Lab Med Online.  2021 Oct;11(4):283-289. 10.47429/lmo.2021.11.4.283.

Evaluation of a Targeted Next-generation Sequencing Assay for BRCA Mutation Screening in Clinical Samples

• Kim MJ
• Song BJ
• Kim SE
• Lee CS
• Im JE
• Oh E
• Lee K
• Cho SI
• Kim KJ
• Park SS
• Seong MW

• KMID: 2526090
• DOI: http://doi.org/10.47429/lmo.2021.11.4.283

Abstract

Background
While several factors contribute to breast cancer pathogenesis, hereditary breast cancer results from a genetic predisposition. Genes associated with hereditary breast cancer may be divided into high- and low-penetrance genes depending on their risk rates. BRCA1 and BRCA2 are typical high-penetrance genes that increase the risk of developing breast and ovarian cancers upon undergoing mutations. This study aimed to evaluate the clinical performance of BRCAaccuTest^TM (NGeneBio, Korea).
Methods
BRCAaccuTest^TM is a reagent used to produce libraries for analyzing BRCA1/2 genes using next-generation sequencing (NGS), which analyzes blood-derived genomic DNA. Libraries with adapters and barcodes compatible with the Illumina platform were produced. The clinical performance of NGS-based BRCAaccuTest^TM in identifying BRCA1/2mutations was compared with that of the traditional Sanger sequencing method. Both NGS and Sanger sequencing were performed in a single laboratory using archival DNA from blood samples of 212 patients with breast cancer.
Results
All target regions amplified were successfully sequenced to obtain a minimum coverage of 20, demonstrating 100% concordance with the pathogenic single-nucleotide variations and small insertions-deletions previously identified by Sanger sequencing.
Conclusions
This study demonstrates the feasibility of using BRCAaccuTest^TM to detect the BRCA1/2 mutations with high accuracy.

Keyword

BRCA; Mutation; Next-generation sequencing; in vitro diagnosis; Hereditary breast and ovarian cancer (HBOC) syndrome

Figure

• 

  Fig. 1 Design of the clinical study. The target number of test samples was determined according to the criteria of 0.99 diagnostic positive/negative agreement, 80% power, and 5% statistical significance. Abbreviations: PPA, positive percent agreement; NPA, negative percent agreement; OA, overall agreement.

• 

  Fig. 2 Quantity and size of the final libraries. (A) The libraries of samples B001 to B207 were quantified and represented in nanomole (nM) quantities in correlation to the amount of input DNA (ng). The black dashed lines indicate the range of the input DNA used in this study. The red dashed arrow represents the average concentration of the library. The blue dashed arrow represents the minimum concentration of the library. (B) The size of each library was individually determined and represented as a dot. The red dashed line indicates the average size (bp) of the library. The blue dashed lines represent the range of the library size (300–500 bp). B021 was excluded owing to an insufficient amount of sample.

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