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Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18
- Source :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, PLoS Genetics, PLoS Genetics, Vol 13, Iss 3, p e1006691 (2017)
- Publication Year :
- 2017
- Publisher :
- Public Library of Science, 2017.
-
Abstract
- Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,802 nucleotides) and structure (V1-[BRCA2_exons_14–20]–V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3’ end of exon 17 (c.7944-7973) and the 5’ end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17–18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA.<br />EAV's lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2013-2016, ISCIII (Fis: PI13/01749) co-funded by FEDER from Regional Development European Funds (European Union), and grant CSI090U14 from the Consejería de Educación (ORDEN EDU/122/2014), Junta de Castilla y León. EFB was supported by a predoctoral fellowship from the University of Valladolid and Banco de Santander (2015-2019).
- Subjects :
- 0301 basic medicine
Cancer Research
Transcription, Genetic
RNA splicing
Exonic splicing enhancer
Biochemistry
Exon
Database and Informatics Methods
0302 clinical medicine
Breast Tumors
RNA Precursors
Medicine and Health Sciences
Small interfering RNAs
Disease
Unclassified variants
Genetics (clinical)
Genetics
Ovarian Neoplasms
Reverse Transcriptase Polymerase Chain Reaction
DNA, Neoplasm
Exons
Genomics
Genomic Databases
Nucleic acids
Splicing assays
Human genes
Oncology
030220 oncology & carcinogenesis
MCF-7 Cells
Female
Sequence Analysis
Research Article
lcsh:QH426-470
Bioinformatics
Ovarian-cancer
Breast Neoplasms
Biology
Guidelines
Research and Analysis Methods
Genome Complexity
Synonymous mutations
Sequence variants
03 medical and health sciences
Genomic Medicine
Ovarian cancer
Sequence Motif Analysis
Breast Cancer
Humans
Breast-cancer susceptibility
RNA, Messenger
Molecular Biology Techniques
Non-coding RNA
Molecular Biology
Ecology, Evolution, Behavior and Systematics
BRCA2 Protein
Breast cancer susceptibility
Base Sequence
Models, Genetic
Alternative splicing
Gene Mapping
Intron
Biology and Life Sciences
Cancers and Neoplasms
Computational Biology
Genome Analysis
Exon skipping
Introns
Gene regulation
lcsh:Genetics
Alternative Splicing
030104 developmental biology
Biological Databases
Polypyrimidine tract
RNA processing
Mutation
Mutagenesis, Site-Directed
Exon Mapping
RNA
RNA Splice Sites
Gene expression
Minigene
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Digital.CSIC. Repositorio Institucional del CSIC, instname, PLoS Genetics, PLoS Genetics, Vol 13, Iss 3, p e1006691 (2017)
- Accession number :
- edsair.doi.dedup.....ea4d38dfd7e5754846dbd74d32d1e401