Dataset Information

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.

ABSTRACT: 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.

SUBMITTER: Bozsik A

PROVIDER: S-EPMC7370166 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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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.

Bozsik Anikó A Pócza Tímea T Papp János J Vaszkó Tibor T Butz Henriett H Patócs Attila A Oláh Edit E

International journal of molecular sciences 20200630 13

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 ...[more]

PMID: 32629901

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Project description:Germline mutations of BRCA1 and BRCA2 predispose individuals to a high risk of breast and ovarian cancer, and elevated risk of other cancers, including those of the pancreas and prostate. BRCA2 mutation carriers may have increased risk of uveal melanoma (UM) and cutaneous melanoma (CM), but associations with these cancers in BRCA1 mutation carriers have been mixed. Here, we further assessed whether UM and CM are associated with BRCA1 or BRCA2 by assessing the presence, segregation and reported/predicted pathogenicity of rare germline mutations (variant allele frequency < 0.01) in families with multiple members affected by these cancers. Whole-genome or exome sequencing was performed on 160 CM and/or UM families from Australia, the Netherlands, Denmark and Sweden. Between one and five cases were sequenced from each family, totalling 307 individuals. Sanger sequencing was performed to validate BRCA1 and BRCA2 germline variants and to assess carrier status in other available family members. A nonsense and a frameshift mutation were identified in BRCA1, both resulting in premature truncation of the protein (the first at p.Q516 and the second at codon 91, after the introduction of seven amino acids due to a frameshift deletion). These variants co-segregated with CM in individuals who consented for testing and were present in individuals with pancreatic, prostate and breast cancer in the respective families. In addition, 33 rare missense mutations (variant allele frequency ranging from 0.00782 to 0.000001 in the aggregated ExAC data) were identified in 34 families. Examining the previously reported evidence of functional consequence of these variants revealed all had been classified as either benign or of unknown consequence. Seeking further evidence of an association between BRCA1 variants and melanoma, we examined two whole-genome/exome sequenced collections of sporadic CM patients (total N = 763). We identified one individual with a deleterious BRCA1 variant, however, this allele was lost (with the wild-type allele remaining) in the corresponding CM, indicating that defective BRCA1 was not a driver of tumorigenesis in this instance. Although this is the first time that deleterious BRCA1 mutations have been described in high-density CM families, we conclude that there is an insufficient burden of evidence to state that the increased familial CM or UM susceptibility is because of these variants. In addition, in conjunction with other studies, we conclude that the previously described association between BRCA2 mutations and UM susceptibility represents a rare source of increased risk.

Project description:Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay-CloneSeq-capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

Dataset Information

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.

Publications

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

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