Originally published as JCO Early Release 10.1200/JCO.2005.02.196 on April 4 2005

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TWIST2 Demonstrates Differential Methylation in Immunoglobulin Variable Heavy Chain Mutated and Unmutated Chronic Lymphocytic Leukemia

Aparna Raval, David M. Lucas, Jennifer J. Matkovic, Kristi L. Bennett, Sandya Liyanarachchi, Donn C. Young, Laura Rassenti, Thomas J. Kipps, Michael R. Grever, John C. Byrd, Christoph Plass

From the Division of Human Cancer Genetics, Division of Hematology-Oncology, Department of Medicine, and Department of Biostatistics, The Ohio State University, Columbus, OH; and Division of Hematology/Oncology, Department of Medicine, University of California, San Diego, CA

Address reprint requests to John C. Byrd, MD, Division of Hematology-Oncology, Starling Loving Hall, Room 302, The Ohio State University, Columbus, OH 43210; e-mail: byrd-3{at}medctr.osu.edu.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V_H) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V_H mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells.

MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells.

RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V_H show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V_H were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression.

CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V_H mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

Supported in part by National Cancer Institute grants CA81534 to the CLL Research Consortium (L.R., T.K., M.R.G., J.C.B., D.L.), CA110496 (J.C.B., K.P., A.R., D.L.) CA93548 (C.P.), P30 CA16058 (C.P., L.J.R., T.J.K., J.C.B.), The Leukemia and Lymphoma Society of America (J.C.B., K.P.), The D. Warren Brown Foundation (J.C.B.). C.P. is a Leukemia and Lymphoma Society Scholar and J.C.B. is a Leukemia and Lymphoma Society Clinical Scholar. A.R is a Leukemia Society Fellow.

C.P. and J.C.B contributed equally to this article.

Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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