Micronucleus assay for Fanconi Anaemia diagnosis with ionising radiation and mitomycin C

Abstract

Introduction: Fanconi Anaemia (FA) is a cancer - prone chromosomal instability disorder characterized by congenital and developmental abnormalities, bone marrow failure, defects in DNA repair and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). Twenty different genetic subtypes of FA have been described and are associated with DNA repair pathways. Mutations in DNA repair genes could be reflected in chromosomal radiosensitivity. Clinical radiosensitivity in FA patients has previously been described. This study aimed to evaluate the in vitro chromosomal radiosensitivity and genomic instability of 14 FA patients and their parents by comparing it to healthy controls using 3 different micronucleus (MN) assays. Materials and Methods: In the cytokinesis block G0 MN assay, heparinised blood in culture medium was irradiated to 2 and 4Gy ionising radiation and immediately stimulated with phytohaemagglutinin (PHA). Cytochalasin B (Cyto B) blocked cytokinesis 23 hours after stimulation. Seventy hours post irradiation, cells were harvested. In the S/G2 MN assay, the cultures were first stimulated with PHA. The cultures were then irradiated 72 hours after stimulation with similar doses as in the G0 MN assay. Cytokinesis was immediately blocked by addition of Cyto B after irradiation. Cells were harvested 8 hours post irradiation. The MMC MN assay was conducted in similar manner as the G0 MN assay; however, the cell damage was induced by the addition of MMC. Slides were stained with acridine orange. Micronuclei were scored using a fluorescent microscope. Results: FA patients demonstrated significantly higher spontaneous and radiation-induced MN frequencies when compared to parents and healthy controls in both the G0 and S/G2 phase. FA patients also exhibited higher MMC-induced chromosomal aberrations. Conclusions: FA patients present with higher chromosomal radiosensitivity and genomic instability when compared to healthy controls. The MMC MN assay is the most sensitive assay to demonstrate differences between FA patients, parents and control