The association of MTHFR A1298C polymorphism with cervical cancer: An Updated Meta-Analysis Involving 2835 Subjects

Background Published data have reported the relationships between MTHFR A1298C polymorphisms and cervical cancer susceptibility. However, the conclusions of these ndings lack consistency. Methods A comprehensive literature search was performed using Web of Science, PubMed, EMBASE, Cochrane library, Wan Fang and CNKI databases. The pooled odds ratios (ORs) with 95% condence intervals (CIs) were used to evaluate the correlation of MTHFR A1298C polymorphism and cervical cancer risk. Fixed-effects or random effects models was adopted according to heterogeneity test. Results A total of nine studies (1145 cases and 1690 controls) were included in this meta-analysis. Pooled data revealed that MTHFR A1298C polymorphism was signicantly associated with an increased risk of cervical cancer in the allele model (P=0.028); the recessive model (P=0.028); and the heterozygous model (P=0.031). Conclusions Our results revealed that MTHFR A1298C polymorphism was associated with risk of cervical cancer.


Background
Cervical cancer is a worldwide public health problem. In spite of being preventable, this disease is still the fourth most common type of cancer among women and the seventh among the general population [1]. Several certain factors appeared to be related with an increased risk of developing cervical cancer are early start of sexual life, multiple pregnancies, diet, smoking, and infection with human papilloma viruses (HPV), among which HPV infection was prominent [2,3]. High-risk subtypes of the human papillomavirus (HPV) cause almost all cervix cancers [4]. However, this does not mean that all HPV infection ultimately cause cervix cancer. It was reported that a single same type HPV infection can only lead to cell immortalization and cannot induce malignant transformation of epithelial cells. HPV infection, with host internal and external environment factors, such as genetic polymorphism leading to susceptibility, the interaction of these factors causes an accumulation of cell damage that eventually leads to cancer.
Folic acid, as a water-soluble B vitamin [5], is essential for proper cellular function. Some studies have identi ed that levels of folate, play a role in cervical carcinogenesis [6,7]. Folic acid is considered to be critical for the biosynthesis of purines and thymidylates which are key factors for normal DNA replication and repair as well as DNA methylation [8]. Once de ciency, it would impair cellular physiology and increase the risk of cervical cancer if it was accompanied by a persistent HPV infection. In addition to exogenous supplements, one of the most vital enzymes as intrinsic factor, named methylenetetrahydrofolate reductase (MTHFR), affect the level of folic acid. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate acid into 5methyltetrahydrofolate acid which is a major circulating form of folate acid. However, the activity of the enzyme was affected by gene polymorphism which was associated with reduced enzyme activity and folate de ciency [9]. There are 2 common types: one is a C-to-T transition at nucleotide 677 (677 C/T) in exon 4, resulting in an alanine-to-valine substitution that affects the catalytic domain of the enzyme, leading to reduced enzyme activity; another is an A-to-C transversion at position 1298 in exon 7 (1298A /C), resulting in a substitution of glutamate with alanine at codon 429 [10]. The later will also lead to low activity to a lesser extent, but the summary is lacking. Therefore, to address this gap, in this study, we assessed and explored the association between MTHFR 1298 A/C with the susceptibility of cervical cancer. Elucidation of an association, if any, might be informative regarding the hypothesis that impaired folate metabolism due to MTHFR polymorphism, resulting in low folate concentration, plays an intensi ed role in the occurrence of cervical cancer with HPV infection.

Search strategy
The meta-analysis was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [11]. Two authors independently searched electronic databases (Web of Science, PubMed, Embase, Cochrane library, WanFang and CNKI from inception to May 2019) for identify relevant articles. Electronic databases were searched using the keywords: "MTHFR", "A1298C", "rs1801131", "polymorphism", "SNP", "variant" and "cervical cancer". The reference lists of the retrieved articles were also searched carefully to identi ed other relevant publications.

Data extraction
Two authors independently extracted the necessary data from each eligible article, and disagreements were reconciled by third author. The following data form each study were extracted: rst author's name, year of publication year, country, ethnicity of population, genotyping methods, number of cases and control, genotyping data, Hardy-Weinberg equilibrium (HWE) in controls.

Quality assessment
The Newcastle-Ottawa Quality Scale (NOS) was used by two authors to evaluate the quality of the studies [12]. A1298C polymorphism and cervical cancer. HWE of the control populations was calculated with Chi-square test and the signi cance level was de ned as p<0.05. The pooled ORs with 95% con dence intervals (CIs) were used to evaluate the correlation of MTHFR A1298C polymorphism and cervical cancer risk. P value < 0.05 was considered statistically signi cant. Heterogeneity across studies was assessed by the Q and I 2 tests. If heterogeneity was detected (P <0.05, I 2 > 50%), the random-effects model was be used to pool ORs. Otherwise, the xed-effects model was adopted. Publication bias was assessed by Begg's funnel plots and Egger's regression test. The sensitivity analysis was performed to evaluate the stability of the pooled results.
Subgroup analyses were performed according to the ethnicity.

Characteristics of Studies
After initial search, a total of 132 articles were identi ed, of which 40 were excluded as duplicates. Of these, 62 records were excluded after screening titles and abstracts. 21 irrelevant studies were excluded after reading full papers. Ultimately, nine articles [13][14][15][16][17][18][19][20][21] were included in this meta-analysis according to inclusion criteria. The literature screening process was shown in Figure 1. Characteristics of the included studies in this meta-analysis are presented in Table 1. Among these studies, 6 studies were performed in Asian populations, 3 studies were conducted in Caucasian populations. The articles were published from 2006 to 2018. Two case-control studies were deviated from the HWE [20,21].

Quantitative Synthesis
The results revealed that MTHFR A1298C polymorphism was signi cantly associated with cervical cancer  Table 2). In the subgroup analysis by ethnicity, no signi cant results were found for Caucasian and Asian population under all ve genetic models (Table 3). There was also signi cant heterogeneity under the all the genetic models. Meta-regression was performed to explore the source of heterogeneity under these genetic models. The confounding factors included the publication year, ethnicity. However, these confounding factors couldn't explain the heterogeneity (P>0.05)

Sensitivity Analysis and Bias Diagnostics
In the current meta-analysis, there was high heterogeneity under recessive, dominant, homozygous and allelic genetic models. Thus, sensitivity analysis was performed by removing one study and re-calculated the pooled ORs and CIs to evaluate stability of the result. After the Gong et al. was removed from the current metaanalysis, the heterogeneity was signi cance decreased while still providing results consistent with our initial ndings ( Figure 7). However, removal of any other studies did not change the results from the original analysis. Therefore, the study of Gong et al. was the high sensitivity study on the pooled OR. Begg's and Egger's test implied that there was no publication bias in the current analysis ( Figure 8; Table 2) Discussion Infection by HPV is not exclusive to cause cervical cancer. Genetic polymorphism is also important in inducing cervicogenesis collectively. Moreover, in distinct populations, the relationship between the risk of cervical tumorigenesis and gene polymorphism was almost various. In our analysis, 1145 cases and 1690 controls from nine publications were involved. The meta-analysis results showed that MTHFR A1298C is a risk factor for cervical cancer, especially in Asian population. As to MTHFR A1298C polymorphism, in previous study, Zhuo et al. [22] published the rst meta-analysis assess the association between MTHFR A1298C gene polymorphisms and cervical cancer risk and did not nd signi cant associations. In contrast, recent meta-analysis by Yi et al. [23], including ve studies, suggested that the signi cant associations between MTHFR A1298C polymorphism and cervical cancer were found among Asians. However, these studies were limited to small sample sizes and high heterogeneity. Therefore, we performed a comprehensive large-scale meta-analysis to investigate these associations. We performed a more comprehensive large-scale meta-analysis (nine studies involving 2835 individuals) to accurately assess these associations.
Folate was indicated a protective effect on cervical epithelial cell [24]. In addition, multiple clinical evidences have reported that evaluated level folate level was inversely related with the risk of cervical cancer [19,25,26].
As the introduction described, folate metabolism is very important for protection of cervical epithelial cell through maintain the methylation, repair, and synthesis of DNA [27]. In physically, folates appear to be essential for cell growth and cell cycle. Once folate de ciency induces lower proliferation rates by increased apoptosis and cell cycle arrest [28]. Thus, this is very negative for normal cell survival, which would promote cell transformation into cancer cells. Some study has shown that low level of serum folate may increase the risk of cervical cancer. Furthermore, it could be potential synergy with HPV infection, promoting cervical cancer development [29]. In our study, we set up the relation of MTHFR, as a key enzyme involving the chemical reaction of vitamin folate, A1298C polymorphism and cervical cancer susceptibility. It is this polymorphism that affects the activity of the enzyme and leads to low folate concentration in serum. To be more speci c, the activity was decreased when a substitution of glutamate with alanine occurred.
In basic research, carriers of variant alleles for MTHFR1298 exhibit genome-wide DNA hypomethylation, which is a characteristic of genome instability [30]. When the activity of MTHFR was decreased or lose, it would lead to relaxation of heterochromatin, a decrease of H3K9me3 levels that often associates with heterochromatin at the centromeric and telomeric regions, and an increase of certain transcripts at the centromeric region. In summary, it would destroy heterochromatin or whole genome stability [31]. Thus, it was not hard to speculate the hazardous of MTHFR A1298C. It has reported that the MTHFR 1298C allele frequency is approximately 20-70% in Asia, 24-46% in Europe, and 0-15% in America [32], which may explain why Asians are strong association. This could give us an appropriate reminder that Asians with carriers of variant alleles for MTHFR1298 may probably more likely to get cervical cancer, if they are infected HPV simultaneously.
In our study, there are still some limitations as follows. Firstly, the sample size was relatively small and may not have provided su cient statistical power. More studies with a larger sample size are required. Meanwhile, it is hoped that the exploration was expanded on the association of MTHFR A1298C polymorphism and tumors of the female reproductive system, including ovarian cancer, which will make the study more broadspectrum and meaningful. Secondly, the subgroup analyses according to age, histological types and other elements were not performed owing to insu cient relevant data. Thus, it is suggested that future study could combine environmental factors, age and genetic polymorphism together to explore the interaction. Thirdly, given that the MTHFR has two types of polymorphisms, whether there is an interaction is unknown.

Conclusions
Our meta-analysis suggested that MTHFR A1298C was associated with cervical cancer development, especially in Asian population. In the future, well-designed and larger-scale studies are necessary to explore the roles of tumor stage, smoking, histology, or gene-gene/gene-environment interactions in the pathogenesis of cervical cancer.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Avaliability of date and meterials
All data are included in this article.

Competing interests
The authors declare that they have no con icts of interests.