Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.
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.
Lyko
Frank
F
Research Group Epigenetics, German Cancer Research Center, Heidelberg, Germany.
Stach
Dirk
D
Brenner
Axel
A
Stilgenbauer
Stephan
S
Döhner
Hartmut
H
Wirtz
Michaela
M
Wiessler
Manfred
M
Schmitz
Oliver J
OJ
D001896:Boron Compounds / Q000737:chemistry; D002853:Chromatography, Liquid; D003596:Cytosine / Q000737:chemistry*; D019175:DNA Methylation; D019075:Electrophoresis, Capillary; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D013050:Spectrometry, Fluorescence
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D003596:Cytosine