septal defect(VSD).As a pilot study,it aims to lay the foundations for investigating the pathogenesis of VSD at protein molecular level,and for developing serum diagnosis technology of VSD.3.To investigate the general distribution and environmental risk factors of neural tube defects in Guangxi province.4.To understand corelationship of health women’s mutations of the methylenetetrahydrofolate reductase(MTHFR) gene in Guangxi and the MTHFR C677T mutation and pregnancy outcomes.5.To assess the knowledge of NTDs of the reproductive-aged women and their compliancy about using folic acid.Spread the knowledge about NTDs to the reproductive-aged women and assess the effects.To make the people attach important to taking folic acid and find a more effective intervention model for NTDs preventing.Methods1.To investigate 115 pairs case-control children and their parents who came from Guangxi province with 1:1 case-control study,we investigated possible risk factors by questionnaire,parents’ blood samples were collected to examine genotype.We adapted PCR-RFLP method to identify MTHFR gene 677 C→T mutation and PCR-ARMS method was used to identify CBS gene 833 T→C mutation.The possible risk factors were analysed by simple and multiple factors Logistic regression,then the interaction between the risk factors of VSD was analysed by Logistic regression,other data were analysed byχ~2 test.2.Serum samples from 56 isolated VSD patients,85 patients with common pediatric diseases and 23 patients with other congenital heart disease(CHD) were detected by CM10 ProteinChip.Data were analyzed by Biomark Wizard software for searching the proteins that were significantly different between serum of VSD and control. Diagnostic model was established by Biomarker Patterns software.3.Investigate the parents of casese and controls by phone or visitation and obtain the material whether they have contacted the relevant risk factor or not,then use the logistic regression model to analyze these material and risk factors.4.Collect 200 women’ blood sample to extract whole blood DNA and examine the MTHFR C677T mutations.Compare the pregnant outcome of three groups.5.Design the investigate questionnaire of NTDs knowledge and investigate the reproductive-aged women which took part in class and which didn’t.Then compare the resuls of two groups.Design the investigate questionnaire of the taking folic acid state of pregnant women and choose women randomly to fill them.Collect 560 pregnant women who took folic acid and 600 pregnant women who did not take it, compare their pregnant outcomes.Analysis the results by x~2 analysis and ANOVA.Results1.115 CHD cases included 46 cases VSD(40.0%)、14 cases PDA(12.2%)、13 cases F4(11.3%)、6 cases ASD(5.2%)、8 cases other simple CHD(7.0%)、28 cases complicated CHD(24.3%).The results showed the percentage of MTHFR C/C、C/T、T/T genotype were 64.8%、29.6%、5.7%respectively,with C and T allele frequency being 79.6% and 20.4%in the healthy population of Guangxi province,meanwhile,the percentage of CBS T/T、T/C、C/C genotype were 42.6%、47.8%、9.6%respectively,with T and C allele frequency being 66.5%and 33.5%.There were significant differences between maternal MTHFR gene C and T allele frequency(P=0.036,OR=1.595, 95%CI:1.029-2.471).There were significant differences between MTHFR gene C and T allele frequency both mother and father(P=0.032,0R=1.724,95%CI:1.043～2.849) for VSD type.The results revealed that 6 factors were related with the occurrence of CHD in offspring:maternal exposures to pesticides in the early stage of pregnancy、suffering from diseases during pregnancy、catching a cold in the early stage of pregnancy、depressed or nervous condition during pregnancy、maternal MTHFR genotype、maternal CB$ genotype;4 factors were related with the occurrence of VSD in offspring:suffering from diseases during pregnancy、using Chinese medicine in the early stage of pregnancy、depressed or nervous condition during pregnancy、maternal MTHFR genotype.There were some interactions between those risk factors of VSD.2.12 proteins were significantly different between serum of 25 Zhuang isolated VSD patients and 36 Zhuang patients with common pediatric diseases.Mass to charge ratio(m/z) 6640.784,8570.154,8698.278,2244.582,5321.868,6441.972 and 7973.310 were up-regulated in serum of VSD,and m/z 2213.579,4604.572,2142.465,9198.582 and 2169.264 were down-regulated in serum of VSD.8 proteins were significantly different between serum of 21 Han isolated VSD patients and 33 Han patients with common pediatric diseases,m/z 6442.233,3322.694,6640.853,5122.828,8702.852, 2746.566 and 8574.304 were up-regulated in serum of VSD,and m/z 2823.419 was down-regulated in serum of VSD.21 proteins were significantly different between serum of 56 isolated VSD patients and 85 patients with common pediatric diseases. m/z 6640.784,8571.079,6441.972,8698.804,3322.549,2128.960,2291.255,2055.652, 2243.846 and 2.99.661 were up-regnlated in serum of VSD,and m/z 2050.946, 9306.537,7783.535,9198.666,2143.745,4604.572,7993.109,2824.602,2230.218, 2083.311 and 9360.675 were down-regulated in serum of VSD.25 proteins were significantly different between serum of 56 isolated VSD patients and 23 patients with other CHD.m/z 8571.079,8698.804,6639.783,6441.008,8889.333,9412.899,9360.333, 4793.333 and 9299.101 were up-regulated in serum of VSD,and m/z 3197.867, 2050.759,2040.154,2824.602,4307.323,3483.920,2961.282,2238.446,2022.592, 2010.471,2311.964,4125.468,2781.738,5308.783,5642.844 and 2260.730 were down-regulated in serum of VSD.25 proteins were significantly different among serum of 56 isolated VSD patients,85 patients with common pediatric diseases and 23 patients with other CHD.m/z were 8570.154,6640.784,6441.972,9360.675,8901.488, 3198.355,2917.269,9262.327,9415.030,2824.394,9198.963,9306.537,4604.572, 3319.479,8691.856,4307.360,5322.994,3269.890,4794.334,2747.381,3483.111, 5309.276,7995.957,3381.451 and 5644.239,respectively.3.In the serum diagnostic model of isolated VSD and patients with common pediatric diseases,the area under receiver operating characteristic curve,accuracy, sensitivity,specificity,positive predictive value and negative predictive value of the intact model were 0.984,96.51%(83/86),97.06%(33/34),96.15%(50/52),94.29% (33/35) and 98.04%(50/51),respectively.The area under receiver operating characteristic curve,accuracy,sensitivity,specificity,positive predictive value and negative predictive value of the terse model were 0.913,88.37%(76/86),97.06% (33/34),82.69%(43/52),78.57%(33/42) and 97.73%(43/44),respectively.In the diagnostic values of these 2 models,except the specificity of the intact model was better than the terse model,others were similar(P＞0.05).(7) In the serum diagnostic model of isolated VSD and patients without VSD,the area under receiver operating characteristic curve,accuracy,sensitivity,specificity,positive predictive value and negative predictive value of the intact model were 0.988,95.00%(95/100),100.00% (34/34),92.42%(61/66),87.18%(34/39) and 100.00%(61/61),respectively.The area under receiver operating characteristic curve,accuracy,sensitivity,specificity, positive predictive value and negative predictive value of the terse model were 0.948, 94.00%(94/100),88.24%(30/34),96.97%(64/66),93.75%(30/32) and 94.12% (64/68),respectively.The diagnostic values of these 2 models were similar(P＞0.05), too.4.The incidence rate of neural tube defects is 0.987%0.Prevalence in country are higher than in city.The result of logistic regression analysis revealed that mother malnutrition,hyperemesis gravidarum,fever and father alcoholism are factors which positively relative to neural tube defects.5.The result of mutation of MTHFR C677T is C\C 59.5%,C/T 34.0%,T/T 6.5%.The difference of birth weight and Apgar abnormal rate of three groups have no significant meaning.6.In the 554 pregnant women,83(15%) women took folic acid everyday from 3 months before pregnancy to 3 months after pregnancy,122(22.2%)women took folic acid after pregnancy,27(4.9%) women took folic acid only when they did not forgot it.The ratio of folic-acid-taking in lower educated subjects level such as only obtain junior middle school and below middle school is the lowest,33.3%and 33.1%;the ratio of the junior college is 48.9%%,the rate of university is 65.0%.7.The rate of reproductive-aged women who know that folic acid can prevent NTDs is 48%,who know taking folic acid must before pregnancy is only 13.4%,who plan to take folic acid before pregnancy is only 23.0%;who know the types of NTDs is 15.0%,who know the possible reasons of NTDs is 26.2%.After we past these information on some reproductive women,those knowledge rates are significant enhanced and over 78.0%.The rate of reproductive-aged women whose knowledge level is junior middle school and below junior middle school and who realize taking folic acid can reduce NTDs is 14.28%.The rate of planning to take folic acid before pregnancy of these women is 7%,however,the ratio of such two situations of university level women are separately 86.89%,73.91%.8.The congenital defects rate of group which took folic acid is 8.96%0,the average birth weight of them is 3237.0g,the premature baby ratio is 6.43%0.These three kinds of data of untaking folic acid group is 13.33%o,3119.3g and 8.50%0,respectively.The difference of average birth weight between two groups is significant.Conclusions1.Maternal MTHFR gene 677 C→T mutation may be one of the reasons of the occurrence of CHD in offspring,the high level of parents’ T allele frequency may be related with the occurrence of VSD in offspring,however,parents’ CBS gene 833 T→C mutation does not appear to be involved in offspring’s CHD.The risk factors of CHD don’t keep the same with VSD type completely.There are interactions between risk factors for the occurrence of VSD,the results suggest that there should be mainly caused by a interaction of two risk factors among all risk factors with the occurrence of other CHD types.2.The proteins among serum of isolated VSD patients,patients with common pediatric diseases and patients with other CHD are different,m/z 6441,6640,8570 and 8700 are possible to be the serum protein biomarkers of VSD.3.The 2 terse models,which were constructed by serum protein profiling,have high accuracy,and show great potential for diagnosis of VSD.4.SELDI-TOF-MS technology is an effective tool for searching the serum protein biomarkers of VSD,and for developing serum diagnosis technology of VSD.5.In order to reduce the incidence rate of neural tube defects,child-bearing aged women should not only increase the folic acid intake but also avoid to exposure under enviromental risk factors.6.The mutations of MTHFR C677T varied between places and people.The result of MTHFR C677T mutation in health pregnant-aged women can provide valuable data for following health care of pregnant women.7.The knowledge regarding of birth defects such as NTDs and using folic acid to prevent it have not been popularized yet among related people.Folic acid has not been widely used.After we spread these knowledge to reproductive-aged women,their knowledge regarding of NTDs and using folic acid to prevent NTDs has been significant improved.8.We suggest that the government should take action and all government departments work cooperatly in order to spread how to preventing NTDs and the benefit of using folic acid to reproductive-aged women widely.Enhance the women’attention about these useful information.Use several methods such as offering NTDs knowledge course to reproductive-aged women,encouraging them to use folic acid and understanding rate of NTDs,and so on.Our ultimate target is to reduce the birth rate of NTDs and other congenital anomalies,improve mother and baby healthas well as the population health quality.更多还原