Diagnostic Screening Workflow for Mutations in the BRCA1 and BRCA2 Genes
Journal Title: Sultan Qaboos University Medical Journal - Year 2015, Vol 15, Issue 1
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 fnancial constraints. Tis 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: Tis 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. Te MPS approach necessitated utilisation of a complementary fragment analysis assay to eliminate apparent false-positives at homopolymeric regions. Variants were fltered on the basis of their frequency and sequence depth. Results: Sanger-based sequencing of PCRamplifed coding regions was successfully achieved. Sensitivity and specifcity 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 specifcity, but required complementary fragment analysis combined with Sanger-based sequencing confrmation in some instances.
Authors and Affiliations
Stella Lai| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand, Clare Brookes| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand, Debra O. Prosser| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand, Chuan-Ching Lan| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand, Elaine Doherty| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand, Donald R. Love| Department of Diagnostic Genetics, LabPLUS, Auckland City Hospital, Auckland, New Zealand
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