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

Autor: Cecilia Zuppi, Giovanni Scambia, Ettore Capoluongo, Flavio Mignone, Maria De Bonis, Giovanni Luca Scaglione, Giulia Canu, Elisa De Paolis, Paola Concolino, Roberta Rizza, Angelo Minucci
Přispěvatelé: Minucci, A, De Paolis, E, Concolino, P, De Bonis, M, Rizza, R, Canu, G, Scaglione, Gl, Mignone, F, Scambia, G, Zuppi, C, Capoluongo, E
Rok vydání: 2017
Předmět:
0301 basic medicine
endocrine system diseases
DNA Copy Number Variations
BRCA1/2 gene
Clinical Biochemistry
Genes
BRCA1
Context (language use)
Biology
Nucleic Acid Denaturation
Biochemistry
Polymerase Chain Reaction
High Resolution Melt
03 medical and health sciences
Exon
0302 clinical medicine
Settore BIO/12 - BIOCHIMICA CLINICA E BIOLOGIA MOLECOLARE CLINICA
Humans
Multiplex
Copy-number variation
skin and connective tissue diseases
Gene
Brca1 gene
Genetics
Competitive PCR-High Resolution Melting
Gene Rearrangement
Ovarian Neoplasms
Massive parallel sequencing
Genome
Human
Biochemistry (medical)
General Medicine
Exons
Reference Standards
Massive Parallel Sequencing
Large genomic rearrangements detection
Competitive PCR-High Resolution Melting Analysi
030104 developmental biology
030220 oncology & carcinogenesis
Female
Laboratories
Zdroj: Clinica chimica acta; international journal of clinical chemistry. 470
ISSN: 1873-3492
Popis: 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 BRCA1 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 BRCA1 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.
Databáze: OpenAIRE
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