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Chronic Lymphocytic Leukemia (CLL) pathogenetic mechanisms have not been fully elucidated yet. However, genetic and epigenetic alterations seem to be involved in the pathogenesis and extensive clinical heterogeneity of the disease. DNA methylation in CpG sites of a gene promoter, which may affect the chromatin structure as well as gene transcriptional activity, is a crucial epigenetic modification in CLL. RAD21 gene is involved in DNA repair and its encoded product acts as basic subunit of the Cohesin protein complex that regulates the cohesion and proper separation of sister chromatids during mitosis or meiosis.

We investigated the methylation status of RAD21 gene promoter and its possible implication in CLL pathogenesis and the formation of CLL cytogenetic aberrations.

The study included 105 CLL patients and 17 healthy donors (controls). Total genomic DNA extraction was performed from bone marrow or peripheral blood samples of all patients and controls. Methylation analysis of RAD21 gene promoter was carried out using the new techology of MethylScreen™ in the CFX96Biorad Real-Time PCR system. For this purpose, we used EpiTect Methyl II PCR Assay which enables us to calculate the methylated and unmethylated fraction after simultaneous digestions with specific restriction enzymes. Karyotypic analysis was performed on unstimulated and stimulated with CpG-oligonucleotide DSP-30 bone marrow cells of CLL patients. FISH analysis was carried out using the commercial CLL set probes for detection of the most common abnormalities of the disease including deletions of 17p13 (TP53), 11q22.3 (ATM) and 13q14.3/13q34.3 (D13S319/13q34) regions and trisomy 12 (CEP 12).

Among the105 CLL patients, 21 patients exhibited a normal karyotype also confirmed by FISH and 84 patients showed chromosome abnormalities detected by karyotypic or/and FISH analysis. Methylation study was successful in all healthy donors and in 101 out of 105 CLL patients. All healthy donors had non-methylated RAD21 gene promoter. On the contrary, 25.74% (26/101) of CLL patients carried >10% cells with methylated CpG islands in RAD21 promoter, which was significantly increased compared to controls (p=0.039, χ²=4.25, df=1). RAD21 methylated cell fraction varied among patients. More specifically, 9.9% of patients (10/101) showed 11-50% methylation rate, 10.89% (11/101) 51-89% and 4.95% (5/101) showed high methylation rate score, >90% of the analyzed cells. Stratification of patients according to cytogenetic findings showed that the promoter of RAD21 was methylated in 28.57% of patients (6/21) with normal karyotypes and 25% of patients (20/80) with abnormal karyotypes. In detail, methylation in RAD21 promoter was present in 33.33% of patients (5/15) with abn(14q32), in 33.33% (4/12) with abn(8), in 31.25% (5/16) with -17/del(17p), in 27.78% (5/18) with trisomy 12, in 25.81% (8/31) with del(13q), in 20% (2/10) with del(6q) and in 12.5% (2/16) with del(11q). Based on karyotypic complexity, RAD21 promoter was methylated in 18.18% (4/22) of patients with a single chromosome aberration, 26.09% (6/23) with two chromosome aberrations and 25.71% (9/35) of patients with complex karyotype (≥3 aberrations).

Methylation of RAD21 gene promoter, which leads to transcriptional inactivation and consequently inhibition of RAD21 expression, seems to be implicated in CLL pathogenesis and the formation of specific chromosome aberrations. Clarification of the epigenetic landscape of CLL may help in the design of new targeted therapeutic agents.