Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk

Abstract

Background: Germline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques.

Methods: Twenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA).

Results: NGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel.

Conclusion: This study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Figure 1

Components of the 25-gene panel. CGH, comparative genomic hybridization; emPCR, emulsion polymerase chain reaction; LIMS, laboratory information management system; LR, large rearrangement; MLPA, multiplex ligation-dependent probe amplification, NGS, next generation sequencing.

Figure 2

The same variant inNBNas detected by Sanger sequencing (A) and NGS (B). Note that the frequency of alleles at variable positions, read depth in independent forward and reverse reads and quality scores can be reviewed here. The heterozygous base change indicated by the arrow in panel A is the same base change selected in the NGS results in panel B. NBN, NBN-associated cancer risk ; NGS, next generation sequencing.

Figure 3

A contiguous deletion involvingEPCAMandMSH2as seen in both microarray CGH (A) and NGS LR (B) views. In the case of the microarray CGH plot the data are on a log₂ scale with wild-type dosage at 0 on the Y-axis. In the case of the NGS dosage plot, the data are on a linear scale with wild-type dosage at 2 on the Y-axis. In both cases, a summary overview is available (top) as well as a zoomed in (bottom) view showing specific probe or amplicon placement. In the summary view, all genes are shown simultaneously with a symbol representing each gene, and exons proceeding 5’ to 3’ across the X-axis. Note that EPCAM exons 2–3 are also included in the microarray CGH assay for normalization purposes but are not tested on the NGS LR assay. NGS LR, next generation sequencing large rearrangements.