Genetic polymorphisms in one-carbon metabolism increased the risk of persistence of pre-neoplastic cervical lesions

Background: Cervical cancer is caused by Human (hr-HPV) infection associated with cofactors that has been analyzed as predictors of cytological abnormalities remission or persistence. These cofactors may be classified as environmental, epigenetic or genetic. Polymorphism in genes of enzymes that act on one-carbon metabolism alter their activity and may be associated with cervical carcinogenesis because they affect DNA synthesis and repair, and gene expression. Therefore, the objective of this study was to analyze the risk of persistence of pre-neoplastic cervical lesions according to genetic polymorphisms involved in one-carbon metabolism. Sample group was divided in Remission (n=60) - presence of pre-neoplastic lesion at first meeting (T1), and normal cytology after six months of follow-up (T2), and Persistence (n=46) presence of pre-neoplastic lesion at T1 and T2. Cervical samples were obtained for cytological analysis (T1 and T2), HPV detection (T1), and evaluation of polymorphism C667T of Methylenetetrahydrofolate Reductase (MTHFR C677T), A2756G of Methionine Synthase (MS A2756G), A66G of Methionine Synthase Reductase (MTRR A66G), double or triple 28 bp tandem repeat in 5'-untranslated enhanced region of Thymidylate Synthase (TSER), and 6 bp deletion at nucleotide1494 in TS 3'-untranslated region (TS3'UTR). Genetic Risk Score (GRS) was calculated for analyze all genetic polymorphism simultaneously. Results: No differences were observed between Remission and Persistence groups of GRS, or genotypic and allelic distribution of MTHFR C677T and MS A2756G polymorphisms. However, higher risk of persistence was observed among women presenting heterozygote genotype - ins/del [OR (IC95%): 3.22 (1.19 – 8.69),


1) BACKGROUND
Persistent high-risk Human Papillomavirus (hr-HPV) infection is the main cause of cervical cancer. However, only 10% of women with viral infection will develop preneoplastic lesions, and less than 1% will progress to cervical cancer. Furthermore, only approximately 30% of high-grade cervical lesions progresses to cancer in uterine cervix, and spontaneous regression occurs in 20-40% of cases (1)(2)(3)(4). Thus, risk of cervical carcinogenesis depends on hr-HPV infection and host-dependent features (5,6). Environmental, genetic and epigenetic cofactors of cervical carcinogenesis has been analyzed as markers for diagnosis, prognosis, and for auxiliary in treatment of pre-neoplastic cervical lesions, since they could be predictors of cytological abnormalities remission or persistence (7)(8)(9)(10).
Several genetic alterations characterize cervical cancer and they may have a substantial impact on risk of cervical carcinogenesis, as genomic instability, chromosomal aberration, and integration of HPV DNA into host genome (4, 7).
Polymorphism in genes of enzymes that act on one-carbon metabolism, such as Methylenetetrahydrofolate Reductase (MTHFR), Methionine Synthase (MS), Methionine Synthase Reductase (MTRR), and Thymidylate Synthase (TS), alter their activity and may be associated with cervical carcinogenesis (11)(12)(13). 4 MTHFR enzyme, whose gene is located on chromosome 1p36.3, is a flavoprotein that acts on folate metabolism, being essential for DNA integrity (14). MTHFR C677T polymorphism consists of cytosine (C) exchange for thymine (T) at nucleotide 677, and results in substitution of Alanine for Valine, leading to decrease in MTHFR activity (14,15). This Single Nucleotide Polymorphism (SNP) may modify the susceptibility to carcinogenesis by modulating the availability of 5,10-methyleneTHF at different points in folate metabolism (16).
MS is a vitamin B12-dependent enzyme, essential to intracellular folate levels maintenance, and catalyzes the methylation of homocysteine to methionine (17,18). MS A2756G polymorphism is caused by an exchange of adenine (A) for guanine (G) at nucleotide 2756, resulting in the substitution of Aspartic Acid for Glycine, close to the binding domain of vitamin B12 (19,20). Van Der Put et al. (1997) suggested that this SNP affects the secondary structure of MS, and has functional consequences. An association between the polymorphic allele (G) of MS and reduction of the number of hypermethylated CpG islands in tumor suppressor genes has been demonstrated (21). Thus, it is possible that the presence of MS A2756G alters the activity of tumor suppressor genes, explaining its association with the development of several types of tumor (22).
MTRR catalyzes the methylation of vitamin B12, that is a cofactor of MS enzyme (23). A66G polymorphism of MTRR enzyme (MTRR A66G) leads to exchange of A by G in nucleotide 66, and to substitution of Isoleucine by Methionine, which results in decrease of MTRR affinity by MS (24). Thus, polymorphic genotype was negatively associated with homocisteinemia, which alters DNA methylation and, consequently, gene expression (23,25).
TS enzyme catalyzes the conversion of deoxyuridine monophosphate (dUMP) to 5 deoxythymidine monophosphate (dTMP), the only de novo source of thymidine for DNA synthesis and repair. TS binds to RNA for repression of translation of its own messenger RNA (mRNA) or other proteins, and can regulate cell cycle progression (26)(27)(28). Moreover, TS expression is an index of cell proliferation and biological malignancy of cancer (29). The polymorphisms most frequently studied are double or triple 28 bp tandem repeat in 5'-untranslated enhanced region (TSER), and 6 bp deletion/insertion at nucleotide 1494 in TS 3'-untranslated region (TS3'UTR). These genetic variations may influence the TS gene expression and the stability of its mRNA, respectively (28).
When the one-carbon metabolism is alter, the integrity of genetic material is compromised due to changes in nucleotide pool and uracil incorporation, leading to DNA instability. In addition, global hypomethylation and site-specific hypermethylation are observed, which lead to activation of proto-oncogenes and silencing of tumor suppressor genes (10,30,31). Therefore, this study evaluated the risk of persistence of pre-neoplastic cervical lesions according to genetic polymorphisms involved in one-carbon metabolism.   To evaluate the association between MTHFR C677T, MS A2756G, and TS3'UTR polymorphisms according to the course of cytological abnormalities, we compared genotype distribution of Remission and Persistence groups ( Table 2).

2) RESULTS
No differences were observed between distribution of MTHFR C677T and MS A2756G, and course of cytological abnormalities (

3) DISCUSSION
Although persistent hr-HPV infection is the main cause of cervical cancer development, genetic alterations may alter the risk of this neoplasia. Thus, genetic markers may be useful in the screening of pre-neoplastic and neoplastic cervical lesions, especially in cases of persistence of HPV infections or recurrent cytological abnormalities (7,32). Moreover, the conventional methods used for screening of cervical cancer are not able to differentiate pre-neoplastic cervical lesions that will regress, or persist and progress. Therefore, the prognosis of individual preneoplastic lesions should be predictable, with the purpose of selecting women with higher risk of persistence and progression, which could decrease the number or unnecessarily treatment of lesions (4).
In this study, five genetic polymorphisms in enzymes that act on one-carbon 11 metabolism were evaluated: MTHFR C677T, MS A2756G, MTRR A66G, TSER, and TS3'UTR. However, distribution of MTRR A66G and TSER were not under Hardy-Weinberg Equilibrium (HWE), which led to their exclusion from further analyzes.
Some studies with Brazilian population also did not present genotypic distribution of this polymorphisms attending HWE (33,34).
Many polymorphisms were identified in TS gene, which is located on chromosome 18p11.32. One of most frequently studied is TS3'UTR, that was related to increased in vitro degradation of mRNA, leading to decrease of expression protein (35). We observed that the presence of polymorphic allele (del) of TS3'UTR increased twice the risk of persistence of cytological abnormalities. Probably it occurred due to decreased synthesis of thymidylate, catalyzed by TS enzyme whose activity is decreased by TS3'UTR polymorphism, leading to DNA uracil incorporation. It results in DNA instability and chromosome damage, crucial events for carcinogenesis (26,35,36).
On the other hands, although MTHFR C677T and MS A2756G polymorphisms have already been associated with the presence of pre-neoplastic and neoplastic lesions in uterine cervix, we did not observe any association of these SNPs with the persistence of cytological abnormalities (11,13).
This was the first study in which the risk of persistence of pre-neoplastic cervical  At the first meeting (T 1 ), an interview was performed for information about sociodemographic and behavioral characteristics, and cervical sample was collected 13 for cytological analysis, genetic polymorphism evaluation, and HPV detection.
After six months of follow-up (T 2 ), 164 women performed the second cytological analysis. From this group, 50 participants presenting normal cytology at T 1 and T 2 , and eight women with normal cytology at T 1 and presence of preneoplastic lesion at T 2 were excluded ( Figure 1). Thus, sample group (n=106) was divided into: Remission (n=60): presence of pre-neoplastic lesion at T 1 , and normal cytology at T 2 ; Persistence (n=46): pre-neoplastic lesion detected at T 1 and T 2 .
Presence of pre-neoplastic lesion was considered when Atypical Squamous (R F L P ) (42). HPV negative samples were also analyzed by conventional PCR with GP5+/GP6+ primers (43).
Sequences of primers, restriction enzymes, and PCR protocols were presented in Supplementary Tables 1, 2, and 3. Table 3 shows the size of DNA fragments that characterize the genotypes of polymorphisms analyzed.

Availability of data and materials:
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests:
There are no compenting interests.     Study flow diagram.

Supplementary Files
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