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

Abstract

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.

Optic Atrophy and Inner Retinal Thinning in CACNA1F-Related Congenital Stationary Night Blindness

“…Recently, two other exonic synonymous variants in CACNA1F (c.646C>T; p.(Leu216Leu) and c.1719G>A; p.(Thr573Thr)) were reported to affect splicing using a minigene approach [42]. In addition, there are reported instances of pathogenic synonymous exonic variants affecting the last nucleotide of an exon leading to a splice site defect in other disorders [86,87]. Although the average macular GCL-IPL thickness in the CACNA1F-subjects was reduced regardless of the mutation class, there appeared to be variability within each mutation class ( Figure 2F).…”

POLE and Mismatch Repair Status, Checkpoint Proteins and Tumor-Infiltrating Lymphocytes in Combination, and Tumor Differentiation: Identify Endometrial Cancers for Immunotherapy

“…After cluster generation and hybridization of sequencing primer, the library pools were sequenced using Hiseq2500 sequencing system (Illumina, Inc, San Diego, CA), with an average depth of coverage >1,000×. The functional effect of mutations was assessed by in silico prediction tools: Non-synonymous single nucleotide variations (SNVs) deemed pathogenic by at least two of the three algorithms (SIFT, PolyPhen-2 and Mutation Taster), and pathogenic mutations in splice sites predicted by both AdaBoost and Random Forest were included for further analysis (14)(15)(16)(17).…”

Prevalence of genetic susceptibility for breast and ovarian cancer in a non-cancer related study population: secondary germline findings from a Swiss single centre cohort

“…A variant was predicted to induce a potential splicing defect (corresponding to the supporting ACMG-AMP "PP3" criterion) if at least three algorithms scored above the relative cut-off values established for the score differences between wild-type and variant. For these difference scores, algorithm-specific thresholds were used as proposed by Tang et al [34] and Baert et al [35] and detailed in table S3 (appendix 3).…”