Molecular Human Reproduction, Vol. 8, No. 10, 952-957,
October 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
Implications on human fertility of the 677C
T and 1298AC polymorphisms of the MTHFR gene: consequences of a possible genetic selection
1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Malaga C.P.29071, 2 Department of Obstetrics and Gynaecology, Clinical University Hospital Lozano Blesa, Zaragoza, 3 Department of Obstetrics and Gynaecology, Clinical University Hospital Carlos Haya, Malaga, Spain and 4 Folkhälsan Institute of Genetics, University of Helsinki, Finland
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Abstract |
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Mutant alleles with the 677C
T and 1298AC polymorphisms of the MTHFR gene, and consequent lower methylentetrahydrofolate reductase enzyme activity, have been related to higher plasma homocysteine levels, which are associated with cardiovascular diseases. We assessed the genotype frequencies, degrees of fertility and homocysteine levels, and discuss a possible genetic selection for the gene polymorphisms studied. A total of 1777 subjects (897 women and 880 men), divided into four age groups, were genotyped by PCR and restriction fragment length polymorphism. The total homocysteine concentration in plasma was determined by fluorescence polarization immunoassay. Based on random pairs and linkage disequilibrium of the two polymorphisms, we estimated the rate of fetal non-viability according to the combinations of these two polymorphisms to be 4.63% for the group >24 years old and 6.31% for the group <24 years old. We detected an increased frequency of mutant alleles in the youngest age group, coincident with a generally increased folate intake by pregnant women in Spain. The genetic selection detected leads to an increase in mutated individuals, the number of whom could increase four-fold over the next 75 years. Although generally reduced in the younger age groups, the homocysteine plasma levels were shown to increase in individuals according to the number of mutations, especially those of the 677T allele. fertility/fetal viability/genetic selection/homocysteine levels/MTHFR gene polymorphisms
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The methylenetetrahydrofolate reductase (MTHFR) gene encodes an enzyme which produces 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for homocysteine, in the synthesis of methionine. Two mutations on the MTHFR gene, 677C
T (Frosst et al., 1995
) and 1298AC (here indicated as 1298ac) (van der Put et al., 1998; Weisberg et al., 1998) have been characterized. The 677CT polymorphism is related to homocysteine and folate levels in different populations (Gudnason et al., 1998). The relationships between MTHFR enzyme deficiency, homocysteine levels and atheroma plaque formation have recently been established in knockout mice (Chen et al., 2001). Several studies have analysed both polymorphisms (van der Put et al., 1998; Weisberg et al., 1998; Chango et al., 2000) but none have found any TT/cc individuals, and very few living persons have been found with three mutations (TT/ac and CT/cc). However Isotalo et al. found all possible genotype combinations, including those with three and four mutations, in an aborted fetal population (Isotalo et al., 2000). This study confirmed that the mutations can be in cis/trans configuration, in contrast to the study of Stegmann who found them to be in linkage disequilibrium (Stegmann, 1999). To date, no studies evaluating the role of these two polymorphisms on human viability have been published. However, analysis of mating for the different genotypes could be applied to quantify the degree of viability associated with these genotype combinations.
In 1998, we suggested the possibility of a genetic selection in Spain in favour of the mutants of the C677CT polymorphism in the MTHFR gene, based on the fact that treatment with vitamins and folates during pregnancy increased the viability of TT homozygous fetuses. This hypothesis was based on the increase in the number of mutated individuals in our population since the mid-1970s. This increase is coincident with the increased intake of vitamins and folates by pregnant women (Muñoz-Moran et al., 1998). Since 1998, the number of individuals in our study has increased, and we have been able to examine their genotype frequencies in four groups of people according to their age: <24, 25–50, 51–75 and >75 years old. We also analysed a second polymorphism from the same gene, 1298ac, as well as total homocysteine (tHcy) levels in plasma and fetal viability, according to the different genotype interactions evaluated. Based on the mild synergic effect of the mutations of the two polymorphisms in decreasing the enzyme activity, we analysed the genotype frequencies by three different groupings: individual polymorphisms, combined genotypes, and by the number of accumulated 0 (CC/aa), 1 (CC/ac and CT/aa ) and 2 mutations (CT/ac,TT/aa and CC/cc ). Subjects with three mutations were not considered because of their low number.
Genetic selection in humans is a difficult phenomenon to detect, since it is usually associated with a slow process of genetic change, the effects of which can only be observed after many generations. Considering that each generation spans a period of 
25 years, our capacity for in-vivo studies is limited to three or four generations.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Subjects
A total of 1777 subjects (897 women and 880 men) were genotyped and divided into four age groups: <24 (n = 523), 25–50 (n = 478), 51–75 (n = 542) and >76 (n = 234) years. The mean ages of the groups were 15.5 ± 11.7, 38.1 ± 12.2, 62.5 ± 10.5 and 88.2 ± 7.1 respectively. A total of 781 individuals (399 women and 382 men),
200 in each age group, were selected for analysis of their plasma tHcy level. Each study group is representative of the entire Spanish population. Individuals were selected randomly from different areas of our province (Malaga, Southern Spain) and from different social statuses, in order to avoid a priori selection bias. All the selected individuals were Caucasian and resident in the study area. The parents and grandparents of those included in the study were also Caucasian and born in Spain, but there was no clustering of the parental and grandparental birth places in any region. The possibility of a founder effect or genetic drift was examined and discounted. To date, no definite data for these polymorphisms in Spain are available. All the selected individuals were also genotyped for the insertion/deletion polymorphism (I/D) of the angiotensin converting enzyme (ACE) gene and M235T polymorphism of the angiotensinogen (Atg) gene, in order to determine whether our adult and young populations were genetically homogeneous. No significant differences were observed for the allele or genotype frequencies of these genes when the younger subjects were compared with the adult population. Furthermore, the I/D polymorphism of the ACE and the M235T polymorphism from the Atg gene showed similar frequencies in our population to those reported by other authors in Spain. We are currently conducting a case–control study with samples from different Spanish regions, but so far have found no differences between subjects >25 years old and our study population (data not published).
The population studied was randomly selected according to age: 0–12 years old: dried blood spots from neonatal screening papers; 10–24 years old: students from primary and secondary schools, and university; individuals 25–50 years old and >51 years were recruited using the Andalusian Health Service identity card. After approval by the University Hospital Ethical Committee, all the subjects were contacted, and from those whose consent was obtained, 10 ml of blood was taken. The investigation in this study conforms with the principles outlined in the Declaration of Helsinki.
Genetic analysis
Genomic DNA was isolated from 200 µl EDTA blood with the QIAamp blood kit (Qiagen) according to the manufacturer’s instructions. Identification of the MTHFR 677CT polymorphism has been described elsewhere (Frosst et al., 1995). Primers produce a 198 bp fragment which includes the C-to-T substitution at nucleotide 677, creating a HinfI recognition sequence. When the mutation is present, HinfI digests the 198 bp into 175 and 23 bp fragments. Detection of the 1298ac polymorphism was performed with the use of primers described elsewhere (van der Put et al., 1998; Weisberg et al., 1998) yielding a 138 bp fragment that is digested into 119 and 19 bp fragments by Fnu4HI in the presence of the C allele. The fragments were analysed by polyacrylamide gel electrophoresis. Furthermore, all the individuals were genotyped for the different alleles of the ACE (II, ID and DD) and M235T Agt polymorphisms (MM, MT and TT) by PCR and restriction fragment length polymorphism.
Total plasma homocysteine analysis
Blood was collected from fasting subjects and placed in vacutainers and tubes containing disodium EDTA for further analysis. After collection, samples were centrifuged (3000 g, 10 min at 4°C ). Plasma was collected and aliquots were stored at –80°C. The plasma tHcy concentration was determined by fluorescence polarization immunoassay using an IMX Abbot analyser (intra- and inter-assay coefficient of variation <8%).
Statistical and mathematical analysis
Compliance of genotype distributions to the Hardy–Weinberg equilibrium was evaluated by 
2 analysis. Student’s t-test was applied to determine the differences in plasma tHcy levels between the various groups. 2-test for contingency tables was used to compare paired frequencies. P < 0.05 was considered to be statistically significant. Values are expressed as the mean ± SD. The statistical analysis was performed with the software package SPSS 10.0.
The genetic selection was calculated for the evolution of the 677CT individual genotypes and for the number of mutations between both polymorphisms 677CT and 1298ac. For the latter, we assumed that 0, 1 and 2 mutations correspond to a locus with two alleles in which 0 and 2 are homozygous and 1 is heterozygous. The genetic selection found can be classified as codominant or incompletely dominant and directional with the heterozygous genotype having an intermediate fitness. For this kind of selection, the most appropriate mathematical model is dq = –hsq(1–q)/ 1–sq(2hp+q) where dq is the change of frequency of the lower fitness allele, s is the fraction of that genotype lost to selection, h is the extent of dominance between 0 for the complete dominance selection against the recessive and 1 for the complete dominance selection against the dominant, and p is the frequency of the higher fitness allele.
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Results |
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We analysed the two previously mentioned genotypes of the MTHFR gene in 1777 subjects divided by age into four groups. A Hardy–Weinberg disequilibrium found between the third (25–50 years) and the fourth (<24 years) groups confirmed the process of dynamic genetic selection in favour of the T allele and against the C allele (Table I
). Grouping the subjects by the number of mutations showed a significant variation in the younger group (<24 years), with an increase in the frequency of subjects with two mutations and a decrease in the groups with no and one mutation (Table I). Analysis of the combined genotypes showed no TT/cc genotype individuals, two with TT/ac and one with CT/cc. The observed frequencies for the TT/aa, CC/cc and CT/ac genotypes increased in the group <24 years old, while the CC/aa and CC/ac genotypes decreased in this group (Table II), and the frequency of the CT/aa genotype was hardly changed.
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We analysed the possible interactions between males and females of the survival genotypes by calculating the probability for mating (Pm) of each couple, and deducing the fetal viability (v) related to these genotypes according to the number of combinations with three or four mutants (Tables III and IV
). The probability of producing non-viable fetuses can be calculated by means of the mathematical approach 
Pm (1–v), where Pm stands for the probability of mating between the different genotypes, calculated by the product of the observed genotype frequencies of the mating couples, and v is the probability, in any interaction, of having viable fetuses for the two polymorphisms of the MTHFR gene. By this approach it can be estimated that there was a mean value of 4.63% of non-viable fetuses for the three older age groups and 6.31% for the youngest group (<24 years).
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In the analysis of plasma tHcy in populations divided by age, we observed that plasma tHcy increased
1 µmol/l per 25 year age increase. We also analysed plasma tHcy levels according to individual genotypes and found a significant increase in tHcy levels for 677TT versus all the other individual genotypes. No remarkable differences have been detected between wild-type and heterozygous subjects of the two polymorphisms elsewhere (Engbersen et al., 1995; Frosst et al., 1995; Harmon et al., 1996; Jacques et al., 1996). However, when plasma tHcy levels were classified according to the combined genotypes and the number of mutations there were differences between subjects in both groups (Table V), showing a generally higher tHcy level with increased numbers of mutations. The number of individuals with three mutations was too low to be analysed statistically.
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To predict the evolution of the allelic frequencies and mutations in future generations, we applied the mathematical quantitative model of selection developed for sexually reproducing diploid organisms described in Materials and methods. The results showed that after applying the calculation to just the 677C
T polymorphism, five generations are needed to reach 90% homozygosity for mutant TT. However, if we make the same prediction according to the occurrence of alleles with either of the 677CT or 1298ac mutations, 90% of the population would have at least two mutations in only three generations (Figure 1).
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Discussion |
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For this kind of selection, the fittest model that can be applied to both population distributions (genotypes and number of mutations) is that of selection against an allele with partial expression in heterozygous individuals. This model describes a faster genetic selection overall when neither of the two alleles are rare in the studied population. From the evolution observed, we can consider that our study population is currently starting the phase of competition between the homozygous genotype of higher fitness (two mutations) and the heterozygous genotype (one mutation) (Figure 1
). The calculated fitness for each type was no mutations = 0.45, one mutation = 0.77 and two mutations = 1.
Although it is difficult, even for us, to accept the dimension of the genetic selection described, the accepted mathematical model applied gave the results reported, which nevertheless must be classified as hypothetical. However, an almost identical evolution pattern was described for a polymorphism of the malate dehydrogenase gene in a Drosophila melanogaster population (Berger, 1971).
There is heterogeneity of the allelic frequencies of the 677CT MTHFR polymorphism between different ethnic groups, and even between regions with the same ethnic group, as demonstrated in Italy (Abbate et al., 1998; Motti et al., 1998). Studies related to age and 677CT polymorphism refer mainly to longevity and no differences have been found for the 677CT genotype frequencies in a population >25 years old. Todesco et al. have found differences in frequencies between individuals older and younger than 60 years (Todesco et al., 1999). Some populations, such as Mexicans (Mutchinick et al., 1999) and Toscanians in Italy (Abbate et al., 1998), show frequencies >30% for the TT homozygous genotype. Other populations, such as Africans, are 0% homozygous for TT and 91% for the wild-type allele (Rajkovic et al., 2000), although African-Americans in the USA have now attained 1–2% for TT and 85% for the C allele (McAndrew et al., 1996). Studies of the evolution of these populations could possibly clarify at which selection phase they are presently in, or whether the proportion of the second polymorphism, 1298ac, exerts a compensating effect in terms of the number of mutations, as in Mexicans in whom the homozygous frequency (cc) in the 1298ac polymorphism is null (Barber et al., 2000).
The finding of a higher fitness for the mutant contrasts with a less efficient enzyme. This is surprising, since fitness is associated with fertility and/or viability. A possible lower fertility in males with the homozygous TT genotype has recently been reported (Bezold et al., 2001).
Isotalo et al. analysed 119 neonatal cord blood samples and 161 fetal tissue samples for MTHFR 677CT and 1298ac mutations to determine whether certain MTHFR genotype combinations were associated with decreased in-utero viability (Isotalo et al., 2000). Mutation analysis demonstrated that all possible MTHFR genotype combinations were represented in the fetal group; 677T and 1298c alleles could occur in either cis or trans configurations. Combined 677CT/1298cc and 677TT/1298cc genotypes, which contained three and four mutant alleles respectively, were not observed in the neonatal group. This suggests decreased viability among fetuses carrying these mutations and a possible selection disadvantage among fetuses with increased numbers of mutant MTHFR alleles. This was the first report to describe the existence of human MTHFR 677CT/1298cc and 677TT/1298cc genotypes and to demonstrate their potential role in compromised fetal viability.
Volcik et al. presented data supporting the conclusion of Isotalo et al. concerning decreased viability among fetuses with the 677TT/1298cc genotype, which they did not observe in the United States and Canadian populations studied (Isotalo et al., 2000; Volcik et al., 2001). Because they observed the 677CT/1298cc genotype in frequencies nearing those expected in three different populations, Volcik et al. concluded that this genotype does not result in a significant selective disadvantage.
However, Isotalo et al. showed that fetal viability, at least for fetuses of a certain age, is much higher for the mutated genotype than for the others, which show no differences in frequencies between neonates and fetuses (Isotalo et al., 2000). In the case of the TT/aa genotype, the difference is one miscarriage per 10 neonates, whilst for the wild-type genotypes there are no differences.
So far, the incidence of the genotype interactions of the MTHFR gene in human fertility and viability has not been estimated. It is possible that the assumption of linkage disequilibrium between these polymorphisms led to the exclusion of this idea, but the Isotalo and Volcik reports demonstrate the cis/trans configuration in this case (Isotalo et al., 2000; Volcik et al., 2001). Our results show an increase for genotype groups with two mutations in the younger group (<24 years) compared with genotype groups with no and one mutation. It could be argued that there have been changes in the TT/aa, CC/cc and CT/ac genotype viability, which were possibly blocked and then unblocked by external factors, such as diet or changes in folate intake, or both, according to our proposed folate-dependent genetic selection (Muñoz-Moran et al., 1998).
A possible explanation for the increased frequencies of the two mutation groups could be the high prevalence of cases of couples with difficulties having children, e.g. CT/ac combined with CT/ac viability = 0.69 (Tables III and IV) (cis/trans model), who, either by greater perseverance, chance or increased folate intake, produce higher than expected frequencies of the alternative genotypes. The increased number of mutants is possibly a compensating effect produced by the low fertility of the couples, in which feasible genotypes with two mutations are replacing the non-viable genotype with three or four mutations, and therefore increasing the number of subjects with two mutations compared with no mutations. But even in this proposal, subjects with one mutation should never decrease, unless we consider the recent proposal of Rosenberg et al. who studied the question of whether the MTHFR 677T alteration has an ancestral origin or has occurred repeatedly (Rosenberg et al., 2002). They analysed the frequency distribution of this mutation along with the 1298ac polymorphism and intronic dimorphisms in white Israelis (Jews and Arabs), Japanese and Ghanaian Africans. Remarkably, the 677T allele was associated with one haplotype in white and Japanese homozygotes. Among the Africans, analysis of maximum likelihood also disclosed an association with the same haplotype, although none of the 174 subjects examined was homozygous for MTHFR 677T. These results suggested that the MTHFR 677T alteration occurred on a founder haplotype that may have had a selective advantage.
Epidemiological studies show that 10% of the population are sterile and that 15% of couples either have problems conceiving or have fewer children than desired. In this latter group the cause is unknown in 15% of cases, so that the genetic interactions of the polymorphisms studied could represent a high percentage in this group.
There is currently no molecular model supporting an increased viability of mutated against wild-type individuals. But if we considered a limited availability of the substrate 5-10 methylenetetrahydrofolate (5,10methyleneTHF), which allele would be more viable on development: the wild-type, with a more efficient MTHFR enzyme, that catalyses the 5,10methyleneTHF to the 5-methylTHF faster, or the mutant allele, which because of its less efficient MTHFR enzyme, produces higher availability of the substrate to thymidilate and purine synthesis? Perhaps in a highly deficient folate state the mutant enzymes could not produce sufficient 5methylTHF, but with a normal or intermediate folate level, mutants could have a higher viability for embryo development.
The implications of this polymorphism on nucleotide synthesis have not yet been determined, but certain data, such as the higher uric acid levels related to mutated subjects (Motti et al., 1998; Zuo et al., 2000), suggest a different nucleotide turnover.
It can be concluded that, accepting the cis/trans mutation model, the genotype interactions of the polymorphisms studied would result in fetal non-viability rates of 4–7%, thus playing a significant role in human fertility. Should this genetic selection continue, the proportion of subjects in our population with two mutations could reach 90% in the next three to five generations, so that the relative infertility rate would grow to 18%, due solely to these genetic combinations. In the mutated individuals, plasma homocysteine levels would increase by 1 µmol/l, despite the overall reduced homocysteine levels observed in the younger age groups. Finally, it should be noted that to obtain functional or epidemiological conclusions, the haplotype, rather than the individual polymorphic genotypes of the polymorphisms studied, should always be considered.
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Acknowledgements |
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We thank Ian Johnstone for help with reviewing the English language of this manuscript. This study was supported by the Spanish Ministry of Education and Culture, Grants SAF99-0130.
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5 To whom correspondence should be addressed. E-mail: engel{at}uma.es
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References |
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Submitted on April 9, 2002; accepted on July 15, 2002.
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