Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

Farber-Katz, Suzette; Hsuan, Vickie; Wu, Sitao; Landrith, Tyler; Vuong, Huy Gia; Xu, Derong; Li, Bing; Hoo, Jayne; Lam, Stephanie; Nashed, Sarah; Toppmeyer, Deborah; Gray, Phillip; Haynes, Ginger; Lu, Hsiao Mei; Elliott, Aaron; Davis, Brigette Tippin; Karam, Rachid

doi:10.3389/fonc.2018.00286

Cited by 23 publications

(34 citation statements)

References 35 publications

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“…The resulting data correlate with the data from previous MANO-B experiments, enabling batch effect adjustment for pathogenicity determination. When a BRCA2 VUS is believed to be related to splicing aberration, its mRNA should be evaluated by RNA sequencing of patient samples 38 . Any aberrant BRCA2 transcripts detected should also be subjected to the ABCD test ( Fig.…”

Section: Resultsmentioning

confidence: 99%

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Ikegami

Ueno

Momozawa

et al. 2020

Preprint

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Keywords: BRCA2, poly(ADP-ribose) polymerase (PARP) inhibitor, variants of unknown 22 significance (VUSs), companion diagnosis, Bayesian hierarchical model 23 24 ABSTRACT 44Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there 45 is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance 46 and they thus remain unactionable. To assess the pathogenicity of variants of unknown 47The BRCA1 and BRCA2 (BRCA) genes encode key proteins in the homology-directed DNA 60 break repair (HDR) pathway, and their inactivation predisposes individuals to cancer 61 development 1 . Germline loss-of-function variants in BRCA markedly increase the risk of early-62 onset breast and ovarian cancer; in such cases, prophylactic oophorectomy and mastectomy 63 and genetic testing for at-risk relatives must be considered 2, 3, 4 . Tumors with pathogenic 64 mutations within BRCA and defective HDR have been shown to be particularly sensitive to 65 platinum-based chemotherapies and PARP inhibitors, the efficacy of which is mediated 66 through synthetic lethality in cancer cells with BRCA loss-of-function 5, 6 . 67 68The American College of Medical Genetics and Genomics (ACMG) standards and guidelines 69 for the interpretation of sequence variants recommend a process for variant classification based 70 on criteria using population, computational, functional, and segregation data 7 . Family-based 71 studies including a multifactorial model of pathology, a cosegregation profile, and the 72 cooccurrence and family history of cancer may exemplify the most reliable methods for 73 classifying BRCA2 gene variants 8, 9 . Nonsense or frameshift mutations within the coding exons 74 of BRCA markedly alter the structures of the protein products and are presumed to confer loss-75 of-function. However, the vast majority of missense variants are individually rare in both 76 general populations and cancer patients, and case-control studies may not have sufficient 77 statistical significance to classify these variants as pathogenic or benign 10, 11, 12 . No current in 78 silico computational prediction algorithm for missense variants is accurate enough when used 79 alone 13 . Thus, the functional evaluation of missense variants of unknown significance (VUSs) 80 is urgently required to improve the interpretation of variants identified by genetic testing and 81 to support clinical decision-making for their carriers 14 . 82Page 5 of 63 While thousands of BRCA1 VUSs have been assessed by recently developed high-throughput 83 functional assays 15, 16, 17 , a few hundred BRCA2 variants have been evaluated by a conventional 84 functional assay for BRCA2, the HDR assay 18, 19 . The HDR assay has three issues requiring 85 improvement: (i) the throughput is quite low, (ii) it uses a hamster cell line with transient 86 expression of BRCA2 cDNA, and most importantly, (iii) it can evaluate only variants in the 87 DNA-binding domain 14, 20 . To overcome these limitations, we propose herein a high-8...

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Section: Resultsmentioning

confidence: 99%

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Ikegami

Ueno

Momozawa

et al. 2020

Preprint

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“…These results were validated using a second orthogonal methodology, CloneSeq, a highly sensitive and specific targeted RNA-seq assay based on cloning of reverse transcription-PCR (RT-PCR) products, followed by massively parallel sequencing of the cloned transcripts. 27 CloneSeq results are displayed as Sashimi plots (Fig. 3b-g and Supplementary Figs.…”

Section: Splicing Profile Helps Detect Mis-splicingmentioning

confidence: 99%

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes

Landrith

Cass

et al. 2020

npj Precis. Onc.

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Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNAsequencing (RNA-seq) to generate a splicing profile of 18 TSGs (APC,

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“…The clinicians who ordered genetic testing from Ambry Genetics as part of patient workup were not directly involved in this study, with the exception of clinicians at 4 participating institutions (ie, Dana-Farber Cancer Institute, Cedars-Sinai Medical Center, Rutgers Cancer Institute of New Jersey, and University of Kansas Cancer Center).When available, family members were also tested. Peripheral blood was collected in PAXgene Blood RNA Tubes (PreAnalytiX) from patients according to manufacturer instructions, and RNA sequencing analysis was performed by CloneSeq as previously described 24. Patients were included in the present…”

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Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer

et al. 2019

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IMPORTANCE Performing DNA genetic testing (DGT) for hereditary cancer genes is now a wellaccepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians' ability to accurately apply strategies for cancer risk reduction and treatment. OBJECTIVE To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition. DESIGN, SETTING, AND PARTICIPANTS Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018. MAIN OUTCOMES AND MEASURES Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach. Author affiliations and article information are listed at the end of this article. Open Access. This is an open access article distributed under the terms of the CC-BY-NC-ND License.

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Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

Cited by 23 publications

References 35 publications

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

High-Throughput Functional Evaluation ofBRCA2Variants of Unknown Significance

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes

Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer

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