Different Gene Methylation Status of the CDKN2B and/ or PDLIM4 as the Result of Comparative Analysis to the Global DNA Methylation in Unsorted Cell Population of Multiple Myeloma Patients

Background: Multiple Myeloma (MM) is a hemato-oncological disease characterized by clonal expansion of malignant plasma cells in the Bone Marrow (BM). Apart from genetic changes, such as point mutations, deletions or translocations, it is well known, that in pathogenesis of MM are also involved epigenetic changes such as DNA methylation. Methylation of both CDKN2B gene, representing an inhibitor of cyclin dependent kinases, and PDLIM4 gene, one of potential tumor suppressor genes engaged in MM evolution, were evaluated in newly diagnosed multiple myeloma patients. Methods: The quantification of the global DNA methylation at 5´-CCGG- 3´sequence using LU minometric Methylation Assay (LUMA) and the colorimetric quantification of the global DNA methylation were performed. Bisulfite-treated DNA in 13 CpGs of a promoter, and 16 CpGs of the first exon of the CDKN2B gene, 9 CpGs of the PDLIM4 gene promoter were analyzed by pyrosequencing. Results: Studied CDKN2B gene regions revealed CpGs methylation in the range 2.8 - 6%, whereas PDLIM4 gene promoter showed increased level of methylated CpGs in the range 13.1 - 27%. We found a strong positive correlation between the global DNA hypomethylation (LUMA) and CDKN2B expression (r = 0. 766, P < 0. 01), and strong negative correlation between global DNA hypermethylation (LUMA) and PDLIM4 promoter methylation level (r = - 0. 994, P < 0. 01). Our data indicate functional unmethylated CDKN2B gene, in contrast to methylated tumor-suppressor PDLIM4 gene in newly diagnosed multiple myeloma patients. Conclusion: In unsorted bone marrow cells of newly diagnosed multiple myeloma patients, the CpG methylation pattern of the studied CDKN2B and PDLIM4 genes varies depending on overall DNA methylation level. Their different methylation status determined in both global DNA hypomethylated and hypermethylatied groups of patients could be related to a followed progression of the multiple myeloma disease. On the base of statistical analysis, the PDLIM4 gene show significantly increased methylation state with negative correlation to the detected DNA methylation level. These methylation changes of the PDLIM4 gene can contribute to pathogenesis of myeloma and its methylation status acts as a prognostic factor.