Molecular Epidemiological Study On Congenital Heart Diseases And Neural Tube Defects

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.