Identification of aberrant methylation of DLC-1 by pyrosequencing in acute lymphoblastic leukemia in children

Abstract

Objective To evaluate the DLC-l gene promoter methylation in acute lymphoblastic leukemia(ALL)in children by methylation specific-PCR(MSP),explore the its prognostic value.Methods Both pyrosequecing and MSP was used to detect DLC-1 gene methylation in bone marrow samples from 34 children with acute lymphoblastic leukemia,5 normal bone marrow samples and acute leukemia T cell line 6T-CET.DLC-1 mRNA expression in cells with、or without treatment with 5-aza-2'-deoxycytidine wag investigated by real time RT-PCR Results MSP analysis showed that DLC-1 promoter methylation Was identified in 21 of bone marrow samples from ALL patients,but was absent in 5 normal bone marrow specimens.These results were completely agreement with pyrosequencing analysis.In additional,the latter can give the quantitative analysis of methylation status in specific CpG sites.No association Was observed between DLC-1 methylation and patient’S clinical-biologic characteristics including age,gender,WBC count,FAB classification,immunology typing and clinical typing(P>0.05)In 18 ALL patients achieving complete remission(CR)witIl DLC-1 methylation,14 relapsed in 12 monthhs,while only 4 relapsed in 1 patients without DLC-1 gene metllylation.Treatment of the cell line 6T-CEM harboring methylation with 5-aza-2'-deoxycytidine increased DLC-1 expression in dose dependent manner. Conclusions This is the first report of high frequency of promoter methylation of DLC-1 in ALL It shows that DLC-1 methylation iS useful for predicting relapse of ALL.Pyrosequencing assay Call provide a sensitive,easy-to-use method for quantitative assessment of DLC-1 methylation. Key words: Leukemia,lymphocytic,chronic; Tumor suppressor proteins; Promoter regions(genetics); Methylation; Polymerase chain reaction