title: Mutational and methylation analysis of the cyclin-dependent kinase 4 inhibitor (p16INK4A) gene in chronic lymphocytic leukemia.
abstract: OBJECTIVE
  Chronic lymphocytic leukemia (CLL) comprises a heterogeneous group of at least two types of disease entities characterized by distinctive clinical, immunophenotypical and genetic features. The molecular mechanisms underlying the pathogenesis and the histological transformation of CLL are not well known. The INK4A/p16, a cyclin dependent kinase inhibitor has been considered as a tumor suppressor gene. Inactivation of this gene by homozygous deletions, mutations and hypermethylation occurs in a variety of human neoplasms. The aim of the present study was to determine the frequency of p16 gene deletions and mutations as well as the methylation status of the same gene in CLL patients.
  
  
  METHODS
  We examined 34 samples from CLL patients by Southern Blotting, Single-Strand Conformation Polymorphism (SSCP), DNA sequencing and Methylation-Specific PCR.
  
  
  RESULTS
  Southern Blot analysis revealed non-rearranged bands in 33/34 cases. Homozygous deletions were not observed in any case. In 1/34 case a rearranged band was detected with EcoRI enzyme. The PCR-SSCP analysis of exons 1 and 3 revealed normal pattern of migration in all cases examined. The analysis of exon 2 revealed abnormal migration pattern in 2/34 cases (5.8%). Sequencing of these cases revealed the presence of the ALA148THR polymorphism. Methylation analysis of p16 gene promoter revealed hypermethylation of CpG islands in 6/34 cases (17.6%).
  
  
  CONCLUSIONS
  These results indicate that genetic alterations of p16 gene are rare events in patients with CLL. The clarification of the role of p16 gene promoter methylation in the pathogenesis and evolution of CLL needs further investigation.
authors.affiliation: Second Department of Internal Medicine, Propaedeutic, University of Athens, ATTIKON University General Hospital, Athens, Greece.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Tsirigotis
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Pappa
authors.forename: Stefanos
authors.initials: S
authors.lastname: Labropoulos
authors.forename: Sotirios
authors.initials: S
authors.lastname: Papageorgiou
authors.forename: Frinda
authors.initials: F
authors.lastname: Kontsioti
authors.forename: John
authors.initials: J
authors.lastname: Dervenoulas
authors.forename: Efstathios
authors.initials: E
authors.lastname: Papageorgiou
authors.forename: Anna
authors.initials: A
authors.lastname: Panani
authors.forename: George
authors.initials: G
authors.lastname: Mantzios
authors.forename: Theofanis
authors.initials: T
authors.lastname: Economopoulos
authors.forename: Sotirios
authors.initials: S
authors.lastname: Raptis
mesh_terms: D018899:CpG Islands; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D005091:Exons; D017353:Gene Deletion; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000209:etiology / Q000235:genetics*; D009154:Mutation; D020125:Mutation, Missense
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16