Fluorescence in situ hybridization as a diagnostic tool for the detection of the FANCA delE12-31 and delE11-17 mutations

[摘要] English: Fanconi anaemia (FA) is a rare autosomal recessive and X-linked disorder characterized by a very high frequency of bone marrow failure and many other manifestations. These include, but are not restricted to, severe birth defects and marked predisposition to malignancies, especially acute myeloid leukaemia and, to a lesser extent, solid tumours (Rodriguez et al, 2005). Cells from FA patients are hypersensitive to agents that produce DNA cross-links, and after in vitro treatment with these agents, display marked chromosome breakage and other cytogenetic abnormalities. FA shows genetic heterogeneity with mutations in any of twelve genes resulting in a similar phenotype. Current diagnostic criteria for FA relies mainly on cytogenetic quantification of chromosomal breakage in response to DEB and/or MMC. The diagnostic value of induced chromosome instability does not appear to be feasible for differentiating between FA carriers and non-carriers, since overlapping in quantitative values between the two groups is common place. In this investigation a population based screening strategy was followed. The method based on fluorescent in situ hybridization (FISH) was applied to allow a rapid and unequivocal identification of two founder Afrikaner FAA gene deletions, in both homozygous and carrier states. Direct labeling by both nick translation and thermal cycling amplification, using dUTP-labeled fluorochromes, resulted in no visible signals after hybridization, even though labeling proved to be successful. This restriction may be ascribed to the relatively small size (1.8kb and 2.3kb, respectively) of the DNA probes. Efficiency of hybridization detection decreases with decreasing probe size and a more sensitive detection method may solve this problem. Indirect labeling by polymerase chain reaction (PCR) amplification using digoxigenin-dUTP (DIG-dUTP) and antibodies (anti-DIG fluorochromes), provides an extremely sensitive method of detection, albeit more time consuming and costly. Bright, clearly defined signals were visualized after hybridization, using fluorescent microscopy. Stringent hybridization conditions, such as formamide contents of the hybridization buffer (70%) and optimal probe concentration (150ng), enhanced target-specificity and reduced background interference to almost none. Predominantly (>70% of interphase nuclei) the number of signals were in agreement with the ploidy of the specific DNA sequence, but the remaining cells revealed a mixture of either one, two or three signals. Target specificity tends to be a problem, especially with the smaller probe. Probes that are too small tend to bind non-specifically and re-hybridize, leaving smaller amounts of probe available for hybridization to the specific target. Even though, after hybridization, both probes resulted in easily visible fluorescent signals, the smaller delE11-17 probe (1.8kb) tended to be more prone to background interference with the signal, and, in addition, less target-specific. Probe hybridization efficiency and background are both influenced by the size of the labeled probe. The length of the probe molecule is critical for probe diffusion and hybridization to the specific target sequence. Probe size should be improved in order to provide a reliable and unequivocal diagnostic tool in the diagnosis of both FA patients and carriers. Longer probes will improve target-specificity and reduce the possibility of hybridization to other complementary regions in the genome. In conclusion, making use of this unique application of FISH offers an effective population directed screening for FA carriers and affected.

[发布机构] University of the Free State