1 Department of Obstetrics and Gynecology and 2 Department of Clinical Chemistry, Klinikum der Universität München, Grosshadern, Munich, Germany
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Abstract |
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Key words: folate metabolism/folic acid deficiency/multiple pregnancy/population genetics/twin rate
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Introduction |
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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 677CT mutation in the MTHFR cDNA which results in the replacement of alanine (GCC), residue 222, by valine (GTC) (Frosst et al., 1995
). 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 6.632.2% (Schneider et al., 1998
).
A higher incidence of T/T homozygotes has been reported among patients with cardiovascular disease (Frosst et al., 1995), venous thrombosis (Arruda et al., 1997
), and neural tube defects (van der Put et al., 1995
) in some, but not all, case-control studies and recently also in mothers of children with trisomy 21 (Down's syndrome) (James et al., 1999
). In colon cancer, in contrast, the mutant genotype appears to have a favourable, protective effect (Chen et al., 1996
).
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., 1989). 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 homocysteinemethionineSAM 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.
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Materials and methods |
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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.
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Results |
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Discussion |
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The birth of twins in humans has been found to be associated with the ethnic origin of the mother and her diet (Nylander, 1981), shows seasonal patterns (Haukioja, 1989
), runs in families, and is influenced by the genotype of the mother (White and Wyshak, 1964
), 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, 1964
; Eskes, 1998
), because the synthesis of methionine and in turn SAM, the universal methyl donor for cellular methylation reactions (Eskes, 1998
; James et al., 1999
), 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, 1990
; Landy and Keith, 1998
). Multivitamin supplementation containing 0.8 mg folate, on the other hand, has been shown to increase the number of multiple pregnancies (Czeizel et al., 1994
).
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., 1998) and may also increase the rate of multiple pregnancies.
In conclusion, our data demonstrate a significant association of the MTHFR 677CT 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.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 19, 2000; accepted on September 7, 2000.