Mol. Hum. Reprod. Advance Access originally published online on December 22, 2005
Molecular Human Reproduction 2006 12(1):51-54; doi:10.1093/molehr/gah252
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Maternal and offspring MTHFR gene C677T polymorphism as predictors of congenital atrial septal defect and patent ductus arteriosus
1Department of Nutrition and Food Hygiene, School of Public Health, Peking University Health Science Center, Beijing and 2The Second Affiliated Hospital of China Medical University, Shenyang, China
3 To whom correspondence should be addressed at: Department of Nutrition and Food Hygiene, School of Public Health, Peking University Health Science Center, Beijing 100083, China. E-mail: liyong{at}bjmu.edu.cn
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Abstract |
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To observe the association of MTHFR gene C677T locus polymorphism with occurrence of congenital heart defects (CHDs), 21 patients with atrial septal defect (ASD), 35 patients with patent ductus arteriosus (PDA), one patient with both conditions combined, and their biological parents were collected as the case group. Another 104 normal individuals and their biological parents without a family history of birth defects were selected as the control group. MTHFR C677T genotypes of each sample were determined by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). The results showed for the occurrence of ASD, the odds ratio (OR) of TT genotype was 4.08 [95% confidence interval (95% CI) = 1.28–13.24] compared with CT genotype. For the occurrence of PDA, the ORs of TT were 3.44 (95% CI = 0.89–16.13) and 2.38 (95% CI = 0.92–6.14) compared with CC and CT genotypes, respectively. Author as meant? Compared with CC + CT genotype combination, the ORs of TT were 3.95 (95% CI = 1.38–11.44) and 2.60 (95% CI = 1.02–6.36) for ASD and PSD respectively. The results also had sex differences and the statistical significance was only observed in male ASD and female PDA. The ORs of T allele carriers were 2.29 (95% CI = 1.08–4.92) and 1.88 (95% CI = 1.02–3.47) compared with C allele for the occurrences of ASD and PDA respectively. The analysis of parents genotype showed that the OR of TT mothers was 2.31 (95% CI = 0.96–5.59, P < 0.05) compared with (CC + CT) for the occurrence of PDA in offspring. So this study could give a clue that MTHFR C677T locus variation was related with occurrence of ASD and PDA, and the carriers of TT genotype and T allele had higher risk of diseases. The mother carrying TT genotype was associated with occurrence of PDA in offspring.
Key words: ASD/family/gene polymorphism/MTHFR/PDA
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Introduction |
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Congenital heart defects (CHDs) are the most prevalent heart disease in infants and children, including mainly atrial septal defect (ASD), ventricular septal defect (VSD), patent ductus arteriosus (PDA), tetralogy of Fallot and other types. Besides a few types of CHDs induced by single gene mutation and chromosome aberration, the majority of CHDs are polygenic diseases affected by both genetic and environmental factors. Periconceptional supplement of multiple vitamins could significantly reduce the incidence of CHDs and neural tube defects (NTDs) (Czeizel and Dudas, 1992
; Shaw et al., 1995; Botto et al., 1996; Berry et al., 1999). Previous studies in pregnant women, who delivered fetuses with NTDs without folate deficiency, showed the abnormality of folate metabolism was the main factor related to the occurrence of NTDs (Mills et al., 1992; Kirke et al., 1993; Li et al., 2000).
Homocysteine (Hcy) is a type of thioalcohol amino acid, which is the comitant metabolic product of methionine remethylation and transsulfuration. 5,10-Methylenetetrahydro-folate reductase (MTHFR) is the key metabolic enzyme of Hcy, it catalyses 5,10-methylene-tetrahydrofolate reduction to 5-methyltetrahydrofolate which as methyl donor induces Hcy remethylation to methionine. The MTHFR gene C677T variation could influence enzyme activity. Studies showed MTHFR enzyme activity of 677TT genotype was 30% of CC genotype, and the activity of CT was 65% of CC. Deficiency of MTHFR could reduce synthesis of 5-methyl-tetrahydrofolate, interrupt the Hcy remethylation to methionine and induce hyperhomocysteinemia. At present, the association studies of MTHFR with CHD were limited with different conclusions (Junker et al., 2001; Wenstrom et al., 2001; Liu et al., 2002; Storti et al., 2003; McBride et al., 2004; Hobbs et al., 2005). As pathogenesis of different types of CHDs are diverse, in this study the relation of MTHFR gene polymorphism with two of the most common types of CHDs, ASD and PDA, were investigated by nuclear family design.
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Materials and methods |
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Subjects
From Chinese Liaoning province 57 CHDs patients and their biological parents, i.e. CHD nuclear families were selected as the case group, including 20 males and 37 females. The average age was 6.2 years old in offspring, 34.4 in fathers and 33.5 in mothers. The CHDs types were diagnosed by cardiovascular specialist with classical clinical manifestation through ultrasound or surgical operation. Cases were identified as 21 (36.8%) simple ASD, 35(61.4%) simple PDA and 1(1.8%) patient with both ASD and PDA.
From the same region, another 104 normal subjects (60 males and 44 females) and their biological parents, i.e. normal nuclear families were selected as the control group. The average age was 8.4 years old in offspring, 35.9 in fathers and 34.8 in mothers. The subjects had no history of CHDs or other deformity through X-ray and ultrasound examination.
In the study, the individuals exposed to diabetes mellitus, phenyl ketonuria, teratogenesis chemicals and X-ray during intrauterine period were excluded. There were no statistical difference between the mothers in each group, in the incidence of upper respiratory tract infection, abnormal pregnancy and delivery history, exposure to pesticide during the early stages of pregnancy. All subjects gave informed written consent, and the study was approved by the Medical Ethics Committee of Peking University.
Blood sample collection
All investigators were trained together before formal investigation. The subjects were requested to complete a registration form, including general information, such as name, age, nationality and obstetric history of the mother, including exposure to drugs, X-rays, pesticides and so on. Venous blood (2 ml) was collected and stored at –70°C for all subjects.
Genotype analysis
Genomic DNA was extracted by salt fractionation method to detect MTHFR C677T genotype by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). In the PCR reaction, the primer sequences were 5'-TGAAGGAGAAGGTGTCTGCGGGA-3' (sense) and 5'-AGGACGGTGCGGTGAGAGTG-3' (anti-sense). PCR reaction conditions were as previously described (Frosst et al., 1995), and Taq DNA polymerase and other reagents were provided from Sino-American Biotechnology Company. PCR products were digested by Hinf I (Sina-American Company) for 4 h at 37°C, separated by 12% polyacrylamide gel electrophoresis (PAGE), identified by 0.5 µg/ml EB dyeing and visualized under UV light. The MTHFR 677CC genotype had only one band: 198 bp, 677CT genotype had three bands: 198 bp, 175 bp and 23 bp, and TT homozygote had two bands: 175 bp and 23 bp.
Statistical analysis
The data was analysed using 
2 test by SPSS and Epi Info software to compare the frequencies of MTHFR C677T allele and genotype between two groups. The odds ratio (OR) was calculated by Epi Info package.
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Results |
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Genetic equilibrium test showed the genotype distribution of the control groups corresponded to Hardy–Weinberg law (P values were 0.704 for control offspring, 0.607 for control fathers and 0.700 for control mothers).
Comparison of the ORs between different genotypes (Table I) showed for ASD that the OR of TT genotype was 4.08 [95% confidence interval (95% CI) = 1.28–13.24] compared with CT genotype. For PDA, the ORs of TT were 3.44 (95% CI = 0.89–16.13) and 2.38 (95% CI = 0.92–6.14) compared with CC and CT genotypes, respectively. All above results were statistically significant (P < 0.05), and in general the genotype distributions of ASD patients were apparently different compared with control offspring (P < 0.05). There was no significant difference between case and control parents for ORs and genotype distributions.
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To further analyse the effect of homozygous mutant (677TT) and taking CC and CT genotypes carriers as one group, the results showed (Table II) the ORs of TT genotype compared with CC + CT were 3.95 (95% CI = 1.38–11.44) and 2.60 (95% CI = 1.02–6.36) for ASD and PSD, respectively. The OR of TT mothers was 2.31 (95% CI = 0.96–5.59, P < 0.05) for the occurrence of PDA in offspring.
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Table III summarizes the frequencies of mutant allele T in ASD and PDA patients were apparently higher than control offspring (P < 0.05), with ORs of T allele carriers 2.29 (95% CI = 1.08–4.92) and 1.88 (95% CI = 1.02–3.47) compared with C allele for the occurrences of ASD and PDA, respectively. The ORs of parents with T allele were not significant compared with C allele carriers (P > 0.05).
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Considering the effects of sex, the ORs of homozygote (TT), compared with (CC + CT) genotype combination, were analysed further in offspring of different sex. The results showed (Table IV) the ORs of TT were 4.70 (95% CI = 1.01–24.39) for the occurrence of ASD in male, and 3.21 (95% CI = 0.92–11.43) for the occurrence of PDA in female with statistical significance.
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Discussion |
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CHDs are very complicated, which could locate in different position and occur in simple or complex appearance, that lead to variety of CHDs. Also for different types of CHDs, the pathological factors might be different. In previous studies, different types of CHDs were always pooled and analysed generally as one group, that produced false conclusions or masked information. With a sufficient sample size, it is necessary to analyse risk factors of CHDs according to different types of CHDs. In this study, the two most common types of CHDs, ASD and PDA, were analysed.
The study showed that in the control group, which could be taken as normal population, the frequencies of MTHFR 677CC, CT and TT genotypes were respectively 19.6–21.4%, 55.3–57.0% and 22.0–24.5%. And the frequencies of allele T and C were 50.5–52.4% and 47.6%–49.5%. The results were similar to other reports about Chinese population frequencies of MTHFR C677T, but the frequency of mutant genotype TT was a little higher than reported in European and American population (Botto and Yang, 2000).
The results showed compared with CC and CT genotypes, the TT genotype was more likely to be born with ASD and PDA, and it remained the same for the allele T carriers. The genotype analysis of parents showed the OR of TT mothers was 2.31 (95% CI = 0.96–5.59, P < 0.05) compared with (CC + CT) for the occurrence of PDA in offspring. The above results suggest that MTHFR C677T locus variation is associated with occurrence of ASD and PDA, and carriers of mutant homozygote TT and allele T were accompanied with high risk of these two types of CHDs. The maternal MTHFR 677TT genotype was associated with high risk of PDA in offspring. The results also indicated a sex difference, and the ORs of TT only in male ASD and female PDA showed statistical significance. As studies showed females were 2–3 times more likely than males to develop PDA, and in this study there were 26 female PDA patients but only 10 male, which may lead to the sex difference of association.
Ralf Junker et al. (2001) studied the relationship of MTHFR C677T gene polymorphism with CHDs, and the results showed TT homozygote was significantly related to congenital heart structure teratogenesis, specially with pulmonary artery stenosis, left heart dysplasia syndrome and coarctation of aorta. Wenstrom et al. (2001) showed the CHDs were associated with high level of Hcy in amniotic fluid and maternal MTHFR C677T variation. But some studies revealed negative results (Storti et al., 2003; McBride et al., 2004; Hobbs et al., 2005). Liu et al. (2002) compared the frequencies of MTHFR 677TT genotype and allele T between CHDs mothers and control mothers (frequencies of TT: case 30%, control 15%; frequencies of T: case 56%, control 53%), and the difference was not significant. These inconsistent results might be due to differences of ethnicity, sample size, study design and so on. In particular, the folate nutritional status was one of the most important factors influencing the Hcy level and occurrence of CHDs, which was contributed to by both environmental and genetic factors. So, despite the positive result of this study, the association of MTHFR C677T gene variation with CHDs need to be approached and confirmed in future studies.
Cardiac development is a very complicated process, involving expression of many genes at different times, space and order, related to cellular migration, differentiation, hyperplasia and acute interaction. Cushion septum is the main structure correlated with compartment separation, which abnormality can lead to atrial and VSD. The formation of cushion septum is mainly associated with local thickening of extracellular matrix and invasion of endocardium epithelial cell transformed by epithelium stroma. Shut-off of ductus arteriosus is mainly associated with neointima that is formulated by precipitation of extracellular matrix and migration of smooth muscle cells. Variation of MTHFR C677T could reduce enzyme activity of MTHFR and induce hyperhomocysteinemia. Some studies thought that hyperhomocysteinemia could inhibit or induce expression of some genes regulating cardiac development, leading to cardiac abnormality. Its concrete mechanisms need to be approached further.
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Acknowledgements |
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Thank you to all participating families and hospitals. This study was supported by grant from the Major State Basic Research Development Program of People’s Republic of China (no. 2001CB510305).
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References |
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Submitted on October 25, 2005; revised on November 24, 2005; accepted on November 28, 2005
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