Instabilidade cromossómica induzida pelo diepoxibutano (DEB) em fibroblastos cutâneos para diagnóstico da anemia de Fanconi

Abstract

Fanconi anemia (FA) is a rare genetic disease, autosomal recessive or X-linked, genetically and phenotypically heterogeneous, characterized by chromosomal instability (CI), bone marrow failure, and predisposition to cancer. The FA is caused by mutations in any one of 15 known genes. Diagnosis based on clinical manifestations can be difficult because of the phenotypic variability, and laboratory tests may be necessary to confirm it, such as the detection of CI induced by diepoxybutane (DEB) in peripheral blood (PB) lymphocyte cultures. In addition, flow cytometry (FC) based quantification of the percentage of cells accumulated at the phase G2 / M of cell cycle, was also described as a complementary test for the diagnosis of FA. However, these tests are not sensitive for the study of mosaicism, a situation where reversion of the AF mutation to a normal phenotype occurs in hematopoietic cells. In those situations the same tests may be performed in fibroblast cultures, where there is no reversion. Nevertheless, these laboratorial procedures were not yet available in the Laboratory of Cytogenetics of the Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto (ICBAS/UP), a reference lab for the study of FA. Therefore, the objectives of the present work were the characterization of the FA population previously studied in this laboratory concerning the percentage of mosaicism, and the implementation of the technique for fibroblast cultures followed by CI detection and study of progression of the cell cycle to G2/M, by FC. Flow cytometry studies were performed in collaboration with the Laboratory of Cytometry from the Haematology Department of Hospital de Santo António, Centro Hospitalar do Porto. Of the 233 patients studied for CI in the PB, 39 were diagnosed as FA and 13% of them as mosaics. This value, per se, justifies the importance of the implementation of the techniques for analysis of CI and cell cycle in fibroblasts. Therefore, implementation was achieved using cutaneous fibroblast samples from 6 normal individuals and 2 patients enrolled for exclusion of FA, in which the diagnosis was not confirmed. The results obtained in the fibroblasts, both for DEB-induced CI and G2/M arrest, are in agreement with results from the literature for controls and non FA patients. In conclusion, it was possible to implement for the first time and successfully the protocol for fibroblast cultures followed by DEB-induced CI analysis and cell cycle analysis by FC. However, the full success of the protocols still requires confirmation with samples of fibroblasts from FA patients. Unfortunately, it was not possible to get those samples during the period of execution of the present work, due to the rarity of the disease.

Date of Award	Sept 2012
Original language	Portuguese
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Beatriz Porto (Supervisor)