Abstract
Background:
It has been proposed that there is a relationship between chromosomal nondisjunction and folate metabolism. Previous studies in mothers of girls with Turner syndrome (TS) and TS patients have suggested that 5,10-methylentetrahydrofolate reductase (MTHFR) C677T and A1298C gene variants might contribute to the risk of TS. However, data in different populations have yielded contradictory results.
Aim:
Here, we design a case-control study to evaluate the MTHFR C677T and A1298C gene variants in TS patients and their mothers as potential risk factors for TS.
Materials and Methods:
Using the TaqMan allelic discrimination assay, the frequency of the MTHFR C677T and A1298C gene variants was compared in two study groups, one of 54 girls with TS versus 93 control girls and another of 49 mothers of girls with TS versus 97 control mothers.
Results:
The allele and genotype frequencies of the MTHFR C677T and A1298C variants were not significantly different nor showed association among TS patients compared with control girls, nor among TS mothers compared with control mothers.
Conclusions:
The risk of having a child with TS did not appear to be associated with the MTHFR C677T and A1298C variants in the studied population.
Introduction
Turner syndrome (TS) results from the complete or partial absence of the second X chromosome in females (Gravholt et al., 2024). The prevalence of TS is 1 out of 2000–2500 live-born female infants (Stochholm et al., 2006), while in Mexico, it occurs in 0.8 per 1000 live births (Flores-Nava et al., 2011). The absence of one X chromosome is more frequently originated from chromosomal nondisjunction (CND) during gametogenesis (free X monosomy) or early embryonic development (mosaic X monosomy) (Gravholt et al., 2019). Although the etiology of CND in TS is multifactorial, some maternal risk factors such as maternal age, short stature, obesity, and folic acid (FA) intake deficiency have been associated with its occurrence (Gravholt et al., 2019).
The C677T and A1298C variants in the 5,10-methylentetrahydrofolate reductase (MTHFR) gene involved in folate metabolism have been associated with defects in folate-dependent homocysteine metabolism, which can result in DNA strand breaks, altered chromosome recombination, loss of pericentromeric structural integrity, DNA hypomethylation, and chromosome segregation (James et al., 1999; Lim et al., 2021; Romero-Bolaño et al., 2024). Based on this evidence, the MTHFR C677T and A1298C variants have also been identified as risk factors for prezygotic CND in mothers of girls with TS (MoTS) (Ismail et al., 2015; Guo et al., 2022; Miljanović et al., 2022). Furthermore, these variants have been associated to somatic CND in girls with TS (gTS) (Santos et al., 2006; Oliveira et al., 2008; Oliveira et al., 2012; Ismail et al., 2015; Guo et al., 2022) due to the high frequency of mosaic X monosomies found among these patients (Gravholt et al., 2024). However, maternal MTHFR variants could have a limited effect on cases of free X monosomy, as most of these patients arise from a paternal CND (Hook and Warburton, 2014). Moreover, this association has shown conflicting results across populations (Bispo et al., 2015; Oliveira et al., 2008) and, to our best knowledge, has not been evaluated in Mexicans. Here, we design a case-control study to evaluate the MTHFR C677T and A1298C variants as potential risk factors for TS in a sample of gTS and their mothers from Western Mexico.
Materials and Methods
This case-control study was conducted during the years 2022–2024 at the “Dr. Juan I. Menchaca” Civil Hospital of Guadalajara (Jalisco, Mexico). All subjects gave informed consent for participation in this study and sample collection, which was approved by our Research Council and Ethics Committee. The case group included 54 gTS. The diagnosis of TS was based on typical clinical features (Gravholt et al., 2024). All of the gTS had a karyotypically confirmed monosomy X. Thirty-three of these patients (61%) had a 45,X karyotype (indicative of a prezygotic event), while 21 (39%) had a 45,X mosaic karyotype (indicative of a postzygotic event) with additional cell lines with other karyotypes (Supplementary Table S1). Forty-nine of their mothers were included as the MoTS group. A random sample of 97 healthy mothers and 93 of their nonmalformed girls were included as a mother’s control group (MoCG) and a girl control group (GiCG), respectively. To increase the power of the study, we aimed for an approximate 1:2 ratio of cases to controls in both study groups. However, the study groups had slightly different sample sizes due to individuals who were unable to provide a blood sample at the time of study enrollment. Clinical data were obtained by reviewing medical files and interviewing mothers of patients with TS and those of controls. Standardized questions on demographic and periconceptional information included the following: Maternal age at conception (years), maternal education (years), self-reported periconceptional use of FA, self-reported pre-pregnancy weight (kg) and height (m), prepregnancy body mass index (kg/m2), and first-trimester exposure to hyperthermia.
With previous written informed consent, both case-control dyads were genotyped for the MTHFR C667T (rs1801133) and A1298C (rs1801131) variants using the TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA). PCRs were performed in a MicroAmp Fast Optical 96-well reaction plate and analyzed on an ABI 7900HT real-time PCR Instrument (Applied Biosystems, Foster City, CA). Sequence Detection System software version 2.4 was used for post-assay data analysis. All genotype analyses were performed by TaqManGenotyper software version 1.4.0.
Allele frequencies and Hardy–Weinberg equilibrium between groups were tested by χ2 test using the data of the control group. Using a multivariate logistic regression analysis, adjusted odds ratios (aOR) and 95% confidence intervals (95% CI) were calculated to estimate the strength of the association between TS and the MTHFR C677T and A1298C genotypes, assuming a dominant model for both the T and C alleles, respectively. Data analysis was performed using the SPSS software (IBM Corp. released 2019; IBM SPSS Statistics, Version 26.0; IBM Corp.; Armonk, NY).
Results
A total of 293 individuals were enrolled in the study. Supplementary Table S2 shows a comparison of maternal characteristics in the study sample. The mean values of maternal age (p = 0.02) and years of education (p = 0.002) were statistically higher in MoTS cases than in the MoCG. These and several other covariates were subsequently included as covariates in the logistic regression analysis.
Table 1 shows the genotypes and allele frequencies for the MTHFR C677T and A1298C variants in the study groups. The distribution for the C677T and A1298C genotypes was in Hardy–Weinberg equilibrium among the study groups, indicating that the sample was representative of the population. Multivariate logistic regression analysis showed no association between the frequency of homozygosity and heterozygosity for the MTHFR C677T and A1298C variants and TS in the studied sample of gTS and MoTS. Only the presence of the 677TT or 1298CC or 677CT/1298AC combined genotype was identified as a risk factor in the MoTS group (aOR = 2.5; 95% CI 1.0–5.9). No additional differences or associations were found.
Logistic Regression Analysis of the Genotype Distribution and Allele Frequencies of MTHFR c677t and a1298c Variants in Patients with Turner Syndrome and Their Mothers Versus Healthy Girls and Their Mothers of the Control Group
Calculated using the Haldane–Anscombe 1/2 correction.
The ORs are adjusted for maternal age, maternal education, use of FA before pregnancy, and first-trimester maternal hyperthermia.
677TT/1298CC and 677TT/1298AC haplotypes were absent in all subjects of the study groups.
aOR, adjusted odds ratio; CI, **Fisher’s exact test; FA, folic acid; TS, Turner syndrome.
Discussion
Maternal and infant MTHFR C677T and A1298C variants have been previously identified as genotypic risks for CND in TS (Santos et al., 2006; Oliveira et al., 2012; Ismail et al., 2015; Guo et al., 2022; Miljanović et al., 2022). However, positive associations between these MTHFR variants and the TS risk have only been observed in certain populations. These associations have been identified in TS populations where the risk alleles are less prevalent, for example, the MTHFR C677T variant has been found in Caucasians, among whom the frequency of the 677 T allele is relatively low (Santos et al., 2006; Miljanović et al., 2022). Similarly, the MTHFR A1298C variant has been found only in TS patients from countries such as Egypt, Brazil, Korea, or China, where the frequency of the MTHFR 1298C allele is notably low (Oliveira et al., 2008; Kim et al., 2011; Ismail et al., 2015; Nefic et al., 2018; Guo et al., 2022). The lack of compensatory genetic mechanisms, or complex interactions including folate or homocysteine status has been argued to explain the apparent population-specificity observed for these variants (Guéant-Rodriguez et al., 2006; Ismail et al., 2015).
It is well-known that the Mexican mestizo population has a very high frequency of the 677 T allele (52%) and of the 677TT genotype (26%) derived from its high Amerindian contribution, whereas the 1298 C allele (14%) and the 1298CC genotype (2%) are rare as a result of its minor European ancestry (Contreras-Cubas et al., 2016; Romero-Bolaño et al., 2024). Although our allele and genotype MTHFR frequencies are concordant with this, we do not find that homozygosity or heterozygosity for the variants C677T and A1298C in the MTHFR gene are associated with TS risk in the studied dyad of TS patients and their mothers (Table 1). Similar negative results reported in other studies (Oliveira et al., 2012; Bispo et al., 2015; Ismail et al., 2015) are against the previous hypothesis that supposed a population specificity for both MTHFR variants (Ismail et al., 2015), suggesting that these variants are not large contributors to the etiology of CND in TS in all populations. This leaves open the possibility that different gene–gene or gene–environment interactions could influence the association (Bispo et al., 2015; Ismail et al., 2015).
TS patients are more likely to have 45,X karyotypes with a retained maternal X than those with a retained paternal X (Hook and Warburton, 2014). Consequently, if chromosomal loss predominantly occurs in the father, maternal MTHFR variants would have a limited or null impact on paternal CND. However, since less than 1% of 45,X conceptuses survive to term, viability is hypothesized to depend on postzygotic mitotic events that generate a cryptic mosaic “rescue” cell line (Hook and Warburton, 2014). Thus, MTHFR variants could impact chromosome segregation during early embryonic mitosis instead of meiosis (Santos et al., 2006; Oliveira et al., 2008; Oliveira et al., 2012; Ismail et al., 2015). Although 39% of our cohort has a mosaic karyotype (Supplementary Table S1), which is indicative of postzygotic events, our findings do not support the association with studied MTHFR variants (Table 1).
The MTHFR C677T and A1298C genotype combination (677TT or 1298CC or 677CT/1298AC) found associated with the MoTS group (Table 1) was cautiously interpreted as a weak association, since its impact on reduction in MTHFR activity, FA, or homocysteine levels, or its effect on methylation patterns is unknown, and also because it requires further confirmation as other studies have not found such a finding (Ismail et al., 2015).
The relatively small sample size of our gTS and MoTS groups constitutes a limitation of this study. Despite the free distribution of FA supplements in Mexico, less than 5% of pregnant women use them. This could explain the very low proportion of women who reported taking FA supplementation before pregnancy in our sample (Supplementary Table S2). This nutritional disadvantage was included as a covariate in the logistic regression analysis. Additionally, we were unable to analyze the impact of FA, homocysteine, or B12 levels on our results, and therefore, this limitation should be considered in future studies.
Conclusions
Our findings do not indicate the association between the MTHFR variants and TS risk in the studied gTS and their mothers. This highlights the need for future studies with large and multiethnic cohorts, considering the effect of other complex interactions.
Footnotes
Acknowledgments
The authors would like to thank the participants for cooperating in this study. They are also grateful to the CRIAC team for their enthusiastic support.
Authors’ Contributions
I.M.R.-F. performed the experiments, recruited the subjects, drew blood samples, and collected the clinical data. L.F.-C., L.B.-M., A.C.-R., S.A.B.-J., and I.C.-Q. performed the experiments and reviewed the article. A.M.C.-M. collected the clinical data and reviewed the article. J.R.C.-R. designed and supervised the study and wrote, reviewed, and edited the article. All authors have read and agreed to the published version of the article.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
No funding was received for this article.
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