Keywords
MTHFD1, Polymorphisms, NSCL/P susceptibility, Meta-analysis
MTHFD1, Polymorphisms, NSCL/P susceptibility, Meta-analysis
Cleft of the lip and/or palate (CL/P) is one of the most common facial malformations1–3 and a societal burden, affecting the patient ability to eat and speak and influencing social integration4. Non-syndromic CL/P, accounting for about 70% of CL/P, is considered closely related to genetic and environmental factors5. Recent studies suggested that using folic acid could reduce the rates of oral clefts6,7 and single nucleotide polymorphisms of some genes such as MTHFR8,9, MTR40 and MTRR involved in the metabolism of folic acid have been associated to high risk of NSCL/P8,9. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), a key gene associated with three sequential enzymatic reactions in the metabolism of folic acid, might play a potential role in the risk of NSCL/P, especially the polymorphism rs2236225 (c.1958G>A)10. Indeed, different observations that linked the polymorphism rs2236225 to the risk of NSCL/P have been reported11,12. The suggestion of a link between rs2236225 polymorphism and susceptibility to NSCL/P might be result of the limitations in sample size, different ethnic populations and other environmental factors. Therefore, we conducted a systematic review and meta-analysis of eligible case-control studies to reveal a more precise connection between the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P.
A systematic search based on the principle of evidence-based medicine13 was performed in PubMed, Web of Science, Cochrane Library, Google Scholar, China National Knowledge Infrastructure (CNKI) and WanFang Database. The final update was made on April 5th, 2015. In line with our knowledge background, the Medical Subject Headings (MESH) terms in PubMed and the known aliases of the genes of interests in the National Center of Biotechnology Information (NCBI), the following terms were used for searching: “cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC” AND “methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD”, which were slightly adjusted to optimize search results (Table S1; PubMed). We didn’t limit the search depending on publication types, data and language. Of course, the review of the published literature was examined carefully and manual search was conducted to avoid missing potential data. Two of the authors (Huaxiang Zhao and Mengqi Zhang) were in charge of the search independently and a third author (Jieni Zhang) conducted a random inspection.
Researches included in our systematic review and meta-analysis meet the following criteria: (1) evaluating the association between the NSCL/P and MTHFD1 polymorphism rs2236225, (2) focusing on humans, (3) case-control studies. Exclusion criteria were: (1) no association between NSCL/P and MTFHD1, (2) not focusing on humans but animal models or in vitro studies, (3) duplication of previous researches, (4) not original literature such as reviews, meta-analyses, comments and editorials.
Data from eligible studies were extracted by two independent researchers (Huaxiang Zhao and Mengqi Zhang) in accordance with the inclusion and exclusion criteria. In case of any discrepancies, the third chief author (Feng Chen) would make a further investigation or bring it into a group-discussion. A special table was used for collecting information from the selected articles and the following entries were recorded: authors (year), country, location of geography, subjects, methods for genotyping, sample size of cases/controls, descriptions of samples rolled in the study, P for HWE (Hardy-Weinberg equilibrium) of control group, whether included in the meta-analysis or not.
A methodological quality assessment adapted from previous studies14–16 was carried on included studies (Table S2). Cases, source of controls, sample sizes and Hardy-Weinberg equilibrium (HWE) were considered as important aspects in this systematic review.
The PRISMA checklist (Supplementary material S3) was used as a protocol in our meta-analysis17. Odd ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the association between the susceptibility to NSCL/P and MTHFD1. Five genetic models were used in the process of pooling the OR and 95% CIs: allelic comparison (A versus G), heterozygote model (AG versus GG), homozygote model (AA versus GG), dominant model (AA + AG versus GG), recessive model (AA versus AG + GG). The significance of the pooled effects was determined by Z-test with P value less than 0.05. The Q-statistic and the I2 test were used to evaluated; P < 0.05 in Q statistic or I2 > 50%18,19, would indicate a significant heterogeneity. When P > 0.05 in Q statistic or I2 < 50%, the fixed pooling model (Mantel-Haenszel) was conducted; if not, the random pooling model (M-H heterogeneity) was used. We also carried subgroup analyses in which different subjects (mothers or children), location of geography (non-Asian countries or Asian countries) were considered potential source of heterogeneity. A sensitivity analysis was conducted by omitting each study in turn to evaluate the single study’s influence on the overall estimation. We used Begg’s funnel plot and Egger’s linear regression test to find out the publication bias of the included studies20–22. The studies with disequilibrium of HWE among control group were added into a supplementary meta-analysis as described previously23. Meanwhile, as for the studies included but not carried into the meta-analysis, to achieve a qualitative analysis we adopted a method described by others24. Results were considered significant when P < 0.05. Stata 12.0 (Stata Corp, College Station, TX, USA) was used for the analysis.
A total of 251 articles resulted from the search described above (PubMed: 86, Web of Science: 8, Google Scholar: 135, Cochrane Library: 0, CNKI: 18, Wanfang: 4). After being imported into EndNote X6 software (Thomson Corporation, Stamford), a screening process was conducted among 102 articles– that is, duplicates were removed using the ‘Discard Duplicates’ function as well as by handwork. Following paper selection by two independent researchers, 15 studies were then thoroughly reviewed. Of these, five studies were excluded, among which two had no control groups25,26, one no relation to MTFHD127, and the other two presented data previously published28,29. Finally, 10 studies that met the criteria were included in the systematic review (Table 1)10–12,30–36 and mathematic data from eight studies were used for reference to carry out the meta-analysis10–12,31–33,35,36. The selection process is shown in Figure 1.
No. | Authors (year) | Country | Geographical location | Subjects | Methods for genotyping | Sample size of case/control group (just for the patients) | Descriptions of samples from study participants | P for HWE* of control group | Whether included in meta- analysis or not | |
---|---|---|---|---|---|---|---|---|---|---|
case | control | |||||||||
1 | Mostowska et al. (2006) | Poland | Europe | Mothers | PCR-RFLPγ | 122 | 82 | The case samples came from healthy mothers of NSCL/P children, while the control group includes samples from healthy mothers of children without NSCL/P. There was no difference between the two groups in their age, habit of smoking. | NMψ | Yes |
2 | Boyles et al. (2008) | Norway | Europe | Mothers and children | MALDI-TOF MSξ | 573 | 763 | 377 cases were CL/P and 196 cases CPO. Most mothers in the case group use supplemental folate during the pregnancy. | NMψ | No |
3 | Mills et al. (2008) | Ireland | Europe | Mothers, fathers and children | PCR-RFLPγ | 1030 | 1000 | 536 were CLP consisted of 494 cases with isolated defects 23 with one additional defect, 18 with multiple defects, and one with Pierre Robin. 426 cases with CPO consisted of 321 isolated defects, 15 with one additional defect, 21 with multiple defects, and 69 with Pierre Robin Sequence. | 0.03 | Yes |
4 | Bufalino et al. (2010) | Brazil | South America | Mothers | PCR-RFLPγ | 106 | 184 | Mothers who smoke, drink and use anti- hypertensives and drugs that could potentially impair the function of folic acids were not included in this study. | 0.66 | Yes |
5 | Mostowska et al. (2010) | Poland | Europe | Children | PCR-RFLPγ | 174 | 176 | The patients with clefts palate only (CPO) were excluded because the researchers thought the pathogenesis of NSCL/P and the CPO was different. | 0.11 | Yes |
6 | Li et al. (2013) | China | Asian | Children | PCR-RFLPγ | 187 | 157 | The patients in the case group consisted of 126 boys and 61 girls. | 0.89 | Yes |
7 | Yuan (2013) | China | Asian | Mothers, fathers and children | PCR-RFLPγ | 150 | 150 | 68 CLO and 82 CLP were enrolled in the case group. | 0.92 | Yes |
8 | Zhao et al. (2013) | China | Asian | Children | PCR-RFLPγ | 294 | 126 | There were 191 CLP and 103 CPO in the patients group. | 0.08 | Yes |
9 | de Aquino et al. (2013) | Brazil | South America | Mothers, fathers and children | Real-Time PCR | 181 | 478 | Patients with clefts palate only (CPO) were excluded. 65 clefts lip only (CLO) and 116 clefts lip and palate (CLP) were included in this study, consisting of 101 males and 80 females. | NMψ | No |
10 | Murthy et al. (2014) | India | Asian | Children | PCR-RFLPγ | 142 | 141 | There were 123 CLP and 19 CPO in the case group. | 0.94 | Yes |
Eventually, all 10 studies containing 6216 samples (2959 cases and 3257 controls) were analyzed in our review. The characteristics of every study can be seen in Table 1. To summarize briefly, there were four studies from European groups, four from Asian groups and two from South American groups, among which two studies focused on the genotype of patients’ mothers only, four on children’ s genotype only and four on both of them. PCR-restriction fragment length polymorphism (PCR-RFLP) was the major method of genotyping, while other techniques had been used as well.
The association between MTHFD1 polymorphism rs2236225 (c.1958G>A) and NSCL/P susceptibility was analyzed through a meta-analysis and qualitative analysis. In the meta-analysis, since significant heterogeneity had been identified by Q-test and I2 statistic in every genetic model, the random effect models were used. Overall, a significant association was not found in any genetic model (A versus G: OR = 1.02, 95% CI 0.86–1.21, PH = 0.010, Figure 2; AG versus GG: OR = 0.97, 95% CI 0.75–1.26, PH = 0.019, Figure 3A; AA versus GG: OR = 1.07, 95% CI 0.70–1.65, PH = 0.005, Figure 3B; AA + AG versus GG: OR = 1.00, 95% CI 0.76–1.31, PH = 0.006, Figure 3C; AA versus AG + GG: OR = 1.05, 95% CI 0.71–1.53, PH = 0.014, Figure 3D). On the other hand, no association was found in the genotypes of children, mothers or fathers in the qualitative analysis30,34.
Next we conducted the subgroup analysis using allelic A versus G model according to the location of geography and subjects (mothers or children). It turned out that there was no significant difference between Asian (OR = 1.03, 95% CI 0.75–1.40, PH = 0.003) or non-Asian population (OR = 1.06, 95% CI 0.86–1.30, PH = 0.118). However, a higher degree of heterogeneity was observed in the Asian countries compared to non-Asian countries (Figure 4A). A similar result was observed in the subgroup analysis between mothers and children. The heterogeneity was much higher in the children group (OR = 0.99, 95% CI 0.72–1.36, PH = 0.001) than in the mothers’ group (OR = 1.11, 95% CI 0.98–1.27, PH = 0.630), while no significant difference was observed in both groups (Figure 4B).
To access the influence of each individual study on the pooled ORs, a sensitivity analysis was performed by omitting each study at a time. The results of sensitivity suggests that no individual study affects the pooled ORs of the associations between MTHFD1 polymorphism rs2236225 (c.1958G>A) and NSCL/P risk under allelic model (Figure 5).
We used the Begg’s funnel plot and Egger’s regression test (both used the allelic A versus G model) to estimate the publication bias. Our results indicate that there is no significant publication bias both in the symmetry of Begg’s funnel plot (P = 0.711, Figure 6) and Egger’s regression test (P = 0.746).
CL/P is one of the most common facial malformations, affecting approximately 1.7/1000 people around the world with ethnic and geographic variation1. Although CL/P is not considered one of the major causes of infant mortality, individuals affected by CL/P it may have difficulties in feeding, speaking, difficult social integration4. Approximately 70% of CL/P cases are considered to be non-syndromic37,38, and their susceptibility has been linked to the expression of various candidate genes through twin studies, familial clustering studies and genome-wide studies39.
Recent studies suggest that using folic acid could reduce the rates of oral clefts6,7. Some genes involved in the metabolism of folic acid such as MTHFR8,9, MTR40, and MTRR41 have been identified. MTHFD1, a crucial gene associated with three sequential enzymatic reactions among 5,10-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate, 10-formyltetrahydrofolate, tetrahydrofolate, might play a potential role in NSCL/P10. However, controversial results about the MTHFD1 polymorphism rs2236225 (c.1958G>A) have been reported in different articles10,12.
In this systematic review, 10 independent case-control studies were included (eight studies for meta-analysis and two studies qualitatively analyzed) containing 6216 samples (2959 cases and 3257 controls). All the eligible studies of meta-analysis and qualitative analysis showed no significant association of MTHFD1 rs2236225 to the risk of NSCL/P, whether in the whole analysis of five model (A versus G, AG versus GG, AA versus GG, AA + AG versus GG, AA versus AG + GG) or in the subgroup of subjects (mothers or children) and the location of geography (non-Asian countries or Asian countries). Meanwhile, high heterogeneity was observed, which might be the reason for the genetic drift and natural selection among different ethnic groups42. Also, small sample size of different studies might be a possible reason for the disparate results. Our findings suggest that the MTHFD1 polymorphism rs2236225 (c.1958G>A) might not be an appropriate biomarker in predicting the susceptibility of an individual to NSCL/P.
Some limitations of this systematic review and meta-analysis should be noted. Firstly, the choice of retrospective studies has its own limitations, as we may encounter selection bias and influence the results of our analysis43. However, a bigger size of cohort study cannot be conducted easily because of the relatively low morbidity44. Secondly, only 10 studies were included in our review, a small sample size that might not provide sufficient evidence to estimate the connections between the MTHFD1 polymorphisms and the risk of NSCL/P.
NSCL/P is also associated with gene-gene and gene-environment interactions45. Although no correlation was observed between MTFHD1 polymorphism rs2236225 (c.1958G>A) and the risk of NSCL/P, in view of MTFHD1 gene’s key role in folic acid metabolism, we cannot draw a definite conclusion that there is no association between MTFHD1 and NSCL/P’s susceptibility. The use of larger sample size studies, different techniques and considering gene-gene or gene-environment interactions should be explored in future investigations.
H Zhao, F Chen, J Lin were responsible for study conception and design of the study. H Zhao, J Zhang, M Zhang acquired the data. H, Zhao, F Chen F, Deng, L Zheng, H Zheng analyzed the data. H Zhao and J Zhang wrote the main manuscript text. Prof. J Lin and Prof. F Chen had full access to all of the data in this review and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have agreed to the final content of the manuscript.
This work was supported by grants 81200762 from National Natural Science Foundation of China; Program for the National Clinical Key Subject, Natural Science Foundation of China (81271183, 81470772); the Medical Scientific Research Project of Chongqing (20141013).
Section/topic | # | Checklist item | Reported on page # |
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Title | |||
Title | 1 | Identify the report as a systematic review, meta-analysis, or both. | 1 |
Abstract | |||
Structured summary | 2 | Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. | 2~3 |
Introduction | |||
Rationale | 3 | Describe the rationale for the review in the context of what is already known. | 4 |
Objectives | 4 | Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). | 5 |
Methods | |||
Protocol and registration | 5 | Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. | none |
Eligibility criteria | 6 | Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. | 5~6 |
Information sources | 7 | Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. | 5 |
Search | 8 | Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. | Table S1 |
Study selection | 9 | State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis). | 5~6 |
Data collection process | 10 | Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. | 5~6 |
Data items | 11 | List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made. | 5~6 |
Risk of bias in individual studies | 12 | Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. | 6 and Table S2 |
Summary measures | 13 | State the principal summary measures (e.g., risk ratio, difference in means). | 6~7 |
Synthesis of results | 14 | Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. | 6~7 |
Risk of bias across studies | 15 | Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). | 6~7 |
Additional analyses | 16 | Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. | 7 |
Results | |||
Study selection | 17 | Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. | 8 and Figure 1 |
Study characteristics | 18 | For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations. | Table 1 |
Risk of bias within studies | 19 | Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12). | 9~10, Table S2 |
Results of individual studies | 20 | For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. | Figure 2 |
Synthesis of results | 21 | Present results of each meta-analysis done, including confidence intervals and measures of consistency. | Figure 2~Figure 4 |
Risk of bias across studies | 22 | Present results of any assessment of risk of bias across studies (see Item 15). |
Table S2, Figure 5~ Figure 6 |
Additional analysis | 23 | Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]). | Figure 5~Figure 6 |
Discussion | |||
Summary of evidence | 24 | Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). | 11~12 |
Limitations | 25 | Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias). | 12 |
Conclusions | 26 | Provide a general interpretation of the results in the context of other evidence, and implications for future research. | 13 |
Funding | |||
Funding | 27 | Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. | 15 |
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
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