Association between MTHFR C677T/A1298C and susceptibility to autism spectrum disorders: a meta-analysis

Background Autism spectrum disorder (ASD) is becoming increasingly prevalent of late. Methylenetetrahydrofolate reductase (MTHFR) has a significant role in folate metabolism. Owing to the inconsistencies and inconclusiveness on the association between MTHFR single nucleotide polymorphism (SNP) and ASD susceptibilities, a meta-analysis was conducted to settle the inconsistencies. Methods For this meta-analysis, a total of 15 manuscripts published up to January 26, 2020, were selected from PubMed, Google Scholar, Medline, WangFang, and CNKI databases using search terms “MTHFR” OR “methylenetetrahydrofolate reductase” AND “ASD” OR “Autism Spectrum Disorders” OR “Autism” AND “polymorphism” OR “susceptibility” OR “C677T” OR “A1298C”. Results The findings of the meta-analysis indicated that MTHFR C677T polymorphism is remarkably associated with ASD in the five genetic models, viz., allelic, dominant, recessive, heterozygote, and homozygote. However, the MTHFR A1298C polymorphism was not found to be significantly related to ASD in the five genetic models. Subgroup analyses revealed significant associations of ASD with the MTHFR (C677T and A1298C) polymorphism. Sensitivity analysis showed that this meta-analysis was stable and reliable. No publication bias was identified in the associations between MTHFRC677T polymorphisms and ASD in the five genetic models, except for the one with regard to the associations between MTHFRA1298C polymorphisms and ASD in the five genetic models. Conclusion This meta-analysis showed that MTHFR C677T polymorphism is a susceptibility factor for ASD, and MTHFR A1298C polymorphism is not associated with ASD susceptibility.

occur as a result of pathophysiological and environmental factors. Folate/homocysteine (Hcy) levels act as a risk factor in ASD [14,15], indicating the involvement of methylenetetrahydrofolate reductase (MTHFR) in ASD. Therefore, MTHFR has been the focal point of investigation on ASD, as inheritance validates the pathophysiological mechanism of ASD [16][17][18].
Correlations between single nucleotide polymorphisms (C677T and A1298C) and susceptibility to ASD are still debatable. A correlation between MTHFR C677T polymorphism and a higher susceptibility to ASD has been reported by Boris et al. [22] among Caucasian children [27]. Guo et al. [31] evidenced that MTHFR C677T polymorphism is a risk factor for ASD among Chinese Han children [31]. El-baz et al. [32] recognized a significant correlation between MTHFR C677T polymorphisms and ASD among Egyptian children [32]. Nonetheless, Dos Santos et al. [28] found no correlation between MTHFR C677T polymorphism and ASD [28]. Studies by Khalil et al. [33] and El-baz et al. [32,34] describe MTHFR A1298C polymorphism to represent a risk factor in correlation with ASD among Egyptian children. On the contrary, Mohammad et al. [35] evidenced that MTHFR A1298C polymorphism variant allele has no link with any independent risk of ASD [35]. In this metaanalysis, updated articles were gathered [26,32,36] to authenticate correlations between MTHFR polymorphism (C677T/A1298C) and susceptibility to ASD.

Search strategy and identification of studies
Scientific literature published before January 26, 2020, in PubMed, Embase, Web of Science, Medline, WanFang datebase, and CNKI database were searched using specific search terms (Supplement file 1). The equivalent Chinese terms were used in the Chinese databases. Moreover, we retrieved related articles from the selected literature references to replenish data that had not been identified in the initial search.
All full-text literature were scrutinized to determine whether the papers to be included.

Selection criteria
The following criteria had to be satisfied by the studies to be incorporated in this meta-analysis: (1) Original studies on the correlation between MTHFR polymorphism (C677T/A1298C) and ASD; (2) Cohort or casecontrol designs; (3) All genotype frequency information is available; (4) Diagnostic criteria of ASD described in the Diagnostic and Statistical Manual of Mental Disorders (4th or 5th edition) [37,38], and/or Childhood Autism Rating Scale [39]. Certain earlier papers referred to the Manual of Mental Disorders (3rd edition) [40]. The exclusion criteria comprised the following: (1) Researches on the correlation between MTHFR polymorphism (C677T/ A1298C) and ASD that are not original; (2) Studies that lack data and complete information; (3) Replicated studies; (4) Review studies.

Data extraction
Two investigators, namely, Yan Li and Shuang Qiu, extracted all the relevant data with the help of a standardized protocol and data collection form. From every qualified study, data such as the name of the first author, year of publication, country, study population (ethnicity), study design, the definition of ASD, sample size of cases and controls, genotyping method, genotype information, and allele frequencies were gathered and documented. Disparities in the study selection were resolved through discussion or consensus with the third investigator (Yawen Liu). The corresponding authors of articles with missing data were emailed for the required data.

Statistical analysis
Odds ratio (OR) and 95% confidence intervals (CI) were deduced to analyse how strongly MTHFR (C677T/ A1298C) polymorphism and the risk of ASD were correlated in the five genetic models, viz., allelic, dominant, recessive, heterozygote, and homozygote. Heterogeneity among studies was assessed through Q-test and I 2 . Random effects model (DerSimonian-Laird methods) [41] was selected to pool data and in case of substantial heterogeneity (Ph < 0.05 and I 2 > 50%); else, fixed effect model (Mantel-Haenszel methods) [42] was chosen. Furthermore, subgroup analyses were stratified according to the state with mandatory fortification of folate, population, sample source, and Hardy-Weinberg equilibrium (HWE). The included studies were tested for HWE in the control group utilizing Chi-square tests. Besides, the stability of the results was tested by performing a sensitivity analysis with the sequential omission of each study. To evaluate the potential publication bias in this metaanalysis, Begg's funnel plot and Egger's test were conducted. Stata version 12.0 (StataCorp LP, College Station, TX, USA) was used to evaluate all analyses, and p < 0.05 was considered to be statistically significant.

Overall results
Upon literature search and critical screening, about 15 studies from 125 articles were included in this metaanalysis, as already discussed in the Methods section ( Fig. 1). A total of 2609 cases and 7496 controls were enrolled from the 15 articles published on the correlation between MTHFR C677T polymorphism and ASD susceptibility. Of those, only nine articles that included 1961 cases and 1652 controls qualified for the evaluation of the link between MTHFR A1298C and ASD as per the selection criteria. The characteristics of each primary study are summarized and presented in Tables 1 and 2.

Association between MTHFR A1298C polymorphism and ASD
Random effect model (P h < 0.05 or I 2 ≥ 50%) was utilized, and no significant correlation between MTHFR A1298C polymorphism and ASD susceptibility in the five genetic models was identified (allelic, dominant, recessive, heterozygote, and homozygote; all p > 0.05; Table 4, Fig.   2b). As per the subgroup analyses, MTHFR A1298C polymorphism was found to be associated with ASD susceptibility among the states without mandatory fortification of folate: allelic model (C vs. A: OR = 1.84, 95% CI = 1.08-3.14, p < 0.05) and dominant model (CC + AC vs. AA: OR = 2.45, 95% CI = 1.16-5.15, p < 0.05). No significant correlation between MTHFR A1298C polymorphism and ASD susceptibility under the other genetic models in any subgroup was found (all p > 0.05) ( Table 4).

Sensitivity analysis and publication bias
The stability of the findings was evaluated through sensitivity analysis conducted by sequentially omitting each study, demonstrating that this meta-analysis is relatively        Fig. 4).
In the present meta-analysis, eight of the selected articles [18,22,32,35,36,43,44,47,50] [32] revealed that MTHFR A1298C polymorphism represented a risk factor in association with ASD. This disagreement may be caused by small samples in the study.
There are several limitations for this study. First, the subgroup analyses of environmental risk factors, sex, and gene-environment interactions were not performed owing to insufficient information. Second, this metaanalysis was mainly focused on Caucasians and Asians, thus limiting the generalization of the findings to other ethnicities. Third, in agreement with the findings of Frustaci et al. [24], Pu et al. [25] and Rai et al. [26], heterogeneity exists in this exploration. Fourth, publication bias was found in the association between MTHFR A1298C polymorphisms and ASD risk.

Conclusion
To conclude, this meta-analysis confirms that C677T polymorphism of MTHFR is remarkably linked with ASD risk. Nevertheless, the findings agree that the A1298C polymorphism of MTHFR is not significantly correlated with ASD. Exploring gene-gene and geneenvironment interactions could throw more light on the genetic link between MTHFR variants and ASD risk.