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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-09122007-091457 |
| Date | 12 September 2007 |
| Creators | Nogabe, Sibongile Joy |
| Contributors | Prof S Jansen, Dr T Pearson, Dr M Theron |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-09122007-091457/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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