1. Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18
- Author
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Eladio Velasco, David J. Sanz, Beatriz Díez-Gómez, Eugenia Fraile-Bethencourt, Valeria Velásquez-Zapata, Alberto Acedo, Universidad de Valladolid, Banco Santander, Ministerio de Economía y Competitividad (España), European Commission, Instituto de Salud Carlos III, and Junta de Castilla y León
- 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 - 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., 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).
- Published
- 2017