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Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by defects in DNA repair, associated with chromosomal instability and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). The FA repair pathway involves complex DNA repair mechanisms crucial for genomic stability. Deficiencies in DNA repair genes give rise to chromosomal radiosensitivity. FA patients have shown increased clinical radiosensitivity by exhibiting adverse normal tissue side-effects. The study aimed to investigate chromosomal radiosensitivity of homozygous and heterozygous carriers of FA mutations using three micronucleus (MN) assays. The G0 and S/G2MN assays are cytogenetic assays to evaluate DNA damage induced by ionising radiation in different phases of the cell cycle. The MMC MN assay detects DNA damage induced by a crosslinking agent in the G0 phase. Patients with a clinical diagnosis of FA and their parents were screened for the complete coding region of 20 FA genes. Blood samples of all FA patients and parents were exposed to ionising radiation of 2 and 4Gy. Chromosomal radiosensitivity was evaluated in the G0 and S/G2 phase. Most of our patients were homozygous for the founder mutation FANCG c.637_643delTACCGCC; p.(Tyr213Lysfs*6) while one patient was compound heterozygous for FANCG c.637_643delTACCGCC and FANCG c.1379G > A, p.(Gly460Asp), a novel missense mutation. Another patient was compound heterozygous for two deleterious FANCA mutations. In FA patients, the G0- and S/G2-MN assays show significantly increased chromosomal radiosensitivity and genomic instability. Moreover, chromosomal damage was significantly elevated in MMC treated FA cells. We also observed an increase in chromosomal radiosensitivity and genomic instability in the parents using 3 assays. The effect was significant using the MMC MN assay. The MMC MN assay is advantageous as it is less labour intense, time effective and has potential as a reliable alternative method for detecting FA patients from parents and controls.

Introduction Fanconi Anaemia (FA) is an autosomal recessive disorder characterized by defects in DNA repair associated with chromosomal instability and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). The clinical manifestation includes congenital and developmental abnormalities and bone marrow failure. FA also has a predisposition to cancer. Fifteen different genetic subtypes of FA have been described. In South Africa, with mixed ethnicity in the population, prevalence of FA ranges between is 1/22 000 for the white Afrikaners to 1/40 000 in the black South Africans. Evidence suggests that FA patients are chromosomally radiosensitive to ionising radiation but with very limited data. The aim of this study is to investigate chromosomal radiosensitivity and genomic instability of homozygous and heterozygous carriers of FA mutations compared to healthy individuals using the micronucleus (MN) assay. Materials and methods For the G0 MN assay, heparinised blood in culture medium was irradiated at 0 Gy (Baseline), 2 Gy and 4 Gy followed by the immediate stimulation of lymphocytes using phytohaemagglutinin (PHA). Cytochalasin B was added 23 hours later to inhibit cytoplasmic division. Cells were harvested 70 hours later. For the S/G2 MN assay, we used the same radiation doses but the culture was only irradiated 72 hours after addition of PHA. To detect DNA damage in the S/G2 phase of the cell cycle, the cells were harvested 8 hours post irradiation. The third assay is similar as the G0 MN assay except the cell damage is induced using MMC. All slides were stained with acridine orange and micronuclei were scored using a fluorescent microscope. Results The micronuclei frequencies are higher in the homozygous carriers compared to the heterozygous carriers and healthy individuals. Conclusions FA mutation carriers show higher chromosomal radiosensitivity and genomic instability compared to healthy individuals.