title: ZAP-70 methylation status is associated with ZAP-70 expression status in chronic lymphocytic leukemia.
abstract: OBJECTIVE
  ZAP-70 expression is a recognized prognostic marker in chronic lymphocytic leukemia (CLL). The aim of this study was to analyze whether the methylation status of the ZAP-70 gene is associated with expression of the ZAP-70 protein.
  
  
  METHODS
  Patients with CLL (n=87), acute lymphoblastic leukemia (n=13), mantle cell leukemia (n=13) and splenic marginal zone lymphoma (n=14) of known immunoglobulin gene mutation (IgVH) status were studied. The methylation status of the 5' region of ZAP-70 was analyzed by combined bisulphite restriction analysis (COBRA), southern blotting and bisulphite sequencing in 10 CLL patients and in normal T/NK and B cells. Further COBRA of a single CpG site located 334bp downstream of the ZAP-70 transcription start site (C-334) was then performed on all patients.
  
  
  RESULTS
  ZAP-70 expression status in CLL and normal peripheral blood lymphocytes is associated with the methylation status of the intron1-exon2 boundary region of ZAP-70 and methylation status of C-334 determined by COBRA is representative of methylation in this region. Of 87 CLL patients, 51/53 ZAP-70 negative patients had methylation at C-334 and 30/32 ZAP-70 positive patients did not have methylation (p<0.0001); a similar association was seen in all other diseases. Median survivals of methylated and unmethylated CLL patients were 211 and 85 months, respectively (p<0.0001).
  
  
  CONCLUSIONS
  Measuring ZAP-70 methylation status at C-334 is a simple and reproducible method for predicting prognosis in CLL, which is closely associated with ZAP-70 expression and IgVH gene mutational status. Methylation of a highly conserved intronic region of ZAP-70 may be responsible for regulation of expression in normal and neoplastic cells.
authors.affiliation: Department of Molecular Biology and Haematology, Royal Bournemouth Hospital, Bournemouth, Dorset, BH7 7DW, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Corcoran
authors.forename: Anton
authors.initials: A
authors.lastname: Parker
authors.forename: Jenny
authors.initials: J
authors.lastname: Orchard
authors.forename: Zadie
authors.initials: Z
authors.lastname: Davis
authors.forename: Michaela
authors.initials: M
authors.lastname: Wirtz
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Schmitz
authors.forename: David
authors.initials: D
authors.lastname: Oscier
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D017124:Conserved Sequence; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics; D006801:Humans; D007438:Introns; D007938:Leukemia / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D012016:Reference Values; D015183:Restriction Mapping; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D015226:Dinucleoside Phosphates; D051746:ZAP-70 Protein-Tyrosine Kinase; C015772:CpG dinucleotide