Methylenetetrahydrofolate reductase C677T (Ala>Val, rs1801133 C>T) polymorphism decreases the susceptibility of hepatocellular carcinoma: a meta-analysis involving 12,628 subjects

Abstract C677T (Ala>Val, rs1801133 C>T), a non-synonymous variant of methylenetetrahydrofolate reductase (MTHFR) gene, has been found to be associated with an impair enzyme activity of MTHFR. The relationship of MTHFR rs1801133 with hepatocellular carcinoma (HCC) has been extensively investigated. However, the findings were conflicting. Recently, more investigations have been conducted on the relationship of MTHFR rs1801133 with HCC. To obtain a more precise assessment on the effect of this non-synonymous variant to the development of HCC, a pooled-analysis was performed. This meta-analysis consisted of 19 independent case–control studies. By using the odds ratio (OR) combined with 95% confidence interval (CI), the relationship of MTHFR rs1801133 with HCC risk was determined. A total of 19 independent case–control studies were included. Finally, 6,102 HCC cases and 6,526 controls were recruited to examine the relationship of MTHFR rs1801133 with HCC risk. In recessive model (TT vs. CC/CT), the findings reached statistical significance (OR, 0.90; 95%CI, 0.82–0.98; P = 0.016). Subgroup analysis also found an association between MTHFR rs1801133 polymorphism and the decreased risk of HCC in hepatitis/virus related patients (recessive model: OR, 0.85; 95%CI, 0.72–0.99; P = 0.035, and allele model: OR, 0.90; 95%CI, 0.81–0.99; P = 0.028). Subgroup analyses indicated that extreme heterogeneity existed in Asian population, larger sample size investigation, hospital-based study and normal/healthy control subgroups. The shape of Begger’s seemed symmetrical. Egger’s linear regression test also confirmed these evaluations. Sensitivity analyses suggested that our findings were stable. In summary, our results highlight that MTHFR rs1801133 polymorphism decreases HCC susceptibility. The relationship warrants a further assessment.


Introduction
In 2018, global cancer statistics estimated that liver malignancy was the fifth most frequent type of cancer incidence among men and the eleven most frequent type among women, about 596,574 and 244,506 new cases diagnosed worldwide, respectively [1]. However, the fatality was the third most frequent type [1]. The etiology of liver cancer (LC) was not well-established. Hepatocellular carcinoma (HCC) is one of the most important primary LC, which comprised almost 80% of LC cases. Some major susceptibility factors (e.g. aflatoxin-contaminated food, superabundant drinking, tobacco consumption, chronic virus infection, higher body mass index and Type 2 diabetes) [2][3][4][5][6] may contribute to the development of HCC. Additionally, hereditary factor has also been suggested to affect the susceptibility for the occurrence of HCC.

Inclusion criteria
In our meta-analysis, the eligible criteria of the included publications were: (1) designed as a case-control study; (2) focusing on the relationship of the MTHFR rs1801133 polymorphism with HCC risk; (3) genotype data could be extracted and (4) publications were compatible with Hardy-Weinberg equilibrium (HWE) in controls.

Exclusion criteria
The criteria for exclusion were as following: (1) publications incompatible with HWE; (2) overlapping data; (3) not case-control study design and (4) only focusing on the relationship of MTHFR rs1801133 polymorphism with HCC survival.

Data extraction
The authors (S. Zhang and J. Jiang) extracted the following data: the surname of first author, publication year, populations studied, country where the investigation was carried out, ratio of sex, age, drinking, positive (%) of hepatitis B surface antigen (HBsAg), genotyping method, the number of participants and MTHFR rs1801133 genotype. If there was conflicting assessment, another reviewer (W. Tang) was invited. During this process, they made a vote to obtain the final decision.

Statistical methods
In the present study, the odds ratios (ORs) combined with 95% confidence intervals (CIs) were harnessed to compare the difference between HCC group and controls. P value (<0.05) was considered statistically significant. The present meta-analysis determined the correlation in four genetic models [e.g. dominant model (TT/CT vs. CC), homozygote model (TT vs. CC), allele model (T vs. C) and recessive model (TT vs. CC/CT)]. Using I 2 metric and Q statistic, the heterogeneity among the eligible case-control studies was evaluated. If P < 0.10 or I 2 > 50%, we defined that there was significant heterogeneity. Thus, the random-effect model was used [30,31]. Otherwise, there was no heterogeneity detected. A fixed-effect model was used to combine the data [32]. The Egger test and Begg's test were used to assess the bias of publication. If P < 0.10, we defined that there was a significant publication bias. By omitting a study one by one and analyzing the remainders, sensitivity analysis was performed to assess the stability of our findings. The distribution of the MTHFR rs1801133 genotype was used to calculate the P value of HWE by using an online software (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) in controls [33][34][35]. STATA 12.0 software (Stata Corp., College Station, Texas) was used to conduct the analysis. In this study, P value was two sided.

Quality assessment of meta-analysis
Two authors (S.Z. and J.J.) independently extracted the data and calculated the quality score of the included case-control studies. The detailed scores were determined by a quality assessment criteria, which were presented in previous studies [36,37]. If the scores were more than 6.0, the investigation had an acceptable quality [38].

Meta-analysis results
In the eligible investigations, the MAF of MTHFR rs1801133 C/T polymorphism was 0.475 in HCC patients (5,670/11,944) and was 0.466 in controls (5,721/12,288). In different race, the MAF of controls was not similar in controls. The MAFs were 0.352 (480/1,362) in mixed populations, 0.328 (214/652) in Caucasians, and that was 0.485 (5,075/10,462) in Asians.

Heterogeneity assessment
In some genetic models, heterogeneity was significant (Table 3). Subgroup analyses indicated that extreme heterogeneity existed in Asian populations, larger sample size investigation, HB study and normal/healthy control subgroups. If we excluded these subgroups in our meta-analysis, the heterogeneity significantly decreased.

Bias evaluation
We used Begger's and Egger's tests to identify the bias of publication among the included investigations. The shape of Begger's test seemed symmetrical (Figure 3). Egger's linear regression test also confirmed these evaluations.

Sensitivity analyses
By sequentially omitting an individual investigation, sensitivity analysis was carried out. This method is considered as a criterion for meta-analysis. The results indicated that the significance of the present study could not be altered by removing any case-control study (Figure 4), suggesting that our findings were stable. Table 2 presents the results of the quality evaluation. Each eligible study had an acceptable quality (scores ≥ 6).

Discussion
Accumulating investigations highlight that MTHFR rs1801133 polymorphism may be associated with the development of HCC. However, the findings of the previous case-control studies were conflicting, with several investigations suggesting a potential relationship, whereas others did not support the correlation. In this investigation, to explore whether the MTHFR rs1801133 polymorphism was implicated in the etiology of HCC, we carried out a pooled-analysis of 19 eligible studies, which recruited 6,102 HCC cases and 6,526 controls. This meta-analysis indicated that the MTHFR rs1801133 polymorphism was a protective factor for the development of HCC in the overall comparison. Compared with the previous study, this pooled-analysis first confirmed the association of MTHFR rs1801133 polymorphism with a decreased risk of HCC.
MTHFR rs1801133 polymorphism locates on 11796321 (NCBI Build 38) of Chromosome 1. Zhu and her/his colleagues first reported that MTHFR rs1801133 polymorphism might confer a risk to HCC [24]. In addition, Cui et al. also suggested that this polymorphism could increase the risk of HCC [20]. However, some case-control studies indicated that the rs1801133 polymorphism in MTHFR gene might decrease the susceptibility of HCC [21,25]. And most studies reported that this SNP in MTHFR gene could not alter the risk of HCC. Thus, the association of MTHFR rs1801133 polymorphism with the susceptibility of HCC was more conflicting. Here, we performed a pooled-analysis of nineteen eligible studies involving 6,102 HCC cases and 6,526 controls to explore the correlation of rs1801133 with the etiology of HCC. The results indicated that this SNP in MTHFR gene could be a protective factor for the occurrence of HCC. Two meta-analyses suggested that rs1801133 was not associated with HCC development [43,44]. Others pooled-analyses reported that MTHFR rs1801133 polymorphism was associated with an increased risk of HCC [45][46][47][48]. Compared with these early meta-analyses, our analysis included more large sample size studies [21,25]. It is worth mentioning that these more recent case-control studies have recruited more participants and reported that rs1801133 polymorphism was a protective factor for the development of HCC. Compared with the most recent meta-analysis [23], the merit of our study was the larger sample size and the detailed subgroup analysis. Combined the eligible studies, we observed that rs1801133 decreased the susceptibility of HCC in the overall comparison.
The quality score was evaluated in our study. Each eligible study had an acceptable quality (scores ≥6). This indicated that our findings were reliable. We also found an association between MTHFR rs1801133 polymorphism and decreased risk of HCC in hepatitis/virus related patients. Of late, in Asian population, some meta-analyses identified that MTHFR rs1801133 polymorphism decreased the risk of colorectal cancer [49,50]. Some publications [51,52] suggested that MTHFR rs1801133 C>T polymorphism (Ala→Val) could promote the level of 5,10-methylene-THF for DNA synthesis, which might be protective to carcinogenesis. In the future, a functional study should be carried out to address how this Ala→Val substitution could decrease the risk of HCC.
Heterogeneity was identified in the overall comparison. In the present study, we conducted subgroup analyses to explore the major source among the eligible studies. Subgroup analysis suggested that major heterogeneity might be due to different populations, sample size, and characteristics of controls.
Some potential limitations should be addressed in this pooled-analysis. First, only published investigations were eligible in our study. Thus, the number of included case-control studies might be inadequate. Second, for lacking of sufficient data, only crude ORs and CIs were calculated. Third, the controls in some of the case-control studies were hepatitis or virus related patients. Fourth, a recent investigation contained some subgroups, we treated them as independent case-control studies. However, in this literature, the same HCC group was used in different stratified analysis. Finally, our study did not focus on the gene-gene and gene-environment interactions.
In summary, the present pooled-analysis highlights that MTHFR rs1801133 polymorphism is a protective factor for the occurrence of HCC, especially in hepatitis/virus related patients. The relationship of MTHFR rs1801133 polymorphism with HCC risk warrants a further determination.