Análise do perfil de metilação do DNA em pacientes com e sem lesão de boca na paracoccidioidomicose

Abstract

Epigenetic changes such as DNA methylation is a specific mechanism for the regulation of gene expression involved in many pathological situations. Altered patterns of DNA methylation are common in cancers and viral infections. Paracoccidioidomycosis (PCM) is a systemic fungal infection caused by a thermo-dimorphic fungus, Paracoccidioides brasiliensis. Brazil is having the largest number of cases in South America. The disease presents primary pulmonary involvement that spreads to oral mucus, skin, reticuloendothelial system, and adrenals. Oral lesions are characterized by erythematous fine granular hyperplasia, and speckled with occasional bleeding called moriform stomatitis. The purpose of this study was to identify profiles of different DNA methylation in patients with and without oral lesions in PCM. The identification of these profiles was based on the strategy of MS-AP-PCR (Methylation-Sensitive arbitrarily Primed PCR). The DNA extracted from whole blood of patients with and without oral lesions was digested with restriction enzymes: Hpa II and Msp I. The digestion products were amplified by PCR (polymerase chain reaction) using arbitrary primers. We obtained a differing profile after amplifying the DNA from patients without mouth lesions compared to the profile of patients with mouth lesions from agarose gel electrophoresis. The differently expressed band was purified and cloned, and the obtained plasmids, P5, P6 and P7, were analyzed by the softwares PhredPhrap and BLASTN tool to obtain mapping data and identify the sequences present in the human genome. The cloned fragment in the plasmid P5 showed similarity with a DNA sequence present in chromosome 20, next to the gene YTHDF1. The fragments cloned in plasmids P6 and P7 showed similarity with the DNA sequences present on chromosome 15, the P6 fragment showed similarity with an intronic sequence from the gene, “RNA binding protein with multiple splicing 2 "(RBPMS2), and the P7 fragment showed similarity with the intronic sequence of the gene "diphthamine biosynthesis 6" (DPH6) ".biosynthesis 6" (DPH6)". The YTHDF1 gene expression in leukocytes was tested by real-time PCR and no significant difference were found in both groups of patients. There is no description in the literature relating the YTHDF1 gene with PCM which has shown the importance of study epigenetic for understanding the cellular mechanisms under normal and pathological conditions.