Diagnostic screening workflow for mutations in the BRCA1 and BRCA2 genes

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. 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. Sanger-based sequencing of PCR amplified coding regions was successfully achieved. Sensitivity and specificity of the combined MPS/homopolymer protocol was determined to be 100% and 99.5%, respectively. 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

Array-based identification of copy number changes in a diagnostic setting. Simultaneous gene-focused and low resolution whole human genome analysis

The aim of this study was to develop and validate a comparative genomic hybridisation [CGH] array that would allow simultaneous targeted analysis of a panel of disease genes and low resolution whole genome analysis. A bespoke Roche NimbleGen 12x135K CGH array [Roche NimbleGen Inc., Madison, Wisconsin, USA] was designed to interrogate the coding regions of 66 genes of interest, with additional widelyspaced backbone probes providing coverage across the whole genome. We analysed genomic deoxyribonucleic acid [DNA] from 20 patients with a range of previously characterised copy number changes and from 8 patients who had not previously undergone any form of dosage analysis. The custom-designed Roche NimbleGen CGH array was able to detect known copy number changes in all 20 patients. A molecular diagnosis was also made for one of the additional 4 patients with a clinical diagnosis that had not been confirmed by sequence analysis, and carrier testing for familial copy number variants was successfully completed for the remaining four patients. The custom-designed CGH array described here is ideally suited for use in a small diagnostic laboratory. The method is robust, accurate, and cost-effective, and offers an ideal alternative to more conventional targeted assays such as multiplex ligation-dependent probe amplification