The methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism is associated with breast cancer subtype susceptibility in southwestern China

Methylenetetrahydrofolate reductase (MTHFR), a folate-dependent enzyme, is reportedly involved in several cancer types. The MTHFR C677T polymorphism influences many biological processes, including tumorigenesis. However, the association between the MTHFR C677T polymorphism and breast cancer (BC) subtypes is not fully understood. In this study, the MTHFR C677T polymorphism was genotyped in 490 individuals with or without BC from southwestern China. Analysis of the association between the MTHFR C677T polymorphism and BC revealed that there was a significant association between the MTHFR C677T polymorphism and triple-negative breast cancer (TNBC) (OR = 2.83, 95% CI: 1.12–9.51, P = 0.0401). Furthermore, the MTHFR C677T polymorphism can also serve as a protective factor in luminal A breast cancer (OR = 0.57, 95% CI: 0.34–0.94, P = 0.0258). Evaluation of the association between the MTHFR C677T polymorphism and clinical characteristics indicated that people who suffered from hypertension had an increased risk for BC (OR = 2.27; 95% CI: 1.08–4.6; P = 0.0264), especially TNBC (OR = 215.38; 95% CI: 2.45–84430.3; P = 0.0317). Our results suggest that the MTHFR C677T polymorphism is significantly associated with susceptibility to luminal B breast cancer and TNBC.


Introduction
Breast cancer (BC) is the most common type of cancer and the leading cause of cancer-related death in women worldwide [1]. More than 90% of breast cancers are not metastatic at the time a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 of diagnosis. For people presenting without metastatic disease, therapeutic goals are tumor eradication and preventing recurrence [2]. Breast cancer is categorized into 4 major subtypes based on unique genetic alterations and the presence or absence of molecular markers for estrogen or progesterone receptors and human epidermal growth factor 2 (Her 2): luminal A, luminal B, Her2 + and triple negative breast cancer (TNBC) [3]. Systemic therapy for both nonmetastatic and metastatic breast cancer is determined by subtype, with the goals of prolonging life and palliating symptoms. As a result, the term "BC" no longer refers to a single disease but rather a heterogeneous group of diseases associated with a diversity of tumor backgrounds/ subtypes.
However, clinically, breast cancer is more aggressive and is associated with a poorer prognosis in younger women than in older women, especially in TNBC patients [4]. Targeted therapies for germline mutations in BRCA1 or BRCA2 and PI3KCA have demonstrated clinical efficacy in breast cancers [5,6]. In this respect, many ongoing studies aim to screen biomarkers as potential predictors of prognosis or response to therapy, which will most likely lead to individualized management of the disease.
Folate (FA), a genetic term for a B-group vitamin (vitamin B9), is important for cell division and homeostasis due to its essential role in the synthesis of S-adenosyl-methionine, the methyl donor required for all methylation reactions in cells [7]. As an important 1-carbon source, folate is involved in many cellular processes, including DNA synthesis, maintenance and epigenetics. Low folate status or insufficient intake has been associated with an increased risk of neural tube defects, cancers and cardiovascular disease [8,9]. Chen et al. demonstrated that folate causes normal cells to turn cancerous [10], and dietary folate intake was associated with DNA methylation in early-stage breast cancer [11]. In addition, Friso et al [12] found that reduced global DNA methylation and lower plasma folate concentration were associated with genotype-dependent methylenetetrahydrofolate reductase (MTHFR).
MTHFR is a folate-dependent enzyme that catalyzes and converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The MTHFR gene is involved in many biological processes, including the regulation of intracellular folate levels, DNA synthesis, methylation and tumorigenesis [13]. There are two well-studied polymorphisms of the MTHFR gene, MTHFR 677C> T and 1298A> C. Large studies have indicated that the MTHFR 677C> T polymorphism may play a role in the development of breast cancer [14,15]. Despite the existence of a variety of data for studies on the association between these polymorphisms and breast cancer, findings of genetic association have been inconsistent for a large body of conditions. Furthermore, previous epidemiological studies indicated C677T and A1298C MTHFR polymorphisms were related to breast cancer susceptibility in Latinos [16]. However, these candidate MTHFR-associated loci have not been investigated in southwestern Chinese populations. Accordingly, the present study was designed to replicate the reported gene SNPs, aiming to identify MTHFR polymorphisms and their correlation with susceptibility to different subtypes of breast cancer. As a result, we found that MTHFR 677C> T exhibited differential expression in breast cancer. Therefore, MTHFR rs1801133 may serve as a novel target indicating lower risk in different subtypes of breast cancer in southwestern Chinese patients.
controls. Subtypes luminal A, luminal B, Her2 + and TNBC were classified by histochemistry. For patient recruitment, controls without a history of cancer were selected based on physical examination in the same region during the same period. Age and sex were used for frequency matching to cases and demographic information, and the clinical characteristics of each participant were collected at the same time.

Statistical analysis
The association of the MTHFR C677T polymorphism with BC incidence was calculated using adjusted odds ratios (AORs) and 95% confidence intervals (CIs) by multivariate logistic regression. Χ 2 tests were used to examine the deviation of genotype frequency in Hardy-Weinberg equilibrium (HWE) and to analyze differences in genotype distribution, clinicopathological features and physicochemical characteristics using the R program (Version 3.61, USA). Overall survival time was calculated from the date of cancer diagnosis to the date of death. A P-value less than 0.05 was considered statistically significant.

Ethics statement
Our study protocol was approved by the Institutional Review Board of Kunming Medical University. All study subjects provided written informed consent to participate in the study. This study conformed to the tenets of the Declaration of Helsinki. All methods applied were performed in accordance with the approved guidelines.

Cases and controls
We recruited a total of 490 BC cases and cancer-free controls. The genotype frequency of MTHFR C677T in the controls and cases was in concordance with Hardy-Weinberg equilibrium (HWE) (P = 0.69 and P = 0.45, respectively). The demographic characteristics of the cases and controls are presented in Table 1.
In this case-control study, the distribution of age was consistently investigated among all cases and controls (P = 0.46). The allelic frequency of MTHFR 677T was 0.44 in the controls and 0.47 in the BC cases. Considering that BC is a complicated disease, many risk factors, such as body weight and menopause, are associated with the occurrence and progression of BC [17]. Therefore, we conducted a stratified analysis on data according to age, diabetes mellitus, presence of hypertension, family history of cancer, menopause, body mass index (BMI), tumor site, tumor size, and lymph node metastasis (LNM) stage to determine whether the MTHFR C677T polymorphism is associated with BC incidence in specific subtypes of the study population, as shown in Table 1.

The MTHFR C677T polymorphism and BC
Considering the small number of MTHFR AA genotypes, the dominant genetic model (CC/ CT+TT) was used to analyze the genotype distribution of the MTHFR C677T polymorphism.  Table 2.

The MTHFR C677T polymorphism and clinical risk of BC
To understand the association between the MTHFR C677T polymorphism and the clinical features of the 4 subtypes of BCs, logistic regression analysis was used to evaluate the association between the MTHFR C677T polymorphism and clinical characteristics (age at diagnosis and menarche, diabetes mellitus, presence of hypertension, cancer family history, menopause, BMI, tumor size, LNM stage, distant metastasis and tumor stage classification), as shown in Table 3. In all BCs, the MTHFR (rs1801133) CT and TT genotypes were associated with elevated blood pressure (OR = 2.27; 95% CI: 1.08-4.6; P = 0.0264). Among the 4 BC subtypes, notably, in luminal B breast cancer, MTHFR CT and TT genotypes showed a strong association with diabetes mellitus (OR = 7.22; 95% CI: 1.56-33.64; P = 0.0092) and a higher BMI (OR = 2.51; 95% CI: 1.1-5.79; P = 0.0289). In addition, for TNBC, the MTHFR677T allele was associated with a very high risk of hypertension (OR = 215.38; 95% CI: 2.45-84430.3; P = 0.0317). There were no significant associations between the MTHFR C677T polymorphism and age at diagnosis or menarche, cancer family history, menopause, tumor size, LNM stage, distant metastasis or tumor stage classification. In addition, the MTHFR C677T polymorphism was not associated with other clinical features of BC in our study.

The MTHFR C677T polymorphism and physicochemical characteristics of BC
The MTHFR gene is involved in many biological processes, including the regulation of intracellular folate levels. Therefore, we analyzed the association of the MTHFR C677T polymorphism with inflammatory, physiological, and biochemical indices in BC patients. The levels of neutrophils, leukocytes, cholesterol, hemoglobin, platelets, albumin, ASTa, ASTb, glucose, cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) were used to assess the association with the MTHFR C677T polymorphism in BC patients (Table 4). In all BC patients, GA and AA genotypes were more  frequent in patients with glucose content exceeding 6.11 than in those with lower glucose content (OR = 2.52; 95% CI: 1.23-4.95; P = 0.0091). However, the association between the MTHFR C677T polymorphism and glucose content disappeared in patients with luminal A, Her2 + or TNBC but appeared to have a strong association with luminal B (OR = 4.37; 95% CI: 1.32-13.54; P = 0.0076). Except for glucose content, other clinical features, as listed in Table 4, were not associated with the MTHFR C677T polymorphism.

The MTHFR C677T polymorphism and BC patient survival
To investigate the association of the MTHFR C677T polymorphism and breast cancer risk, we conducted a five-year survival analysis. Our results revealed that BC patients with the MTHFR C677T polymorphism (CT+ TT) exhibited a relatively shorter survival time than 677CC carriers (HR, 1.97; 95% Cl, 1.03-3.77; P = 0.041) (Fig 1).

Discussion
In the present study, we investigated whether the MTHFR C677T polymorphism is related to the occurrence of different subtypes of BC. We found that the MTHFR C677T polymorphism was substantially associated with luminal B and TNBC susceptibility and exerted a protective effect against the risk of luminal A. Moreover, this study indicated that TNBC patients with the MTHFR C677T polymorphism are associated with hypertension, and the same gene polymorphism in luminal B breast cancer patients may be associated with certain clinical characteristics, such as diabetes mellitus and increased BMI. To our knowledge, this study provides evidence that MTHFR gene polymorphisms are associated with susceptibility to different BC subtypes and influence relevant clinicopathological features.
Some studies have reported inconsistent data for the significance of MTHFR 677C> T, although the MTHFR 677T allele was reported to be a potential genetic risk factor for increased BC susceptibility, as determined in a recent meta-analysis [16]. These results suggest that MTHFR plays a vital role in the etiology and progression of breast cancer. Furthermore, the MTHFR 677C> T polymorphism decreases MTHFR production, which correlates with increased plasma Hcy and decreased plasma FA levels [7]. Many epidemiological studies have demonstrated that dietary folate intake exerts a protective effect against the risk of breast cancer [18,19]; thus, folate intake insufficiency leads to a high risk of BCs because it may induce disturbances in human chromosomal DNA replication and DNA repair [20].  TNBC (ER − /PR − /HER2 − ) accounts for approximately 10-20% of all breast cancers. It is regarded as an aggressive disease that affects a young patient population and for which effective targeted therapy is not yet available [4]. As two main luminal-like subclasses, luminal A and luminal B were identified by their estrogen and progesterone status and gene expression profiles [21]. In our study, we found that the MTHFR 677C> T polymorphism plays opposite roles in TNBC and luminal A as a high-risk or a protective factor, respectively, related to different estrogen/progesterone statuses, gene expression and mechanisms of breast carcinogenesis. Therefore, the molecular mechanisms of the function between the MTHFR 677T allele and TNBC or luminal-like breast cancer subtypes are intriguing and warrant further investigation.
The association between the MTHFR 677C> T polymorphism and the clinical feature of BC remains unclear. There is considerable evidence linking low folate status with an increased risk of adverse events, including gestational hypertension [22], and the common C677T polymorphism in the gene encoding the folate-metabolizing enzyme MTHFR is implicated in the development of hypertension [23,24]. Meanwhile, over half of older patients diagnosed with cancer have chronic conditions, such as hypertension, diabetes mellitus and dyslipidemia [25]. Breast cancer patients with diabetes, in particular, have an increased risk of all-cause mortality [26] and potentially breast cancer recurrence and mortality [27]. Our data showed that the MTHFR genotype 677C> T was associated with clinical features, such as diabetes mellitus and BMI in luminal B and hypertension in TNBC, indicating that the MTHFR 677C>T polymorphism may be an important risk factor affecting luminal B and TNBC. The potential role of the MTHFR 677T allele in BC clinical features requires further verification through animal experiments.
The MTHFR 677C> T polymorphism was associated with glucose content in all BC patients, especially in luminal B patients, as shown in Table 4. This finding is consistent with the high diabetes mellitus risk in luminal B patients, indicating that the MTHFR 677C> T polymorphism may play a vital role in the balance of glucose in luminal B breast cancer. Regarding the mechanism, some studies have indicated that low-dose folic acid supplementation caused by MTHFR genotypes 677C> T reduce serum homocysteine levels but increases glucose concentrations [28]; unfortunately, insulin resistance has been found to be significantly associated with tumor progression in luminal B subtype breast cancer in postmenopausal women [29]. Therefore, we speculate that increased glucose concentrations induced by MTHFR genotypes 677C> T may be associated with luminal B tumorigenesis and progression.
Additionally, several limitations should be acknowledged in this study. First, a limited number of MTHFR C677T polymorphism samples were studied, and the population of this study was limited to the difference between regions in southwestern China. Second, the sample sizes of both populations were not equivalent, especially for the southwestern Chinese population. Population characteristics were biased, such as the age of the samples or the ratio of males to females. Third, the way the MTHFR C677T polymorphism in the MTHFR gene affects the development and subtypes of BC is still unclear. Information regarding the MTHFR C677T polymorphism and clinical characteristics or other causal relationships in BC patients is lacking and remains to be further investigated. Furthermore, despite its association with the MTHFR C677T polymorphism in BC subtypes, the role of rs1801133 in the regulation of folate consumption or glucose deserves further study in the future. The causation of different BC subtypes is the complex multifactorial interplay of genetic and environmental factors, but here, we focused on genetic factors without taking into account certain environmental factors associated with BC subtypes, such as tobacco and alcohol intake and physical activities. Last, although the results of our study provide evidence for the MTHFR C677T polymorphism in the MTHFR gene as a potential biomarker for use in BC subtype analysis, a prospective study in a larger cohort of ethnically diverse patients is warranted to validate these findings.
Generally, in Han Chinese individuals in southwestern China, the MTHFR 677C> T polymorphism may not predispose to each BC subtype, but it may significantly increase the risk of susceptibility in luminal B breast cancer and triple-negative breast cancer.