Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay.
CALPAIN 3
CAPN3
LGMD2A
LGMDR1
exonic
minigene
missense variant
neuromuscular disease
splicing
Journal
Human mutation
ISSN: 1098-1004
Titre abrégé: Hum Mutat
Pays: United States
ID NLM: 9215429
Informations de publication
Date de publication:
10 2020
10 2020
Historique:
revised:
01
07
2020
received:
05
04
2020
accepted:
09
07
2020
pubmed:
16
7
2020
medline:
1
4
2022
entrez:
16
7
2020
Statut:
ppublish
Résumé
Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often-overlooked splicing effect of missense variants, we developed the functional assay ("minigene") for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon-intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning-based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the "minigene" functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics.
Substances chimiques
Muscle Proteins
0
CAPN3 protein, human
EC 3.4.22.-
Calpain
EC 3.4.22.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1797-1810Informations de copyright
© 2020 Wiley Periodicals LLC.
Références
Anderson, L. V., Davison, K., Moss, J. A., Richard, I., Fardeau, M., Tomé, F. M., … Beckmann, J. S. (1998). Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A. The American Journal of Pathology, 153(4), 1169-1179.
Barp, A., Laforet, P., Bello, L., Tasca, G., Vissing, J., Monforte, M., … Carlier, R. Y. (2020). European muscle MRI study in limb girdle muscular dystrophy type R1/2A (LGMDR1/LGMD2A). Journal of Neurology, 267(1), 45-56.
Bonnet, C., Krieger, S., Vezain, M., Rousselin, A., Tournier, I., Martins, A., … Tosi, M. (2008). Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. Journal of Medical Genetics, 45(7), 438-446. https://doi.org/10.1136/jmg.2007.056895
Brnich, S. E., Abou Tayoun, A. N., Couch, F. J., Cutting, G. R., Greenblatt, M. S., Heinen, C. D., … Clinical Genome Resource Sequence Variant Interpretation Working Group (2019). Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Medicine, 12(1), 3.
Cheng, J., Nguyen, T. Y. D., Cygan, K. J., Çelik, M. H., Fairbrother, W. G., Avsec, Ž., & Gagneur, J. (2019). MMSplice: Modular modeling improves the predictions of genetic variant effects on splicing. Genome Biology, 20(1), 48.
Cooper, D. N., Krawczak, M., Polychronakos, C., Tyler-Smith, C., & Kehrer-Sawatzki, H. (2013). Where genotype is not predictive of phenotype: Towards an understanding of the molecular basis of reduced penetrance in human inherited disease. Human Genetics, 132(10), 1077-1130.
de Paula, F., Vainzof, M., Passos-Bueno, M. R., de Cássia, M., Pavanello, R., Matioli, S. R., … Zatz, M. (2002). Clinical variability in calpainopathy: What makes the difference? European Journal of Human Genetics: EJHG, 10(12), 825-832.
Desmet, F.-O., Hamroun, D., Lalande, M., Collod-Béroud, G., Claustres, M., & Béroud, C. (2009). Human Splicing Finder: An online bioinformatics tool to predict splicing signals. Nucleic Acids Research, 37(9), e67.
Du, J., Sudarsanam, M., Pérez-Palma, E., Ganna, A., Francioli, L., Iqbal, S., … Lal, D. (2019). Variant Score Ranker-a web application for intuitive missense variant prioritization. Bioinformatics, 35(21), 4478-4479.
Duguez, S., Bartoli, M., & Richard, I. (2006). Calpain 3: A key regulator of the sarcomere? FEBS Journal, 273(15), 3427-3436. https://doi.org/10.1111/j.1742-4658.2006.05351.x
Duno, M., Sveen, M.-L., Schwartz, M., & Vissing, J. (2008). cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark. European Journal of Human Genetics: EJHG, 16(8), 935-940.
Ellingford, J. M., Thomas, H. B., Rowlands, C., Arno, G., Beaman, G., Gomes-Silva, B., … Black, G. C. M. (2019). Functional and in-silico interrogation of rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders. bioRxiv, 781088. https://doi.org/10.1101/781088
Fairbrother, W. G., Holste, D., Burge, C. B., & Sharp, P. A. (2004). Single nucleotide polymorphism-based validation of exonic splicing enhancers. PLOS Biology, 2(9), e268. https://doi.org/10.1371/journal.pbio.0020268
Fanin, M., Fulizio, L., Nascimbeni, A. C., Spinazzi, M., Piluso, G., Ventriglia, V. M., … Angelini, C. (2004). Molecular diagnosis in LGMD2A: Mutation analysis or protein testing? Human Mutation, 24(1), 52-62.
Fardeau, M., Eymard, B., Mignard, C., Tomé, F. M. S., Richard, I., & Beckmann, J. S. (1996). Chromosome 15-linked limb-girdle muscular dystrophy: Clinical phenotypes in Reunion Island and French metropolitan communities. Neuromuscular Disorders, 6(6), 447-453. https://doi.org/10.1016/s0960-8966(96)00387-2
Fokkema, I. F. A. C., Taschner, P. E. M., Schaafsma, G. C. P., Celli, J., Laros, J. F. J., & den Dunnen, J. T. (2011). LOVD v.2.0: The next generation in gene variant databases. Human Mutation, 32(5), 557-563.
Gaildrat, P., Killian, A., Martins, A., Tournier, I., Frébourg, T., & Tosi, M. (2010). Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods in Molecular Biology, 653, 249-257.
Gault, C. M., Martin, F., Mei, W., Bai, F., Black, J. B., Barbazuk, W. B., & Settles, A. M. (2017). Aberrant splicing in maize reveals a conserved role for U12 splicing in eukaryotic multicellular development. Proceedings of the National Academy of Sciences of the United States of America, 114(11), E2195-E2204.
Gelb, B. D., Cavé, H., Dillon, M. W., Gripp, K. W., Lee, J. A., Mason-Suares, H., … ClinGen RASopathy Working Group (2018). ClinGen's RASopathy Expert Panel consensus methods for variant interpretation. Genetics in Medicine, 20(11), 1334-1345. https://doi.org/10.1038/gim.2018.3
Havrilla, J. M., Pedersen, B. S., Layer, R. M., & Quinlan, A. R. (2019). A map of constrained coding regions in the human genome. Nature Genetics, 51(1), 88-95.
Hu, Z., Yu, C., Furutsuki, M., Andreoletti, G., Ly, M., Hoskins, R., … Brenner, S. E. (2019). VIPdb, a genetic Variant Impact Predictor Database. Human Mutation, 40(9), 1202-1214.
Ioannidis, N. M., Rothstein, J. H., Pejaver, V., Middha, S., McDonnell, S. K., Baheti, S., … Sieh, W. (2016). REVEL: An Ensemble Method for predicting the pathogenicity of rare missense variants. American Journal of Human Genetics, 99(4), 877-885.
Jagadeesh, K. A., Paggi, J. M., Ye, J. S., Stenson, P. D., Cooper, D. N., Bernstein, J. A., & Bejerano, G. (2019). S-CAP extends pathogenicity prediction to genetic variants that affect RNA splicing. Nature Genetics, 51(4), 755-763.
Jaganathan, K., Kyriazopoulou Panagiotopoulou, S., McRae, J. F., Darbandi, S. F., Knowles, D., Li, Y. I., … Farh, K. K.-H. (2019). Predicting splicing from primary sequence with deep learning. Cell, 176(3), 535-548.
Karczewski, K. J., Francioli, L. C., Tiao, G., Cummings, B. B., Alföldi, J., Wang, Q., … MacArthur, D. G. (2019). Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. Genomics (No. biorxiv;531210v1; p. 677). bioRxiv.
Karolchik, D., Hinrichs, A. S., Furey, T. S., Roskin, K. M., Sugnet, C. W., Haussler, D., & Kent, W. J. (2004). The UCSC Table Browser data retrieval tool. Nucleic Acids Research, 32(Database issue), D493-D496.
Kelly, M. A., Caleshu, C., Morales, A., Buchan, J., Wolf, Z., Harrison, S. M., … Funke, B. (2018). Adaptation and validation of the ACMG/AMP variant classification framework for MYH7-associated inherited cardiomyopathies: Recommendations by ClinGen's Inherited Cardiomyopathy Expert Panel. Genetics in Medicine, 20(3), 351-359.
Kergourlay, V., Raï, G., Blandin, G., Salgado, D., Béroud, C., Lévy, N., … Bartoli, M. (2014). Identification of splicing defects caused by mutations in the dysferlin gene. Human Mutation, 35(12), 1532-1541.
Krahn, M., Biancalana, V., Cerino, M., Perrin, A., Michel-Calemard, L., Nectoux, J., … Cossée, M. (2019). A National French consensus on gene lists for the diagnosis of myopathies using next-generation sequencing. European Journal of Human Genetics: EJHG, 27(3), 349-352.
Lee, K., Krempely, K., Roberts, M. E., Anderson, M. J., Carneiro, F., Chao, E., … Karam, R. (2018). Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants. Human Mutation, 39(11), 1553-1568.
Lu, Z.-X., Jiang, P., & Xing, Y. (2012). Genetic variation of pre-mRNA alternative splicing in human populations. Wiley Interdisciplinary Reviews: RNA, 3(4), 581-592.
Mort, M., Sterne-Weiler, T., Li, B., Ball, E. V., Cooper, D. N., Radivojac, P., … Mooney, S. D. (2014). MutPred Splice: Machine learning-based prediction of exonic variants that disrupt splicing. Genome Biology, 15(1), R19.
Nascimbeni, A. C., Fanin, M., Tasca, E., & Angelini, C. (2010). Transcriptional and translational effects of intronic CAPN3 gene mutations. Human Mutation, 31(9), E1658-E1669.
Ohno, K., Takeda, J. I., & Masuda, A. (2018). Rules and tools to predict the splicing effects of exonic and intronic mutations. Wiley Interdiscip Rev RNA, 9(1). https://doi.org/10.1002/wrna.1451
Ono, Y., Ojima, K., Shinkai-Ouchi, F., Hata, S., & Sorimachi, H. (2016). An eccentric calpain, CAPN3/p94/calpain-3. Biochimie, 122, 169-187.
Ono, Y., Shimada, H., Sorimachi, H., Richard, I., Saido, T. C., Beckmann, J. S., … Suzuki, K. (1998). Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A. The Journal of Biological Chemistry, 273(27), 17073-17078.
Oza, A. M., DiStefano, M. T., Hemphill, S. E., Cushman, B. J., Grant, A. R., Siegert, R. K., … ClinGen Hearing Loss Clinical Domain Working Group (2018). Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Human Mutation, 39(11), 1593-1613.
Park, E., Pan, Z., Zhang, Z., Lin, L., & Xing, Y. (2018). The expanding landscape of alternative splicing variation in human populations. American Journal of Human Genetics, 102(1), 11-26.
Puppo, F., Dionnet, E., Gaillard, M.-C., Gaildrat, P., Castro, C., Vovan, C., … Lévy, N. (2015). Identification of variants in the 4q35 gene FAT1 in patients with a facioscapulohumeral dystrophy-like phenotype. Human Mutation, 36(4), 443-453.
Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J., & Kircher, M. (2019). CADD: Predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Research, 47(D1), D886-D894.
Richard, I., Roudaut, C., Saenz, A., Pogue, R., Grimbergen, J. E., Anderson, L. V., … Beckmann, J. S. (1999). Calpainopathy-A survey of mutations and polymorphisms. American Journal of Human Genetics, 64(6), 1524-1540.
Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., … ACMG Laboratory Quality Assurance Committee (2015). Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine, 17(5), 405-424.
Rowlands, C. F., Baralle, D., & Ellingford, J. M. (2019). Machine learning approaches for the prioritization of genomic variants impacting pre-mRNA splicing. Cells, 8(12), 1513. https://doi.org/10.3390/cells8121513
Savisaar, R., & Hurst, L. D. (2017). Estimating the prevalence of functional exonic splice regulatory information. Human Genetics, 136(9), 1059-1078.
Stephenson, J. D., Laskowski, R. A., Nightingale, A., Hurles, M. E., & Thornton, J. M. (2019). VarMap: A web tool for mapping genomic coordinates to protein sequence and structure and retrieving protein structural annotations. Bioinformatics, 35(22), 4854-4856.
Théry, J. C., Krieger, S., Gaildrat, P., Révillion, F., Buisine, M.-P., Killian, A., … Tosi, M. (2011). Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes. European Journal of Human Genetics: EJHG, 19(10), 1052-1058.
Tournier, I., Vezain, M., Martins, A., Charbonnier, F., Baert-Desurmont, S., Olschwang, S., … Tosi, M. (2008). A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects. Human Mutation, 29(12), 1412-1424.
Turunen, J. J., Niemelä, E. H., Verma, B., & Frilander, M. J. (2013). The significant other: Splicing by the minor spliceosome. Wiley Interdisciplinary Reviews: RNA, 4(1), 61-76.
Woolfe, A., Mullikin, J. C., & Elnitski, L. (2010). Genomic features defining exonic variants that modulate splicing. Genome Biology, 11(2), R20.
Ye, Q., Campbell, R. L., & Davies, P. L. (2018). Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation. Journal of Biological Chemistry, 293(11), 4056-4070. https://doi.org/10.1074/jbc.ra117.001097
Yeo, G., & Burge, C. B. (2004). Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. Journal of Computational Biology, 11(2-3), 377-394.
Zhou, X., Edmonson, M. N., Wilkinson, M. R., Patel, A., Wu, G., Liu, Y., … Zhang, J. (2016). Exploring genomic alteration in pediatric cancer using ProteinPaint. Nature Genetics, 48(1), 4-6.