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1. Assessment of branch point prediction tools to predict physiological branch points and their alteration by variants

Author

Raphaël Leman, Hélène Tubeuf, Sabine Raad, Isabelle Tournier, Céline Derambure, Raphaël Lanos, Pascaline Gaildrat, Gaia Castelain, Julie Hauchard, Audrey Killian, Stéphanie Baert-Desurmont, Angelina Legros, Nicolas Goardon, Céline Quesnelle, Agathe Ricou, Laurent Castera, Dominique Vaur, Gérald Le Gac, Chandran Ka, Yann Fichou, Françoise Bonnet-Dorion, Nicolas Sevenet, Marine Guillaud-Bataille, Nadia Boutry-Kryza, Inès Schultz, Virginie Caux-Moncoutier, Maria Rossing, Logan C. Walker, Amanda B. Spurdle, Claude Houdayer, Alexandra Martins, and Sophie Krieger

Subjects
Branch point, Prediction, RNA, Benchmark, HSF, SVM-BPfinder, Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract

Abstract Background Branch points (BPs) map within short motifs upstream of acceptor splice sites (3’ss) and are essential for splicing of pre-mature mRNA. Several BP-dedicated bioinformatics tools, including HSF, SVM-BPfinder, BPP, Branchpointer, LaBranchoR and RNABPS were developed during the last decade. Here, we evaluated their capability to detect the position of BPs, and also to predict the impact on splicing of variants occurring upstream of 3’ss. Results We used a large set of constitutive and alternative human 3’ss collected from Ensembl (n = 264,787 3’ss) and from in-house RNAseq experiments (n = 51,986 3’ss). We also gathered an unprecedented collection of functional splicing data for 120 variants (62 unpublished) occurring in BP areas of disease-causing genes. Branchpointer showed the best performance to detect the relevant BPs upstream of constitutive and alternative 3’ss (99.48 and 65.84% accuracies, respectively). For variants occurring in a BP area, BPP emerged as having the best performance to predict effects on mRNA splicing, with an accuracy of 89.17%. Conclusions Our investigations revealed that Branchpointer was optimal to detect BPs upstream of 3’ss, and that BPP was most relevant to predict splicing alteration due to variants in the BP area.

Published
2020
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2. Supplementary Data Tables S1-S4 from Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12

Author

Pascaline Gaildrat, Alexandra Martins, Maaike P.G. Vreeswijk, Thierry Frebourg, Claude Houdayer, Dominique Stoppa-Lyonnet, Harry Vrieling, Lisa Golmard, Fabienne M.G.R. Calléja, Charlotte Grout, Aurélie Drouet, Maud Privat, Paul Vilquin, Sarab Lizard, Violaine Bourdon, Myriam Bronner, Virginie Guibert, Françoise Bonnet-Dorion, Omar Soukarieh, Hélène Tubeuf, Capucine Delnatte, Françoise Révillion, Johanna Sokolowska, Nadia Boutry-Kryza, Mélanie Léone, Virginie Caux-Moncoutier, Marine Guillaud-Bataille, Sophie Krieger, Sandrine M. Caputo, Romy L.S. Mesman, and Laëtitia Meulemans

Abstract

Supplementary Tables S1-S4. Table S1. Description of primers used in this study. Table S2. Variants selected in BRCA2 exon 12 and its flanking intronic regions. Table S3. Overview of bioinformatics predictions and experimental data obtained for the 40 selected BRCA2 exon 12 variants. Table S4. Clinical and family data of patients carrying BRCA2 exon 12 spliceogenic variants.

Published
2023
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3. Supplementary Tables from Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System

Author

Alexandra Martins, Shyam K. Sharan, Pascaline Gaildrat, Thierry Frebourg, Claude Houdayer, Dominique Stoppa-Lyonnet, Angela R. Solano, Laurence Venat-Bouvet, Chrystelle Colas, Séverine Audebert-Bellanger, Fátima Vaz, Pascal Pujol, Danièle Muller, Hélène Larbre, Violaine Bourdon, Françoise Bonnet-Dorion, Myriam Vezain, Daniela Di Giacomo, Aurélie Drouet, Omar Soukarieh, Eileen Southon, Susan Reid, Linda Cleveland, Marine Guillaud-Bataille, Capucine Delnatte, Nadia Boutry-Kryza, Mélanie Léoné, Françoise Révillion, Laëtitia Meulemans, Alice Fiévet, Gaia Castelain, Julie Hauchard, Virginie Caux-Moncoutier, Sophie Krieger, Julie Rondeaux, Teresa Sullivan, Sandrine M. Caputo, and Hélène Tubeuf

Abstract

Supp Tables S1-S6 (including variant classification, bioinformatics predictions versus experimental data, primer sequences, summary of biological, clinical, tumoral, familial and multifactorial data, and description of statistical analysis approaches)

Published
2023
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4. Data from Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System

Author

Alexandra Martins, Shyam K. Sharan, Pascaline Gaildrat, Thierry Frebourg, Claude Houdayer, Dominique Stoppa-Lyonnet, Angela R. Solano, Laurence Venat-Bouvet, Chrystelle Colas, Séverine Audebert-Bellanger, Fátima Vaz, Pascal Pujol, Danièle Muller, Hélène Larbre, Violaine Bourdon, Françoise Bonnet-Dorion, Myriam Vezain, Daniela Di Giacomo, Aurélie Drouet, Omar Soukarieh, Eileen Southon, Susan Reid, Linda Cleveland, Marine Guillaud-Bataille, Capucine Delnatte, Nadia Boutry-Kryza, Mélanie Léoné, Françoise Révillion, Laëtitia Meulemans, Alice Fiévet, Gaia Castelain, Julie Hauchard, Virginie Caux-Moncoutier, Sophie Krieger, Julie Rondeaux, Teresa Sullivan, Sandrine M. Caputo, and Hélène Tubeuf

Abstract

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.

Published
2023
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5. Supplementary Data from Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12

Author

Pascaline Gaildrat, Alexandra Martins, Maaike P.G. Vreeswijk, Thierry Frebourg, Claude Houdayer, Dominique Stoppa-Lyonnet, Harry Vrieling, Lisa Golmard, Fabienne M.G.R. Calléja, Charlotte Grout, Aurélie Drouet, Maud Privat, Paul Vilquin, Sarab Lizard, Violaine Bourdon, Myriam Bronner, Virginie Guibert, Françoise Bonnet-Dorion, Omar Soukarieh, Hélène Tubeuf, Capucine Delnatte, Françoise Révillion, Johanna Sokolowska, Nadia Boutry-Kryza, Mélanie Léone, Virginie Caux-Moncoutier, Marine Guillaud-Bataille, Sophie Krieger, Sandrine M. Caputo, Romy L.S. Mesman, and Laëtitia Meulemans

Abstract

Fig. S1 to S10. Figure S1. Variant selection from human variation databases; Figure S2. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of presumed LoF variants; Figure S3. Bioinformatics predictions of 3'/5' ss alterations for variants located at position IVS{plus minus}1/2 of BRCA2 exon 12; Figure S4. Bioinformatics analysis of variants predicted to alter 3'/5' ss of BRCA2 exon 12 (â^†MES {less than or equal to} -15%); Figure S5. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in minigene assays of variants predicted to alter 3'/5' ss (A) or ESR (B); Figure S6. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in control and patient lymphoblastoid cell lines; Figure S7. RT-PCR analysis of BRCA2 exon 12 splicing patterns in puromycin- or mock-treated lymphoblastoid cell lines of control individuals and patients carrying the c.6844G>T or c.6901G>T nonsense variants; Figure S8. Capillary electrophoresis analyses of BRCA2 exon 12 splicing patterns in variant-expressing mESC; Figure S9. Quantitation of BRCA2 protein expression in mESC; Figure S10. Sensitivity of BRCA2 variants to cisplatin and PARP inhibitors.

Published
2023
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6. Data from Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12

Author

Pascaline Gaildrat, Alexandra Martins, Maaike P.G. Vreeswijk, Thierry Frebourg, Claude Houdayer, Dominique Stoppa-Lyonnet, Harry Vrieling, Lisa Golmard, Fabienne M.G.R. Calléja, Charlotte Grout, Aurélie Drouet, Maud Privat, Paul Vilquin, Sarab Lizard, Violaine Bourdon, Myriam Bronner, Virginie Guibert, Françoise Bonnet-Dorion, Omar Soukarieh, Hélène Tubeuf, Capucine Delnatte, Françoise Révillion, Johanna Sokolowska, Nadia Boutry-Kryza, Mélanie Léone, Virginie Caux-Moncoutier, Marine Guillaud-Bataille, Sophie Krieger, Sandrine M. Caputo, Romy L.S. Mesman, and Laëtitia Meulemans

Abstract

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications.Significance:This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

Published
2023
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7. SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing

Author

Raphaël Leman, Béatrice Parfait, Dominique Vidaud, Emmanuelle Girodon, Laurence Pacot, Gérald Le Gac, Chandran Ka, Claude Ferec, Yann Fichou, Céline Quesnelle, Camille Aucouturier, Etienne Muller, Dominique Vaur, Laurent Castera, Flavie Boulouard, Agathe Ricou, Hélène Tubeuf, Omar Soukarieh, Pascaline Gaildrat, Florence Riant, Marine Guillaud‐Bataille, Sandrine M. Caputo, Virginie Caux‐Moncoutier, Nadia Boutry‐Kryza, Françoise Bonnet‐Dorion, Ines Schultz, Maria Rossing, Olivier Quenez, Louis Goldenberg, Valentin Harter, Michael T. Parsons, Amanda B. Spurdle, Thierry Frébourg, Alexandra Martins, Claude Houdayer, and Sophie Krieger

Subjects
sequence variants, machine learning, SPiP, Genetics, RNA, splicing predictions, Genetics (clinical)
Abstract

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5’/3’ splice sites, branch sites or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on machine learning approach, comprehensive assessment of variant effect on different splicing motifs. We gathered a curated set of 4,616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. Bayesian analysis provided us the number of control variants, i.e. variants without impact on splicing, to mimic the deluge of variants from high throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, significantly better than 0.965 spliceAI for the same dataset. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: [https://sourceforge.net/projects/splicing-prediction-pipeline/](https://sourceforge.net/projects/splicing-prediction-pipeline/)

Published
2022
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8. Calibration of pathogenicity due to variant-induced leaky splicing defects by using BRCA2 exon 3 as a model system

Author

Gaia Castelain, Capucine Delnatte, Pascaline Gaildrat, Myriam Vezain, Laëtitia Meulemans, Nadia Boutry-Kryza, Teresa Sullivan, Dominique Stoppa-Lyonnet, Julie Hauchard, Pascal Pujol, Thierry Frebourg, Séverine Audebert-Bellanger, Daniela Di Giacomo, Danièle Muller, Aurélie Drouet, Hélène Tubeuf, Susan W. Reid, Julie Rondeaux, Mélanie Léoné, Violaine Bourdon, Françoise Bonnet-Dorion, Françoise Révillion, Hélène Larbre, Shyam K. Sharan, Omar Soukarieh, Sandrine M. Caputo, Alice Fiévet, Laurence Venat-Bouvet, Virginie Caux-Moncoutier, Alexandra Martins, Fátima Vaz, Angela R. Solano, Claude Houdayer, Eileen Southon, Chrystelle Colas, Marine Guillaud-Bataille, Linda Cleveland, Sophie Krieger, Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques (GPMCND), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche de l'Institut Curie [Paris], Institut Curie [Paris], Université Paris sciences et lettres (PSL), National Cancer Institute Frederick, Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), Normandie Université (NU)-UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN), Unité de génétique et biologie des cancers (U830), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de médecine oncologique [Gustave Roussy], Institut Gustave Roussy (IGR), Département de biologie et pathologie médicales [Gustave Roussy], Centre Régional de Lutte contre le Cancer Oscar Lambret [Lille] (UNICANCER/Lille), Université de Lille-UNICANCER, Hospices Civils de Lyon (HCL), Centre hospitalier universitaire de Nantes (CHU Nantes), Actions for OnCogenesis understanding and Target Identification in ONcology (ACTION), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Institut Jean Godinot [Reims], UNICANCER, Centre Paul Strauss, CRLCC Paul Strauss, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Maladies infectieuses et vecteurs : écologie, génétique, évolution et contrôle (MIVEGEC), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), Hôpital Morvan - CHRU de Brest (CHU - BREST ), Hôpital Dupuytren [CHU Limoges], Centro de Educación Médica e Investigaciones Clínicas (CEMIC), and This work was funded by a translational research grant from the French National Cancer Institute and the Direction Générale de l’Offre des Soins (INCa/DGOS, AAP/CFB/CI), by the OpenHealth Institute, the Groupement des Entreprises Fran¸caises dans la Lutte contre le Cancer (Gefluc), the Fédération Hospitalo-Universitaire (FHU) Normandy Centre for Genomic and Personalized Medicine (NGP), the European Union and Region Normandie as well as by the Intramural Research Program from the Center for Cancer Research of the NCI, NIH. Europe gets involved in Normandie with European Regional Development Fund (ERDF). H. Tubeuf was funded by a CIFRE PhD fellowship (#2015/0335) from the French Association Nationale de la Recherche et de la Technologie (ANRT) in the context of a public–private partnership between INSERM and Interactive Biosoftware and by two short-term fellowship from EMBO (#3436) and Cancéropôle Nord-Ouest. J. Hauchard and G. Castelain were sponsored by the French INCa.

Subjects
0301 basic medicine, Cancer Research, endocrine system diseases, In silico, Genes, BRCA2, [SDV.CAN]Life Sciences [q-bio]/Cancer, Breast Neoplasms, Biology, MESH: Protein Isoforms, Article, 03 medical and health sciences, Exon, Mice, 0302 clinical medicine, Protein-fragment complementation assay, Animals, Humans, Protein Isoforms, MESH: Animals, Genetic Predisposition to Disease, skin and connective tissue diseases, Gene, MESH: Mice, Genetics, Ovarian Neoplasms, MESH: Humans, MESH: Alternative Splicing, MESH: Genetic Predisposition to Disease, RNA, Exons, MESH: Ovarian Neoplasms, Alternative Splicing, 030104 developmental biology, Oncology, 030220 oncology & carcinogenesis, RNA splicing, Female, Haploinsufficiency, MESH: Exons, MESH: Female, MESH: Breast Neoplasms, MESH: Genes, BRCA2, Minigene
Abstract

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.

Published
2020
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9. Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12

Author

Romy L. S. Mesman, Marine Guillaud-Bataille, Mélanie Léoné, Capucine Delnatte, Aurélie Drouet, Alexandra Martins, Hélène Tubeuf, Laëtitia Meulemans, Omar Soukarieh, Pascaline Gaildrat, Violaine Bourdon, Nadia Boutry-Kryza, Sophie Krieger, Myriam Bronner, Fabienne Calléja, Johanna Sokolowska, Maud Privat, Dominique Stoppa-Lyonnet, Harry Vrieling, Maaike P.G. Vreeswijk, Françoise Révillion, Sandrine M. Caputo, Claude Houdayer, Sarab Lizard, Charlotte Grout, Françoise Bonnet-Dorion, Lisa Golmard, Virginie Caux-Moncoutier, Paul Vilquin, Virginie Guibert, Thierry Frebourg, Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques (GPMCND), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Center for Human and Clinical Genetics, Leiden University Medical Center (LUMC), Service de Génétique Oncologique, Institut Curie [Paris], Université Paris sciences et lettres (PSL), Institut Gustave Roussy (IGR), Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Léon Bérard [Lyon]-Hospices Civils de Lyon (HCL), Hospices Civils de Lyon (HCL), Service de Génétique [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Laboratoire d'Oncologie Moléculaire Humaine, Centre Régional de Lutte contre le Cancer Oscar Lambret [Lille] (UNICANCER/Lille), Université Lille Nord de France (COMUE)-UNICANCER-Université Lille Nord de France (COMUE)-UNICANCER, Service de génétique médicale - Unité de génétique clinique [Nantes], Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Validation et identification de nouvelles cibles en oncologie (VINCO), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Nutrition-Génétique et Exposition aux Risques Environnementaux (NGERE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Service d'Oncologie Génétique, de Prévention et Dépistage, Service de Biopathologie [CHRU Montpellier], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre Jean Perrin [Clermont-Ferrand] (UNICANCER/CJP), UNICANCER, Imagerie Moléculaire et Stratégies Théranostiques (IMoST), Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de génétique et biologie des cancers (U830), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de génétique [Rouen], CHU Rouen, Normandie Université (NU)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Universiteit Leiden-Universiteit Leiden, Université de Lille-UNICANCER-Université de Lille-UNICANCER, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), UNICANCER-Université Lille Nord de France (COMUE)-UNICANCER-Université Lille Nord de France (COMUE), Centre hospitalier universitaire de Nantes (CHU Nantes), Imagerie Moléculaire et Stratégies Théranostiques - Clermont Auvergne (IMoST), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne (UCA), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Service de biochimie [CHU Trousseau], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Trousseau [APHP]

Subjects
0301 basic medicine, Genetics, Cancer Research, media_common.quotation_subject, Nonsense, Alternative splicing, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biology, 3. Good health, 03 medical and health sciences, Exon, 030104 developmental biology, 0302 clinical medicine, Oncology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, 030220 oncology & carcinogenesis, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], RNA splicing, splice, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Allele, Gene, media_common, Minigene
Abstract

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell–based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. Significance: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

Published
2020
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10. Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort

Author

Gérald Le Gac, Alexandra Martins, Pascaline Gaildrat, Florence Coulet, Françoise Revillon, Antoine Rousselin, Sandrine M. Caputo, Marine Guillaud-Bataille, Chandran Ka, Mélanie Léoné, Nadia Boutry-Kryza, Virginie Caux-Moncoutier, Claude Férec, Capucine Delnatte, Danielle Muller, Grégoire Davy, Amanda B. Spurdle, Maria Rossing, Raphaël Leman, Yann Fichou, Michael T. Parsons, Barbara Wappenschmidt, Violaine Bourdon, Joanna Sokolowska, Nicolas Sevenet, Claude Houdayer, M.-P. Audrézet, Brigitte Bressac-de Paillerets, Françoise Bonnet-Dorion, Sylvie Mazoyer, Gaia Castelain, Sophie Krieger, Laurent Castera, Etienne Rouleau, Centre Régional de Lutte contre le Cancer François Baclesse [Caen] (UNICANCER/CRLC), Normandie Université (NU)-UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN), Génomique et Médecine Personnalisée du Cancer et des Maladies Neuropsychiatriques (GPMCND), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique, génomique fonctionnelle et biotechnologies (UMR 1078) (GGB), EFS-Université de Brest (UBO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Brestois Santé Agro Matière (IBSAM), Université de Brest (UBO), Unité de génétique et biologie des cancers (U830), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5), Université Sorbonne Paris Cité (USPC), Institut Curie [Paris], Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Léon Bérard [Lyon]-Hospices Civils de Lyon (HCL), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Validation et identification de nouvelles cibles en oncologie (VINCO), Institut Bergonié [Bordeaux], UNICANCER-UNICANCER-Université Bordeaux Segalen - Bordeaux 2-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique (Biologie pathologie), Département de biologie et pathologie médicales [Gustave Roussy], Institut Gustave Roussy (IGR)-Institut Gustave Roussy (IGR), University Hospital of Cologne [Cologne], IT University of Copenhagen (ITU), Centre Paul Strauss, CRLCC Paul Strauss, Laboratoire d'oncogénétique moléculaire, Unite d'Oncogenese Virale, Centre Régional de Lutte contre le Cancer Oscar Lambret [Lille] (UNICANCER/Lille), Université de Lille-UNICANCER-Université de Lille-UNICANCER, QIMR Berghofer Medical Research Institute, Nutrition-Génétique et Exposition aux Risques Environnementaux (NGERE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Service de Génétique Clinique [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Génétique épidémiologique et moléculaire des pathologies cardiovasculaires, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), Service de Génétique médicale [CHU Pitié-Salpêtrière], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre hospitalier universitaire de Nantes (CHU Nantes), Etablissement Français du Sang Bretagne, EFS, Laboratoire de Génétique Moléculaire et d'Histocompatibilité [Brest], Hôpital Morvan [Brest]-Centre Hospitalier Régional Universitaire de Brest (CHRU Brest), COMBE, Isabelle, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), UNICANCER-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), UNICANCER, Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), IT University of Copenhagen, Institut Gustave Roussy (IGR), Service d'Oncologie Génétique, de Prévention et Dépistage, Laboratoire d'Oncologie Moléculaire Humaine, Université Lille Nord de France (COMUE)-UNICANCER-Université Lille Nord de France (COMUE)-UNICANCER, CHU Pontchaillou [Rennes], Université de Caen Normandie - UFR Santé (UNICAEN Santé), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU), Centre René Gauducheau, and CRLCC René Gauducheau

Subjects
0301 basic medicine, International Cooperation, RNA Splicing, In silico, Spice, Breast Neoplasms, Computational biology, Biology, Sensitivity and Specificity, Set (abstract data type), 03 medical and health sciences, Exon, 0302 clinical medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, Consensus sequence, RNA and RNA-protein complexes, Humans, Computer Simulation, 030304 developmental biology, BRCA2 Protein, Ovarian Neoplasms, Internet, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], BRCA1 Protein, Intron, Computational Biology, Genetic Variation, Reproducibility of Results, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], In vitro, 030104 developmental biology, 030220 oncology & carcinogenesis, RNA splicing, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Female, RNA Splice Sites, RNA characterisation and manipulation, Corrigendum, Computational Methods, Optimal decision
Abstract

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

Published
2018
Full Text
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11. Skipping Nonsense to Maintain Function: The Paradigm of

Author

Laëtitia, Meulemans, Romy L S, Mesman, Sandrine M, Caputo, Sophie, Krieger, Marine, Guillaud-Bataille, Virginie, Caux-Moncoutier, Mélanie, Léone, Nadia, Boutry-Kryza, Johanna, Sokolowska, Françoise, Révillion, Capucine, Delnatte, Hélène, Tubeuf, Omar, Soukarieh, Françoise, Bonnet-Dorion, Virginie, Guibert, Myriam, Bronner, Violaine, Bourdon, Sarab, Lizard, Paul, Vilquin, Maud, Privat, Aurélie, Drouet, Charlotte, Grout, Fabienne M G R, Calléja, Lisa, Golmard, Harry, Vrieling, Dominique, Stoppa-Lyonnet, Claude, Houdayer, Thierry, Frebourg, Maaike P G, Vreeswijk, Alexandra, Martins, and Pascaline, Gaildrat

Subjects
Adult, Aged, 80 and over, BRCA2 Protein, Male, Exons, Middle Aged, Polymorphism, Single Nucleotide, Recombinant Proteins, Pedigree, Alternative Splicing, Mice, Loss of Function Mutation, Cell Line, Tumor, Animals, Hereditary Breast and Ovarian Cancer Syndrome, Humans, Female, Genetic Predisposition to Disease, Embryonic Stem Cells, Aged
Abstract

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced

Published
2019

12. FGFRI and PLAT genes and DNA amplification at 8p12 in breast and ovarian cancers

Author

Charles Theillet, José Adelaide, Geneviève Louason, Françoise Bonnet-Dorion, Jocelyne Jacquemier, Jalila Adnane, Michel Longy, Dionisi Katsaros, Piero Sismondi, Patrick Gaudray, and Daniel Birnbaum

Subjects
Cancer Research, Gene Expression, Breast Neoplasms, Filaggrin Proteins, Biology, Fibroblast growth factor, chemistry.chemical_compound, Gene duplication, Gene expression, Genetics, Humans, Receptor, Fibroblast Growth Factor, Type 1, Receptor, Gene, Ovarian Neoplasms, Chromosomes, Human, Pair 11, Gene Amplification, Receptor Protein-Tyrosine Kinases, DNA, Neoplasm, Dna amplification, Receptors, Fibroblast Growth Factor, Fibroblast Growth Factors, chemistry, Tissue Plasminogen Activator, Cancer research, Female, Plasminogen activator, DNA, Chromosomes, Human, Pair 8
Abstract

Several chromosomal regions are found to be consistently amplified in human breast cancers. For two of these regions, 8p12 and 10q26, we previously reported the amplification of genes encoding FGF receptors, FGFRI/FLG and FGFR2/BEK, in about 12% of breast tumors. The PLAT gene, encoding the tissue-type plasminogen activator, is also located close to or within the 8p12 region. In the present study, we show that both FGFRI and PLAT can be amplified in breast as well as ovarian carcinomas. FGFRI amplification was detected in 14.5% of breast and 7.8% of ovarian tumors, whereas PLAT was found to be amplified in 15.6% and 19.4% of the tumors, respectively. Each gene could be amplified independently of the other. These data raised the question of which gene is selected for amplification at 8p12. In most cases, the levels of expression of FGFRI and PLAT in breast tumors were comparable to their level of expression in normal mammary tissue. However, FGFRI was expressed above the normal level in a certain number of cases. This gene could be a good candidate as "driver" of the 8p12 amplification, but it cannot account for all complex molecular events taking place in this region.

Published
1993

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