Predicting the Pathogenic Potential of BRCA1 and BRCA2 Gene Variants Identified in Clinical Genetic Testing.

OBJECTIVES: Missense variants are very commonly detected when screening for mutations in the BRCA1 and BRCA2 genes. Pathogenic mutations in the BRCA1 and BRCA2 genes lead to an increased risk of developing breast, ovarian, prostate and/or pancreatic cancer. This study aimed to assess the predictive capability of in silico programmes and mutation databases in assisting diagnostic laboratories to determine the pathogenicity of sequence-detectable mutations. METHODS: Between July 2011 and April 2013, an analysis was undertaken of 13 missense BRCA gene variants that had been detected in patients referred to the Genetic Health Services New Zealand (Northern Hub) for BRCA gene analysis. The analysis involved the use of 13 in silico protein prediction programmes, two in silico transcript analysis programmes and the examination of three BRCA gene databases. RESULTS: In most of the variants, the analysis showed different in silico interpretations. This illustrates the interpretation challenges faced by diagnostic laboratories. CONCLUSION: Unfortunately, when using online mutation databases and carrying out in silico analyses, there is significant discordance in the classification of some missense variants in the BRCA genes. This discordance leads to complexities in interpreting and reporting these variants in a clinical context. The authors have developed a simple procedure for analysing variants; however, those of unknown significance largely remain unknown. As a consequence, the clinical value of some reports may be negligible.

Diagnostic Screening Workflow for Mutations in the BRCA1 and BRCA2 Genes.

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 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. CONCLUSION: In comparison to traditional Sanger-based 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.

Diagnostic genetics at a distance: von hippel-lindau disease and a novel mutation.

Genetic testing at a distance is commonplace where members of a family with a segregating germline mutation are geographically separated. For the most part, this challenge is addressed through the intervention of health professionals in taking and/or processing blood samples for subsequent couriering of DNA to a referral laboratory. In some circumstances, however, the collecting of pivotal clinical material may involve direct patient involvement. We describe such a situation where noninvasive saliva samples were provided by members of a family manifesting Von Hippel-Lindau (VHL) disease. The analysis identified a novel mutation in the VHL gene that was used to exclude other family members as being at risk of VHL disease.

Characterising stutter in forensic STR multiplexes.

Stutter is an artefact seen when amplifying short tandem repeats and typically occurs at one repeat unit shorter in length than the parent allele. In forensic analysis, stutter complicates the analysis of DNA profiles from multiple contributors, known as mixed profiles, a common profile type. Consequently it is important to both understand and predict stutter behaviour in order to improve our understanding of the resolution and interpretation of these profiles. Whilst stutter is well recognised and documented, little information is available that identifies and quantifies what influences the formation of stutter. In this work we use a novel approach to examine this. We have used synthetic oligonucleotides comprising multiple repeat units to test; the influence of repeat number, the influence of repeat sequence and the impact of interruptions to the repeat sequence length. Using multiple replicates allows detailed statistical analysis. We have confirmed a linear relationship between stutter ratio and repeat number. We have shown that increased A-T content increases stutter ratio and that interruptions in repeating sequences decreased stutter ratios to levels similar to the longest uninterrupted repeat stretch. We also found that there was no relationship between stutter ratio and repeat number for a repeat unit with an A-T content of 1/4 and that half of the interrupted repeat sequences stuttered significantly less than their longest uninterrupted repeat stretches. We have applied the knowledge gained to examine specific features of the loci present in the AmpFlSTR(®) SGM Plus(®) multiplex kit used in our laboratory.