title: Quantitative DNA methylation analysis identifies a single CpG dinucleotide important for ZAP-70 expression and predictive of prognosis in chronic lymphocytic leukemia.
abstract: OBJECTIVE
  Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.
  
  
  METHODS
  High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.
  
  
  RESULTS
  Through this comprehensive analysis, we identified a small area in the 5' regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.
  
  
  CONCLUSIONS
  Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5' regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.
authors.affiliation: German Cancer Research Center, Heidelberg, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Claus
authors.forename: David M
authors.initials: DM
authors.lastname: Lucas
authors.forename: Stephan
authors.initials: S
authors.lastname: Stilgenbauer
authors.forename: Amy S
authors.initials: AS
authors.lastname: Ruppert
authors.forename: Lianbo
authors.initials: L
authors.lastname: Yu
authors.forename: Manuela
authors.initials: M
authors.lastname: Zucknick
authors.forename: Daniel
authors.initials: D
authors.lastname: Mertens
authors.forename: Andreas
authors.initials: A
authors.lastname: Bühler
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Oakes
authors.forename: Richard A
authors.initials: RA
authors.lastname: Larson
authors.forename: Neil E
authors.initials: NE
authors.lastname: Kay
authors.forename: Diane F
authors.initials: DF
authors.lastname: Jelinek
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kipps
authors.forename: Laura Z
authors.initials: LZ
authors.lastname: Rassenti
authors.forename: John G
authors.initials: JG
authors.lastname: Gribben
authors.forename: Hartmut
authors.initials: H
authors.lastname: Döhner
authors.forename: Nyla A
authors.initials: NA
authors.lastname: Heerema
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
authors.forename: Christoph
authors.initials: C
authors.lastname: Plass
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016000:Cluster Analysis; D018899:CpG Islands; D019175:DNA Methylation; D018572:Disease-Free Survival; D005260:Female; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D012045:Regulatory Sequences, Nucleic Acid; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics* / Q000378:metabolism
chemical_list: D007143:Immunoglobulin Heavy Chains; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human