Angelo Minucci1, Elisa De Paolis2, Paola Concolino2, Maria De Bonis2, Roberta Rizza2, Giulia Canu2, Giovanni Luca Scaglione2, Flavio Mignone3, Giovanni Scambia4, Cecilia Zuppi2, Ettore Capoluongo5. 1. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Teaching and Research Hospital "Agostino Gemelli" Foundation, Rome, Italy. Electronic address: angelo.minucci@virgilio.it. 2. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Teaching and Research Hospital "Agostino Gemelli" Foundation, Rome, Italy. 3. Department of Science and Innovation Technology (DISIT), University of Piemonte Orientale, Alessandria, Italy. 4. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Teaching and Research Hospital "Agostino Gemelli" Foundation, Rome, Italy. 5. Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Teaching and Research Hospital "Agostino Gemelli" Foundation, Rome, Italy. Electronic address: ettoredomenico.capoluongo@unicatt.it.
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 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.
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.
Authors: Maria De Bonis; Elisa De Paolis; Maria Elisabetta Onori; Giorgia Mazzuccato; Antonio Gatto; Pietro Ferrara; Pietro Manuel Ferraro; Andrea Urbani; Angelo Minucci Journal: Mol Biol Rep Date: 2021-04-17 Impact factor: 2.316
Authors: Giorgia Mazzuccato; Maria De Bonis; Vittoria Carboni; Claudia Marchetti; Andrea Urbani; Giovanni Scambia; Ettore Capoluongo; Anna Fagotti; Angelo Minucci Journal: Mol Biol Rep Date: 2020-05-28 Impact factor: 2.316
Authors: Angelo Minucci; Giovanni Scambia; Maria De Bonis; Elisa De Paolis; Concetta Santonocito; Anna Fagotti; Ettore Capoluongo; Paola Concolino; Andrea Urbani Journal: Mol Biol Rep Date: 2020-12-12 Impact factor: 2.316
Authors: Angelo Minucci; Paola Concolino; Maria De Bonis; Alessandra Costella; Ida Paris; Giovanni Scambia; Ettore Capoluongo Journal: Hum Genome Var Date: 2018-04-20