Reassessment of MTRR rs1801394 Polymorphism and Neural Tube Defects Risk

To editor: Neural tube defects (NTDs) occur in the central nervous system and can be caused by genetic and environmental factors. NTDs are serious congenital disabilities resulting from incomplete closure of the neural tube during early pregnancy, affecting the brain and spinal cord. The most common NTDs are spina bifida and anencephaly, with folic acid deficiency being a major risk factor. According to studies, congenital problems typically manifest within the first 28 days of pregnancy. NTDs are the second most frequent congenital disabilities in humans, occurring at a rate of 0.5 to 5.0 per 1000 births.1 The methionine synthase reductase (MTRR) gene encodes the enzyme methionine synthase reductase, which regenerates functional methionine synthase through reductive methylation using S-adenosylmethionine. It acts as an activation partner for methionine synthase (MTR), assisting in the conversion of homocysteine (Hcy) to methionine with the help of cobalamin, and subsequently regenerating MTR.2 The 66-base pair region of the MTRR gene harbors an A-to-G transition (66A > G), resulting in the substitution of isoleucine with methionine (I22M) and potentially leading to MTRR deficiency in patients.3 The flavin mononucleotide-binding domain of the MTRR enzyme interacts with MTR, disrupting its attachment to the MTR-cobalamin complex and slowing Hcy remethylation.4 This study addresses a gap in the literature by identifying a 2013 meta-analysis5 that reported the MTRR A66G polymorphism significantly contributes to NTDs. An updated meta-analysis6 in 2015 confirmed these findings across diverse populations. However, both studies relied on maternal samples from mothers of children affected by NTDs. In contrast, our study includes both parents and their NTD-affected offspring as the study population, providing a more comprehensive analysis. This forms the rationale for our research. We conducted a systematic search for studies on NTDs, MTRR, genes, polymorphisms, single nucleotide polymorphisms, and genetic variations using PubMed, Google Scholar, and Embase. The meta-analysis included only English-language articles meeting specific criteria: case-control designs focused on the association between the MTRR gene and NTDs in parents and fetal health, providing data on genotype and allele frequencies, 95% confidence interval (CI), and P for odds ratio (OR). The Newcastle-Ottawa Scale was used for quality assessment. Studies that did not meet these criteria or lacked sufficient data were excluded, and data extraction was conducted systematically (Supplementary Fig. 1, https://links.lww.com/MFM/A71). Information on allelic frequencies and genotypes for case and control participants was collected from the reviewed publications. When genotypic data were unavailable, we estimated them using allelic frequencies. Studies lacking meaningful data from both groups were excluded. We extracted information from each study, including the PubMed ID, study design, publication year, first author, sample size, ethnicity, Hardy-Weinberg equilibrium (HWE) score, and language. Statistical analyses were performed using Review Manager 5.4, with a significance threshold of P < 0.05. Heterogeneity was assessed using the Q statistic and I2 metric, with P < 0.1 considered significant. A random-effects model was used to calculate the OR and 95% CI for the association between gene polymorphism and NTDs. The overall OR and CI were visualized using a forest plot, and potential publication bias was examined using a funnel plot. The analysis of the MTRR rs1801394 polymorphism in relation to NTDs was conducted using five genetic models: allelic (A vs. G; I2 = 72%), homozygote (AA vs. GG; I2 = 59%), heterozygote (AG vs. GG; I2 = 0%), dominant (AG + GG vs. AA; I2 = 76%), and recessive (AA + AG vs. GG; I2 = 0%). Based on heterogeneity (I2), the random-effects model yielded the following results: for the allelic model, OR = 0.89, 95% CI: 0.64, 1.23 (P = 0.46); for the homozygote model, OR = 1.35, 95% CI: 0.71, 2.54 (P = 0.36); and for the dominant model, OR = 1.10, 95% CI: 0.66, 1.85 (P = 0.71). In the fixed-effects analysis, the heterozygote model showed OR = 0.84, 95% CI: 0.61, 1.17 (P = 0.31), and the recessive model demonstrated OR = 1.29, 95% CI: 0.94, 1.77 (P = 0.11). These results apply to both children with NTDs and their parents, as illustrated in Supplementary Figure 2, https://links.lww.com/MFM/A71. All heterogeneity results were calculated using the Mantel-Haenszel method. Supplementary Figure 3, https://links.lww.com/MFM/A71 shows that Begg’s and Egger’s tests indicated no evidence of publication bias. Supplementary Table 1, https://links.lww.com/MFM/A71 provides detailed information on all included studies, including case and control characteristics. A sensitivity analysis was performed using the online tool MetaGenyo to assess variations in the MTRR gene (rs1801394). This analysis examined the impact of removing individual studies, including those violating HWE, on the pooled ORs. As shown in Supplementary Figure 4, https://links.lww.com/MFM/A71, the original findings remained consistent, and no single study significantly influenced the overall results. In this study, we investigated the relationship between the MTRR gene and NTDs. Our analysis revealed no significant association between the MTRR rs1801394 polymorphism and NTD risk across various genetic models. The ORs were close to 1, with P > 0.05, indicating no statistically significant risk. No significant association was found in the heterozygote model either. While some models exhibited high heterogeneity, the results were consistent across studies, with no evidence of publication bias. Although previous studies on the MTRR gene and NTDs have reported mixed and sometimes controversial findings—ranging from significant associations in the Han Chinese population to no apparent role in Ethiopian mothers—our meta-analysis concluded that there is no substantial relationship between this gene and NTDs. Further research with larger and more consistent study populations is needed to validate these findings and clarify the potential role of this polymorphism in NTD risk. Acknowledgment Thanks to the Chettinad Academy of Research and Education for their continuous support and encouragement. Funding None. Author Contributions Jethendra Kumar Muruganantham wrote the contents and edited the figures and tables of this manuscript. Ramakrishnan Veerabathiran designed the study, edited the contents of this manuscript, and approved the manuscript for submission. All authors read and approved the final manuscript. Conflicts of Interest None. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files.

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