Gene Polymorphisms Involved in Folate Metabolism and DNA Methylation with the Risk of Head and Neck Cancer

Background: Folate is essential for DNA synthesis, repair, and methylation. Polymorphisms in genes associated with folate metabolism may alter these processes and, consequently, modulate cancer development. Aim: We aimed to assess DNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), DNMT3B -579G/T (rs2424909), DHFR 19-pb ins/del (rs70991108), SHMT1 1420C/T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033) polymorphisms with risk of head and neck cancer. Methods: A case-control study was conducted in 1,086 Brazilian individuals. Real-time and conventional polymerase chain reactions-PCR were performed for genotyping the polymorphisms. Results: The single nucleotide polymorphism (SNP), DNMT3B -283T/C, revealed a higher risk of head and neck squamous cell carcinoma (HNSCC) when compared with the C group in the codominant (p< 0.001), dominant (p<0.001), and overdominant (p= 0.001) models for T/C and C/C genotypes. DNMT3B -149C/T and DNMT3B -579G/T revealed no association between groups in any model. The DHFR 19-pb ins/del polymorphism protected against HNSCC development compared to the C group by the codominant (p< 0.001), dominant (p< 0.001), and overdominant (p< 0.001) models. In the TYMS, the 3R/3R genotype had a protective effect against HNSCC development compared with the C group by the recessive models (p= 0.009). In contrast, SHMT1 1420 C/T presented no association between the HNSCC and C groups. DHFR 19-pb ins/del polymorphisms protected against oral cavity cancer (p= 0.003), and only TYMS-28 3R/3R decreased the risk of tumor progression (p= 0.023). In the Kaplan–Meier curve, an association was found between DHFR ins/ins and TYMS -28 3R carriers with respect to relapse-free time; further, DNMT3B -579 T and TYMS-28 2R/2R carriers had longer survival times. Conclusion: DNMT3B -283T/C is associated with higher risk, whereas DHFR 19-pb ins/del and TYMS 28 3R/3R protect against head and neck cancer. We also highlighted the association of TYMS 3R/3R genotype carriers with relapse-free cancer protection and survival time.

Introduction carcinogens, thereby forming adducts of DNA that are not recognized during DNA replication and repair (Wang et al., 2017).
Folate or vitamin B plays a pivotal role in one-carbon metabolism, and generally low levels can be associated with the absence of methylation and DNA repair, thereby promoting carcinogenesis (Pieroth et al., 2018). Thus, several functional gene polymorphisms linked to folate metabolism can cause an imbalance in methylation reactions, as well as free nucleotides for DNA synthesis and repair (Coppedè et al., 2019).
Thus, considering the contradictory results and genetic heterogeneity of the Brazilian population, it is important to evaluate the role of certain folate polymorphisms for susceptibility to head and neck carcinogens. Here, we aimed to investigate the association of polymorphisms DNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), DNMT3B -579G/T (rs2424909), DHFR 19pb ins/del (rs70991108), SHMT1 1420C/T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033) with the risk of head and neck cancer.

Ethics Statement
The Research Ethics Committee of São José do Rio Preto Medical School (FAMERP) in São José do Rio Preto, São Paulo, Brazil, approved this study (Registration Number 013/2012), and written informed consent for the collection of biological material was obtained from all individuals.
Individuals were classified as having early stage (T0, T1, and T2; N0 and M0) and advanced stage tumors (T3 and T4; N1, N2, N3, and M1) based on their tumor, node, and metastasis (TNM) staging. The case and control group participants were interviewed to obtain their demographic and lifestyle data. Those who smoked more than 100 cigarettes in their lifetime were considered as tobacco consumers and those who consumed four doses of alcohol per week were considered as alcohol consumers.

Discussion
In this study, we found that the sociodemographic characteristics and risk factors indicated that the male gender, advanced age, smoking, and alcohol consumption were associated with greater susceptibility to cancer development when compared to the control group. Head and neck cancer has a multifactorial etiology and involves various risk factors.
In most countries, the rate at which men are affected is approximately two to five times higher than that of women, and this difference is presumably related to the adverse effects of carcinogens such as alcohol and tobacco, more commonly observed among men. Excessive consumption of carcinogens can affect nutrient absorption by the intestine, causing major nutritional deficiencies, and can modify the metabolic pathways, such as that of folate, which is essential for purine and pyrimidine synthesis and DNA methylation (Rettig and D'Souza, 2015). Moreover, the consumption of two or more cigarette packs along with four or more alcoholic drinks per day increases the risk of developing this neoplasia by 35-fold (Canova et al., 2010). Furthermore, the elderly are prone to cancer risk, with a median diagnosis age of approximately 60-70, which is higher than the median age considered at risk in this study (Cohen et al., 2018).
Numerous studies have explored the association of DNMT3B polymorphisms and cancer risk but yielded conflicting results. One meta-analysis suggests that DNMT3B -283T/C and DNMT3B -579G/T may play a protective role against different types of cancers. Moreover, in the subgroup analysis, DNMT3B -579G/T appeared to contribute to decreased risk of lung and colorectal cancer, whereas DNMT3B -149C/T was associated with a decreased risk of head and neck cancer (Zhang et al., 2015). Another systematic evaluation of cancer risk demonstrated that DNMT3B -149C/T, DNMT3B -283T/C, and DNMT3B -579G/T polymorphisms were observed as protective factors against cancer in the Asian population (Duan et al., 2015); however, in our study, the DNMT3B -149C/T and -579G/T SNPs did not contribute to the risk of head and neck cancer, whereas DNMT3B -283T/C might be a risk factor for head and neck carcinogenesis. Li et al., (2016) demonstrated that DNMT3B -283T/C (rs6087990) has a potential effect on gastric cancer initiation.
We found that DHFR 19-pb ins/del (rs70991108) and TYMS 28-bp tandem repeat (rs34743033) polymorphisms have a protective effect against head and neck cancer. Our study is the first to evaluate the association between a DHFR 19pb deletion polymorphism and the risk of this neoplasia. Evaluation in other cancer types reveals different results. Jokic et al., (2011) andLiu et al., (2013) demonstrated that DHFR deletion was not associated with colon cancer risk. In contrast, Xu et al., (2007) reported an increased breast cancer risk in women with this gene deletion. Corroborating our findings, one study evaluated the DHFR 19-pb ins/del (rs70991108) polymorphism in mother/child dyads in acute lymphoblastic leukemia onsetlatency and demonstrated a good prognosis for carrier patients with homozygous deletion (Tisato et al., 2019).
The role of TYMS 28-bp tandem repeat polymorphisms was not associated with acute lymphoblastic leukemia and lung cancer risk development (Oosterom et al., 2018;Stanisławska-Sachadyn et al. 2019); however, a study in the Brazilian population revealed increased risk association between the 2R/2R and 2R/3R variants in sporadic and hereditary breast cancer development, which was not in accordance with the results of our study (da Silva Nogueira et al., 2012).
In our study, the SHMT1 1420C/T (rs1979277) polymorphism had no association with head and neck cancer risk; however, it was associated with tumor progression. The human SHMT1 gene is located at chromosome 17p11.2; the cytosolic isoform and its coenzyme, vitamin B6, catalyze the reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate that provide one-carbon units during pyrimidine and purine syntheses (Coppedè et al., 2019). A meta-analysis revealed that no association was found between SHMT1 1420C>T (rs1979277) and the overall risk of cancer; however, in the subgroup analysis, significant associations with a protective effect were found in colorectal cancer and in the Asian population (Wang et al., 2014).
A preliminary study by our research group also found no risk association with head and neck cancer in the Brazilian population (Succi et al., 2014), which was in accordance with the results obtained for gastric cancer (Kim et al., 2016), colorectal cancer (Komlósi et al., 2010), non-Hodgkin lymphoma (Skibola et al., 2004), lung cancer (Wang et al., 2014), and breast cancer (Lissowska et al., 2007). According to Cheng et al., (2008), mutations in SHMT1 leading to an aberrant protein can be compensated for by the wild-type SHMT2 gene that encodes an isoform with the same function.
This is the first study to demonstrate an association between survival and relapse-free times. Individuals with wild-type genotype, DNMT3B -579 G/G, have longer survival times in relation to DNMT3B -579 T carriers, and this is also true for TYMS-3R carriers with respect to the TYMS-2R/2R genotype. Moreover, this last polymorphism protects TYMS-3R/3R genotype carriers against cancer recurrence. In vitro and in vivo studies have been carried out to verify the functional consequences of the variable number of tandem repeat polymorphism. In general, the TYMS alleles contain two or three copies of repeats (2R and 3R), and the TS genes with triple sequence have higher expression levels than those with double sequence, and thus have transcriptional activity with the 3R sequence, which can be three to four times greater than that with the 2R carriers (Gusella and Padrini, 2007).
A previous study that involved a smaller number of head and neck cancer patients evaluated survival and relapse-free time for the DNMT3B 46359C/T and SHMT1 1420C/T polymorphisms, but failed to demonstrate any statistically significant association between the wild-type and polymorphic carriers (Succi et al., 2014). In the present study, no association was observed between the SHMT1 1420C/T polymorphism and survival time and relapse-free cancer.
Thus, our findings reveal that DNMT3B -283T/C is associated with cancer risk, whereas DHFR 19-pb ins/ del and TYMS 28-bp tandem repeat polymorphisms have a protective effect against head and neck cancer in relation to that in the control group. Furthermore, we highlight the association of the TYMS 3R/3R genotype with relapse-free cancer protection and the increased survival time of these patients in relation to those with the TYMS 2R allele.