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Breast Cancer Research and Treatment

, Volume 165, Issue 3, pp 765–770 | Cite as

Detection of BRCA1 gross rearrangements by droplet digital PCR

  • Elena V. Preobrazhenskaya
  • Ilya V. Bizin
  • Ekatherina Sh. Kuligina
  • Alla Yu. Shleykina
  • Evgeny N. Suspitsin
  • Olga A. Zaytseva
  • Elena I. Anisimova
  • Sergey A. Laptiev
  • Tatiana V. Gorodnova
  • Alexey M. Belyaev
  • Evgeny N. Imyanitov
  • Anna P. SokolenkoEmail author
Brief Report

Abstract

Purpose

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs.

Materials and methods

We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon.

Results

141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5–7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs.

Conclusion

Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

Keywords

Large genomic rearrangements Droplet digital PCR BRCA1 mutation 

Abbreviations

BC

Breast cancer

ddPCR

Droplet digital polymerase chain reaction

LGRs

Large genomic rearrangements

MLPA

Multiplex ligation-dependent probe amplification

NGS

Next generation sequencing

OC

Ovarian cancer

Notes

Acknowledgements

This work has been supported by the Russian Science Foundation (Grant 14-25-00111).

Compliance with ethical standards

Conflict of interest

The authors declares that they have no conflict of interest.

Supplementary material

10549_2017_4357_MOESM1_ESM.pdf (284 kb)
Supplementary material 1 (PDF 284 kb)
10549_2017_4357_MOESM2_ESM.pdf (892 kb)
Supplementary material 2 (PDF 891 kb)

References

  1. 1.
    Bougie O, Weberpals JI (2011) Clinical considerations of BRCA1- and BRCA2-Mutation carriers: a review. Int J Surg Oncol 2011:374012PubMedPubMedCentralGoogle Scholar
  2. 2.
    Li X, You R, Wang Xinwei et al (2016) Effectiveness of prophylactic surgeries in BRCA1 or BRCA2 mutation carriers: a meta-analysis and systematic review. Clin Cancer Res 22:3971–3981CrossRefGoogle Scholar
  3. 3.
    Iyevleva AG, Imyanitov EN (2016) Cytotoxic and targeted therapy for hereditary cancers. Hered Cancer Clin Pract 14:17CrossRefGoogle Scholar
  4. 4.
    Puget N, Torchard D, Serova-Sinilnikova OM et al (1997) A 1-kb Alu-mediated germ-line deletion removing BRCA1 exon 17. Cancer Res 57:828–831PubMedGoogle Scholar
  5. 5.
    Schouten JP, McElgunn CJ, Waaijer R et al (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30:e57CrossRefGoogle Scholar
  6. 6.
    Hogervorst FB, Nederlof PM, Gille JJ et al (2003) Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method. Cancer Res 63:1449–1453PubMedGoogle Scholar
  7. 7.
    Sluiter MD, van Rensburg EJ (2011) Large genomic rearrangements of the BRCA1 and BRCA2 genes: review of the literature and report of a novel BRCA1 mutation. Breast Cancer Res Treat 125:325–349CrossRefGoogle Scholar
  8. 8.
    Ewald IP, Ribeiro PL, Palmero EI, Cossio SL, Giugliani R, Ashton-Prolla P et al (2009) Genomic rearrangements in BRCA1 and BRCA2: a literature review. Genet Mol Biol 32:437–446CrossRefGoogle Scholar
  9. 9.
    Ramus SJ, Harrington PA, Pye C et al (2007) Screening for the BRCA1-ins6kbEx13 mutation: potential for misdiagnosis. Mutation in brief #964.Online. Hum Mutat 28:525–526CrossRefGoogle Scholar
  10. 10.
    Feliubadaló L, Lopez-Doriga A, Castellsagué E et al (2013) Next-generation sequencing meets genetic diagnostics: development of a comprehensive workflow for the analysis of BRCA1 and BRCA2 genes. Eur J Hum Genet 21:864–870CrossRefGoogle Scholar
  11. 11.
    Casilli F, Di Rocco ZC, Gad S et al (2002) Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments. Hum Mutat 20:218–226CrossRefGoogle Scholar
  12. 12.
    Concolino P, Mello E, Minucci A et al (2014) Advanced tools for BRCA1/2 mutational screening: comparison between two methods for large genomic rearrangements (LGRs) detection. Clin Chem Lab Med 52:1119–1127CrossRefGoogle Scholar
  13. 13.
    Barrois M, Bièche I, Mazoyer S et al (2004) Real-time PCR-based gene dosage assay for detecting BRCA1 rearrangements in breast-ovarian cancer families. Clin Genet 65:131–136CrossRefGoogle Scholar
  14. 14.
    Sokolenko AP, Bogdanova N, Kluzniak W et al (2014) Double heterozygotes among breast cancer patients analyzed for BRCA1, CHEK2, ATM, NBN/NBS1, and BLM germ-line mutations. Breast Cancer Res Treat 145:553–562CrossRefGoogle Scholar
  15. 15.
    Iyevleva AG, Suspitsin EN, Kroeze K et al (2010) Non-founder BRCA1 mutations in Russian breast cancer patients. Cancer Lett 298:258–263CrossRefGoogle Scholar
  16. 16.
    Ratajska M, Brozek I, Senkus-Konefka E et al (2008) BRCA1 and BRCA2 point mutations and large rearrangements in breast and ovarian cancer families in Northern Poland. Oncol Rep 19:263–268PubMedGoogle Scholar
  17. 17.
    Rudnicka H, Debniak T, Cybulski C et al (2013) Large BRCA1 and BRCA2 genomic rearrangements in Polish high-risk breast and ovarian cancer families. Mol Biol Rep 40:6619–6623CrossRefGoogle Scholar
  18. 18.
    Rudnicka H, Masojc B, van de Wetering T et al (2014) First recurrent large genomic rearrangement in the BRCA1 gene found in Poland. Cancer Epidemiol 38:382–385CrossRefGoogle Scholar
  19. 19.
    Sokolenko AP, Rozanov ME, Mitiushkina NV et al (2007) Founder mutations in early-onset, familial and bilateral breast cancer patients from Russia. Fam Cancer 6:281–286CrossRefGoogle Scholar
  20. 20.
    Van der Stoep N, van Paridon CD, Janssens T et al (2009) Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner. Hum Mutat 30:899–909CrossRefGoogle Scholar
  21. 21.
    Bankevich A, Nurk S, Antipov D et al (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477CrossRefGoogle Scholar
  22. 22.
    Bogdanova N, Helbig S, Dörk T (2013) Hereditary breast cancer: ever more pieces to the polygenic puzzle. Hered Cancer Clin Pract 11:12CrossRefGoogle Scholar
  23. 23.
    Sokolenko AP, Suspitsin EN, Kuligina ESh et al (2015) Identification of novel hereditary cancer genes by whole exome sequencing. Cancer Lett 369:274–288CrossRefGoogle Scholar
  24. 24.
    Afghahi A, Kurian AW (2017) The changing landscape of genetic testing for inherited breast cancer predisposition. Curr Treat Options Oncol 18:27CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Elena V. Preobrazhenskaya
    • 1
  • Ilya V. Bizin
    • 2
  • Ekatherina Sh. Kuligina
    • 1
  • Alla Yu. Shleykina
    • 1
  • Evgeny N. Suspitsin
    • 1
    • 3
  • Olga A. Zaytseva
    • 1
  • Elena I. Anisimova
    • 4
  • Sergey A. Laptiev
    • 5
  • Tatiana V. Gorodnova
    • 1
  • Alexey M. Belyaev
    • 1
  • Evgeny N. Imyanitov
    • 1
    • 3
    • 5
    • 6
  • Anna P. Sokolenko
    • 1
    • 3
    • 7
    Email author
  1. 1.N.N. Petrov Institute of OncologySt.-PetersburgRussia
  2. 2.Peter the Great St.-Petersburg Polytechnic UniversitySt.-PetersburgRussia
  3. 3.St.-Petersburg State Pediatric Medical UniversitySt.-PetersburgRussia
  4. 4.Leningrad Regional Oncology CenterSt.-PetersburgRussia
  5. 5.Pavlov First St.-Petersburg State Medical UniversitySt.-PetersburgRussia
  6. 6.I.I. Mechnikov North-Western Medical UniversitySt.-PetersburgRussia
  7. 7.Laboratory of Molecular OncologyN.N. Petrov Institute of OncologySt.-PetersburgRussia

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