MTHFD1 c.1958G>A and TCN2 c.776G>C polymorphisms of folate metabolism genes and their implication for oral cavity cancer

Head and neck cancer is the sixth most commonly diagnosed cancer, making it a serious and growing health problem [1]. The term head and neck cancer encompasses a very broad group of cancers caused by tumor growth in parts of the upper respiratory tract, such as the nasal cavity, paranasal sinuses, oral cavity, pharynx, and larynx [2]. The symptoms of head and neck cancer differ depending on the location of origin of the cancer. Common symptoms are hoarseness, ulceration, pain, and weight loss for cancers of the larynx, oral cavity, oropharynx, and hypopharynx, respectively [3]. The cancers are classified within the same group due to the similarity of the diagnostic and therapeutic tools used for their detection; they are classified according to the anatomically based TNM classification of malignant tumors [4]. More than 90% of head and neck cancers are squamous cell carcinoma (HNSCC) [1]. Other malignant neoplasms are diagnosed much less frequently, most often they are salivary gland tumors or even more rarely soft tissue tumors or lymphomas.

In 2018, tumors of the lip and oral cavity accounted for 2% of all malignant tumors registered worldwide (354,864 cases) and contributed to 177,384 deaths that year [4]. Epidemiological evidence indicates that the worldwide incidence of head and neck cancer increases with age, occurring most often in people over 50 years old, and they are more common in men than women. The risk of cancer is caused mainly by exposure to tobacco and alcohol abuse [1, 5]. Infection with human papilloma virus [6], environmental factors [7], dietary habits [8], and poor oral hygiene [9] also play a role in the pathogenesis of head and neck cancer. Intensive worldwide, multicenter research has been undertaken to discover new prognostic and treatment predictive biomarkers for these cancers [10, 11, 12, 13].

Carcinogenesis is a complex and multistage process which involves the conversion of normal cells into tumor cells. Oncogenic transformation occurs following mutations that change the function of specific genes, such tumor suppressor genes or oncogenes, and their encoded protein products. Important changes in the transcriptional and translational regulation of genes also occur in the process of malignant transformation [14]. Recently, much attention has been given to epigenetic changes that influence the regulation of gene expression without affecting the sequence of nucleotides. These phenomena are reversible under the action of chemicals and act through two routes: the epigenetic modification of gene expression by DNA methylation and the remodeling of chromatin by the alteration of histone proteins [15]. DNA methylation is essential for normal cell growth and differentiation. That process occurs during X-chromosome inactivation, genomic imprinting, cell death, and cell cycle control. In mammalian cells, cytosine residues of CpG sites are methylated by methyltransferase (DNMT) at position C5 [16]. The methylation process plays an important role in genome stability. Disorders of methylation patterns may become useful biological markers associated with many forms of tumor. Therefore, despite being difficult to clearly define and with no well-defined mechanism of action, epigenetic phenomena are of great potential clinical importance [17].

DNA methylation is a biochemical process of adding a methyl group to the substrate. The following enzymes take part in that process: methylenetetrahydrofolate reductase (MTHFR) [18], methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) [19], methionine synthase (MTR) [20], methionine synthase reductase (MTRR) [21], transcobalamin 2 (TCN2) [22], methyltransferase betaine – homocysteine (BHMT) [23], methyltransferase betaine – homocysteine 2 (BHMT2) [24] and synthase β – cystathionine (CBS) [25]. Numerous studies implicate abnormal methylation in cancer. Aberrant process may be related to the occurrence of a polymorphism in the gene encoding the corresponding protein.

MTHFD1 is an enzyme that performs three different activities (5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, and 10-formylotetrahydrofolate synthetase). Each of these catalyzes the interconversion of tetrahydrofolate. That is a substrate for the biosynthesis of methionine, thymidylate, and purine nucleotides. The most analyzed polymorphism of the MTHFD1 gene, c.1958G>A, is located within the domain responsible for 10-formyltetrahydrofolate synthetase activity. The polymorphism results in the substitution of glutamine (Gln) with arginine (Arg) at codon 653 [19].

TCN2 belongs to the vitamin B₁₂ – binding protein family and allows cobalamin to be transported into the cell [26]. Vitamin B₁₂ is a co-factor for the enzyme MTR, which catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate. That chemical as well as methionine are converted by MTHFR from 5,10-methylenetetrahydrofolate. Methionine may be then converted to S-adenosylmethionine, an important methyl donor for the methylation process, including DNA methylation. MTR links the methylation and synthesis of purine and pyrimidine pathways via generation of the methyl donor methionine and synthesis of tetrahydrofolate, respectively [27]. Both low MTR activity and low vitamin B₁₂ level are determinants of DNA hypomethylation [28]. The most common TCN2 polymorphism is the replacement of guanine to cytosine at position 776; this change will replace the amino acid arginine (Arg) with proline (Pro) at position 259 in the protein sequence of TCN2. This change in the TCN2 protein sequence affects the ability of the protein to bind to vitamin B₁₂ and disrupts the binding of the transcobalamin-vitamin B₁₂ receptor complex. The TCN2 c.776G>C genotype has a significant influence on the cellular delivery system of vitamin B₁₂ [29]. Healthy subjects with low vitamin B₁₂ status manifest reduced levels of homocysteine [30].

Our previous study [31] showed that the TCN2 gene polymorphism modulates the risk of oral cancer, but the number of patients and controls were limited. Recent studies have examined whether polymorphisms within the MTHFD1 and TCN2 genes correlate with increased risk of oral cavity cancer and may be associated with cancer risk, tobacco smoking, alcohol consumption and gender.

Multiple logistic regression was used to determine the effect of the interaction between genetic polymorphisms and risk factors associated with head and neck cancer. The model includes gender (reference: female), the consumption of tobacco products (reference: nonsmoking), and alcohol consumption (reference: nondrinkers). The frequencies of the analyzed polymorphisms among OCSCC patients and controls were evaluated using the Hardy-Weinberg (HW) equilibrium test. The distribution of the alleles was analyzed with the Yates chi-square test. Results are shown as odds ratio (OR) and 95% confidence intervals (95% CI). P-values were calculated as two-sided. Means were compared using a t-test or analysis of variance. Probabilities were considered significant at p values less than 0.05 (Statistica, Tulsa, Oklahoma, USA).

The genotype and allele distributions of the MTHFD1 c.1958G>A and TCN2 c.776G>C gene polymorphisms are summarized in Table 3. The observed genotype frequencies of MTHFD1 c.1958G>A and TCN2 c.776G>C in the control group were in agreement with the Hardy-Weinberg equilibrium (HWE).

For the MTHFD1 c.1958G>A polymorphism, GG, GA, and AA genotype frequencies were 12.5, 75.6, and 11.9%, respectively, for the subjects who had suffered from cancer and 26.5, 55 and 18.5%, respectively, for control patients. The 1958 G allele frequencies were 49% among the subjects who had suffered from cancer and 54% among the control patients, while the 1958 A allele frequencies were 51% among subjects who had suffered from cancer and 46% among controls.

For the TCN2 c.776G>C polymorphism, GG, CG, and CC genotype frequencies were 34.5%, 48.7%, and 16.8% for the experimental group, and 17%, 35.5%, and 47.5%, respectively, for the control. In the gene TCN2, 776G allele frequencies were 59% among the subjects who had suffered from cancer and 35% among the controls, while 776C allele frequencies were 41% among subjects who had suffered from cancer and 65% among controls.

The results of comparison between groups showed that tobacco, alcohol use, and male gender (p < 0.0001) were predictors of the disease (Table 4).

The data of the interaction between genotype and tobacco smoking, alcohol consumption, and gender with respect to the risk for oral cavity cancer are shown in Table 5. The presence of the MTHFD1 c.1958G>A variant genotype may increase the risk of oral cavity cancer for smokers (p= 0.01) and males (p= 0.003). On the other hand, the protective effect was observed for TCN2 c.776G>C variant genotype for smokers (p < 0.0001), alcohol consumption (p< 0.0001), and males (p = 0.003).

Malignant tumors have a significant impact on public health and constitute a significant economic burden on the population due to lost productivity and the costs associated with illness and treatment. It is expected that this problem will grow as the population ages [34].

He ad and neck cancers are classified according to their location. Risk factors, diagnosis, and treatment may vary depending on the disease subtype. The head and neck region of the body is located close to other important life organs and systems. Cancers of the head and neck can damage the brain, spinal cord, and nerves, as well as the sense organs responsible for contact with the outside world. The tumors may also affect the endocrine organs, such as the thyroid or pituitary gland. Regardless of whether a tumor is malignant or benign, it often directly affects the performance of several structures simultaneously [2].

Non-healing ulcers, new or fast-growing growths, or warts on the skin, mouth, or nose can develop in response to anxiety and need to be investigated to determine whether they pose a threat to health or life. Particular attention should be paid to frequently occurring headaches, dizziness and changes in temperament and mood. In addition, frequent nosebleeds, ear or neck aches, persistent hoarseness, and voice alterations can be symptoms of cancer, as can problems with chewing and swallowing, choking, and the presence of ulcers in the mouth and blood in the saliva. Other symptoms may include numbness in the face or tongue, tooth loss, or a well-fitting prosthesis becoming mismatched [35].

Al though genetic and environmental factors take part in the pathogenesis of head and neck cancer, the mechanisms underlying the process remain unclear. However, host genetic factors are the important determinants of the pathophysiology of the disease. Polymorphisms may be potentially used as a biomarker to evaluate cancer risk. Sequence variants in Toll-like receptor genes, which mediate the innate immune response defense against microorganisms, have been linked to head and neck cancer risk [36]. Moreover, strong evidence confirms that alterations in the genes associated with DNA methylation may contribute to carcinogenesis. Hypermethylation promotes gene silencing, and hypomethylation of a promoter can make it susceptible to induction by oncogenes [37].

The aim of the study was to determine the relationship between the occurrence of two polymorphisms of genes encoding selected enzymes associated with the methylation process, viz. MTHFD1 (rs2236225) and TCN2 (rs1801198), and the risk of developing cancer of the head and neck. This study included a group of 439 patients with histologically confirmed oral cavity cancer and a control group of 200 individuals without cancer.

Data indicate the presence of 30,000 CpG dinucleotides, also known as CpG islands, in the human genome; most of which are located in promotor regions. Under normal conditions, they remain unmethylated. Methylation of those structures leads to different diseases, including carcinogenesis [38]. Hazra et al. [39] found that aberrant methylation of the promoter CpG island may contribute to colorectal cancer development by silencing tumor suppressor genes. The inactivation of tumor suppressor genes by hypermethylation plays a key role in the process of malignant transformation [40].

MTHFR c. 677C>T gene polymorphism may be a risk factor for thyroid cancer. Furthermore, MTR c. 2756A>G is associated with tumor extent and aggressiveness [41]. Moreover, DNMT3B c. -283T>C gene polymorphism is linked to a higher risk of head and neck cancer [42]. The available literature data demonstrates the impact of the MTHFD1 c.1958G>A gene polymorphism on the appearance of neural tube birth defects in children [43, 44] and loss in the second trimester of pregnancy [45]. Data indicate that SNPs in the MTHFD gene coexist with folate and homocysteine concentration disorders in families with congenital heart disease [46]. Kempisty et al. reported a higher prevalence of schizophrenia and bipolar disorder among patients with 1958AA and 1958AG genotypes [47], whereas Sutherland et al. found no evidence for an association of the MTHFD1 c.1958G>A polymorphism with migraine susceptibility [48]. The MTHFD1 gene polymorphism is also regarded as the most common risk factor for cancer. Stevens et al. [49] noted an increased risk of developing malignant breast cancer in postmenopausal women who were carriers of the T and C alleles in the presence of at least one variant of the MTHFD1 c.677C>T and MTHFD1 c.1298A>C, respectively. Moreover, breast cancer patients who were carrying A allele for the MTHFD1 c.1958G>A polymorphism showed a higher frequency of tumor CpG island hypermethylation [50]. Wang et al. reported a connection between the presence of several mutated variants of this gene and the increased risk of gastric cancer [51]. Another study found the MTHFD1 c.1958G>A polymorphism to be related with a lower probability of event-free survival in children with lymphoblastic leukemia [52]. Despite expectations, MTHFD1c.1958G>A polymorphism has been found not to be a risk factor for prostate cancer [53]; however, MTHFD1 c.1958G>A SNP was associated with the emergence of squamous cancer cells in the head and neck in alcohol abusers and smokers [54].

It has been found that the presence of the TCN2 c.776CG+GG variant was related to a higher risk of occurrence of other disease entities such as colorectal adenoma [55] and Crohn disease [56]. The TCN2 776GG variant increased the susceptibility of individuals to peripheral nerve dysfunction [57]. Our earlier results indicate that the CG and GG genotypes and the G allele increase the likelihood of oral cancer [31]. Protein encoded by the TCN2c.776G>C G allele is associated with lower vitamin B₁₂ transport from blood to tissues [30], and patients with GG genotype had significantly lower concentrations of holotranscobalamin [58].

In conclusion, the presence of the MTHFD1 c.1958G>A polymorphism increases the risk of head and neck cancer associated with tobacco smoking and male sex, whereas TCN2 c.776G>C variant has protective effects with tobacco smoking, alcohol consumption, and male sex.