Hypermethylation of the p15/INK4B Gene in Fanconi Anemia.

Abstract Fanconi anemia (FA) is an inherited bone marrow failure syndrome with multiple complementation groups, characterized by genomic instability and predisposition to MDS and AML. Recent evidence indicates that multiple FA proteins are involved in DNA repair. Thus, increased genetic damage and secondary dysregulation of cell proliferation, differentiation and apoptosis are thought to play important roles in the development of bone marrow failure and subsequent progression to MDS/AML. However, little is known about molecular abnormalities responsible for these hematological disorders. Numerous studies indicated that epigenetic silencing of p15/INK4B, an inhibitor of cyclin-dependent kinases, plays an important role in the pathogenesis of MDS and AML. In the present study, we examined methylation status of 5′ CpG islands of the p15 gene in bone marrow mononuclear cells of FA patients, using methylation-specific PCR (MSP) and combined bisulfite restriction analysis (COBRA). Bone marrow samples were analyzed in 10 patients and serially studied in 4 of them. Hypermethylation of the p15 promoter region was detected in 5 patients (50%). This group included 3 patients with MDS: FA28-1 with refractory anemia (RA), FA87 with RAEB (RA with excess of blasts), and FA88 with later development of RA and progression to RAEB; whereas myelodysplasia was not observed in 2 patients (FA89, FA90). In two cases (FA88, FA90), p15 hypermethylation became negative during their courses, perhaps because of decreased myeloid cells. On the other hand, none of 5 patients without p15 hypermethylation had MDS. These results suggest that p15 hypermethylation is associated with development of MDS and occurs in the early phase of clonal evolution in the disease. Methylation status of p15 may be a useful prognostic factor of FA. Patient Age at onset (year old) Time from onset (month) Cytopenia MDS Cytogenetic abnormalities p15 methylation MSP b p15 methylation COBRA c a siblings, b MSP: methylation specific PCR, c COBRA: combined bisulfite restriction analysis, d ND: not determined FA28-1a 5 128 severe RA − − + 133 severe RA − + ++ FA87 8 252 severe RAEB + + +++ FA88 5 31 moderate − − + +++ 45 severe RA + − − 58 severe RAEB + + + FA89 5 49 mild − − + + 56 severe − − + + FA90 2 2 mild − − + ++ 31 severe − − − − FA28-2a 5 51 mild − − − NDd FA28-3a 3 12 mild − − − NDd FA47 3 15 mild − − − NDd FA68 5 46 moderate − − − NDd FA91 5 129 mild − − − NDd