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Abstract

Objectives: Screening for mutations in large genes is challenging in a molecular diagnostic environment. Sanger-based DNA sequencing methods are largely used; however, massively parallel sequencing (MPS) can accommodate increasing test demands and financial constraints. This study aimed to establish a simple workflow to amplify and screen all coding regions of the BRCA1 and BRCA2 (BRCA1/2) genes by Sanger-based sequencing as well as to assess a MPS approach encompassing multiplex polymerase chain reaction (PCR) and pyrosequencing. Methods: This study was conducted between July 2011 and April 2013. A total of 20 patients were included in the study who had been referred to Genetic Health Services New Zealand (Northern Hub) for BRCA1/2 mutation screening. Patients were randomly divided into a MPS evaluation and validation cohort (n = 10 patients each). Primers were designed to amplify all coding exons of BRCA1/2 (28 and 42 primer pairs, respectively). Primers overlying known variants were avoided to circumvent allelic drop-out. The MPS approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were filtered on the basis of their frequency and sequence depth. Results: Sanger-based sequencing of PCRamplified coding regions was successfully achieved. Sensitivity and specificity of the combined MPS/homopolymer protocol was determined to be 100% and 99.5%, respectively. Conclusion: In comparison to traditional Sangerbased sequencing, the MPS workflow led to a reduction in both cost and analysis time for BRCA1/2 screening. MPS analysis achieved high analytical sensitivity and specificity, but required complementary fragment analysis combined with Sanger-based sequencing confirmation in some instances.


Keywords

Massively Parallel Sequencing BRCA1 Gene BRCA2 Gene HBOC Syndrome Detection heterozygote.

Article Details

How to Cite
Lai, S., Brooke, C., Prosser, D. O., Lan, C.-C., Doherty, E., & Love, D. R. (2015). Diagnostic Screening Workflow for Mutations in the BRCA1 and BRCA2 Genes. Sultan Qaboos University Medical Journal, 15(1), 58–70. Retrieved from https://journals.squ.edu.om/index.php/squmj/article/view/1989

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