Mirna Edith Morales-Marín1
Omar Náfate-López2Amalia Guadalupe Gómez-Cotero3
Miguel Angel Cid-Soto4
Humberto Nicolini1*
Xochitl Helga Castro-Martínez1*- 1Laboratorio de Genómica de las Enfermedades Psiquiátricas y Neurodegenerativas, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
- 2Unidad de Neuropsiquiatria Infantil, "Dr Manuel Velasco Suárez”, Hospital de Especialidades Pediátricas del Centro Regional de Alta Especialidad, Tuxtla Gutierrez, Chiapas, Mexico
- 3Centro de Investigación en Ciencias de la Salud, Unidad Santo Tomás, Instituto Politécnico Nacional, Mexico City, Mexico
- 4Consorcio de Oncogenómica, Instituto Nacional de Medicina Genómica, Mexico City, Mexico
Using genetic approaches to study autism spectrum disorder (ASD) is essential to understanding the etiology of the condition. The C677T variant has emerged as a risk factor, and here we present the first association study of this variant in a Mexican population with ASD. Our objective was to assess the variant MTHFR C677T (rs1801133) in a group of Mexican patients with ASD through a case-control association analysis. We found no significant association of MTHFR C677T and ASD, with no rate differences between cases and controls (C vs T: odds ratio = 0.9698, 95% confidence interval = 0.7773–1.21, P = 0.7858). Results of this and other studies evaluating the link between ASD and this variant have been controversial. Our findings suggest that other ancestry-related factors may play a role.
Introduction
Autism spectrum disorder (ASD) is described by the Diagnostic and Statistical Manual of Mental Disorders, 5th ed., as a group of neurodevelopmental disorders characterized by repetitive patterns of behavior and difficulties in establishing communication and social interaction. The global prevalence is 1:160 live births, and ASD can be classified into different levels of severity based on previously described activities or interests (1, 2). ASD is a group of entities that is increasing, the CDC has reported a prevalence of 1:36 children have ASD at the age of 8 years (3). A single study has been reported in Mexico with epidemiological data on this condition, with a prevalence of 1:116 individuals in the City of León, Guanajuato (4).
This group of disorders is called a spectrum because of the range of symptoms and their intensity and co-occurrence or not with intellectual disability. Symptoms begin to manifest at very early ages. The exact etiology remains poorly understood but involves both genetic and environmental factors (5). Several risk factors have been described, including paternal age, intrauterine exposure to maternal infection or some medications, intestinal microbiota profile, cesarean section, environmental contaminants, among others (6). As well as certain protective factors such as the intake of vitamin D, folic acid, breastfeeding (6–8). Early detection of ASD and timely intervention are important for supporting personal development, facilitating independence, and reducing family and socioeconomic impact. Currently, no molecular diagnostic testing is available for ASD, and diagnosis is made using psychiatric scales.
The human Methylenetetrahydrofolatereductase gene (MTHFR), located in chromosome region 1p36.3, encodes an enzyme involved in folate metabolism. Several single nucleotide variants (SNVs) on MTHFR have been linked to enzyme defects and different human diseases, including occlusive vascular disease, neural tube defects, colon cancer, and acute leukemia (9–12). Decreased MTHFR protein activity may affect the one-carbon metabolism cycle, which in turn might impair cortical and hippocampal neurogenesis during development and affect brain maturation and function (13). It has been reported that patients with ASD show a deficiency of the enzyme methylenetetrahydrofolate reductase and that after supplementation with traditional psychopharmacological treatment they had improvement in symptoms (14). The C677T SNV, among the most studied and clinically significant MTHFR variants, is located in exon 4 and involves the replacement of a cytosine by a thymine, leading to the substitution of valine for alanine at codon 222 (Zhang YX et al., 2022). Although previous reports have found an association of C677T SNVs with ASD (15–19) (Pu D et al., 2013; Rai V 2016; Wei H et al., 2021; Li Y et al., 2020; Sadeghiyeh T et al., 2019), most of these studies have been performed in populations with European or Asian ancestry, and admixed Latino American populations are strongly underrepresented. To bridge this gap, here we evaluated the association of the C677T SNV with ASD in a Mexican population.
Material and methods
Sample population
The study included 179 patients with ASD selected through the CMIGA consortium (Consorcio Mexicano de Investigación en Genética del Autismo) (20) and 1483 unaffected individuals. ASD participants were ages 2–18 years, and their parents agreed to their participation in the study. All ASD participants and their parents underwent an Autism Diagnostic Interview (21) and signed an informed consent and assent for minors. The control group was composed of 1483 individuals, which belong to the MxGDAR (Mexican Genomic Database for Addiction Research) cohort. This cohort is an epidemiological sample composed of 3393 healthy individuals recruited in Mexico from the 2016 National Survey of Drug, Alcohol, and Tobacco Use; where the 1483 individuals used as control subjects showed nor psychiatric symptoms neither consumption of risky substances (22). Thus, the use of this 1483 samples as control group discard any bias introduce if apparently healthy subjects were used as control group. Although the MxGDAR cohort overrepresented female individuals, our analysis showed no significant differences in the genotype and allele frequencies of rs1801133 variant. This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics and Research Committee at the INMEGEN. The distribution of cases and controls is shown in Table 1.
Table 1. Distribution of cases and controls.
DNA extraction and genotyping
Genomic DNA was extracted from blood lymphocytes or mouth swab using a commercial kit following the manufacturer’s protocol (Qiagen, Puregene). The genotype of the MTHFR C677T polymorphism in the studied sample was obtained from genotyping data generated with the Illumina Infinium® PsychArray-24 v1.0 microarrays (Illumina, San Diego, California, USA) in the cohorts MxGDAR and CMIGA. This array contains 593,260 gene variants associated with predisposition and risk for developing psychiatric or neurodevelopmental conditions such as autism.
Statistical analysis
Statistical analysis to assess the relationship between MTHFR C677T and ASD was performed using Plink v1.07 software (23). The Chi square test was used for group comparison between ASD participants and unaffected controls. Odds ratios and 95% confidence intervals were calculated, and p < 0.05 was set as indicating significance.
Results
The most frequent genotype for both groups was CT, at 49% for ASD participants and 46% for controls (Table 2). No significant differences were observed between the groups for the MTHFR C677T variant (C vs T: OR = 0.9698, 95% CI = 0.7773–1.21, p = 0.7858) (Table 3). The control group results were consistent with Hardy–Weinberg equilibrium for the examined gene variant (p = 0.01219).
Table 2. MTHFR C677T genotype and allelic distribution in patients with ASD and control groups.
Table 3. Comparation of C677T allele frequency between ASD individuals and healthy controls.
Discussion
A recent meta-analysis comprising 2609 individuals with ASD and 7496 unaffected participants from 15 previously published studies found a significant association between the MTHFR C677T variant and ASD in all genetic models evaluated (i.e., allelic, heterozygote, homozygote, dominant, and recessive), highlighting this variant as an important genetic risk factor for ASD (18). That meta-analysis included populations of European, East Asian, South Asian, Latin American, and African ancestry. An independent meta-analysis found a significant association of the MTHFR C677T variant with ASD in populations of European ancestry and in a population with Asian ancestry (16). Another meta-analysis evaluating the association of six candidate genes with ASD (MTHFR, SLC25A12, OXTR, RELN, 5-HTTLPR, and SHANK) identified the C677T variant as a consistent risk factor for ASD (17).
Similar to our findings, however, a previous study in a Han Chinese population of 201 children with ASD and 200 unaffected controls showed no significant association between MTHFR C677T and ASD or ASD severity (24). Likewise, this variant was not significantly associated with ASD in two independent studies in children, one with 151 ASD participants and 100 unaffected individuals in Brazil, and the other with 98 ASD participants and 70 unaffected individuals in Turkey (25, 26).
The frequency of the C677T variant in public genomic databases such as dbSNP changes depending on ethnic group. Its frequency in admixed Latino American groups is 0.45, whereas in the European population it is 0.34, and in South Asian individuals, it is 0.16 (27). Allele frequency can be modified by environmental and geographical factors, and ASD has a multifactorial etiology, so that the association of the C677T variant with ASD could be strongly dependent on other factors related to the ancestry of the population. Likewise, Kang J. et al., 2024 observed a genetic correlation between inflammatory bowel disease and osteoporosis in European populations but not in East Asian populations (28). Taking together, these findings indicate that the presence of other genetic factors, including genetic dominance, pleiotropy, fitness trade-offs, sign epistasis, and genetic linkage between loci interfere in the association of genetic variants with pathological conditions. Supporting this hypothesis, a study carried out on Mexican Mestizos and a diverse group of Native Americans from Mexico reported a frequency similar to that observed in our analysis, as well as the highest frequency worldwide for the variant allele in Mexican Amerindians (29). An important limitation of our study is the small sample size, and our findings must be validated using a larger population.
Data availability statement
The data presented in the study are deposited in the EVA repository, accession number PRJEB101865.
Ethics statement
The studies involving humans were approved by ethics committee, Instituto Nacional de Medicina Genómica. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.
Author contributions
XC: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. MM: Funding acquisition, Investigation, Writing – review & editing. ON: Resources, Writing – review & editing. AG: Resources, Writing – review & editing. HN: Conceptualization, Project administration, Supervision, Writing – review & editing. MC: Formal Analysis, Software, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by CONACYT FOSSIS 262115 and INMEGEN.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 5th Ed. Ciudad de Méxio, México: American Psychiatric Association (2013).
2. Home . Available online at: https://www.who.int/home.
3. Maenner MJ, Warren Z, Williams AR, Amoakohene E, Bakian AV, Bilder DA, et al. Prevalence and characteristics of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2020. MMWR Surveill Summ. (2023) 72:1–14. doi: 10.15585/mmwr.ss7202a1, PMID: 36952288
4. Fombonne E, Marcin C, Manero AC, Bruno R, Diaz C, Villalobos M, et al. Prevalence of autism spectrum disorders in guanajuato, Mexico: the leon survey. J Autism Dev Disord. (2016) 46:1669–85. doi: 10.1007/s10803-016-2696-6, PMID: 26797939
5. Lord C, Brugha TS, Charman T, Cusack J, Dumas G, Frazier T, et al. Autism spectrum disorder. Nat Rev Dis Primers. (2020) 6:5. doi: 10.1038/s41572-019-0138-4, PMID: 31949163
6. Kreiman BL and Boles RG. State of the art of genetic testing for patients with autism: A practical guide for clinicians. Semin Pediatr Neurol. (2020) 34:100804. doi: 10.1016/j.spen.2020.100804, PMID: 32446438
7. Emberti Gialloreti L, Mazzone L, Benvenuto A, Fasano A, Alcon AG, Kraneveld A, et al. Risk and protective environmental factors associated with autism spectrum disorder: evidence-based principles and recommendations. J Clin Med. (2019) 8:217. doi: 10.3390/jcm8020217, PMID: 30744008
8. Ristori MV, Mortera SL, Marzano V, Guerrera S, Vernocchi P, Ianiro G, et al. Proteomics and Metabolomics Approaches towards a Functional Insight onto AUTISM Spectrum Disorders: Phenotype Stratification and Biomarker Discovery. Int J Mol Sci. (2020) 21:6274. doi: 10.3390/ijms21176274, PMID: 32872562
9. Xie SZ, Liu ZZ, Yu JH, Liu L, Wang W, Xie DL, et al. Association between the MTHFR C677T polymorphism and risk of cancer: evidence from 446 case-control studies. Tumour Biol. (2015) 36:8953–72. doi: 10.1007/s13277-015-3648-z, PMID: 26081619
10. Li Y, Pei YX, Wang LN, Liang C, Tang YL, Zhang XL, et al. MTHFR-C677T gene polymorphism and susceptibility to acute lymphoblastic leukemia in children: A meta-analysis. Crit Rev Eukaryot Gene Expr. (2020) 30:125–36. doi: 10.1615/CritRevEukaryotGeneExpr.2020033468, PMID: 32558492
11. Marques M, Alves F, Leitão M, Rodrigues C, and Ferreira JT. Methylenetetrahydrofolate reductase polymorphisms as risk factors for retinal venous occlusive disease: A literature review. Eur J Ophthalmol. (2021) 31:884–91. doi: 10.1177/11206721211000647, PMID: 33715478
12. Tabatabaei RS, Fatahi-Meibodi N, Meibodi B, Javaheri A, Abbasi H, Hadadan A, et al. Association of fetal MTHFR C677T polymorphism with susceptibility to neural tube defects: A systematic review and update meta-analysis. Fetal Pediatr Pathol. (2022) 41:225–41. doi: 10.1080/15513815.2020.1775734, PMID: 32536242
13. Zhang Y-X, Yang L-P, Gai C, Cheng C-C, Z-y G, Sun H-M, et al. Association between variants of MTHFR genes and psychiatric disorders: A meta-analysis. Front Psychiatry. (2022) 13:976428. doi: 10.3389/fpsyt.2022.976428, PMID: 36061291
14. Khan S and Naeem A. MTHFR deficiency in biological siblings diagnosed with autism and attention-deficit hyperactivity disorder (ADHD): A report of two cases. Cureus. (2023) 15:e36294. doi: 10.7759/cureus.36294, PMID: 37073207
15. Pu D, Shen Y, and Wu J. Association between MTHFR gene polymorphisms and the risk of autism spectrum disorders: a meta-analysis. Autism Res. (2013) 6:384–92. doi: 10.1002/aur.1300, PMID: 23653228
16. Rai V. Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility. Metab Brain Dis. (2016) 31:727–35. doi: 10.1007/s11011-016-9815-0, PMID: 26956130
17. Wei H, Zhu Y, Wang T, Zhang X, Zhang K, and Zhang Z. Genetic risk factors for autism-spectrum disorders: a systematic review based on systematic reviews and meta-analysis. J Neural Transm (Vienna). (2021) 128:717–34. doi: 10.1007/s00702-021-02360-w, PMID: 34115189
18. Li Y, Qiu S, Shi J, Guo Y, Li Z, Cheng Y, et al. Association between MTHFR C677T/A1298C and susceptibility to autism spectrum disorders: a meta-analysis. BMC Pediatr. (2020) 20:449. doi: 10.1186/s12887-020-02330-3, PMID: 32972375
19. Sadeghiyeh T, Dastgheib SA, Mirzaee-Khoramabadi K, Morovati-Sharifabad M, Akbarian-Bafghi MJ, Poursharif Z, et al. Association of MTHFR 677C>T and 1298A>C polymorphisms with susceptibility to autism: A systematic review and meta-analysis. Asian J Psychiatr. (2019) 46:54–61. doi: 10.1016/j.ajp.2019.09.016, PMID: 31614268
20. Morales-Marín ME, Castro Martínez XH, Centeno Cruz F, Barajas-Olmos F, Náfate López O, Gómez Cotero AG, et al. Differential DNA methylation from autistic children enriches evidence for genes associated with ASD and new candidate genes. Brain Sci. (2023) 13:1420. doi: 10.3390/brainsci13101420, PMID: 37891789
21. Lord C, Rutter M, and Le Couteur A. Autism Diagnostic Interview-Revised: a revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders. J Autism Dev Disord. (1994) 24:659–85. doi: 10.1007/BF02172145, PMID: 7814313
22. Martínez-Magaña JJ, Genis-Mendoza AD, Villatoro Velázquez JA, Bustos-Gamiño M, Juárez-Rojop IE, Tovilla-Zarate CA, et al. Genome-wide association study of psychiatric and substance use comorbidity in Mexican individuals. Sci Rep. (2021) 11:6771. doi: 10.1038/s41598-021-85881-4, PMID: 33762635
23. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. (2007) 81:559–75. doi: 10.1086/519795, PMID: 17701901
24. Zhang Z, Yu L, Li S, and Liu J. Association study of polymorphisms in genes relevant to vitamin B12 and folate metabolism with childhood autism spectrum disorder in a han chinese population. Med Sci Monit. (2018) 24:370–6. doi: 10.12659/msm.905567, PMID: 29348398
25. dos Santos PA, Longo D, Brandalize AP, and Schüler-Faccini L. MTHFR C677T is not a risk factor for autism spectrum disorders in South Brazil. Psychiatr Genet. (2010) 20:187–9. doi: 10.1097/YPG.0b013e32833a2220, PMID: 20440228
26. Sener EF, Oztop DB, and Ozkul Y. MTHFR gene C677T polymorphism in autism spectrum disorders. Genet Res Int. (2014) 2014:698574. doi: 10.1155/2014/698574, PMID: 25431675
27. Sherry ST, Ward M, and Sirotkin K. dbSNP-database for single nucleotide polymorphisms and other classes of minor genetic variation. Genome Res. (1999) 9:677–9. doi: 10.1101/gr.9.8.677, PMID: 10447503
28. Kang J, Wu X, Li Y, Zhao S, Wang S, and Yu D. Association between inflammatory bowel disease and osteoporosis in European and East Asian populations: exploring causality, mediation by nutritional status, and shared genetic architecture. Front Immunol. (2024) 15:1425610. doi: 10.3389/fimmu.2024.1425610, PMID: 39136019
29. Contreras-Cubas C, Sánchez-Hernández BE, García-Ortiz H, Martínez-Hernández A, Barajas-Olmos F, Cid M, et al. Heterogenous distribution of MTHFR gene variants among mestizos and diverse amerIndian groups from Mexico. PloS One. (2016) 11:e0163248. doi: 10.1371/journal.pone.0163248, PMID: 27649570
Keywords: MTHFR, C677T, genetic variant, ASD, Mexican population
Citation: Morales-Marín ME, Náfate-López O, Gómez-Cotero AG, Cid-Soto MA, Nicolini H and Castro-Martínez XH (2025) MTHFR C677T is not associated with autism spectrum disorder in a Mexican cohort. Front. Psychiatry 16:1452940. doi: 10.3389/fpsyt.2025.1452940
Received: 21 June 2024; Accepted: 31 March 2025;
Published: 17 November 2025.
Edited by:
Antonio M. Persico, University of Modena and Reggio Emilia, ItalyReviewed by:
Alfredo Bellon, Penn State Milton S. Hershey Medical Center, United StatesDaniel Campbell, Michigan State University, United States
Rossella Tupler, University of Modena and Reggio Emilia, Italy
Copyright © 2025 Morales-Marín, Náfate-López, Gómez-Cotero, Cid-Soto, Nicolini and Castro-Martínez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xochitl Helga Castro-Martínez, eGNhc3Ryb0Bpbm1lZ2VuLmdvYi5teA==; Humberto Nicolini, aG5pY29saW5pQGlubWVnZW4uZ29iLm14