Clonality studies and N-ras and p53 mutation analysis of hematopoietic cells in Fanconi anemia

Patients with Fanconi anemia (FA) have an extraordinary predisposition to acute myelogenous leukemia (AML). The genetic mechanisms underlying the neoplastic transformation of FA hematopoietic cells are unknown. In this study, we have investigated the molecular features of hematopoiesis in the course of FA at different stages of the disease, including aplastic anemia, myelodysplastic syndrome (MDS), and AML. The analysis focused on defining the clonality status of FA hematopoiesis as well as the putative involvement of N-ras, a dominantly acting oncogene, and p53, a tumor suppressor gene, which are known to play a role in human hematopoietic tumors. Clonality of hematopoiesis was assessed by testing X-chromosome Inactivation at the DXS255 locus, which displays different methylation patterns according to the activation status of the corresponding X homolog. Five out of seven FA cases analyzed for clonality displayed monoclonal hematopoiesis, including one case at the aplastic anemia stage, three cases with MDS and one with AML. Mutations of the N-ras and p53 genes were studied by a combination of single strand conformation polymorphism (SSCP) analysis and direct sequencing of the PCR product in the bone marrow and/or peripheral blood of 18 FA patients (seven with aplastic anemia, seven with MDS, four with AML). Only normal N-ras and p53 sequences were detected in all cases analyzed. These results suggest that monoclonal hematopoiesis is a frequent finding in the course of FA and may precede the onset of neoplasia in some cases. The genetic mechanisms underlying FA-associated leukemogenesis appear to be independent of N-ras and p53 mutations, which are relatively frequent events in myeloid tumors associated with other hematologic disorders.