TY - JOUR

T1 - Multiplex ddPCR assay for screening copy number variations in BRCA1 gene

AU - Oscorbin, Igor

AU - Kechin, Andrey

AU - Boyarskikh, Uljana

AU - Filipenko, Maxim

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Purpose: Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene. Methods: In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status. Results: DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1. Conclusions: We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.

AB - Purpose: Germinal and somatic rearrangements in BRCA1 gene play a significant role in carcinogenesis of breast and ovarian cancer. The present study is dedicated to the development of multiplex droplet digital PCR (ddPCR) assay for detecting large deletions and duplications in the BRCA1 gene. Methods: In-house tetraplex ddPCR assay for BRCA1 gene analysis was used for testing of DNA samples with BRCA1 status. Results: DNA specimens were purified from 24 individuals. The presence of BRCA1 rearrangements in samples was confirmed by a commercial MLPA-based kit. An amplitude-based multiplex ddPCR assay was developed: 8 multiplexes, each containing primers and probes to amplify 3 BRCA1 exons and 1 reference gene (ALB or RPP30). A novel assay demonstrated 100% concordance with the commercial MLPA-based kit, identifying 9 specimens with different deletions in BRCA1, 1 with duplication, and 14 with the wild-type BRCA1. Conclusions: We have designed a simple, precise, and cost-effective assay for BRCA1 rearrangement testing, based on ddPCR. The developed assay is the first multiplex ddPCR-based test that provides results in accordance with MLPA and can be used for routine clinical screening.

KW - BRCA1

KW - CNV

KW - Digital PCR

KW - Gene rearrangements

KW - Multiplex amplification

UR - http://www.scopus.com/inward/record.url?scp=85071724862&partnerID=8YFLogxK

U2 - 10.1007/s10549-019-05425-3

DO - 10.1007/s10549-019-05425-3

M3 - Article

C2 - 31482362

AN - SCOPUS:85071724862

VL - 178

SP - 545

EP - 555

JO - Breast Cancer Research and Treatment

JF - Breast Cancer Research and Treatment

SN - 0167-6806

IS - 3

ER -