Dataset Information

Diagnostic mRNA splicing assay for variants in BRCA1 and BRCA2 identified two novel pathogenic splicing aberrations.

ABSTRACT: Background:Pathogenic variants in BRCA1 and BRCA2 cause hereditary breast and ovarian cancer. Screening of these genes has become easily accessible in diagnostic laboratories. Sequencing and copy number analyses are used to detect pathogenic variants, but also lead to identification of variants of unknown clinical significance (VUS). If the effect of a VUS can be clarified, it has direct consequence for the clinical management of the patient and family members. A splicing assay is one of several tools that might help in the classification of VUS. We therefore established mRNA analyses for BRCA1 and BRCA2 in the diagnostic laboratory in 2015. We hereby report the results of mRNA analysis variants in BRCA1 and BRCA2 after three years. Methods:Variants predicted to alter splicing and variants within the canonical splice sites were selected for splicing analyses. Splicing assays were performed by reverse transcription-PCR of patient RNA. A biallalic expression analysis was carried out whenever possible. Results:Twenty-five variants in BRCA1 and BRCA2 were analyzed by splicing assays; nine showed altered transcripts and 16 showed normal splicing patterns. The two novel pathogenic variants in BRCA1 c.4484?+?3 A?>?C and c.5407-10G?>?A were characterized. Conclusions:We conclude that mRNA analyses are useful in characterization of variants that may affect splicing. The results can guide classification of variants from unknown clinical significance to pathogenic or benign in a diagnostic laboratory, and thus be of direct clinical importance.

SUBMITTER: Wangensteen T

PROVIDER: S-EPMC6532242 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Diagnostic mRNA splicing assay for variants in <i>BRCA1</i> and <i>BRCA2</i> identified two novel pathogenic splicing aberrations.

Wangensteen Teresia T Felde Caroline Nangota CN Ahmed Deeqa D Mæhle Lovise L Ariansen Sarah Louise SL

Hereditary cancer in clinical practice 20190522

<h4>Background</h4>Pathogenic variants in <i>BRCA1</i> and <i>BRCA2</i> cause hereditary breast and ovarian cancer. Screening of these genes has become easily accessible in diagnostic laboratories. Sequencing and copy number analyses are used to detect pathogenic variants, but also lead to identification of variants of unknown clinical significance (VUS). If the effect of a VUS can be clarified, it has direct consequence for the clinical management of the patient and family members. A splicing a ...[more]

PMID: 31143303

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Project description:ImportanceThe clinical importance of genetic testing of BRCA1 and BRCA2 in breast, ovarian, prostate, and pancreatic cancers is widely recognized. However, there is insufficient evidence to include other cancer types that are potentially associated with BRCA1 and BRCA2 in clinical management guidelines.ObjectiveTo evaluate the association of BRCA1 and BRCA2 pathogenic variants with additional cancer types and their clinical characteristics in 100 914 individuals across 14 cancer types.Design, setting, and participantsThis case-control analysis to identify cancer types and clinical characteristics associated with pathogenic variants in BRCA1 and BRCA2 included DNA samples and clinical information from 63 828 patients with 14 common cancer types and 37 086 controls that were sourced from a multi-institutional hospital-based registry, BioBank Japan, between April 2003 and March 2018. The data were analyzed between August 2019 and October 2021.Main outcomes and measuresGermline pathogenic variants in coding regions and 2 bp flanking intronic sequences in BRCA1 and BRCA2 were identified by a multiplex polymerase chain reaction-based target sequence method. Associations of (likely) pathogenic variants with each cancer type were assessed by comparing pathogenic variant carrier frequency between patients in each cancer type and controls.ResultsA total of 65 108 patients (mean [SD] age at diagnosis, 64.1 [11.6] years; 27 531 [42.3%] female) and 38 153 controls (mean [SD] age at registration, 61.8 [14.6] years; 17 911 [46.9%] female) were included in this study. A total of 315 unique pathogenic variants were identified. Pathogenic variants were associated with P < 1 × 10-4 with an odds ratio (OR) of greater than 4.0 in biliary tract cancer (OR, 17.4; 95% CI, 5.8-51.9) in BRCA1, esophageal cancer (OR, 5.6; 95% CI, 2.9-11.0) in BRCA2, and gastric cancer (OR, 5.2; 95% CI, 2.6-10.5) in BRCA1, and (OR, 4.7; 95% CI, 3.1-7.1) in BRCA2 in addition to the 4 established cancer types. We also observed an association with 2 and 4 other cancer types in BRCA1 and BRCA2, respectively. Biliary tract, female breast, ovarian, and prostate cancers showed enrichment of carrier patients according to the increased number of reported cancer types in relatives.Conclusions and relevanceThe results of this large-scale registry-based case-control study suggest that pathogenic variants in BRCA1 and BRCA2 were associated with the risk of 7 cancer types. These results indicate broader clinical relevance of BRCA1 and BRCA2 genetic testing.

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

Diagnostic mRNA splicing assay for variants in BRCA1 and BRCA2 identified two novel pathogenic splicing aberrations.

Publications

Diagnostic mRNA splicing assay for variants in <i>BRCA1</i> and <i>BRCA2</i> identified two novel pathogenic splicing aberrations.

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