Hum. Reprod. Advance Access published online on October 25, 2007
Human Reproduction, doi:10.1093/humrep/dem337
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Genotypes of the C677T and A1298C polymorphisms of the MTHFR gene as a cause of human spontaneous embryo loss
1 Department of Biochemistry, Hospital Costa del Sol, Marbella, Spain 2 Department of Biochemistry and Molecular Biology, University of Malaga, Bulevar Louis Pasteur s/n, 29071 Malaga, Spain 3 Department of Pathologic Anatomy, Hospital Materno-Infantil Carlos Haya, Malaga, Spain 4 Department of Fertility, Clinica Rincón, Malaga, Spain
5 Correspondence address. E-mail: engel{at}uma.es
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Abstract |
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BACKGROUND: Polymorphisms C677T and A1298C of the MTHFR gene have been implicated in fetal viability. In this study, we determined the allele and genotype frequencies of these polymorphisms in different populations, including spontaneous abortion (SA) fetal tissues, with the objective of evaluating their impact on fetal viability.
METHODS: 342 samples of fetal tissues, selected from SA occurring during the 1980s, 230 samples from subjects born in the 1980s and a third set of samples from 204 subjects born in the 1950s, were genotyped by using TaqMan probes.
RESULTS: The wild CC genotype of the C677T polymorphism showed a strong protective effect against abortion (0.03 in SA versus 0.47 in 1950s and 0.43 in 1980s) (P < 0.0001). Genotypes of three mutations in the combinations of polymorphisms for C677T and A1298C showed a very low frequency in the living population; however, the three mutations genotypes were over expressed in the SA group (0.02 in 1950s; 0.03 in 1980s and 0.17 in SA) (P < 0.0001). Samples with four mutations (n = 2) were found only in the SA group.
CONCLUSIONS: There is no linkage disequilibrium between C667T and A1298C polymorphisms. Fetal viability is directly related to the CC genotype as a protector while the three and four mutation MTHFR genotypes appear to be a determinant on fetal non-viability and SA.
Key words: MTHFR/MTR/polymorphism/folate/spontaneous abortion
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Introduction |
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The methylenetetrahydrofolate reductase gene (MTHFR) encodes an enzyme that produces 5-methyltetrahydofolate, which is the methyl donor to homocysteine for synthethizing methionine. Two single nucleotide polymorphisms (SNP's) of the MTHFR gene, the C677T (Frosst et al., 1995
) and the A1298C (Weisberg et al., 1998), as well as the A2756G of the methyltransferase gene (MTR), have been studied in several populations (Weisberg et al., 1998). In most studies, certain haplotypes of the polymorphisms C677T and A1298C from the MTHFR gene were not found. This is the case of the combination 677TT/1298CC, which is the four-mutation-genotype. But even subjects with three mutations, such as 677TT/1298AC or 677CT/1298CC, were at very low frequencies in live populations. Stegmann et al. (1999) proposed a linkage disequilibrium between polymorphisms C677T and A1298C of the MTHFR gene after analysing 23 German families. This study verified that the mutations 677T and 1298C were never found in the cis configuration. However, Isotalo et al. (2000) obtained, in fetal samples, a high percentage of genotypes with three and four mutations, far above the hazard for crossing-over possibilities. Volcik et al. (2001) presented data supporting the conclusion of Isotalo et al. (2000) concerning a decreased viability among fetuses with the 677TT/1298CC genotype, which they did not observe in North American and Canadian populations. As they observed, in three different populations, the 677CT/1298CC (three mutations) genotype at frequencies nearing those expected, Volcik et al. (2001) concluded that this genotype does not represent any significant selective disadvantage.
Zetterberg et al. (2002) examined the distribution of the C677T and A1298C polymorphisms in 80 fetal tissue samples from spontaneous abortions (SAs) occurring between the sixth and twentieth week of pregnancy, compared with 125 healthy blood donors. Only 1 of the 80 SA embryos had the wild type genotype combination, 677CC/1298AA, as compared with 19 of 125 controls. Combined genotypes with three or four mutant alleles were not detected in any of the groups, suggesting complete linkage disequilibrium between the two polymorphisms. Zetterberg et al. (2002) concluded from the data that the effect of one or more MTHFR mutated allele may be detrimental during embryogenesis when the folate requirement is high.
Finally, Ogino and Wilson (2003) calculated the haplotype frequencies of the polymorphisms C677T and A1298C in pooled general populations derived from data published in 16 articles. They found that most 677T and 1298C alleles were associated with 1298A and 677C alleles, respectively. They propose that there may be an increased frequency of the very rare cis 677T/1298C haplotype in some parts of the UK and Canada, possibly due to a founder effect. As we proposed (Reyes-Engel, 2002), if there is no linkage disequilibrium, the prevalence of the mutations of these two polymorphisms could have a very important role in human fertility.
In the present study, we investigated the association between MTHFR and MTR polymorphisms and fetal viability.
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Material and Methods |
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The study was approved by the Ethics Committee at the University Hospital ‘Virgen de la Victoria’ (Málaga). All samples were obtained with the written consent of the subjects. The study group consisted of 342 fetal tissue samples from SAs, obtained from the Department of Pathology of the University Hospital Carlos Haya (Málaga). These samples were selected by prior checking of the clinical history and with the inclusion criteria of fetal tissue being histological confirmed as from SA of
3 months (11 ± 1.70 weeks) and unknown etiology. The criteria to establish the unknown etiology were the clinical history of the father and mother and the histological analysis of the fetuses; by these criteria anencephalus, hydrotropic degeneration, known chromosomal anomalies, acraneous, leporine lip malformation, multiple malformations and amniotic band syndrome were all discarded. A second population consisted of 230 caucasian subjects born in Malaga in the 1980s with a mean age of 22 ± 1.58 and a third population consisted of 204 caucasian subjects born in Malaga in the 1950s with a mean age of 54 ± 12.5. These latter two populations would be the coetaneous (1980s) and previous (1950s) generations of the study group because one of the inclusion criteria was to be born in Malaga and at least a second generation Andalusian.
The fetal samples were extracted from the archival formalin-fixed, paraffin-embedded tissue sections. Genomic DNA of fetal tissue was extracted by the method described by Coombs et al. (1999). Genomic DNA of the second and third groups was extracted from peripheral leukocytes by standard procedures with the Kit AquaPure Genomic DNA Blood (Bio-Rad, Spain).
Genotyping was performed on three SNP's: a1298C and C677T (MTHFR) and A2756G (MTR) by real-time PCR using allele specific Taqman® probes and primers described by Ulvik and Ueland (2001) and the following optimized protocol of 45 cycles: 10 s—94°C, 40 s—54°C, 15 s—72°C. The PCR mix (25 µl total volume) consisted of 5 µl of genomic DNA, 0.35 µl of A2756G sense primer, 0.62 µl of A2756G anti-sense primer, 0.85 µl Taqman® probe FAM, 0.43 µl Taqman® probe TET, 20 µl PCR-buffer iQ-SupermixTM (Bio-Rad, Spain), containing 100 mM KCl, 40 mM Tris–HCl, (pH 8.4) 1.6 mM dNTP (dATP, dCTP, dGTP and dTTP), iTaq polymerase (50 units/ml) and 6 mM MgCl2, in 17.75 µl H2O.
All samples were genotyped and allelic and genotypic frequencies were compared. Differences were analysed statistically by Chi square test or Fisher's exact test to calculate statistical significance that was considered significant for P < 0.001 and very significant for P < 0.0001. Odds ratios (OR) were also calculated for a 99% confidence interval (CI).
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Results |
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Single genotypes
When polymorphisms were analysed independently, genotypes and allelic frequencies of the C677T polymorphisms of the MTHFR showed significant differences between the SA group and the other populations. The 677CC genotype showed a very low frequency in the SA population compared with the control groups. In contrast, the frequencies of mutated genotypes (677CT and 677TT) were higher in the SA group. These differences were statistically significant (Table I).
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Odd ratios showed a strong protection against abortion by the CC genotype, 1 abortion for every 22 births (OR = 0.044), whereas the mutated genotypes showed a significant risk of abortion (OR = 3.2 for CT and 1.9 for TT) (Table I).
C677T/A1298C genotypes
These genotypes showed statistically significant differences between the SA population and the other groups. These differences were found when the homozygous 677CC was present. All of the CC genotypes were much lower in SA than in the living population (P < 0.001). We also saw that protection is maintained in any combination with 677CC genotype (OR < 1 in all cases).
From the point of view of the number of mutations, a progressive increase of the OR value was observed. Genotypes of the MTHFR combination showed an increased risk of abortion as the number of mutations rose, with a maximum of three mutations (OR = 5.7). The 677CC/1298TT genotype could not be compared because it was only found in the fetal population (Table II).
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C677T/A2756G genotypes
This combination showed the same pattern of influence due to the 677CC genotype, with significantly reduced frequencies of the 677CC/2756AA and 677CC/2756AG genotypes in SA. CT/AA, CT/AG and TT/AA genotypes were higher in the SA population (P < 0.001 for CTAA and TTAA), showing risk patterns with an OR > 2.
The proportion of subjects with one or two mutations was significantly higher in the SA population. However, genotypes with three and four mutations did not show a high OR, probably because of a low frequency of the 2756GG genotype in the general population (Table II).
A1298C/A2756G genotypes
In this combination of genotypes, there were no statistically significant differences when genotypes or number of mutations were compared (Table II).
C667T/A1298C/A2756G genotypes
In these genotypes, as it has been described previously, the 677CC genotype of the MTHFR gene is in a minor proportion in SA group compared with the others. These differences were statistically significant in three of the nine combinations with CC genotype, and the OR was inferior to 0.5 in all cases in which it could be determined statistically.
Combinations with the 677CT genotype are in the major proportion of the SA population in seven of the nine combinations.
An increased frequency in the SA population was found for all genotypes containing the 677TT genotype.
In relation to the number of mutations, it is important to emphasize the decreased number of subjects with zero mutations in fetal group (P < 0.0001), and the increased number of subjects with two, three (P < 0.001) or four mutations in the fetal group. This fact gives rise to a progressive increase of the OR according to the number of mutations going from a value of 0.07 for zero mutations to 1.9 for five mutations (Table III).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionFundingAuthor RolesAcknowledgementsReferences |
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Two polymorphisms of the MTHFR gene, the C677T and A1298C have been extensively studied in different populations (Ogino and Wilson, 2003
). Some conclude that there is a linkage disequilibrium between both polymorphisms (Stegmann et al., 1999; Zetterberg et al., 2002) as there are no live subjects with four mutations (677TT/1298CC) and a very low number with three mutations (677TT/1298AC and 677CT/1298CC). Other studies carried out on abortion samples demonstrated higher frequencies of three and four mutation samples (Isotalo et al., 2000; Volcik et al., 2001), which cast doubt on the linkage disequilibrium. The importance of elucidating this question is the following: if there is no linkage disequilibrium between both polymorphisms, it means that a combination of three and four mutations is possible but it means that fetal viability is minimal or null, and this could represent one of the main causes of SA. In this study, we analysed a group of SA samples and a coetaneous live populations both from 1980s. We also did the analysis of the previous generation, which gave us not only the perspective of the genotype evolution between two generations, but also the expected frequencies of this population, which should match with the 1980s population.
The proposal is that if we find in the fetal population the alleles absent in the live populations in such a way that they present a normal mendelian pattern, there would not be any linkage between the two markers.
In this study, the contrast in the frequencies between both populations and the fetal one shows that the wild-type genotype (CC) of the C677T polymorphism of the MTHFR gene exerts a protection against SA, whatever its combination may be.
The three-mutation genotypes (677CT/1298CC, 677TT/1298CA) of the two MTHFR polymorphisms appear to cause a high risk of abortion. A genotype of four mutations can be considered as a non-viable genotype, since we have not found it in any live subject out of 468. However, the three and four mutations genotypes were found in the fetal population at expected and sometimes at higher than expected frequencies from the Hardy–Weinberg equilibrium. This fact suppresses the possibility of linkage disequilibrium between both polymorphisms in our population.
In contrast, when the combination of polymorphisms C677T (MTHFR) and A2756G (MTR) is analysed, genotypes with three and four mutations do not show any significant change in the fetal population, although the protector effect against abortion of the genotypes with the wild type 677C polymorphism is maintained. As for fetal viability, mutations of the MTR gene do not constitute any abortion risk.
The results obtained in our population disclaim the linkage disequilibrium between the alleles of the polymorphism C677T and A1298C of the MTHFR gene as proposed by Stegmann et al. (1999). In conclusion, the difference lays in the fact that this author does not study a fetal population. Isotalo et al. (2000) and Volcik et al. (2001) gave similar results to us, but the fetal population of Isotalo et al. (2000) was very heterogeneous in age and ethiology. Volcik et al. (2001) obtained unexpected results because of a high frequency of the 677CT/1298CC genotype (three mutations) in live populations. Zetterberg et al. (2002) reported a very similar study to us with the same inclusion criteria for fetal population, but on a smaller number of samples. The results of Zetterberg et al. (2002) match our results only on the protector effect of the wild-type genotype 677CC/1298AA, for the MTHFR gene, which is considered as a strong protector against abortion. Nevertheless, in this study, genotypes of three and four mutations were not found. They concluded that there is linkage disequilibrium between the above-mentioned polymorphisms. But this study showed genotypic frequencies that are difficult to understand. Only 1 677CC/1298AA genotype was found in a SA population of 84, allowing them, like us, to classify this genotype as a strong protector. However, with the addition of only one mutation of the A1298C as with the 677CC/1298AC genotype, there becomes a strong risk, yet the 677CC/1298CC genotype, with two mutations, becomes more seldom again, indicating a protector effect. On the other hand, this strong effect of one mutation of the A1298C polymorphism would mean a functional effect for this polymorphism, which has not been reported. Recently, Bae et al. (2007) reported in a study of a Korean fetal population that the 677CC genotype of the MTHFR gene is at risk of abortion that does not coincide with the protection found by Zetterberg et al. (2002) and the present study. Usually, these contradictory results between different reports could be related to distinct technical approaches due to genotyping methods, different allele distributions in diverse populations, different nutritional and folate supplementation between populations, and/or to unknown causes referred to as genomic peculiarities that could give a variety of results. In our study, we consider the fact that one of the groups, the combination of the A1298C polymorphism of the MTHFR gene and the MTR polymorphism, gives no deviation of the frequencies between the populations as an excellent internal control to avoid bias.
In our previous report Reyes-Engel et al. (2002), we estimated, under the hypothesis of no linkage disequilibrium, that the fetal non-viability in our population due to the interaction of the C677T and A1298C polymorphisms was around 6%. In the present study, we confirm this hypothesis by analysing a fetal population of the 1980s compared with a coetaneous living population and that corresponding to its parents. From these results, it can be concluded that there is no linkage disequilibrium between both polymorphisms and that it could be possible to quantify the degree of fetal viability of each couple with the analysis of the combination of the polymorphisms C677T and A1298C of the MTHFR gene.
In fetal samples, the frequency of the CC genotype of the C677T polymorphism is extremely low and frequencies of genotypes with three and four mutations (C677T and 1298C) are very high. The prevalence of chromosomal alterations in clinical SA are reported to be around 50% (Speroff and Fritz, 2006). Both prevalences are clearly overlapped. These results suggest that chromosomal alterations and the MTHFR polymorphisms studied are related.
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Funding |
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Ministerio de Educación y Ciencia (Spain) (BFU2004-01571).
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Author Roles |
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Samples and data acquisition, conception and design, revision—G.C.
Statistical analysis, drafting and revision—A.M.-O.
Sample processing, revision—A.J.J.
Coordination, sample acquisition, bibliographic search, revision—M.J.G.
Statistical analysis, revision—A.R.P.
Sample and data acquisition, revision—F.M.
Data analysis, design, revision—M.R.
Guarantor, interpretation of data, conception and design, drafting, coordination, revision—A.R.-E.
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Acknowledgements |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionFundingAuthor RolesAcknowledgementsReferences |
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We thank Sarah Van Haelst for help with reviewing the English language of this manuscript.
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References |
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Submitted on July 23, 2007; resubmitted on September 17, 2007; accepted on September 26, 2007.
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