Fluorescence in situ hybridization as a diagnostic tool for the detection of the FANCA delE12-31 and delE11-17 mutations
| dc.contributor.advisor | Pearson, T. | |
| dc.contributor.advisor | Theron, M. | |
| dc.contributor.advisor | Jansen, S. | |
| dc.contributor.author | Nogabe, Sibongile Joy | |
| dc.date.accessioned | 2015-09-29T07:34:16Z | |
| dc.date.available | 2015-09-29T07:34:16Z | |
| dc.date.copyright | 2005-11 | |
| dc.date.issued | 2005-11 | |
| dc.date.submitted | 2005-11 | |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | Afrikaans: Fanconi anemie (FA) is ‘n seldsame, outosomaal resessief oorgeërfde toestand, gekenmerk deur ‘n hoë voorkoms van beenmurgversaking asook verskeie ander defekte. Laasgenoemde sluit erge geboortegebreke en ‘n verhoogde neiging to maligniteite soos akute miëloiëde leukemie in, maar is nie noodwendig daartoe beperk nie (Rodriques et al., 2005). Die selle van FA pasiënte is hipersensitief teenoor agense wat DNA-kruisverbindings vorm. In vitro behandeling hiermee veroorsaak ‘n verhoogde insidensie van chromosoombreuke en ander sitogenetiese veranderinge. FA toon ‘n genetiese heterogeniteit, met mutasies in enige van elf gene wat ‘n ooreenstemmende fenotipe tot gevolg het. Die teenswoordige diagnostiese kriteria vir FA berus hoofsaaklik op die sitogenetiese kwantifisering van chromosoombreuke uitgelok deur DEB en/of MMC. Diagnosties is geïnduseerde chromosoomonstabiliteit egter nie ‘n betroubare maatstaf vir die onderskeid tussen FA draers en nie-draers nie, angesien oorvleueling in kwantitatiewe waardes tussen die twee groepe te groot is. ‘n Strategie wat sifting van ‘n bevolkingsgroep ten doel het, is met hierdie ondersoek gevolg. ‘n Tegniek gebaseer op Fluoressensie In Situ Hibridisasie (FISH) is aangewend vir vinnige diagnose van en ondubbelsinnige onderskeid tussen draers en aangetastes van twee Afrikaner FAA geendelesies. Direkte merking deur beide kerfvertaling en hittesiklus-amplifisering deur gebruik van dUTP-gemerkte fluorochrome, het na hibridisasie geen waarneembare seine opgelewer nie. Merking was oënskynlik suksesvol. Die relatiewe klein grootte van die DNA peilers (1.8kb en 2.3kb, onderskeidelik), is waarskynlik hiervoor verantwoordelik. Die effektiwiteit van hibridisasie daal met afname in grootte van die peiler. Hierdie probleem kan aangespreek word deur gebruik te maak van ‘n meer sensitiewe tegniek vir waarneming van die peiler. Indirekte merking deur middel van van polimerase kettingreaksie (PCR)-amplifisering en die gebruik van digoksigenien-dUTP (DIG-dUTP) en antiliggame (anti-DIG fluorochrome), is ‘n hoogs sensitiewe opsporingstegniek, alhoewel tydrowend en duur. Na hibridisasie het die seine helder en duidelik afgebaken onder die fluoressensiemikroskoop vertoon. Die toepas van streng hibridisasiekriteria soos 70% formamiedinhoud van die hibridisasiebuffer en 150ng as optimale peilerkonsentrasie, verhoog teikenspesifisiteit en verminder agtergrondsteurnis tot feitlik niks. In meeste gevalle (>70% van die interfase kerne) was die aantal seine in ooreenstemming met die ploiëdie van die spesifieke DNA volgorde, terwyl die oorblywende selle ‘n mengbeeld van een, twee of drie seine vertoon het. Teikenspesifisitiet was veral problematies by die kleiner peiler. Peilers wat te klein is neig om nie-spesifiek te bind of te herhibridiseer, wat kleiner hoeveelhede vir hibridisasie laat om aan die spesifieke teiken te bind. Alhoewel dit maklik was om seine van beide peilers na hibridisasie waar te neem, was die kleiner delE11–17 peiler (1.8kb) meer onderhewig aan agtergrondsteuring, asook minder teikenspesifiek. Die effektiwiteit van peilerhibridisasie sowel as agtergrondsteurnis word beide deur die grootte van die gemerkte peiler beïnvloed. Die lengte van die peilermolekuul is van kritiese belang vir peilerverspreiding en hibridisasie aan die spesifieke teikenvolgorde. Die grootte van die peiler is van deurslaggewende belang in die daarstelling van ‘n betroubare en ondubbelsinnige diagnostiese tegniek vir die diagnose van beide FA pasiënte en draers. Deur van langer peilers gebruik te maak, kan teikenspesifisiteit vehoog en die kanse vir hibridisasie aan ander komplementêre streke verminder word. 78 Die slotsom is dat FISH op ‘n unieke wyse aangewend kan word om ‘n bevolkingsgerigte sifting vir FA draers en aangetasdes op koste-effektiewe manier uit te voer. | af |
| dc.description.sponsorship | University of the Free State, Faculty of Health Sciences, Research Committee | en_ZA |
| dc.description.sponsorship | Medical Research Council | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/1301 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Fanconi anaemia | en_ZA |
| dc.subject | FAA | en_ZA |
| dc.subject | Fish | en_ZA |
| dc.subject | Founder mutations | en_ZA |
| dc.subject | DelE12- 31 | en_ZA |
| dc.subject | DelE11-17 | en_ZA |
| dc.subject | PCR | en_ZA |
| dc.subject | Afrikaner population | en_ZA |
| dc.subject | Fanconi's anemia -- Molecular aspects | en_ZA |
| dc.subject | Fanconi's anemia -- Genetic aspects | en_ZA |
| dc.subject | Children -- Diseases -- South Africa | en_ZA |
| dc.subject | Dissertation (M.Med.Sc. (Medical Science))--University of the Free State, 2005 | en_ZA |
| dc.title | Fluorescence in situ hybridization as a diagnostic tool for the detection of the FANCA delE12-31 and delE11-17 mutations | en_ZA |
| dc.type | Dissertation | en_ZA |