Mutational and methylation analysis of the cyclin-dependent kinase 4 inhibitor (p16INK4A) gene in chronic lymphocytic leukemia.
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.
Tsirigotis
Panagiotis
P
Second Department of Internal Medicine, Propaedeutic, University of Athens, ATTIKON University General Hospital, Athens, Greece.
Pappa
Vassiliki
V
Labropoulos
Stefanos
S
Papageorgiou
Sotirios
S
Kontsioti
Frinda
F
Dervenoulas
John
J
Papageorgiou
Efstathios
E
Panani
Anna
A
Mantzios
George
G
Economopoulos
Theofanis
T
Raptis
Sotirios
S
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
D019941:Cyclin-Dependent Kinase Inhibitor p16