Analysis of CpG Island DNA Methylation of p15^INK4b and RIL in Bone Marrow Samples of Patients with Monoclonal Gammopathies

Introduction: Adding a methyl group on the cytosine residues of CpG dinucleotides (CpGs) is a basic phenomenon in DNA methylation. This modification belongs to a group of epigenetic changes that are thought to play one of the key roles in tumor development and progression. DNA hypermethylation occurs mainly in CpG islands of the promoter region in tumor-supressor (TS) genes, which leads to gene silencing. On the other hand, global DNA hypomethylation leads to a genomic instability. Many specific genes are epigenetically changed in individual malignant diseases and these genes are intensively studied by the scientific community. According to the recent research in plasma cell neoplasms, the global genic hypomethylation is predominant in contrast to hypermethylation events. Epigenetic changes of TS genes may worsen prognosis, drug response and microenvironment interaction in multiple myeloma patients. Our aim was to assess the methylation level of the RIL and p15^INK4b gene. RIL protein acts as suppressor of cell proliferation and it sensitizes tumor cells to apoptosis, on the other hand, the p15 ^INK4bplays an important role in inhibiting the cell cycle progression in G1 phase.

Methods: Our updated study includes 72 bone marrow aspirate-samples from 68 patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS). 68 samples were acquired at the time of the diagnosis and four patients with MM were assessed also in remission after chemotherapy. Extracted DNA after bisulfite modification was examined by pyrosequencing method (Pyromark Q96, Qiagen, Germany) and the selected gene regions – RIL promotor, p15^INK4b promotor and p15 ^INK4b exon were analysed. Nine CpGs in the RIL promoter, thirteen CpGs in the p15^INK4b promoter and sixteen CpGs in the p15^INK4bexon were studied. Average methylation level (MtL) was generated and samples were sorted according to the mean MtL (%) into four groups for the RIL gene (less than 10% MtL, 11-19% MtL, 21-50% and more than 50%). We used commercially available unmethylated DNA and methylated DNA control. Statistical analysis was performed using Pyromark Software and Wilcoxon rank sum test.

Results: Out of the four groups for the methylation level of the RIL gene promoter, the groups with 11-19% MtL and 21-50% MtL, detected significant and highly significant differences respectively at both p-values - p-value ≤0.05 and p-value ≤0.01. There were only 2 samples with MtL level over 50% in patients with active MM, precluding valid statistical analysis. In both regions of the p15 ^INK4b gene, i.e. the promoter and the exon, we did not find any significant differences between the methylation levels - mean MtL ranged from 3% to 6% in p15^INK4b promotor and from 3% to 12% in p15 ^INK4b exon. The time-dependend analysis of the DNA methylation level changes in four patients assessed at the time of diagnosis and in remission of the disease after chemotherapy course revealed a significant decrease of CpGs methylation in all MM patients reaching therapeutic response.

Conclusions: Updated data on the epigenetic analysis in patients with monoclonal gammopathies confirmed an important role of the RIL gene in the epigenetic network acting in pathogenesis of MM. The presented results suggest possible prognostic value and therapeutic target in patients with MM. In contrast, the p15 ^INK4b gene known as a frequent focus of epigenetic modifications in many tumors, did not show statistically significant differences in our study. Our results indicate that for DNA methylation analyses of MM or MGUS patients, the RIL gene could be more useful marker than the p15 ^INK4b gene. Moreover, the decrease of the methylation level in patients undergoing systemic chemotherapy might be associated with treatment response, suggesting its potential prognostic value.

The paper was supported by the grant IGA MZ CR NT14393.