Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients

Abstract

Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

Characterization of DNA Demethylation Effects Induced by 5-Aza-2′-Deoxycytidine in Patients with Myelodysplastic Syndrome

“…The validity of the results obtained by this kind of analysis has been confirmed in a number of investigations with diverse experimental backgrounds (see for example, refs. 13,14).…”

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

“…However, with regards to the IgV H mutation status, Lyko and colleagues, 14 using a different quantitative method and a chi-squared statistics, have earlier identified an estimated cut-off value in global cytosine methylation that maximized the differences in the distribution of IgV H mutated and un-mutated cases. The same statistics applied to our data set allowed to identify 23.8% (p = 0.0223) and 86.1% (p = 0.0107) as cut-off values for Alu and SAT-α methylation levels, respectively (data not shown).…”

“…Although a reduced overall 5-Methyl-Cytosine (5mC) content has previously been demonstrated in CLL, 14,15 the role of repetitive element methylation has been scarcely investigated in this disease. 23 In the current study, we quantified the methylation levels of Alu, LINE-1 and SAT-α repetitive sequences in a panel of seven healthy donors and 77 untreated B-CLLs in early stage disease, using the Pyrosequencing technology.…”

“…12 Repetitive elements, mainly the interspersed repeated DNA sequences, contain numerous CpG dinucleotides; therefore, the methylation status of these sequences is relevant to the understanding of the global DNA methylation pattern. 13 Global or gene-specific hypomethylation, [14][15][16][17] as well as hypermethylation affecting putative tumor-suppressor genes, [18][19][20][21][22] …”

Epigenetics and chronic lymphocytic leukemia

“…In other words, less genomic DNA methylation is associated with increased mutations and genomic instability. The DNA from patients with CLL are globally hypomethylated when compared with peripheral blood mononuclear cells from healthy volunteers [18][19][20]. This has been confirmed by two groups, using two different methods of measurement.…”