Analysis of the PTEN gene hypermethylation profile and correlation with the clinical and pathological factors in renal cell carcinoma

Abstract

Despite identification of prognostic factors in renal cell carcinoma, tumors with the same staging, treatment and histological sub-type present distinct evolutions. Molecular biology procedures have sought to identify new prognostic factors able to better stratify patients regarding a greater or lesser risk of recurrence and submit them to treatments based upon molecular targeted therapies. PTEN (phosphatase with tensin homology deleted in chromosome 10) is a tumor suppressor gene acting on control of cell migration, proliferation and apoptosis. PTEN may be inactivated by mutation, deletion and hypermethylation in different solid neoplasm. DNA methylatioin corresponds to an epigenetic event in which occurs addition of the radical methyl (CH3) to carbon 5 of the nitrogenous bases Cytosine (C), located at 5' from the Guanines (G) through action of the DNA methyltransferase enzymes. The methyl radical occupies the gene's promoting region preventing formation of a primary transcript, bringing about silencing. Analysis of the hypermethylation profile of certain genes has been tested regarding its prognostic capability by means of a correlation with clinical pathological factors, besides being the target of therapies aimed to reverse the phenotype and normalize cell growth. In the analysis of the PTEN gene methylation profile, existence of a pseudogene located in chromosome 9p12 must be taken into account. It presents 98% of the same nucleotide sequences as the true PTEN and interferes with analysis of results. For ZYSMAN et al. 2002, the correct choice of the primer to be used to assess methylation of PTEN is important to avoid false positive results by means of a correct differentiation of the true and of the pseudogene. Frequency of hypermethylation of the CpG islands in the promoter region of PTEN gene in renal cell carcinoma (RCC) remains unknown. Research of the PTEN methylation will be carried out in this study by using the pyrosequencing technique. The pyrosequencing technique is a method that analyzes quantitatively and in real time, incorporation of nucleotides through release of pyrophosphate by luminometric detection. The DNA segments to be analyzed will be manually extracted from paraffin embedded blocks and then they will be submitted to real time PCR reaction with beta actin gene to confirm the DNA quality. After DNA extraction, the material will be treated with bisulfite and another real time PCR will be realized to confirm the integrity of DNA and the complete treatment with bisulfite. Methylated cytosine in the original DNA remains unaffected with the use of bisulfite, while the not methylated cytosines are converted to uracil. Later, the material will be hybridized with PTEN primer to complement the DNA sequence under study and methylation of gene will be analyzed by pyrosequencing. Use of pyrosequencing in the research of methylation has some advantages in relation to other techniques: 1) control the quality of the reaction after treatment with biosulfite; 2) analyze methylation sites adjacent to the principal site, identifying as well, partially methylated sequences; 3) confirm the correct position of the primer in the DNA molecule modified through the nucleotide sequence generated around the methylation site. Data obtained from medical charts were typed and filed in a data bank using the "Excel 7.0 for Windows" program. Data will be updated as for current follow-up time and cases of renal neoplasm operated in the institution, from 2005 to 2008 will be added, totaling approximately 200 cases. For statistical analysis the software Statistical Package for Social Science (SPSS) for Windows will be used. (AU)