Research On Correlation Of Micronutrients During Periconceptional And Maternal-fetal MTHFR Gene Polymorphism With Congenital Heart Disease

1. Background and Objection1.1 BackgroundCongenital heart disease (CHD), also is called Congenital Heart Defects(CHDs), is a set of malformation caused by abnormal fetal cardiovascular development in fetal period,which characterized by the heart and great vessels in the shape and structure abnormalities or the route should be closed after birth automatically, is the greatest and the most common birth defects in all congenital malformations, and has gradually become one of the important cause of death in infants.Common clinical types include ventricular septal defect,atrial septal defects,patent ductus arteriosus, tetralogy of Fallott pulmonary stenosis,aortic stenosis, and complete transposition of the great arteries etc. CHD was divided into isolated simple CHD group and complex CHD group (CHD with other system malformations). Worldwide, CHD had been the most common type of birth defects,the incidence of which was about 4%o to 10%o, more than one million infants born every year.In our country, CHD was the top of birth defects,the incidence of live birth babies was 8%o to 10%o, prenatal morbidity was 10%o to20%o;Some of the patients with CHD will die in infancy, and the survival children with CHD need more medical care and surgery, which will bring heavy mental and economic burden for families and society .Worldwide, CHD had been the most common type of birth defects,the incidence of which was about 4%o to 10%o, more than one million infants born every year.In our country, CHD was the top of birth defects,the incidence of live birth babies was 8%o to 10%o, prenatal morbidity was 10%o to20%o; The results Maternal and child health monitoring of China show that the CHD children has to be number one in all defects, and is one of the two death causes in children younger than five years old. The average incidence of CHD of Shenzhen from 2009 to 2013 was 7.25%o, with a slight rising trend.The incidence in 2009 was 5.44‰,and increased to 10.53‰ in 2013. CHD often result in miscarriage, still birth, stillbirth and neonatal death.Some of the patients with CHD will die in infancy, and the survival children with CHD need more medical care and surgery, which will bring heavy mental and economic burden for families and society.The U.S. Centers for Disease Control and Prevention reported that about 9% to 18% of CHD resulted from gene or chromosomal abnormalities,the other caused by a combination of genetic and environmental risk factors. The reasons such as poverty and malnutrition during pregnancy were more common, especially in low and middle income countries. There are many research on CHD risk factors in our country and abroad, risk factors on CHD is complex, including genetic and non-genetic factors. The present study results confirmed the genetic factors, environmental factors and polygenic inheritance has certain relevance with CHD, especially confirmed that the genetic factors and environmental factors interact with each other,and is the main reason for the formation of congenital heart disease. Non-genetic factors, such r environment factors before or during pregnancy of mother become the focus of current research on CHD risk factors. Previous studies have shown that, the mother suffering from virus, radioactive irradiation and some drugs during pregnancy, especially in early pregnancy, can significantly increase the risk of congenital heart disease of. Smoking, drinking alcohol during pregnancy, contacting environment risk for (such as chemicals, ionizing radiation, harmful gas, etc.) may increase the risk of birth to children with congenital heart disease.Many studies in our country and abroad have found that the phenomenon of unreasonable nutrient intake during pregnancy is common survey, and the condition of nutriment shortage is also not completely the same in different place. The trace elements play an important role in physiological functions and life activities. The lack of nutrition elements of pregnant women can lead to fetal growth restriction, even lead to miscarriage, premature birth, and even deformity. Trace elements are closely associated with fetal growth and development. Understanding the deficiency condition of trace elements of pregnant women and the change rule in different periods is not only related to the health of pregnant women, also have relationship to the normal development of the fetus. According to the current existing evidence, malnutrition of mother during pregnancy, especially the lack of some micronutrients closely related to children born bad ending. A lot of experimental studies have shown that the lack of certain nutrients associated with congenital heart disease. Iron and zinc deficiency is common during pregnancy. It has confirmed that the supplement of iron/folic acid during pregnancy can significantly reduce the early neonatal death[13]. Animal experiments show that in the sensitive period of rat embryonic development, iron deficiency can cause embryonic rat cardiac enlargement, iron nutritional status played a key role on the cardiovascular system development. Zinc plays an important role in keeping enzymes and proteins activities of body, also can adjust the growth, development and differentiation of embryo, animal experiments show that zinc deficiency can lead to pregnancy embryo intrauterine growth retardation and structural abnormalities.Zinc deficiency during pregnancy can interfere with the development of fetal central nervous system, severe cases can cause the deformation of the central nervous system. In pregnant women, the lack of iron may cause a continuous declination of the hemoglobin oxygen capacity, lead to chronic hypoxia phenomena on fetus or pregnant women, then affect the growth and development of fetal, and increase the neonatal mortality.Zinc can inhibit the use of iron, iron obstacles involved in hematopoiesis mechanism, which makes human body resistant iron deficiency anemia. Adverse pregnancy outcome caused by zinc deficiency cannot be ignored, show the embryo intrauterine growth retardation and related structural abnormalities, including the heart, lung, brain and genitourinary malformations.Zinc deficiency in the heart can cause a series of developmental anomalies including ventricular defect, aortic stenosis, ventricular septal defect, cardiac outflow tract malformation and dysplasia of unknown artery.Iron and zinc deficiency can affect the growth of the heart, and trace element act on each other, and trace element deficiency can lead to various types of cardiac anomalies. Methionine synthetase homocysteine metabolism is one of the three key enzyme in the process of, It can be affected by Hcy levels and metabolism and increase the risk of cardiovascular disease. The trace elements may influence each other, the lack of trace elements may result in multiple types of cardiac abnormalities.Our hospital as shenzhen maternity and child care, is the main maternal and child health care and delivery hospital, it’s necessary to understand its data change trend and basic epidemiological characteristics of congenital heart disease, congenital heart disease patients in our hospital and mother’s clinical data were retrospectively analyzed. Through the questionnaire survey to understand their diet and environmental risk factors during pregnancy of the normal pregnant women and the women pregnant with congenital heart disease children,screening out the possible risk factors for congenital heart disease, screening out prevention and control of risk factors. Knowing the intake and the defficiengcy of trace element of shenzhen of gestational age women is goog to guide the rational diet during pregnancy. Understanding lack common elements during pregnancy, analysis ing these nutrients differences in mothers pregnant with CHD children and mothers pregnant with nomal children,then screening out those micronutrients who are associated with congenital heart disease,can be benefit to make intervention measures of shenzhen or provide scientific basis for the establishment of policy, to be better to help to do a good job of primary prevention of congenital heart disease.Heart development is a very complex process in embryonic development, cardiovascular development need about 800 genes, and the normal development of the heart requires more than 400 genes, either genetic abnormalities or in different time, space, successively appeared abnormal expression and interaction are likely to lead to CHD.5,10-methylenetetrahydrofolate reductase MTHFR gene C677T polymorphism is related to folate metabolism, and may be congenital heart disease susceptibility genes.Human MTHFR gene location on chromosome 1 p36.3, the entire coding area have about 1980 bp, contain 11 exon, the cDNA sequence length is about 2.2 KB. The gene C677T and A1298C is the most common SNPS loci. Their mutation will lead to the heat resistance and reduced activity of MTHFR, The reduced MTHFR activity can lead to less compound of 5,10-methylenetetrahydrofolate reductase,, that make homocysteinemia cannot be methyl into methionine and pile up.Studies have shown that high homocysteine as cardiac toxicity factor,,it induce and change the number and position of the cell apoptosis by stimulating certain signaling channel and regulation system, which induced the occurrence of cardiac abnormalities. Hagoort thought high HCY levels in mother is one of the risk factors of neonatal congenital heart disease (CHD), elevated serum HCY levels was mainly affected by genetic and nutritional factors. Studies have found that 677 C/T MTHFR gene mutation is the main cause of HCY levels,and a remarkably positive correlation existed. Meta analysis of Li (19 articles including 4219 cases and 123 controls) 20 points out that MTHFR 677 c> T polymorphism is the risk factors of CHD (OR= 1.26,95% CI:1.06～1.51, P=0.009). Huang [301 made a case-control study in patients with tetralogy of Fallot (TOF)and pointed out that the MTHFR 677 homozygous genotype is a risk factor for TOF (OR=1.81,95% CI:1.15～2.84, P=0.010). The parent-child pair analysis of case-control study of MTHFR A1298C genotypes were tested and statisticed by Karen E,and he found that mother A1298C heterozygote can raise children’s risk of aortic stenosis (OR= 2.90,95% CI:1.22 6.86).At home and abroad,study of MTHFR gene is mainly focused on breast cancer, spontaneous abortion, male infertility, down’s syndrome and congenital heart disease, etc. MTHFR gene research is mainly focus on MTHFRrs1801133 G677 C and MTHFR rs1801131 A1298C two loci, and because of the polymorphism of MTHFR gene loci exist different geographic and racial differences, and the influence on fetal congenital heart disease of MTHFR gene polymorphisms of mother is still not very clear. Therefore, it is necessary to detect the polymorphism of MTHFR gene between mother and fetal.,know the relationship of MTHFR gene polymorphism and congenital heart disease.By this,it is expected to detect MTHFR gene polymorphism in the mother’s blood, screen out the people at high risk of CHD first,to offer help for prenatal screening and risk assessment of CHD.1.2 ObjectiveThe retrospective analysis was carried out in this study to analysis their data of CHD fetal and their mother in our hospital from 2010.1 to 2014.12, understand the change trend and basic data of congenital heart disease epidemiology characteristics; Choosing congenital heart disease in our hospital monitoring data of the mother and the mother of normal data between January 2013 and June 2014 Randomly, understand the environment around pregnancy pregnant time,to explore the relationship between environment factors and the risk of congenital heart disease (CHD). Knowing the change rule of trace elements in whole blood of pregnant women in different pregnant of shenzhen,it can provide guidance for pregnancy reasonable diet and nutrition supplements during pregnant; The detection of whole blood iron, zinc and methionine synthetase content and maternal-fetal MTHFR gene polymorphism in congenital heart disease fetal and their mother and nomal fetal and their mother in our hospital, analysising the dependency pertinence correlation between these substance and CHD, provide the theory basis for risk prediction and the prevention of congenital heart disease, and, hope that can help screening high-risk groups through dectecting maternal whole blood iron, zinc, methionine synthetase maternal-fetal MTHFR gene polymorphism of fetal congenital heart disease screening.2. Materials and Methods2.1 MaterlsEpidemiological studies:we retrospectively analyzed their basis information of fetal of 1131 cases of congenital heart disease who were induced labor delivery and their mother, the types of defects, and so on between January 2010 and December 2010 in our hospital. We randomly choosed pregnant women who did antenatal care in our hospital and lived in shenzhen more than 1 year from January 2013 to June 2014, women who pregnant CHD fetal were named case group and women who pregnant normal fetal were named the control group. According to 1:2 matching, matching conditions:the age difference within 2 year-old, difference of gestational weeks within 2 weeks,live in the same area of shenzhen, the same nation(Han). Questionnaire survey was conducted by face-to-face or telephone follow-up,465 questionnaires were collected. We inputed data by EpiData 3.1 software, analysised these factors by SPSS20.0 software with single factor and multiple factors conditional logistic regression analysis, the age of two groups of pregnant women, pregnancy phase comparison, there were no statistically significant difference (P>0.05), comparable.There was no statistically significant difference (P>0.05) of maternal age, gestational age in the groups.Laboratory studies:we randomly choosed 2000 cases of pregnant women who did antenatal examination in our hospital as study group,200 cases of normal non pregnant women of child-bearing age women in our hospital as control group, and compare trace element values of study group and control group. Sixty fetus with congenital heart diseases and their mothers were recruited into the maternal experiment group and the fetal experiment group respectively, while sixty normal fetus and their mothers were were divided into the maternal control group and the fetal control group severally, All experimental subjects were from the cases of induced labor or delivery pregnant women and fetus from the Maternal and Child Health Hospital of Shenzhen between January 2014 and July 2015. According to 1:1 matching, matching conditions followed the former.There was no statistically significant difference (P> 0.05) of maternal age, gestational age in the groups.2.2 Methods2.21 There were a total of 1131 cases children who were induced labor with congenital heart disease in shenzhen maternity and child care system between January 2010 and December 2014 in our hospital, we collected information of CHD fetal and their mothers, analyzed the number of birth defects monitoring in our hospital within 5 years of and the tendency of the number of congenital heart disease data and basic epidemiological features of congenital heart disease data of fetal and his mother (including pregnant women’s household type, cultural degree, age distribution, inferior pregnancy; data such as gestational age, gender, and outcome. CHD was divided into isolated simple CHD group and complex CHD group (CHD with other system malformations),of which the prognosis, social and demographic characteristics and the complications of pregnancy were analyzed.2.22 A 1:2 matched case-control study was used. First of all, we need obtained knowledge and consent of the respondents, Questionnaire survey was conducted by face-to-face or telephone follow-up by unified training investigators, knowing the surround environment and diet during pregnancy, and so on and so forth, questionnaire contents include basic situation about pregnant women and her husband (age, educational level, occupation, family economic conditions and living environment, etc.), environmental risk factors exposed during pregnancy (pregnancy tobacco exposure, exposure to radiation, ionizing radiation, animal feed, or drugs), emotion, negative life events during pregnancy and nutrition during pregnancy and so on. The case group and the control group surveys are completed by the same investigators. The object of study were elected strictly in accordance with the cases and controls screening criteria.2.23 Our first object of blood drawing are 2000 cases of normal pregnant women and 200 normal non-pregnant women in child-bearing age. Routine disinfection should be done before drawing blood, phlebotomize 3 ml in the special trace element in vitro, seal set 4℃ refrigerator under test. Tested the samples in 3 days by atomic absorption spectrophotometry.In induced labor or childbirth,gathering the maternal venous blood 6ml from two groups,3ml of blood into the the heparin anticoagulation tubes then kept in-80℃ refrigerator, to be used for detecting of the whole blood concentration of iron and zinc,3ml of blood into the drying tube,to 3000 r/heart 10 minutes after the separation serum after being and its natural solidification 30 min, then kept in-80℃ refrigerator, to be used for detecting of serum concentration of methionine synthetase; The other 2ml venous blood was kept using EDTA anticoagulant and 5ml amniotic fluid was collected in induced labor or delivery,kept in -80℃ refrigerator,to be used for detecting MTHFR genetic polymorphisms.2.24 Detect the whole blood concentration of iron and zinc by using inductively coupled plasma mass spectrometry system(ICP-MS).2.25 Detect the content of methionine synthase by enzyme-linked immunosorbent assay.2.26 Detect the maternal-fetal MTHFR gene polymorphism (loci677,1298) by using PCR sequencing technology.2.27 Creating data documents on the Excel after verification and arrangement,data were analyzed by the SPASS20.0 statistical software. We do normality inspection on measurement data first, normal distribution data use two independent samples test, measurement data were expressed as mean±standard deviation, non-normal distribution data use two independent samples nonparametric test, expressed with a median (interquartile range) [M] (Q); Count data were expressed as rate, the two samples were compared using Chi-square/Fisher test and adopt single factor Logistic regression analysis on possible risk factors to select the first meaningful variables, introducing the single factor analysis of meaningful to multi-factor logistic regression analysis (alpha= 0.05, alpha out= 0.05), only the data P<0.05 can be used for the statistics significance.3. Results3.1 The average incidence of CHD was 13.22%o,with a slight rising trend,92.22% of CHD were diagnosed by ultrasound,and 52.25% were diagnosed before delivery.The proportions of the age≥35 years old,temporary household register, high school and technical secondary school culture level of the pregnant women whose offspring with CHD were 15.21%, 46.77% and 52.75% respectively;Moreover,The majority of CHD was singletons, male, born in the latter half of the year.The most common cardiovascular malformations were ventricular septal defect,patent ductus arteriosus atrial septal defect,and,the incident was 5.6%o,2.32%oand1.17%orespectively.There was about 22.19% of CHD with other system malformations,which mainly were nervous system,musculoskeletal system, chromosome abnormality,. Compared simple cardiac malformations and complex cardiac-malformations.We found that there exited significant differences in culture degree,the number of pregnancies, gestational age, prognosis, diagnosis time and the way of diafnosis(P<0.05).3.3 The lack rate of trac element iron in pregnancy is highest,up to 8.25%,the second is zinc,up to7.4%.The lack of trace element is differentduring different pregnancy.Zinc have a priority during early pregnancy, iron have a priority during middle and late pregnancy.Iron deficiency rate went up higher with the increasing pregnancy week.Constituent ration of iron deficiency among three groups was statistically significant after statistic processsing(X2=49.9, P<0.000).Calcium deficiency rate is the highest in early group.Zinc deficiency rate is the highest in middle group.Iron deficiency rate is the highest in late group.Anaemia rate is 7.05% in experimental groups.The rate of prevent miscarriages is 30.2%and the rate of folic acid intake is 91.75%.,we compared zinc、iron and methionine synthetase content in CHD group and normal group,and found that content of each substance in the CHD group were lower than normal group, and the differences were statistically significant (P<0.05). The rate of iron lack is the first is 41.679%(25/60), to be number one and compared with control group,the difference was statistically significant (P =0.010).3.4 In Amniotic fluid samples, the homozygous mutant (TT) frequency of MTHFR rs1801133 in CHD group was 41.67%(25/60), the control group was 20.00%(12/60), the difference between the two groups was statistically significant (chi-square value=6.604, P =0.010), polymorphic loci form edifference between the two groups was statistically significant (chi-square value=6.604, P=0.037). In CHD group, homozygous mutant (CC) frequency of MTHFR rs1801131 was 5.00%(3/60), the control group was 1.67%(1/60), there was no statistically significant difference between the two groups (chi-square value= 1.034, P=0.309), polymorphic loci form difference between the two groups have no significant statistical difference (chi-square value=1.310, P=0.519).In blood samples, the homozygous mutant (TT) frequency of MTHFR rs1801133 in CHD group was 20.00% (12/60), the control group was 5.00%(3/60), differences between the two groups was statistically significant (chi-square value=6.171, P=0.013), polymorphic loci form edifference between the two groups was statistically significant (chi-square value=6.176, P=0.046). In case group homozygous mutant (CC) frequency of MTHFR rs1801131 was 18.33.00%(11/60), the control group was 5.00%(3/60), the difference between the two groups was statistically significant (chi-square value= 5.175, P=0.023), polymorphic loci form difference between the two groups was statistically significant (chi-square value= 10.139, P=0.006).4. Conclusion4.1 The incidence of congenital heart disease in our hospital from 2010 to 2014 as a whole is on the rise. Ultrasonic examination, the temporary type, high school or technical secondary school, single child born, male, the second half year have priorities; the most common cardiovascular malformation type are ventricular septal defect (VSD), the arterial catheter was not closed (PDA), atrial septum defect (ASD); The most common other system malformations were nervous system, bone of limbs, chromosome abnormality; Compared simple cardiac malformations and complex cardiac-malformations,we found that there exited significant differences in culture degree,the number of pregnancies, gestational age, prognosis, diagnosis time and the way of diafnosis. To understand the infection situation and characteristics, improve inspection equipment and technology, enhance the publicity on prenatal care and prenatal diagnosis knowledge, reduce the incidence of heart first and birth rate.4.2 Single factor analysis showed that mother’s cultural degree, father’s cultural degree fever, mother got fever in the early stages of pregnancy, pregnancy medication preparation, mother took pills in 3 months before pregnant, milk intake during pregnancy, eggs intake during pregnancy, soy intake during pregnancy, seafood intake during pregnancy, pregnancy supplement compound vitamins, calcium supplementation during pregnancy, radiation exposure during pregnancy, ionizing radiation exposure during pregnancy, negative life events during pregnancy, family economic condition, anemia during pregnancy may have a certain correlation with offspring CHD; Multi-factor analysis showed that radiation exposure during pregnancy, ionizing radiation exposure during pregnancy, anemia is a risk factor for CHD offspring during pregnancy, and the degree of mother culture, pregnant preparation, milk intake during pregnancy, pregnancy multivitamins, calcium supplementation during pregnancy, family economic condition can reduce the risk of CHD.4.3 trace elements deficiency gave priority to iron and zinc of Shenzhen women during pregnancy. In different pregnancy, the condition of lack of trace element is different, zinc deficiency had priority in early pregnancy, and iron deficiency in middle and late pregnancy.The content of iron, zinc and methionine synthetase in CHD group was lower than normal group, the lack rate of iron was number one in CHD group.4.4 MTHFR gene mutation can affect a key enzyme of folic acid-methionine metabolism, MTHFR can affect the HCY levels and metabolic processes leading to the occurrence of congenital heart disease, mutant frequencies of two loci homozygous C677 T and A1298C of MTHFR gene in CHD group are higher than the control group, it hint that two gene loci TT and CC homozygous mutations may be congenital heart disease susceptibility genes.