65ª Reunião Anual da SBPC

C. Ciências Biológicas - 8. Genética - 6. Genética

The Influence of MTHFR C677T polymorphism on global genomic DNA methylation in oral cells MTHFR C677T polymorphism and DNA methylation.

Isabela Tatiana Sales de Arruda - Universidade Federal da Paraíba Darlene Camati Persuhn - Universidade Federal da Paraíba Naila Francis Paulo de Oliveira - Universidade Federal da Paraíba

INTRODUÇÃO:

DNA methylation is one epigenetic modification, mediated by DNA methyltransferases dependent of methyl donor and SAM is the primary methyl group donor for most biological methylation reactions (Chiang et al. 1996). This is provided by the methionine cycle. The availability of methyl groups for DNA methylation reactions is mainly regulated by the MTHFR activity. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methyleneTHF to 5-methylTHF that can be used in methionine cycle to generate SAM (Schwann and Rozen, 2001). Several common single nucleotide polymorphisms (SNP) of the one-carbon enzymes have been identified. In particular, the MTHFR C677T polymorphism (also known as rs1801133, p.Ala222-Val) - substitution of C to T at nucleotide 677 in exon 4, leads to the amino acid alanine being replaced valine - is the commonest of the MTHFR gene and produce a thermolabile variant of the MTHFR resulting in a decrease of 30% in this enzyme activity (Sharp and Little 2004). So, there has been a speculation that this polymorphism induces hypomethylation and then activate proto-oncogenes, and that could explain the association with the MTHFR C677T polymorphism and cancer (Izmirli, 2012).

OBJETIVO DO TRABALHO:

The aim of the present study was to assess the influence of MTHFR C677T polymorphism on genomic global DNA methylation in oral cells in young subjects with no systemic disorder neither with clinical signs of any visible change in oral mucosa.

MÉTODOS:

This study was approved by the Institutional Review Board of the Federal University of Paraiba. Volunteers were informed about the nature of the research. The sample of recruited subjects has between 19-25 years old, male and female and healthy. Subjects were included in three categories according to MTHFR Genotypes: MTHFR 677CC (normal homozygous); CT (heterozygous); TT (affected homozygous). The oral cells were collected by mouthwash with dextrose 3% and centrifuged at 3000rpm for 10 minutes (Trevilatto and Line 2000). DNA was purified using DNAzol® (Invitrogen). MTHFR C677T genetic polymorphism was detected by PCR-RFLP (Arruda et al., 1997). The PCR products of 198 bp were digested with endonuclease HinfI, producing a 175 and a 23 bp fragments if the polymorphism was present. These fragments were resolved by electrophoresis in 6% polyacrylamide gel with Gel Red (Biotium) staining. The global DNA methylation levels in epithelial oral cells were obtained with an ELISA based commercial kit (MDQ1, Imprint® Methylated DNA Quantification Kit, Sigma Aldrich). DNA at a concentration of 150 ng. The global DNA methylation levels were compared for each group using Kruskal-Wallis test at significant level of 5%, using the statistical program GraphPad Prism 5.0.

RESULTADOS E DISCUSSÃO:

All groups (54 subjects aged 19–25 years) were characterized according to MTHFR genotype being CC (normal homozygous) (n=17); CT (heterozygous) (n=19); TT (affected homozygous) (n=18). We detected in oral epithelial cells up to 55% and 47% for CC and CT genotypes and up to 34% for TT genotype. The three groups did not differ by age and global methylation level by gender (p=0.75; Kruskal-Wallis). We observed a trend towards lower degree of methylation in individuals with TT genotype in comparison to CC and CT groups. MTHFR plays a central role in biotransformation of folate to form SAM, the universal methyl donor in cells (Schwann and Rozen, 2001). DNA methylation is dependent of SAM and thus it has been suggested that genetic alterations in MTHFR like polymorphism may influence the epigenome (Sharp and Little, 2004). Global hypomethylation increases oncogene activations (Feinberg, 2007). This is the first study to evaluate this association in oral epithelial cells and we did not detected significant differences among the genotype. We observed difficulty of finding a direct correlation between polymorphisms and deregulation in DNA methylation. The data show that the diet may be more decisive in DNA methylation profile than MTHFR polymorphism (McKay et al., 2012).

CONCLUSÕES:

This study evaluated the polymorphism impact in the profile of global DNA methylation and no significant differences between groups was detected, although a tendency for a decrease in the percentual of global DNA methylation in individuals with TT genotype was observed. However, the small sample size precludes any definitive conclusions to be drawn. The findings of this pilot study require confirmation in a larger study with serum folate dosage and combinations of one-carbon enzymes and DNMT polymorphisms.

Palavras-chave: DNA methylation, Epigenetic, MTHFR polymorphism.