Molecular screening of Coloured South African breast cancer patients for the presence of BRCA mutations using high resolution melting analysis

[摘要] English: The populations of South Africa (SA) exhibit a rainbow of genetic diversity due to the high contribution of ancestral genetic admixture. The economic structure of this third world country has limited the exploration of this genetic diversity with regards to familial breast cancer (BC) testing. As the SA Coloured woman has a lifetime risk of 1 in 22 to develop BC, the main aim of the study involved targeting the highly penetrant genes BRCA1 and BRCA2 for comprehensive mutation analysis. This was done in order to determine the range of variants and mutations present within BC patients representing this group.In order to perform such a comprehensive screen, High Resolution Melting Analysis (HRMA) was optimised and validated for use in conjunction with the protein truncation test (PTT), genotyping assays using real-time based PCR, single stranded conformational analysis (SSCP) and DNA Sanger sequencing to determine the presence of potential disease-causing mutations. A total of 229 Coloured BC patients were included based on a specific selection criteria. This criteria included being affected with BC and either having a positive family history of the disease, or an early age at onset (<45 years) or bilateral disease. All male BC patients were included.Twelve different pathogenic or class 5 mutations were detected for a total of 33 patients. These mutations were identified using genotyping analysis, PTT and HRMA. These mutations were confirmed using Sanger sequencing. These mutations included all three the Afrikaner founder mutations, together with the Xhosa/Coloured mutation detected for the Xhosa and Coloured population residing in the Western Cape.A total of 50 variants were identified using HRMA, ranging from single base changes to a 12bp deletion occurring within the coding region of BRCA2. The clinical significance of these variants were classified using computer-based analysis. Variants of unknown significance (VUS) were investigated using a multiple evidence-based approach in order to confirm their clinical status. These included using the BIC, ClinVar, the ENIGMA guidelines, and the 1000 Genomes project database. This was done in order to investigate whether the variant was novel or allocated to a specific population cluster. The majority of the variants was class 1 polymorphisms, exhibiting normal variation. The portfolio of variants reflected 300 years of admixture between the Bantu-speaking Black African populations of the North Western Cape province, the European settlers and the slaves from the East as global, Eastern and African polymorphisms were observed.Numerous new pathogenic mutations were identified, ranging from likely pathogenic (class 4) to class 5. Many of these mutations proved to be restricted to the southern tip of SA. Based on these results, recommendations can be made regarding the composition of targeted mutation panels for the diagnostic testing of SAC BC patients and their families.

[发布机构] University of the Free State