Analysis of Telomeric Proteins Expression in Plasma Cell Disorders

Telomeres are specialized DNA-protein complexes that play an essential role in maintaining chromosomal integrity and stability. Their progressive shortening with each cell division can be compensated by the enzyme telomerase, a specialized ribonucleoprotein complex that includes an RNA template (TERC) and a reverse transcriptase catalytic subunit (hTERT). In addition to telomerase, telomere length (TL) maintenance depends on the shelterin complex, a core of telomere-binding proteins that protect telomeric ends. Among them, TRF1 and TRF2 (telomeric repeat-binding factor 1 and 2) are negative regulators of TL. On the other hand, TANK1 (tankyrase-like protein 1) (positive regulator) interacts with and ADP-ribosylates TRF1, displacing it from telomeric DNA. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. In this study, we evaluated the association between the expression of telomeric genes TRF1, TRF2, TANK1 and hTERT in patients with MM and MGUS. Results were correlated with clinicopathologic characteristics of patients. Fifty-seven patients: 32 MM (21 males; mean age: 70.5 years; range: 52–86 years; 53% stage III) and 25 MGUS (9 males; mean age: 66.4 years; range: 39–82) were studied. All patients provided informed consent and the study was approved by the Ethics Committee of our Institution. RNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls. Gene expression was quantified by real time PCR, using Taq- Man methodology. GAPDH (Gliceraldehide-3-phosphate dehydrogenase) was used as housekeeping gene to normalize the expression of each target gene. The expression levels of TRF1, TRF2, TANK1 and hTERT mRNA were significantly different in cases respect to controls. In patients, lower levels of TRF1, with statistical differences between MM and MGUS, and higher expression of TRF2, TANK1 and hTERT were observed (Table 1). In both pathologies, significant correlation between TRF2 and hTERT were found (r=0.70; p=0.0001 and r=0.36; p=0.07, for MM and MGUS, respectively). Furthermore, a high significant association between TANK1 expression and that of TRF2 and hTERT in MM was observed (r=0.80, p=0.0001; r= 0.45, p=0.009, respectively).

Table 1. Summary of the TRF1, TRF2, TANK1 and hTERT mRNA levels in MM and MGUS

For a better analysis of hTERT expression, patients were divided into two groups: hTERT mRNA<1 (A) and >1 (B). In MM, the mean level of expression was: 0.04±0.001 for group A (n=21) and 7.5±4.9 for group B (n=11), whereas in MGUS, the mean values observed were 0.28±0.001 (n=17) and 5.8±0.78 (n=8), respectively (p=0.0001). Analysis of hTERT and clinical parameters showed a significant correlation in LDH (r=0.77; p=0.005) and creatinine (r=0.73; p=0.01) for MM group B, and in b₂ microglobulin (r=0.74; p=0.05) for MGUS group B. Our findings showed an association between telomeric proteins and plasma cell disorders. Differences in TRF1 expression between MM and MGUS would suggest that this gene may play a role in evolution of MGUS to MM. Finally, the correlation between hTERT and clinical characteristics would indicate that the expression of this gene could be a possible prognostic factor in these pathologies.