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

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          Abstract

          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.

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          The Expanding Landscape of Alternative Splicing Variation in Human Populations

          Alternative splicing is a tightly regulated biological process by which the number of gene products for any given gene can be greatly expanded. Genomic variants in splicing regulatory sequences can disrupt splicing and cause disease. Recent developments in sequencing technologies and computational biology have allowed researchers to investigate alternative splicing at an unprecedented scale and resolution. Population-scale transcriptome studies have revealed many naturally occurring genetic variants that modulate alternative splicing and consequently influence phenotypic variability and disease susceptibility in human populations. Innovations in experimental and computational tools such as massively parallel reporter assays and deep learning have enabled the rapid screening of genomic variants for their causal impacts on splicing. In this review, we describe technological advances that have greatly increased the speed and scale at which discoveries are made about the genetic variation of alternative splicing. We summarize major findings from population transcriptomic studies of alternative splicing and discuss the implications of these findings for human genetics and medicine.
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            ENIGMA--evidence-based network for the interpretation of germline mutant alleles: an international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes.

            As genetic testing for predisposition to human diseases has become an increasingly common practice in medicine, the need for clear interpretation of the test results is apparent. However, for many disease genes, including the breast cancer susceptibility genes BRCA1 and BRCA2, a significant fraction of tests results in the detection of a genetic variant for which disease association is not known. The finding of an "unclassified" variant (UV)/variant of uncertain significance (VUS) complicates genetic test reporting and counseling. As these variants are individually rare, a large collaboration of researchers and clinicians will facilitate studies to assess their association with cancer predisposition. It was with this in mind that the ENIGMA consortium (www.enigmaconsortium.org) was initiated in 2009. The membership is both international and interdisciplinary, and currently includes more than 100 research scientists and clinicians from 19 countries. Within ENIGMA, there are presently six working groups focused on the following topics: analysis, clinical, database, functional, tumor histopathology, and mRNA splicing. ENIGMA provides a mechanism to pool resources, exchange methods and data, and coordinately develop and apply algorithms for classification of variants in BRCA1 and BRCA2. It is envisaged that the research and clinical application of models developed by ENIGMA will be relevant to the interpretation of sequence variants in other disease genes. © 2011 Wiley Periodicals, Inc.
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              The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non‐coding RNA and synonymous mutations

              Abstract Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor‐specific mutations not only in protein‐coding sequences but also in non‐coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this “dark matter” of the genome. Malignancy‐driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5′‐UTR and 3′‐UTR. Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non‐coding RNA, such as microRNA, lncRNA, and lincRNA. A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR‐21 as well as tumor suppressor genes such as TP53/p53, APC , BRCA1, or RB1 can be affected by these alterations. In summary, coding‐independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non‐coding or allegedly silent mutations in tumorigenesis.
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                Author and article information

                Contributors
                Journal
                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                2234-943X
                27 July 2018
                2018
                : 8
                : 286
                Affiliations
                [1] 1Translational Genomics Laboratory, Ambry Genetics , Aliso Viejo, CA, United States
                [2] 2Department of Bioinformatics, Ambry Genetics , Aliso Viejo, CA, United States
                [3] 3Department of Research and Development, Ambry Genetics , Aliso Viejo, CA, United States
                [4] 4Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey , New Brunswick, NJ, United States
                Author notes

                Edited by: Paolo Peterlongo, IFOM - The FIRC Institute of Molecular Oncology, Italy

                Reviewed by: Eladio Andrés Velasco, Instituto de Biología y Genética Molecular (IBGM), Spain; Paolo Radice, Istituto Nazionale dei Tumori (IRCCS), Italy

                *Correspondence: Rachid Karam rkaram@ 123456ambrygen.com

                This article was submitted to Cancer Genetics, a section of the journal Frontiers in Oncology

                Article
                10.3389/fonc.2018.00286
                6072868
                30101128
                b2ffe285-b457-4e8b-9598-90fe8770eafc
                Copyright © 2018 Farber-Katz, Hsuan, Wu, Landrith, Vuong, Xu, Li, Hoo, Lam, Nashed, Toppmeyer, Gray, Haynes, Lu, Elliott, Tippin Davis and Karam.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 16 March 2018
                : 09 July 2018
                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 35, Pages: 13, Words: 7228
                Categories
                Oncology
                Original Research

                Oncology & Radiotherapy
                genetic testing,hereditary breast and ovarian cancer,hboc,brca1,brca2,rna,splicing,ngs
                Oncology & Radiotherapy
                genetic testing, hereditary breast and ovarian cancer, hboc, brca1, brca2, rna, splicing, ngs

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