Molecular screening for the presence of large deletions or duplications in BRCA using Multiplex ligation-dependent probe amplification in South Africa

[摘要] 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.

[发布机构] University of the Free State