Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants in<i>BRCA1</i>and<i>BRCA2</i>genes

Montalban, Gemma; Bonache, Sandra; Moles‐Fernández, Alejandro; Gadea, Neus; Tenés, Anna; Torres-Esquius, Sara; Carrasco, Estela; Balmañà, Judith; Dı́ez, Orland; Gutiérrez‐Enríquez, Sara

doi:10.1002/humu.23882

Cited by 12 publications

(6 citation statements)

References 52 publications

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“…Additionally, it is worth stating that the main purpose of this work was not the clinical classification of the variants assessed but rather to investigate how to improve the in silico identification of deep intronic splicing variants. Qualitative analysis can only detect aberrant transcripts, but additional quantification of the functional transcripts, cosegregation data or other functional assays are needed to classify the variants that induce pseudoexons [42,43].…”

Section: Discussionmentioning

confidence: 99%

Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes

Moles‐Fernández¹,

Domènech-Vivó²,

Tenés

et al. 2021

Cancers

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The contribution of deep intronic splice-altering variants to hereditary breast and ovarian cancer (HBOC) is unknown. Current computational in silico tools to predict spliceogenic variants leading to pseudoexons have limited efficiency. We assessed the performance of the SpliceAI tool combined with ESRseq scores to identify spliceogenic deep intronic variants by affecting cryptic sites or splicing regulatory elements (SREs) using literature and experimental datasets. Our results with 233 published deep intronic variants showed that SpliceAI, with a 0.05 threshold, predicts spliceogenic deep intronic variants affecting cryptic splice sites, but is less effective in detecting those affecting SREs. Next, we characterized the SRE profiles using ESRseq, showing that pseudoexons are significantly enriched in SRE-enhancers compared to adjacent intronic regions. Although the combination of SpliceAI with ESRseq scores (considering ∆ESRseq and SRE landscape) showed higher sensitivity, the global performance did not improve because of the higher number of false positives. The combination of both tools was tested in a tumor RNA dataset with 207 intronic variants disrupting splicing, showing a sensitivity of 86%. Following the pipeline, five spliceogenic deep intronic variants were experimentally identified from 33 variants in HBOC genes. Overall, our results provide a framework to detect deep intronic variants disrupting splicing.

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Section: Discussionmentioning

confidence: 99%

Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes

Moles‐Fernández¹,

Domènech-Vivó²,

Tenés

et al. 2021

Cancers

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“…To estimate the relative proportions of each transcript, semi-quantitative fluorescent RT-PCRs were performed using a FAM-labeled primer under standard conditions, except that 26 cycles were herein applied [ 14 , 27 , 28 ]. Fluorescent products were run with LIZ-1200 Size Standard by Macrogen (Seoul, Korea) and analyzed using Peak Scanner software V1.0 (Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

Bueno-Martínez

Sanoguera-Miralles

Valenzuela-Palomo

et al. 2021

Cancers

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RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G > A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T > C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

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“…No biological samples were available for carriers of c.165C>T, c.316þ6T>A, and c.316þ6T>G. Besides LCL and PAXgene samples from healthy individuals, two additional controls were used in this experiment: (i) a LCL from a patient harboring an unequivocal pathogenic Alu insertion in the middle of exon 3 (c.156_157insAlu) known to cause total exon skipping (33) and LCLs from four carriers of the neutral variant c.68-7T>A, known to lead to mild exon skipping (19). The biallelic splicing patterns of BRCA2e3 in patient biological samples were analyzed by semiquantitative RT-PCR, a common approach used in clinical settings (34), and compared with those generated from equivalent RNA samples of control individuals (Fig. 2A and B; Supplementary Table S1).…”

Section: Confirmation Of Variant-induced Spliceogenicity In Rna Samples From Patients With Hbocmentioning

confidence: 99%

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System

et al. 2020

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◥BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons.Significance: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

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Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants inBRCA1andBRCA2genes

Cited by 12 publications

References 52 publications

Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes

Role of Splicing Regulatory Elements and In Silico Tools Usage in the Identification of Deep Intronic Splicing Variants in Hereditary Breast/Ovarian Cancer Genes

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System

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