1 Genetic Epidemiology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia and 2 Psychology Department, Free University, Amsterdam, Netherlands
3 To whom correspondence should be addressed at: Queensland Institute of Medical Research, 300 Herston Rd, Herston, QLD 4006, Australia. e-mail grantM{at}qimr.edu.au
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Abstract |
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Key words: DZ twins/MTHFR/polymorphisms/twinning frequency
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Introduction |
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Folate-dependent homocysteine metabolism is critical for female fertility and early embryonic development. Folic acid is essential for DNA replication and cellular methylation reactions. Abnormal metabolism of folate and homocysteine are associated with neural tube defects (NTDs) (Fleming, 2001) and recurrent abortions (Nelen et al., 1997
). Several studies (Garabedian and Fraser, 1994
; Kallen et al., 1994
; Whiteman et al., 2000
) have documented a positive association between NTD and the frequency of twins. Dietary supplementation around conception with folic acid alone, or in combination with other vitamins, reduces the incidence and recurrence of NTDs (Lumley et al., 2000
; Fleming, 2001
). Recent studies provide no support for suggestions of an increased frequency of twins following folate supplementation (Czeizel and Dudas, 1992
; Lumley et al., 2000
; Ericson et al., 2001
; Li et al., 2003
).
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, and catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is essential for the subsequent conversion of homocysteine to methionine and functioning of the methylation cycle (Hobbs et al., 2000; van der Put et al., 2001
). Decreased production of 5-methyltetrahydrofolate may lead to increased homocysteine concentrations and impaired methylation of proteins and DNA. During pregnancy, folate requirements increase, making folate deficiency more likely, and consequently MTHFR variants may have an effect on developing embryo(s).
Two common single nucleotide polymorphisms (SNPs) in MTHFR (C677T and A1298C) reduce enzyme activity (Frosst et al., 1995; van der Put et al., 1998
; Weisberg et al., 1998
) and are associated with both the twinning phenotype and variation in embryo survival. A casecontrol study investigated the C677T polymorphism in mothers with dichorionic twin pregnancies, and found a lower frequency of the 677T allele amongst mothers of twins compared with women who gave birth to singletons (Hasbargen et al., 2000
). These authors suggested that the 677T allele of MTHFR is protective against multiple pregnancies. The 677T allele is also associated with an increased risk of NTD (Botto and Yang, 2000
). Analyses of MTHFR genotypes in spontaneous abortion reported decreased embryo viability in fetuses carrying the 677T and 1298C alleles (Isotalo et al., 2000
; Volcik et al., 2001
; Zetterberg et al., 2002
). Carrying one or more variant alleles may be detrimental during early embryogenesis when folate requirements are high (Zetterberg et al., 2002
). Any effect of the 677T allele to reduce multiple pregnancy in mothers of twins may act through reduced survival of twin embryos, or through some other mechanism. The aim of the present study was to investigate the role of MTHFR variants in twinning by studying transmission of MTHFR alleles in families of mothers of DZ twins and in a population sample of adolescent twins.
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Materials and methods |
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In addition, the SNPs were typed in 1861 individuals from 462 families of DZ and monozygotic (MZ) adolescent twins recruited from schools in the Brisbane and surrounding areas of south-eastern Queensland (Zhu et al., 1999). Samples were collected from twins, siblings and their parents.
Study protocols were reviewed and approved by the Bancroft Centre Human Research Ethics Committee. Participation was voluntary and each patient provided their informed consent.
Genomic DNA was extracted (Miller et al., 1988) from peripheral venous blood samples. Zygosity of the twins was determined by differences in sex, eye colour or hair colour, and by typing nine independent microsatellite markers (AmpFLSTR® Profiler PlusTM; Applied Biosystems, Foster City, CA, USA) in equivocal cases. The probability of dizygosity given concordance of all markers in the panel was <103.
The C677T polymorphism (ValAla) in exon 4 of MTHFR (GenBank accession number rs1801133) was typed by PCR-RFLP in the MODZT families using a protocol and primers as described previously (Hasbargen et al., 2000
). The C677T polymorphism in the adolescent twin families and the A1298C polymorphism (Glu
Ala) (GenBank accession number rs1801131) in both populations were genotyped using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). PCR products of 114 bp and 115 bp for the C677T and A1298C polymorphisms were amplified using the primer pairs CCCGAAGCAAGGAGCTTTG, AAAGCGGAAGAATGTGT CAGC and CCTGAAGAGCAAGTCCCCC, CCGGTTTGGTTCT CCCG respectively. HinfI or MboII restriction enzyme digestion and agarose gel electrophoresis were used to identify DNA controls required for each polymorphism in the Sequence Detection System (SDS) allelic discrimination assays. Using the standard protocol for the SDS assay, fluorescently labelled probes used for C677T polymorphism were: 5'-(VIC)-CTGCGGGAGCCGATTTCATCAT-6-carboxy-tetramethyl-rhodamine(TAMRA)-3' and 5'-6-carboxy fluorescein-(FAM)-CTGCGGGAGTCGATTTCATCATCA-TAMRA-3' to detect the C and T alleles respectively. Probes used for the A1298C polymorphism were 5'-(VIC)-CAAAGACACTTTCTTCACTGGT CAGCTCC-TAMRA-5' and 5'-FAM-CAAAGACACTTGCTTCA CTGGTCAGCTC-TAMRA-3' to detect the A and C alleles respectively. The final concentration of reagents in the PCR mix (reaction volume 15 µl) was 1 x TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nmol/l Primers, 175 nmol/l FAM probes and 200 nmol/l VIC probes. The reaction mix was added to 15 ng of genomic DNA that had been pre-dried in 96-well plates. Following optimization of each assay in the ABI Prism 7700 SDS, PCR reactions were amplified on ABI GeneAmp 9700 PCR machines for 2 min at 50°C, 10 min at 95°C, followed by 45 two-step cycles of 15 s at 95°C and 1 min at 62°C. End-point detection genotype analysis was performed on amplified samples using the ABI PRISM 7700 software using standard procedures.
The program Sib-pair was used to calculate allele and genotype frequencies, and perform two-parent, one-locus transmission/disequilibrium tests (TDT) to examine allele transmission to MODZT. The one-locus TDT was also used to examine paternal and maternal allele transmission to male and female offspring individually in the adolescent twin families. A two-locus TDT was performed using GENEHUNTER (Kruglyak et al., 1996) and TDTHAP (Clayton and Jones, 1999
). The transmission of haplotypes was tested for the mother of a pair of DZ twins in MODZT families, and for a twin in the adolescent twin families. Segregation distortion and parent of origin effects were also tested for.
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Results |
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Discussion |
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In some previous studies, individuals with 677TT/1298CC or 677CT/1298CC genotypes were not observed, and the 677TT/1298AC genotype was rare (van der Put et al., 1998; Weisberg et al., 1998
; Zetterberg et al., 2002
). These groups concluded that the alleles only occur in the trans configuration. In the present study, there was strong LD between the two variants consistent with the short distance (1902 bp) between the variable bases in the MTHFR DNA sequence. The 677T/1298C allele was observed, but the haplotype was rare and found in only a small number of families from the Netherlands.
Several studies have reported effects of MTHFR genotypes on fetal viability (Isotalo et al., 2000; Volcik et al., 2001
; Zetterberg et al., 2002
). It is not clear whether specific alleles at individual SNPs in MTHFR or the combined presence of one or more variant alleles at the C677T and A1298C SNPs predispose to embryo or fetal loss during pregnancy (Isotalo et al., 2000
; Volcik et al., 2001
; Zetterberg et al., 2002
). MTHFR is a homodimer and may be subject to complex regulation (Goyette et al., 1998
). Recent biochemical studies of MTHFR compared the wild-type enzyme with purified variant forms of the C677T (Ala222Val) and A1298C (Glu429Ala) variants (Yamada et al., 2001
), and activities of the wild-type or A1298C variant enzymes were indistinguishable. The C677T variant protein dissociated more easily into monomers and lost its flavin adenine dinucleotide (FAD) cofactor on dilution, leading to the loss of enzyme activity (Yamada et al., 2001
). The results of these studies supported the association of the C677T variant with increased risk of disease. Association with A1298C may result from direct effects of the variant or through LD with the C677T and/or other variants.
The C677T variant is associated with a moderate risk of NTD (Botto and Yang, 2000), giving an increased odds ratio of 1.6 for infants homozygous for the 677T allele. Higher frequencies of variant MTHFR alleles were observed in fetal samples and spontaneous abortions compared with control samples (Isotalo et al., 2000
; Zetterberg et al., 2002
), although the pattern of allele differences was not the same in each study. Twins may be at higher risk for embryo loss, and mothers with dichorionic twin pregnancies had a lower frequency of the 677T allele compared with women who gave birth to singletons (Hasbargen et al., 2000
). In the present larger study, the transmission disequilibrium of haplotypes was tested (given the high LD between the polymorphisms in MTHFR), and no evidence was observed for an association between haplotypes at MTHFR and twinning phenotype in MODZT. The allele frequency for the 677T allele in the mothers of twins (0.35) was similar to that in the mothers of singletons in a previous study (Hasbargen et al., 2000
). The allele frequency was not significantly different from that in fathers of MZ and DZ twins (0.36) from the adolescent twin families. Population studies do not show evidence for fathers contributing to variation in either DZ or MZ twins (Lewis et al., 1996
). The present study had good power to detect association between MTHFR and twinning. The odds ratio for transmission of the 677T variant to mothers of twins was 0.87, with a tight confidence interval (0.701.07).
Haplotype transmission was also considered to MZ or DZ twins, but no evidence was seen for any effects of MTHFR genotype on the survival of twin embryos. The present sample comprised individuals who survived the entire pregnancy. Calculations based on the data of others (Zetterberg et al., 2002) suggested that large deviations in adult haplotype frequencies caused by embryo loss from abortion would not be expected. No difference was observed in haplotype transmission that might have been caused by embryo or fetal loss.
Periconceptional folate supplementation has been strongly recommended to reduce the incidence of NTDs, but a meta-analysis found that the reported increase in the frequency of twins following folate supplementation was not significant (Lumley et al., 2000). Recent results from a large population-based cohort study in China found no evidence for effects of folic acid supplementation on twinning frequency (Li et al., 2003
), though this population generally has a low frequency of twins. Based on the results of the present studies, it can be concluded that variation in twinning frequency is not associated with genotype at MTHFR in mothers of twins, or with the loss of specific MTHFR genotypes during twin pregnancies.
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Acknowledgements |
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References |
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Submitted on May 18, 2003; accepted on July 13, 2003.