The number of dichorionic twin pregnancies is reduced by the common MTHFR 677C->T mutation

Uwe Hasbargen1,3, Peter Lohse2 and Christian J. Thaler1

1 Department of Obstetrics and Gynecology and 2 Department of Clinical Chemistry, Klinikum der Universität München, Grosshadern, Munich, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In multiple pregnancies, demands for folic acid are considerably increased. The most common inborn error of folate metabolism is mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the synthesis of a thermolabile variant of the enzyme with impaired catalytic activity which leads to reduced 5-methyltetrahydrofolate (5-methyl-THF) and mildly elevated homocysteine plasma concentrations when folate status is inadequate. To investigate whether the number of offspring is influenced by this mutation, we determined the frequency of the 677C->T substitution in 156 singleton and 40 twin mothers with dichorionic placentation. The T allele frequency in singleton (0.30) and twin mothers (0.16) was significantly different (P = 0.011). Mothers with the 677C->T mutation had a 2.28 times lower risk of having a twin pregnancy than those without (95% confidence interval = 1.18–4.66; P = 0.008). Our observation would explain, at least in part, the hereditary trait of multiple gestations and is in agreement with the ethnic distribution pattern of the T allele which has been found to be inversely correlated with the incidence of dichorionic twins. Our findings suggest that the MTHFR 677C->T mutation interferes with human brood size, probably by influencing the proliferation of rapidly dividing embryonic and maternal cells.

Key words: folate metabolism/folic acid deficiency/multiple pregnancy/population genetics/twin rate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Folate deficiency is probably the most common vitamin deficiency worldwide, particularly in areas where poverty and malnutrition are common, and women are more frequently affected than men, especially when they are pregnant or lactating. In these cases, an exogenous supply is important to prevent deficiency symptoms and to promote normal growth of the embryo, as folate deficiency during pregnancy is associated with fetal hypotrophy, preterm delivery, congenital malformations, developmental abnormalities, and teratogenicity. The association between inadequate folate status of the mother and neural tube defects has led to the general fortification of the food supply with folate in certain countries, e.g. the USA.

A key enzyme of folate metabolism is methylenetetrahydrofolate reductase (MTHFR) which catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-methyl-THF), the latter providing the methyl group for the conversion of homocysteine to methionine catalysed by methionine synthetase. The combined action of the two enzymes supplies single carbon units for downstream DNA, protein, and lipid methylation reactions which use S-adenosylmethionine (SAM) as their intracellular methyl donor. Failure to generate enough methionine, either as a consequence of folate deficiency, a decrease in MTHFR enzymic activity, or both, leads to a depletion of the SAM pool, compromises essential methylation steps, and exposes the body to the toxicity of homocysteine.

Severe MTHFR deficiency is a rare inborn error of folate metabolism with ~50 cases reported worldwide which results in hyperhomocysteinaemia, homocystinuria, hypomethioninaemia, and DNA hypomethylation. Patients present with developmental delay, occlusive vascular disease, thromboembolism, and neurological symptoms (for review, see Rosenblatt, 1995; Green and Miller, 1999; Rosenblatt and Whitehead, 1999).

However, much more common is mild MTHFR deficiency due to the synthesis of a thermolabile variant of the enzyme with reduced catalytic activity. The gene defect results in mildly elevated homocysteine plasma concentrations and decreased 5-methyl-THF concentrations when folate status is inadequate. DNA sequence analysis of affected patients has shown that this thermolability is caused by a 677C->T mutation in the MTHFR cDNA which results in the replacement of alanine (GCC), residue 222, by valine (GTC) (Frosst et al., 1995Go). The mutation is very heterogeneously distributed among different ethnic races, the prevalence of the homozygous mutant genotype ranging from 0% in Africa to 13.2% in South America, corresponding with a T allele frequency of 6.6–32.2% (Schneider et al., 1998Go).

A higher incidence of T/T homozygotes has been reported among patients with cardiovascular disease (Frosst et al., 1995Go), venous thrombosis (Arruda et al., 1997Go), and neural tube defects (van der Put et al., 1995Go) in some, but not all, case-control studies and recently also in mothers of children with trisomy 21 (Down's syndrome) (James et al., 1999Go). In colon cancer, in contrast, the mutant genotype appears to have a favourable, protective effect (Chen et al., 1996Go).

In multiple pregnancies, the rapid growth of the embryos during the first weeks of life results in an increased requirement for folic acid (Tremblay et al., 1989Go). The MTHFR 677C->T mutation further increases dependence on folic acid for adequate homocysteine remethylation to methionine, because 5-methyl-THF is the required co-substrate in the remethylation reaction catalysed by the enzyme methionine synthase, and affected individuals have to increase their folate intake in order to overcome decreased MTHFR activity. We believed that this mutation could affect the incidence of multiple gestations by altering homocysteine–methionine–SAM metabolism in the presence of suboptimal folate concentrations. This prompted us to initiate a cohort study in order to determine the prevalence of the T allele in women with spontaneous singleton and dichorionic twin pregnancies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study population
Between February 1998 and March 2000, genotypes were determined of 215 Caucasian women who delivered a total of 274 babies (156 singleton and 59 twin pregnancies) at our hospital. Selection criteria for all participants were a first trimester ultrasound for exact dating, the absence of any assisted reproductive technique (ART) to achieve the current pregnancy, and dichorionic placentation documented by histopathological examination in the case of twin pregnancies. Monochorionic twin pregnancies were viewed as singleton pregnancies and were therefore excluded from the data analysis. A total of 40 twin pregnancies matched the criteria, while 19 twins had to be excluded (six monochorionic, 12 ART, one monochorionic + ART). Controls were recruited continuously from patients with singleton pregnancies who were delivered at our hospital between December 1998 and March 1999.

Ethical considerations
Written informed consent was obtained from all participants and the study was carried out according to the guidelines of the 1975 Declaration of Helsinki.

DNA isolation and amplification by the polymerase chain reaction
Genomic DNA was extracted from white blood cells using the Qiagen blood mini kit (Qiagen, Hilden, Germany) and amplified with primer pair MT-1 (5'-TTGAGGCTGACCTGAAGCACTTG-3'; modified according to Schneider et al., 1998) and MT-2 (5'-AGGACGGTGCGGTGAGAGTG-3'; Frosst et al., 1995) for 40 cycles at 95°C for 20 s, 65°C for 20 s, and 72°C for 20 s.

Restriction fragment length polymorphism analysis
Polymerase chain reaction (PCR) products were digested with the restriction endonuclease HinfI (5'-G/ANTC-3'; New England BioLabs, Beverly, MA, USA) and analysed on 2% low melting point agarose (Gibco BRL Life Technologies, Rockville, MD, USA) gels.

Statistical analysis
Maternal and gestational age, and abortion rate are expressed as the mean value ± SD. Gravidity and parity are expressed as median and range. Differences were analysed using the Wilcoxon two-sample test. Comparisons of categorized data were made using the two-sided, non-parametric Fisher's exact test (SPSS for Windows 8.0; SPSS Inc. Chicago, IL, USA). P < 0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The characteristics of mothers with singleton and dichorionic twin pregnancies are shown in Table IGo. There were no significant differences between the two groups regarding maternal age, gravidity, parity, and rate of spontanous abortions. The only exception was the mean gestational age at birth which was 26.4 days lower in twin pregnancies when compared with singleton pregnancies. This is expected since multiple pregnancies usually tend to be of shorter duration.


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Table I. Patient characteristics. Values are given as mean ± SD or median with the range shown in parentheses
 
In Table IIGo, the frequencies of the C/C, C/T, and T/T genotypes in singleton and twin mothers meeting the study criteria are compared. Overall, homozygosity for the T allele was observed in 8.7% (n = 17), compound heterozygosity in 37.8% (n = 74), and homozygosity for the C allele in 53.6% (n = 105) of the subjects. Heterozygosity for the 677C->T mutation occurred in 41.7% of the controls and in 22.5% of the twin mothers, while the homozygous C/C genotype was observed in 48.7% of the mothers with singleton pregnancies and in 72.5% of the mothers with dichorionic twins. The difference was significant [relative risk = 2.27; 95% confidence interval (CI) 1.11–4.96; P = 0.016]. The frequency of the T/T genotype was also lower in twin mothers (5.0 versus 9.6%), but this difference was not statistically significant.


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Table II. MTHFR 677C->T mutation: genotype in mothers with singleton and dichorionic twin pregnancies
 
The increase of the T allele frequency in singleton mothers was even more pronounced when the combined prevalence of the C/T and T/T genotypes in singleton pregnancies (51.3%) and in twin pregnancies (27.5%) was compared (Table IIGo). Accordingly, the T allele frequencies of singleton (0.30) and twin mothers (0.16; relative risk = 1.96; 95% CI = 1.11–3.62; P = 0.011) also differed significantly (Table IIIGo). Mothers with the 677C->T mutation therefore had a 2.28 times lower risk (CI = 1.18–4.66; P = 0.008) of having a twin pregnancy than those without this mutation (Table IIGo). The mutation showed no association with regard to maternal age, gravidity, parity, and rate of spontaneous abortions (Table IVGo).


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Table III. MTHFR 677C->T mutation: allele frequencies in mothers with singleton and dichorionic twin pregnancies
 

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Table IV. Patient characteristics with regard to MTHFR genotype. Values are given as mean ± SD or median with the range shown in parentheses
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we observed a significant difference in the prevalence of the 677C->T mutation of the MTHFR gene in mothers with singleton versus dichorionic twin pregnancies (Tables II and IIIGoGo). The T allele frequency in singleton mothers (0.30) was comparable with published data for central Europe (Table VGo). In twin mothers, in contrast, the T allele occurred with a frequency of only 0.16. This effect was mainly due to a reduced prevalence of the compound heterozygous C/T genotype in mothers with dichorionic twins (22.5 versus 41.7% in the control group). Classification of the subjects according to genotype did not reveal any significant differences between the two groups when maternal age, gravidity, parity, and gestational age were compared (Table IVGo).


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Table V. T allele frequencies and twin rates in different races
 
A review of published data on ethnic and geographical differences in the incidence of twin pregnancies and in the prevalence of the T allele also revealed a characteristic pattern of low T allele frequencies corresponding with high rates of dichorionic twins. As shown in Table VGo, the rate of twin births in ethnic groups with low T allele frequencies, e.g central Africans (0.07) is relatively high (32.1 per thousand), while it is low (6.0 per thousand) in populations with high T allele frequencies, e.g. eastern Asians (0.40). The percentage of monochorionic twin pregnancies, in contrast, is nearly identical in all populations worldwide (Anderson, 1990Go).

The birth of twins in humans has been found to be associated with the ethnic origin of the mother and her diet (Nylander, 1981Go), shows seasonal patterns (Haukioja, 1989Go), runs in families, and is influenced by the genotype of the mother (White and Wyshak, 1964Go), suggesting that twinning is heritable and that the twinning rate is influenced by nutrition. Hence, our results may (for the first time) offer an explanation for these independent observations which were made over the last 40 years. The data show that a common 677C->T nucleotide substitution in the MTHFR gene, which results in a thermolabile variant of the enzyme with reduced catalytic activity, is significantly more prevalent in women with singleton pregnancies. MTHFR catalyses the reduction of 5,10-methylene-THF to 5-methyl-THF and 5-methyl-THF serves as the methyl donor for the methylation of homocysteine to methionine. In MTHFR 677C->T hetero- and homozygotes, enzymic activity is impaired. The mutation increases a subject's dependence on folic acid for adequate homocysteine remethylation to methionine, because affected individuals require more folate than C/C homozygotes for thermolabile MTHFR to function adequately. Therefore, relative folic acid deficiency or long-standing suboptimal folate nutrition may adversely affect pregnancy outcome in subjects with the C/T and T/T genotypes (Hibbard, 1964Go; Eskes, 1998Go), because the synthesis of methionine and in turn SAM, the universal methyl donor for cellular methylation reactions (Eskes, 1998Go; James et al., 1999Go), is decreased. This promotes DNA hypomethylation, strand breakage in genomic DNA, and chromosomal fragility as well as impaired DNA synthesis and altered regulation of gene expression. Consequently, it could be speculated that, in pregnant mothers with low folate concentrations and impaired MTHFR catalytic activity, methyl groups are in short supply. This could influence DNA, lipid, and protein methylation reactions in rapidly growing embryonic and maternal cells and, e.g. result in the undetected loss of a dichorionic co-twin, which reportedly is a common outcome of early multiple gestations (Anderson, 1990Go; Landy and Keith, 1998Go). Multivitamin supplementation containing 0.8 mg folate, on the other hand, has been shown to increase the number of multiple pregnancies (Czeizel et al., 1994Go).

Food fortification with folate is advocated and has been put into practice in several countries especially in order to prevent neural tube defects. Based on our results, we anticipate that this practice may lead to changes in MTHFR genotype frequencies as has been recently demonstrated for a young Spanish population (Munoz Moran et al., 1998Go) and may also increase the rate of multiple pregnancies.

In conclusion, our data demonstrate a significant association of the MTHFR 677C->T mutation with the risk for dichorionic twin pregnancies, suggesting that the T substitution protects against multiple gestations. This observation is in agreement with the worldwide geographic and ethnic distribution pattern of the T allele when compared with twin rates and may, at least in part, explain the hereditary trait of multiple gestations and the interplay of genetic and nutritional factors in the birth of dichorionic twins.


    Acknowledgments
 
We would like to thank all patients for their willingness to participate in this study. We are also indebted to Dr Sabine Fuchshuber for patient recruitment, to Heike Ruebsamen and Michaela Beck for technical assistance, and to Dr Dorothea Nagel for help with the statistics.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Klinikum der Universität München, Grosshadern, Marchioninistrasse 15, D-81377 Munich, Germany.E-mail: hasbargen{at}gyn.med.uni-muenchen.de Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 19, 2000; accepted on September 7, 2000.