Diagnostic Screening Workflow for Mutations in the BRCA1 and BRCA2 Genes

Objectives: screening for mutations in large genes is challenging in a molecular diagnosticenvironment. 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.