Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of <i>BRCA</i>
 Gene Unclassified Variants

Vallée, Maxime; Sera, Tonya L. Di; Nix, David A.; Paquette, A. M.; Parsons, Michael T.; Bell, Russel N.; Hoffman, Andrea; Hogervorst, Frans B.L.; Goldgar, David E.; Spurdle, Amanda B.; Tavtigian, Sean V.

doi:10.1002/humu.22973

Cited by 52 publications

(77 citation statements)

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“…NA, not applicable: multifactorial classification not assigned as the combined odds of causality were insufficient (≥0.5 and ≤2) to derive a posterior probability of pathogenicity (Vallee et al., ).…”

Section: Resultsmentioning

confidence: 99%

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

et al. 2018

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The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early‐onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.‐287C>T and PAX5 binding to BRCA2:c.‐296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

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Section: Resultsmentioning

confidence: 99%

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

et al. 2018

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“…Variant c.8023A>G, which induced ex18del309, had previously been classified as pathogenic [21] (BIC and UMD databases), so this transcript disrupts BRCA2 function. Also, c.8331G>A might be an important risk allele as abnormal transcripts almost reach 60%, which is the suggested threshold for severe splicing aberrations [51]. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), [52] 12 spliceogenic variants were 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) and 8 as 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), under the splicing viewpoint (S4 Table).…”

Section: Discussionmentioning

confidence: 99%

Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

et al. 2017

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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 (1,806 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.

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“…Priors assesses whether pathogenicity of a missense variant is more likely to originate from the amino acid substitution or a splicing defect (Vallée et al., ). Of all exonic variants included in this study Priors gave the highest score for impaired splicing (0.34) for BRCA1 c.4674A > G & BRCA2 c.517G > C, and both variants lead to aberrant splicing.…”

Section: Discussionmentioning

confidence: 99%

Thorough in silico and in vitro cDNA analysis of 21 putativeBRCA1andBRCA2splice variants and a complex tandem duplication inBRCA2allowing the identification of activated cryptic splice donor sites inBRCA2exon 11

et al. 2018

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For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

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Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation of BRCA Gene Unclassified Variants

Cited by 52 publications

References 100 publications

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18

Thorough in silico and in vitro cDNA analysis of 21 putativeBRCA1andBRCA2splice variants and a complex tandem duplication inBRCA2allowing the identification of activated cryptic splice donor sites inBRCA2exon 11

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