|Ahead of print publication
Association of MTHFR 677C>T polymorphism with breast cancer risk: A case–control study and meta-analysis
Harmesh Lal1, Bhavna Sharma1, Vasudha Sambyal1, Kamlesh Guleria1, Neeti Rajan Singh2, Manjit Singh Uppal2, Mridu Manjari3, Meena Sudan4
1 Department of Human Genetics, Human Cytogenetics Laboratory, Guru Nanak Dev University, Amritsar, Punjab, India
2 Department of Surgery, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India
3 Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India
4 Department of Radiotherapy, Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, Punjab, India
|Date of Submission||28-Jul-2020|
|Date of Decision||15-Sep-2020|
|Date of Acceptance||25-Dec-2020|
|Date of Web Publication||23-Oct-2021|
Department of Human Genetics, Human Cytogenetics Laboratory, Guru Nanak Dev University, Amritsar, Punjab
Source of Support: None, Conflict of Interest: None
Background and Objectives: Breast cancer is a complex, multifactorial disease that arises as a result of interactions between multiple genes and environmental factors. Methylenetetrahydrofolate reductase (MTHFR) is a low susceptibility gene, involved in folate metabolism. It assists in conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which further leads to DNA methylation. 5,10-methylenetetrahydrofolate assists in conversion of uracil to thymine and purine synthesis for DNA synthesis. MTHFR 677C>T polymorphism alters the activity of MTHFR enzyme potentially effecting DNA repair and synthesis, hence a potential risk for cancer like breast cancer. Hence, the present study was conducted to evaluate association of MTHFR 677C>T polymorphism and breast cancer in Punjabi population. Moreover, a meta-analysis was conducted to address the same.
Materials and Methods: A total of 247 breast cancer patients and 247 controls were selected from Punjabi population for analysis using PCR-RFLP method. For meta-analysis, 67 studies were selected, and allele contrast, homozygous, heterozygous, dominant, and recessive models were used to evaluate the association between MTHFR 677C>T and breast cancer.
Results: The frequencies of CC, CT, and TT genotype were 68.4% versus 74.5%, 28.7% versus 23.5%, and 2.9% versus 2.0% in patients and controls, respectively. There was no significant difference found. In meta-analysis, significant association was found in overall and Asian population while no significant association was found in Caucasians.
Interpretation and Conclusions: MTHFR 677C>T polymorphism is not a risk factor for breast cancer in Punjabi population. Inconsistency with the meta-analysis can be due to ethnic diversity.
Keywords: Breast cancer, meta-analysis, methylenetetrahydrofolate reductase, polymorphism
|How to cite this URL:|
Lal H, Sharma B, Sambyal V, Guleria K, Singh NR, Uppal MS, Manjari M, Sudan M. Association of MTHFR 677C>T polymorphism with breast cancer risk: A case–control study and meta-analysis. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=329051
| > Introduction|| |
Breast cancer, with a worldwide incidence rate of 2.1 million in 2018, is the most frequent cancer and the leading cause of cancer death among women. It is also the leading cause of disability-adjusted life years among women worldwide.
Breast cancer is a complex and multifactorial disease that arises as a result of interaction between environmental and genetic risk factors and is highly heterogeneous in nature., Age at menarche and menopause, reproduction history, high level of estrogen, age, body mass index (BMI), alcohol consumption, cigarette smoking, ionizing radiation exposure, family history of the disease, and genetic background are risk factors of this cancer.
Low susceptibility genes in interaction with environmental factors are the important cause of the development of cancer. Methylenetetrahydrofolate reductase (MTHFR) is a low susceptibility gene mapped to chromosome 1p36.3 which encodes MTHFR enzyme involved in the folate metabolism pathway. It leads to irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate which is the primary form of circulatory folate. 5-methyltetrahydrofolate is involved in remethylation of homocysteine to methionine. Methionine is a precursor for the synthesis of “S-adenosylmethionine” which is involved in the methylation of DNA. 5,10-methylenetetrahydrofolate is involved in purine synthesis and conversion of uracil to thymine for DNA synthesis. Folate is required for DNA methylation, DNA synthesis, and repair; therefore, its deficiency has been related to the development of various cancers, including breast cancer.
Polymorphisms in the genes involved in folate metabolism pathway have potential to influence the pathway. MTHFR 677C>T polymorphism is a polymorphism that causes substitution of alanine to valine at 222 amino acid position that leads to thermolabile enzyme with reduced activity. Homozygous mutant (TT) has 30% activity while heterozygous (CT) has 65% activity compared to wild-type homozygous. As this polymorphism can affect DNA repair and synthesis, hence, it is a potential candidate for cancer risk. There are several published studies that have investigated the association between MTHFR 677C>T polymorphism and breast cancer susceptibility but have given contradictory results.
In India, breast cancer is the most frequent and leading cause of cancer death among women. The total number of new breast cancer cases diagnosed in India comprises 15.46% of all cancer cases in both sexes and 27.7% of all cancer cases in females. In Punjab, in North India, increased incidence of cancer has been reported and the age-adjusted rate of breast cancer incidence ranges from 17.3 to 37.3 per 100,000. Besides this, Punjabi women have been reported to be anemic which can be due to folate deficiency. Therefore, this case–control study was carried to investigate the association between MTHFR 677C >T and breast cancer susceptibility in Punjab region.
In previous studies from India, four studies showed significant association of T allele with increased breast cancer,,,, one showed significant association of the minor “T” allele with decreased breast cancer, three studies showed nonsignificant association.,, There is no published study yet which investigates the association of MTHFR 677C>T with breast cancer from Punjab. Meta-analysis, being a robust tool to examine inconsistencies in the results due to multiple factors, was conducted in all eligible studies to address the same.
| > Materials and Methods|| |
The present study involved 247 confirmed breast cancer patients diagnosed at tertiary care hospital and 247 age-, gender-, and habitat-matched volunteers as control for analysis. Control group consists of unrelated healthy individuals with no family history of any cancer in at least three generations and no history of any other chronic disease. All subjects were from Punjabi population. Predesigned questionnaire was used to collect information regarding personal history, family history, disease history, etc., Informed consent was obtained from all the participants, and the study was carried out under the guidelines of institutional ethical committee, as per the Declaration of Helsinki.
Analysis of MTHFR 677C>T polymorphism
Five milliliters of venous blood from each subject was collected in 0.5 M EDTA vial and stored at − 20°C until further analysis. DNA was extracted from blood by standard phenol-chloroform method. Analysis of polymorphism was done by PCR-RFLP method. Amplification of genomic DNA sequence was achieved by specific set of primers which generate 203 bp long PCR product. The PCR products were digested with Hinf I (NEB) restriction digestion enzyme. C to T substitution created a site for Hinf I enzyme which produced 173 bp and 30 bp long fragments on digestion [Figure 1].
|Figure 1: Photograph of 2% EtBr stained agarose gel electrophoresis showing HinfI digested products. (lane 1, 4, 5 = CC; lane 2 = TT; lane 3, 6 = CT; M = 100 bp molecular marker)|
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Genotypic and allelic frequencies were calculated, and Chi-square goodness-of-fit test was used to test whether the distribution of control sample was in accordance with the Hardy–Weinberg equilibrium (HWE). Odds ratio of allelic and genotypic distribution along with 95% of confidence interval was used to assess the association between polymorphism and breast cancer risk. Comparison of genotypic data of cases and controls was done by Chi-square test. P < 0.05 was considered statistically significant.
Eligible studies analyzing the association between MTHFR 677C>T and breast cancer were retrieved by searching PubMed, Google Scholar, Scopus, and Web of Science databases. The keywords used for retrieval were “breast cancer,” “breast carcinoma,” “breast neoplasms” in combination with “MTHFR,” “methylenetetrahydrofolatereductase,” “C677T,” “Ala222Val,” “rs1801133.” The references of retrieved studies were also searched to identify relevant studies.
Inclusion and exclusion criteria
The inclusion criteria for eligible studies were as follows: (1) case–control studies published in peer-reviewed journal, (2) investigating the association of MTHFR C677T polymorphism with breast cancer, (3) genotype data of cases and controls were complete, (4) genotype distribution of control must comply with the HWE, and (5) studies published only in English. The exclusion criteria were as follows: (1) studies with overlapped data, (2) no detailed information of genotype data, (3) meta-analysis and reviews, and (4) genotypic data not in HWE.
First author's name, year of publication, country, ethnicity, sample size, and genotypic and allelic frequencies were extracted from each study. Different ethnicities were categorized as Asians, Caucasians, and Africans. Studies with multiple ethnicities were categorized as mixed.
Chi-square test between observed and expected genotypic distribution was used to check the Hardy–Weinberg distribution in control samples only. Odds ratios with 95% confidence interval were used to evaluate association between MTHFR 677C>T and breast cancer risk. P ≤ 0.05 was considered statistically significant. Five genetic models: homozygote model (TT vs. CC), heterozygote model (CT vs. CC), dominant model (CT + TT vs. CC), recessive model (TT vs. CT + CC), and allele model (T vs. C) were used to evaluate association between MTHFR 677C>T polymorphism and breast cancer in overall and subgroup analysis. Subgroup analysis was conducted on the basis of ethnicity and source of controls. Cochran's Chi-square–based Q-statistic was used to assess the heterogeneity among studies, and quantification of heterogeneity was assessed by I2 test (I2 ranges from 0% to 100%, with higher value representing the higher heterogeneity). Random effect model was used, to incorporate the between studies' heterogeneity in calculation, if P < 0.05 or I2 >50%; otherwise, fixed effect model was used for meta-analysis., Estimation of potential publication bias was carried out by funnel plot, Begg's, and Egger's test., The Package “Meta: An R package of meta-analysis” in R Studio version 1.1.463, RStudio PBC, 250 Northern Ave, Boston, MA 02210, USA was used to conduct meta-analysis of all related studies.
| > Results|| |
In case–control study, 247 breast cancer patients (243 females, 4 males) and 247 age-, gender-, and habitat-matched healthy controls were screened. 60 (24.3%) subjects belonged to urban area and 184 (74.5%) belonged to rural area while 3 (1.2%) subjects were from suburban area in both cases and controls. Mean age (in years) of the cases and controls was 46.79 ± 11.54 and 46.39 ± 13.23, respectively. For cases and controls, the mean BMI (kg/m2) was 25.71 ± 5.33 and 26.41 ± 4.57 while the mean Waist Hip Ratio (WHR) was 0.96 ± 0.07 and 0.96 ± 0.06, respectively. In cases, 139 subjects were vegetarian and 104 were nonvegetarian, while in controls, 142 subjects were vegetarian and 101 were nonvegetarian. There was no significant difference in age, BMI, WHR, and diet between cases and controls.
The genotypic and allelic frequencies of cases and controls are presented in [Table 1]. Distribution of genotypic frequency in controls was in accordance with HWE (P = 0.86). The frequency of CC genotype was lower in cases (68.4%) as compared to controls (74.5%). The CT and TT genotypes were higher in cases than that in the controls, but difference was statistically nonsignificant.
|Table 1: Distribution of MTHFR 677C>T allelic and genotypic frequencies among breast cancer cases and controls|
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Following the inclusion and exclusion criteria, 67 case–control studies including the present study were included in this meta-analysis [Supplementary Table 1].,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, At first, 119 studies, without including the present study, were selected by searching different databases. Out of these studies, 24 studies were excluded because these were not case–control studies, 7 studies were excluded due to incomplete data, 12 studies were not in HWE, 4 studies reported duplicated data, 1 study was retracted, 2 studies was removed due to publication bias, and 2 studies were removed because they reported ambiguous data. The total number of cases and controls included in this meta-analysis was 23,440 and 27,880, respectively. Of these 67 studies, 27 were carried out among Asian, 29 were Caucasian, and 11 were among mixed population. In 28 studies, controls were recruited from general population, 29 studies were hospital-based studies, while there were no data on source of controls in 10 studies.
Pooled data showed a moderately significant heterogeneity in all models. Meta-analysis in four genetic models, i.e., allele contrast, homozygote, dominant and recessive model, showed a mild but significant association of MTHFR 677C>T polymorphism with breast cancer risk. Forest plot has been drawn for the allele contrast model to estimate the association of MTHFR 677C>T polymorphism with the risk of breast cancer [Figure 2]. In cumulative meta-analysis with random effects model, the association of T allele with breast cancer risk became significant with addition of Gao et al.'s study and remained significant after this. [Table 2] summarizes the results for pooled analysis.
|Table 2: Summary for odds ratio with 95% confidence interval, P value for heterogeneity, I2, and P values for Begg's and Egger's test for publication bias in different genetic models|
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In Asians, significant association was found between increased breast cancer risk and MTHFR 677C>T polymorphism in all models, except in heterozygote and dominant models. In Caucasians, association was found to be nonsignificant. A significant heterogeneity was found in Asians subgroup. On stratification by the source of control group, population-based studies showed significant heterogeneity in allele contrast model, homozygote model, and recessive model. Association between MTHFR 677C>T polymorphism and breast cancer risk was nonsignificant in recessive model with random effects; otherwise, the association was found to be significant in all other model. In hospital-based studies, significant association was found in homozygote and recessive model. Hospital-based studies showed comparatively more heterogeneity than population-based studies.
In studies comprising Indian population, significant high heterogeneity between studies was found in allele contrast, heterozygote, and dominant model. Homozygote and recessive models showed lack of heterogeneity and high risk of breast cancer was found in these models although the association was still statistically nonsignificant. [Table 3] summarizes the results of subgroup analysis.
|Table 3: Summary for odds ratios with 95% confidence interval, P value for heterogeneity, and I2 value in subgroup analysis|
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Publication bias and sensitivity analysis
Funnel plot [Figure 3], Begg's, and Egger's tests were used to carry out the estimation of publication bias in all genetic model. Sensitivity analysis was performed to analyze the stability of result by omitting one study at a time and excluding the studies with less than 100 subjects in one of the groups. No change was materially observed, indicating the stability of result.
| > Discussion|| |
Folate metabolism pathway is involved in the biosynthesis of purines, thymidylates for repair, and synthesis of DNA. Hence, folate deficiency can compromise nucleotide synthesis and lead to uracil misincorporation and chromosome breakage. Folate metabolism pathway is also involved in the synthesis of S-adenosylmethionine, the universal methyl donor for DNA methylation. Compromising the synthesis of S-adenosylmethionine can lead to hypomethylation, potentially influence the gene expression, and facilitate carcinogenesis., Generally, folate prevents the development of tumors before established preneoplastic lesions, but it would improve tumorigenesis after the establishment of lesions.
MTHFR is a low susceptibility gene in the folate metabolism pathway, and MTHFR 677C>T polymorphism is a common polymorphism which leads to thermolabile enzyme with reduced activity. Therefore, this polymorphism has potential to influence the pathway, hence various cancers including breast cancer. Previously, many studies were conducted to evaluate the association between MTHFR 677C>T polymorphism and breast cancer in the past, but the results were inconclusive. For instance, CT genotype was found to be associated with 1.5-fold increase in breast cancer risk in population from England, but European multicenter study reported nonsignificant association. Similarly, for TT genotype, Ergul et al. reported correlation with increased breast cancer risk, but Cam et al. reported no association with risk in Turkish population.
Furthermore, association of this polymorphism with breast cancer can be modified by folate intake and other nutrients intake., As women in Punjab have been reported to be anemic which can be because of folate deficiency, 247 cases and 247 controls from the Punjab region were analyzed for MTHFR 677C>T polymorphism in the present study, and we found no significant association of MTHFR 677C>T polymorphism with breast cancer risk. In previous studies from the Indian population, two studies reported nonsignificant association of polymorphism with breast cancer., Another study from South India reported higher frequencies of CT genotype and T allele in controls than cases but nonsignificant association. Hence, the present study is in agreement with these previous studies.
On the other side, CT genotype and T allele have been reported to be associated with increased breast cancer risk from Uttar Pradesh and Jammu and Kashmir region, in North India., In another study, TT genotype was found to be associated with increased breast cancer risk in the South Indian population. The authors suggest that relation with high risk could be due to thermolabile enzyme which has tendency to lose its active dimer form with loss in the FAD-binding capacity and decreased specific activity. In another study from Jammu and Kashmir, T allele reported to be associated with protection, although the sample size for study was small.
Inconsistency in the results of the different studies can be due to different lifestyle, dietary pattern, source of subjects, environmental factors, and ethnicity. Meta-analysis is a robust tool to examine these inconsistencies. Several meta-analyses have been published for the purpose of investigating the role of MTHFR 677C>T polymorphism and breast cancer risk whose details are given in [Supplementary Table 2].
First meta-analysis was published in 2006 which showed no association of MTHFR 677C>T polymorphism with breast cancer risk. Similar results were observed in two other meta-analyses., In a meta-analysis comprises 39 studies, T allele and TT genotype were significantly correlated with increased breast cancer risk, but here some studies were missing from analysis. Besides this, meta-analysis included an overlapped study. Zhang et al., Qi et al., Yu and Chen, Li et al., Zhong et al., and Yan et al. pooled 37, 41, 51, 57, 59, and 23 studies, respectively, and suggested a significant correlation of TT genotype with increased breast cancer susceptibility.,,,,, Kumar et al. in their meta-analysis of 75 studies also reported the increased breast cancer risk associated with T allele and TT genotype in overall population. Xie et al., Gonzales et al., Zhang et al., and Mo et al. pooled 68, 83, 82, and 85 studies, respectively, and suggested a significant association of MTHFR 677C>T with breast cancer risk.,,, However, these meta-analyses involved many studies that deviated from HWE.
Liang et al. suggested association of T allele and TT genotype with increased breast cancer susceptibility in the Chinese population; however, this meta-analysis based on 22 studies had multiple issues which were further resolved by another meta-analysis, although the results remained the same. On the other hand, significant protection was found in Chinese Han population with high significant heterogeneity in dominant model. T allele and TT genotype were also associated with increased breast cancer risk in Turkish and Latino population., On the other hand, pooled analysis of 61 studies reported nonsignificant association of MTHFR 677C>T polymorphism with breast cancer susceptibility which was contradictory to previous meta-analyses.
Present meta-analysis pooling 67 studies, excluded the studies which deviated from HWE for the purpose of stringent inclusion criteria, were undertaken which indicated the significant association of MTHFR 677C>T polymorphism with breast cancer susceptibility. In subgroup analysis, MTHFR 677C>T was found to be significantly associated with breast cancer risk in Asians. In another published meta-analysis, T allele and TT genotype were found to be associated with the mild increase in breast cancer risk in Asians and authors suggested that MTHFR 677C>T polymorphism contributes to overall risk of breast cancer. Compared to the present meta-analysis, previously published meta-analyses either had included fewer studies or studies which deviated from HWE. As Asian studies showed significant between studies' heterogeneity and mainly consist of studies on the Chinese population, a pooled analysis based on studies on the Indian population was conducted. In this analysis, the association was found to be nonsignificant which was in agreement with the present case–control study.
As inconsistency can arise due to the source of controls, subgroup analysis based on the source of control was conducted to achieve more accurate results. The present study suggests that hospital-based studies contribute more heterogeneity than population-based studies. Furthermore, sensitivity analysis of excluding studies with less than 100 subjects in either of the group and excluding one study at a time to identify the study affecting the results had been performed to increase the robustness of the results.
The present meta-analysis is the updated meta-analysis till the date which investigates the association of MTHFR 677C>T polymorphism with breast cancer risk. However, present meta-analysis and case–control study had some limitations: (1) gene–gene and gene–environment interaction had not been analyzed; (2) only single polymorphism of a gene is considered. Furthermore, other limitation for meta-analysis was that only studies published in English were included.
| > Conclusions|| |
The present case–control study suggested that MTHFR 677C>T polymorphism was not associated with breast cancer risk in the population of Punjab region. Although present meta-analysis indicated a positive correlation of polymorphism with breast cancer risk, it showed significant between studies' heterogeneity which indicated that association may differ between different populations.
We are highly thankful to all the subjects for their valuable participation. Financial assistance from UGC under UPE and CPEPA scheme sanctioned to Dr. Vasudha Sambyal and Dr. Kamlesh Guleria and DST-FIST, PURSE grant, and CSIR-SRF to Harmesh Lal is highly acknowledged.
Financial support and sponsorship
Financial assistant from UGC under UPE and CPEPA scheme, DST-FIST, PURSE grant, and CSIR supported the study.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]