Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: A new approach to assess quantitative status of BRCA1 gene in a reference laboratory

Giovanni Scambia, Angelo Minucci, Elisa De Paolis, Paola Concolino, Maria De Bonis, Roberta Rizza, Giulia Canu, Giovanni Luca Scaglione, Cecilia Zuppi, Ettore Domenico Capoluongo, Flavio Mignone

Risultato della ricerca: Contributo in rivistaArticolo in rivista

15 Citazioni (Scopus)

Abstract

Aim of the study: Evaluation of copy number variation (CNV) in BRCA1/2 genes, due to large genomic rearrangements (LGRs), is a mandatory analysis in hereditary breast and ovarian cancers families, if no pathogenic variants are found by sequencing. LGRs cannot be detected by conventional methods and several alternative methods have been developed. Since these approaches are expensive and time consuming, identification of alternative screening methods for LGRs detection is needed in order to reduce and optimize the diagnostic procedure. The aim of this study was to investigate a Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) as molecular tool to detect recurrent BRCA1 LGRs. Material and methods: C-PCR-HRMA was performed on exons 3, 14, 18, 19, 20 and 21 of the BRCAI gene; exons 4, 6 and 7 of the ALB gene were used as reference fragments. Results: This study showed that it is possible to identify recurrent BRCA1 LGRs, by melting peak height ratio between target (BRCA1) and reference (ALB) fragments. Furthermore, we underline that a peculiar amplicon-melting profile is associated to a specific BRCAI LGR. All C-PCR-HRMA results were confirmed by Multiplex ligation-dependent probe amplification. Conclusions: C-PCR-HRMA has proved to be an innovative, efficient and fast method for BRCA1 LGRs detection. Given the sensitivity, specificity and ease of use, c-PCR-HRMA can be considered an attractive and powerful alternative to other methods for BRCA1 CNVs screening, improving molecular strategies for BRCA testing in the context of Massive Parallel Sequencing.
Lingua originaleEnglish
pagine (da-a)83-92
Numero di pagine10
RivistaClinica Chimica Acta
Volume470
DOI
Stato di pubblicazionePubblicato - 2017

Keywords

  • Competitive PCR-High Resolution Melting
  • Large genomic rearrangements detection

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