Molecular screening for the presence of large deletions or duplications in BRCA using Multiplex ligation-dependent probe amplification in South Africa
| dc.contributor.advisor | Van der Merwe, N. C. | |
| dc.contributor.advisor | Dajee, B. K. | |
| dc.contributor.author | Moeti, Pakiso James | |
| dc.date.accessioned | 2017-01-23T10:26:09Z | |
| dc.date.available | 2017-01-23T10:26:09Z | |
| dc.date.issued | 2016-07 | |
| dc.description.abstract | English: Germline BRCA gene mutations are associated with hereditary breast and ovarian cancers (OVC). Identification of these mutations greatly improves the preventive strategies and management of patients affected with the disease. The large majority of alterations identified within BRCA1 and BRCA2 are point mutations and small insertions/deletions. However, an increasing number of large genomic rearrangements (LGRs) are internationally being reported. Their contribution to familial breast cancer risk varies for different populations, for in some countries it represents a founder mutation (such as the Netherlands), whereas in others this type of mutation is totally absent. The main objective was to optimize and validate this new technique for use within the diagnostic laboratory and to screen various South African (SA) population groups for the presence of these larger genomic rearrangements present within BRCA1 and BRCA2. A total of 129 patients, who tested negative for the presence of smaller pathogenic BRCA1 or BRCA2 mutations were included in the study. The patients represented the Black, Indian and Coloured populations of South Africa. The selection criteria included being affected with breast cancer, have a minimum of one other family member affected with the disease or an early age at onset (diagnosed before the age of 45). Genomic DNA was extracted from peripheral blood samples. Multiplex Ligation-dependent probe amplification (MLPA) was performed using the SALSA® MLPA® probemixes P002-C1, SALSA® MLPA® P002-D1 and SALSA® MLPA® P087-C1 for BRCA1 and SALSA® MLPA® probemixes P045-B3 and SALSA® MLPA® P077-A3 for BRCA2. The data obtained were analyzed by using the GeneMarker® software v 2.6.4. Screening for the presence of LGRs within BRCA1 and BRCA2, did not reveal any genomic rearrangements present within these genes. Although no patients were identified that carried this type of deletions or duplications, the use of the five and two for BRCA2, of which one represented the confirmation set) were successfully validated for use on the diagnostic platform. The data furthermore highlighted the dramatic effect that small deletions or duplications within these genes might have when situated within the critical ligation site of the specific probe set. The presence of these smaller mutations could result in false positive results. The results of this study serve as a warning to pathology laboratories within SA, as the most common Afrikaner founder mutation situated within BRCA2 exon 17 affects the ligation of the probe set for exon 17. The presence of this mutation resulted in a reduced signal for exon 17, therefore a false positive result. This places emphasis on the confirmation of all potential positive results by using an alternative method or different probemix in order to prevent reporting of a false positive result. The data gathered corresponded to that of previous SA studies and supported the tentative hypothesis that LGRs do not seem to play a significant role within the various SA populations. It does not contribute significantly to the familial BC risk within SA. | en_ZA |
| dc.description.abstract | Afrikaans: Oorerflike mutasies in die BRCA gene word geassosieer met familiële bors en ovariële karsinoom. Die identifisering van hierdie tipe mutasies kan voordele vir die aangetaste pasiënt inhou, rakende voorkomende strategieë en behandeling. Alhoewel die meerderheid van hierdie veranderinge in BRCA1 en BRCA2 enkel basispaar mutasies en klein invoegings of delesies is, word al hoe groter herrangskikkings in die genoom al meer in die internasionale literatuur beskryf. Die bydrae wat hierdie groter herrangskikkings maak tot die algehele oorerflike borskanker risiko, variëer. In sekere lande (soos Nederland) verteenwoordig die groter herrangskikkings ‘n stigterseffek, terwyl dit feitlik afwesig is in ander. Die doel van die studie was om die gebruik van die nuwe tegniek (Multiplex Ligation-dependent probe amplification of MLPA) te optimiseer en te valideer, sodat dit met vertroue gebruik kan word om pasiënte van die Suid-Afrikaanse (SA) populasies te sif vir die teenwoordigheid van hierdie tipe mutasies binne BRCA1 en BRCA2. ʼn Totaal van 129 borskanker pasiënte is ingesluit in hierdie studie. Hierdie pasiënte het negatief getoets vir die teenwoordigheid van kleiner siekte-veroorsakende veranderinge in hierdie twee gene. Die pasiënte was verteenwoordigend van die Swart, Indiër en Kleurling bevolking van SA. Die pasiënt moes aangetas wees met borskanker, ten minste een ander aangetaste familielid in die familie hê of gediagnoseer wees voor ouderdom 45. Genomiese DNA is geëkstraheer vanuit volbloed. Die MLPA tegniek is uitgevoer deur gebruik te maak van vyf verskillende stelle peilstukke, drie vir BRCA1 en twee vir BRCA2 (SALSA® MLPA® P002-C1, SALSA® MLPA® P002-D1 en SALSA® MLPA® P087-C1 vir BRCA1; en SALSA® MLPA® P045-B3 en SALSA® MLPA® P077-A3 vir BRCA2). Die data is verwerk deur gebruik te maak van GeneMarker® sagteware (weergawe 2.6.4). Sifting vir die teenwoordigheid van LGRs binne BRCA1 en BRCA2, het geen positiewe resultate opgelewer nie. Hoewel geen pasiënte geïdentifiseer is wat oor groter herrangskikkings beskik nie, is die gebruik van die vyf verskillende ondersoekstelle suksesvol ge-optimiseer en ge-implementeer vir gebruik in die diagnostiese laboratorium. Die studie beklemtoon egter die drastiese effek wat klein delesies of duplikasies binne hierdie gene kan hê wanneer die spesifieke mutasie in die ligeringsgebied van die peilstukke geleë is. Die teenwoordigheid van hierdie kleiner mutasies kan tot vals positiewe MLPA resultate lei. Die resultate van hierdie studie dien as 'n waarskuwing aan patologie laboratoriums binne SA, aangesien die mees algemene Afrikaner stigtersmutasie in BRCA2 ekson 17 in so ‘n gebied geleë is. Die teenwoordigheid van hierdie mutasie in die ligeringsgebied lei tot ʼn verlaging in die sein vir ekson 17, met ander woorde ʼn vals positiewe MLPA uitslag. Dit plaas klem op die feit dat alle positiewe MLPA resultate bevestig moet word deur gebruik te maak van ʼn alternatiewe metode of ʼn tweede ondersoek stel. Sodoende sal foutiewe positiewe uitslae voorkom word. Die data verkry uit hierdie studie stem ooreen met die van vorige SA studies en ondersteun die tentatiewe hipotese dat LGRs nie ‘n belangrike rol in die verskillende Suid-Afrikaanse bevolkings speel nie. Die teenwoordigheid van hierdie tipe herrangskikkings dra nie beduidend by tot die familiële risikos vir oorerflike borskanker in SA nie. | af |
| dc.identifier.uri | http://hdl.handle.net/11660/5392 | |
| 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 | Dissertation (M.Med.Sc. (Human Genetics))--University of the Free State, 2016 | en_ZA |
| dc.subject | South Africa | en_ZA |
| dc.subject | Familial breast cancer | en_ZA |
| dc.subject | Large genomic rearrangements | en_ZA |
| dc.subject | Multiple ligation dependent probe amplification | en_ZA |
| dc.subject | Mutations | en_ZA |
| dc.subject | BRCA1 | en_ZA |
| dc.subject | BRCA2 | en_ZA |
| dc.subject | Breast -- Cancer -- Genetic aspects | en_ZA |
| dc.title | Molecular screening for the presence of large deletions or duplications in BRCA using Multiplex ligation-dependent probe amplification in South Africa | en_ZA |
| dc.type | Dissertation | en_ZA |