Diagnostic screening workflow for mutations in the BRCA1 and BRCA2 genes

الملخص EN

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.