Detecting splicing patterns in genes involved in hereditary breast and ovarian cancer

Davy, Grégoire; Rousselin, Antoine; Goardon, Nicolas; Castéra, Laurent; Harter, Valentin; Legros, Angélina; Müller, Etienne; Fouillet, Robin; Brault, Baptiste; Henriques, Anna Smirnova; Lemoine, Frédéric; Grange, Pierre de la; Guillaud‐Bataille, Marine; Caux‐Moncoutier, Virginie; Houdayer, Claude; Bonnet, Françoise; Blanc-Fournier, Cécile; Gaildrat, Pascaline; Frébourg, Thierry; Martins, Alexandra; Vaur, Dominique; Krieger, Sophie

doi:10.1038/ejhg.2017.116

Cited by 53 publications

(70 citation statements)

References 45 publications

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“…To accomplish this, BRCA1/2 genes were used as controls since extensive analysis of the splice isoforms repertoire of these genes was previously conducted using PCRbased techniques 21,22 and recently also by targeted RNA-seq. 31 Our results show that the approach used in our study was able to identify almost all previously described BRCA1/2 splicing events, i.e. 93% of the splicing events were detected with our standard analysis.…”

Section: Discussionsupporting

confidence: 55%

“…Overall our method allowed to detect more known events than a previous targeted RNA-seq study. 31 Nevertheless, we did not find 2 BRCA1 and 1 BRCA2 junctions described in that study. So, we aligned the raw reads to the already known events not found by the STAR aligner and visually inspected the outcome.…”

Section: Performance Testcontrasting

confidence: 59%

“…We detected 46 and 36 alternative splicing events (Tables 1 and 2) with expression levels ranging from 0.02-6% to 0.05-61% of the reference RAD51C and RAD51D exon-exon junctions, respectively (see Supporting Information Methods for details on the estimation strategy). Of the alternative splicing events, 14 and 11 events detected in RAD51C and RAD51D, respectively, were not previously described in Ensembl, Gencode or Davy et al 31 It is noteworthy that in 3 of 4 samples a frameshift isoform of RAD51D lacking exon 3 was more abundant than the reference transcript (isoform 1) and the isoform containing a downstream alternative exon 3 (Supporting Information Fig. S5).…”

Section: Rad51c and Rad51d Splicing Eventsmentioning

confidence: 86%

“…Intron 17 contains a rare GC donor splice site, 32 but we were able to detect the normal 17-18 exon-exon junction, as well as the 18-19 junction, and other GC-donor splice sites. The donor site of exon 20C (previously described as 20B 31 ) was also detected with 40 reads. It is unclear why the STAR aligner did not detect these events in our data.…”

Section: Performance Testmentioning

confidence: 99%

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Targeted RNA‐seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes

Brandão

Mensaert

López‐Perolio

et al. 2019

Intl Journal of Cancer

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A subset of genetic variants found through screening of patients with hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome impact RNA splicing. Through target enrichment of the transcriptome, it is possible to perform deep‐sequencing and to identify the different and even rare mRNA isoforms. A targeted RNA‐seq approach was used to analyse the naturally‐occurring splicing events for a panel of 8 breast and/or ovarian cancer susceptibility genes (BRCA1, BRCA2, RAD51C, RAD51D, PTEN, STK11, CDH1, TP53), 3 Lynch syndrome genes (MLH1, MSH2, MSH6) and the fanconi anaemia SLX4 gene, in which monoallelic mutations were found in non‐BRCA families. For BRCA1, BRCA2, RAD51C and RAD51D the results were validated by capillary electrophoresis and were compared to a non‐targeted RNA‐seq approach. We also compared splicing events from lymphoblastoid cell‐lines with those from breast and ovarian fimbriae tissues. The potential of targeted RNA‐seq to detect pathogenic changes in RNA‐splicing was validated by the inclusion of samples with previously well characterized BRCA1/2 genetic variants. In our study, we update the catalogue of normal splicing events for BRCA1/2, provide an extensive catalogue of normal RAD51C and RAD51D alternative splicing, and list splicing events found for eight other genes. Additionally, we show that our approach allowed the identification of aberrant splicing events due to the presence of BRCA1/2 genetic variants and distinguished between complete and partial splicing events. In conclusion, targeted‐RNA‐seq can be very useful to classify variants based on their putative pathogenic impact on splicing.

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Section: Discussionsupporting

confidence: 55%

Section: Performance Testcontrasting

confidence: 59%

Section: Rad51c and Rad51d Splicing Eventsmentioning

confidence: 86%

Section: Performance Testmentioning

confidence: 99%

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Targeted RNA‐seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes

Brandão

Mensaert

López‐Perolio

et al. 2019

Intl Journal of Cancer

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“…Isoform Δ18 is a minor event detected only in HUVH carrier (SF 7.2%) and in HUVH/HCSC controls (SF 4.2% and 1.4%, respectively); isoform Δ16‐18 was only detected in HUVH due to primer location and displayed no notable differences between carrier and controls (SF 13.1% and 11.1%, respectively); and putative isoform ▼17q 224 was only detected in HCSC samples, although not in all RT‐PCR assays, and showed higher levels in HCSC carrier (SF 17.1%) compared to controls (1.2%; Figure a). Two previous studies detected Δ17,18 and Δ18 in control lymphoblastoid cell lines (LCLs) and normal breast tissue, with Δ17,18 being more abundant (Davy et al., ; Fackenthal et al., ). Normalized CE data from full‐length transcript (FL) showed a two‐fold reduction in carriers compared to controls, suggesting that the variant allele is not producing FL (Figure b).…”

mentioning

confidence: 98%

Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing:BRCA2c.7976+5G > T as a case study

Montalban¹,

Fraile-Bethencourt

López‐Perolio

et al. 2018

Human Mutation

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Many BRCA1 and BRCA2 (BRCA1/2) genetic variants have been studied at mRNA level and linked to hereditary breast and ovarian cancer due to splicing alteration. In silico tools are reliable when assessing variants located in consensus splice sites, but we may identify variants in complex genomic contexts for which bioinformatics is not precise enough. In this study, we characterize BRCA2 c.7976 + 5G > T variant located in intron 17 which has an atypical donor site (GC). This variant was identified in three unrelated Spanish families and we have detected exon 17 skipping as the predominant transcript occurring in carriers. We have also detected several isoforms (Δ16-18, Δ17,18, Δ18, and ▼17q ) at different expression levels among carriers and controls. This study remarks the challenge of interpreting genetic variants when multiple alternative isoforms are present, and that caution must be taken when using in silico tools to identify potential spliceogenic variants located in GC-AG introns.

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Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer

et al. 2019

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IMPORTANCE Performing DNA genetic testing (DGT) for hereditary cancer genes is now a wellaccepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians' ability to accurately apply strategies for cancer risk reduction and treatment. OBJECTIVE To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition. DESIGN, SETTING, AND PARTICIPANTS Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018. MAIN OUTCOMES AND MEASURES Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach. Author affiliations and article information are listed at the end of this article. Open Access. This is an open access article distributed under the terms of the CC-BY-NC-ND License.

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Detecting splicing patterns in genes involved in hereditary breast and ovarian cancer

Cited by 53 publications

References 45 publications

Targeted RNA‐seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes

Targeted RNA‐seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes

Characterization of spliceogenic variants located in regions linked to high levels of alternative splicing:BRCA2c.7976+5G > T as a case study

Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer

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