Novel BRCA1 Large Genomic Rearrangements in Italian Breast/Ovarian Cancer Patients

Background Genetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients. Methods We first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products. Results In our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6–99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6–99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16–17, 21–22 and 20, have been characterized. Conclusions Our study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.

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Complex Characterization of Germline Large Genomic Rearrangements of the BRCA1 and BRCA2 Genes in High-Risk Breast Cancer Patients—Novel Variants from a Large National Center

International Journal of Molecular Sciences ◽

10.3390/ijms21134650 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4650

Author(s):

Anikó Bozsik ◽

Tímea Pócza ◽

János Papp ◽

Tibor Vaszkó ◽

Henriett Butz ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Brca1 Mutation ◽

Transcript Level ◽

Genomic Rearrangements ◽

Breast Cancer Patients ◽

Brca1 And Brca2 ◽

Large Genomic Rearrangements ◽

Amplification Method

Large genomic rearrangements (LGRs) affecting one or more exons of BRCA1 and BRCA2 constitute a significant part of the mutation spectrum of these genes. Since 2004, the National Institute of Oncology, Hungary, has been involved in screening for LGRs of breast or ovarian cancer families enrolled for genetic testing. LGRs were detected by multiplex ligation probe amplification method, or next-generation sequencing. Where it was possible, transcript-level characterization of LGRs was performed. Phenotype data were collected and analyzed too. Altogether 28 different types of LGRs in 51 probands were detected. Sixteen LGRs were novel. Forty-nine cases were deletions or duplications in BRCA1 and two affected BRCA2. Rearrangements accounted for 10% of the BRCA1 mutations. Three exon copy gains, two complex rearrangements, and 23 exon losses were characterized by exact breakpoint determinations. The inferred mechanisms for LGR formation were mainly end-joining repairs utilizing short direct homologies. Comparing phenotype features of the LGR-carriers to that of the non-LGR BRCA1 mutation carriers, revealed no significant differences. Our study is the largest comprehensive report of LGRs of BRCA1/2 in familial breast and ovarian cancer patients in the Middle and Eastern European region. Our data add novel insights to genetic interpretation associated to the LGRs.

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