title: TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Human Cancer Genetics, Department of Medicine, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Aparna
authors.initials: A
authors.lastname: Raval
authors.forename: David M
authors.initials: DM
authors.lastname: Lucas
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Matkovic
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Bennett
authors.forename: Sandya
authors.initials: S
authors.lastname: Liyanarachchi
authors.forename: Donn C
authors.initials: DC
authors.lastname: Young
authors.forename: Laura
authors.initials: L
authors.lastname: Rassenti
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kipps
authors.forename: Michael R
authors.initials: MR
authors.lastname: Grever
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.forename: Christoph
authors.initials: C
authors.lastname: Plass
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D001402:B-Lymphocytes / Q000473:pathology; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D015973:Gene Expression Regulation, Leukemic; D015972:Gene Expression Regulation, Neoplastic; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics*; D011379:Prognosis; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D027041:Somatic Hypermutation, Immunoglobulin; D014157:Transcription Factors / Q000235:genetics*; D016896:Treatment Outcome; D051879:Twist-Related Protein 1
chemical_list: D000964:Antimetabolites, Antineoplastic; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012097:Repressor Proteins; C486274:TWIST2 protein, human; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine