{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T10:18:07Z","timestamp":1768558687306,"version":"3.49.0"},"reference-count":35,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2016,11,10]],"date-time":"2016-11-10T00:00:00Z","timestamp":1478736000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of University and Research","award":["PON03PE_00060_2 and PON03PE_00060_7 (Campania - Bioscience)"],"award-info":[{"award-number":["PON03PE_00060_2 and PON03PE_00060_7 (Campania - Bioscience)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants (MYBPC3-c.506-2A&gt;C, MYBPC3-c.906-7G&gt;T, MYBPC3-c.2308+3G&gt;C, SCN5A-c.393-5C&gt;A, and ACTC1-c.617-7T&gt;C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the MYBPC3-c.506-2A&gt;C mutation in mRNA from the peripheral blood of one of the patients affected by hypertrophic cardiac myopathy revealed the loss of the canonical splice site and the use of an alternative splicing site, which caused the loss of the first seven nucleotides of exon 5 (MYBPC3-G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed that MYBPC3-c.2308+3G&gt;C and SCN5A-c.393-5C&gt;A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either MYBPC3-c.906-7G&gt;T or ACTC1-c.617-7T&gt;C. In conclusion, functional in vitro analysis of the effects of potential splicing mutations can confirm or otherwise the putative pathogenicity of non-coding mutations, and thus help to guide the patient's clinical management and improve genetic counseling in affected families.<\/jats:p>","DOI":"10.3390\/ijms17111883","type":"journal-article","created":{"date-parts":[[2016,11,10]],"date-time":"2016-11-10T10:51:39Z","timestamp":1478775099000},"page":"1883","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":31,"title":["Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3487-7743","authenticated-orcid":false,"given":"Giulia","family":"Frisso","sequence":"first","affiliation":[{"name":"CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy"},{"name":"Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universit\u00e0 di Napoli Federico II, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicola","family":"Detta","sequence":"additional","affiliation":[{"name":"CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pamela","family":"Coppola","sequence":"additional","affiliation":[{"name":"CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy"},{"name":"Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universit\u00e0 di Napoli Federico II, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7774-2799","authenticated-orcid":false,"given":"Cristina","family":"Mazzaccara","sequence":"additional","affiliation":[{"name":"CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy"},{"name":"Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universit\u00e0 di Napoli Federico II, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Pricolo","sequence":"additional","affiliation":[{"name":"Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universit\u00e0 di Napoli Federico II, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonio","family":"D\u2019Onofrio","sequence":"additional","affiliation":[{"name":"Dipartimento di Cardiologia, Seconda Universit\u00e0 degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8291-9517","authenticated-orcid":false,"given":"Giuseppe","family":"Limongelli","sequence":"additional","affiliation":[{"name":"Dipartimento di Cardiologia, Seconda Universit\u00e0 degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raffaele","family":"Calabr\u00f2","sequence":"additional","affiliation":[{"name":"Dipartimento di Cardiologia, Seconda Universit\u00e0 degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2346-3564","authenticated-orcid":false,"given":"Francesco","family":"Salvatore","sequence":"additional","affiliation":[{"name":"CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy"},{"name":"IRCCS-Fondazione SDN, 80143 Napoli, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2016,11,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1900","DOI":"10.1016\/j.febslet.2005.02.047","article-title":"Are splicing mutations the most frequent cause of hereditary disease?","volume":"579","author":"Audit","year":"2005","journal-title":"FEBS Lett."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1136\/jmg.2004.029538","article-title":"Splicing in action: Assessing disease causing sequence changes","volume":"42","author":"Baralle","year":"2005","journal-title":"J. Med. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1101\/gr.183483.114","article-title":"Actionable exomic incidental findings in 6503 participants: Challenges of variant classification","volume":"25","author":"Amendola","year":"2015","journal-title":"Genome Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2125","DOI":"10.1093\/hmg\/ddu733","article-title":"Comparison and integration of deleteriousness prediction methods for nonsynonymous snvs in whole exome sequencing studies","volume":"24","author":"Dong","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1002\/humu.20901","article-title":"Prediction and assessment of splicing alterations: Implications for clinical testing","volume":"29","author":"Spurdle","year":"2008","journal-title":"Hum. Mutat."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Petersen, S.M., Dandanell, M., Rasmussen, L.J., Gerdes, A.M., Krogh, L.N., Bernstein, I., Okkels, H., Wikman, F., Nielsen, F.C., and Hansen, T.V. (2013). Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in danish colorectal cancer patients. BMC Med. Genet., 14.","DOI":"10.1186\/1471-2350-14-103"},{"key":"ref_7","first-page":"2675","article-title":"The missing puzzle piece: Splicing mutations","volume":"6","author":"Lewandowska","year":"2013","journal-title":"Int. J. Clin. Exp. Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1016\/j.cell.2009.02.011","article-title":"RNA and disease","volume":"136","author":"Cooper","year":"2009","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1111\/j.1399-0004.2009.01190.x","article-title":"A child cohort study from southern italy enlarges the genetic spectrum of hypertrophic cardiomyopathy","volume":"76","author":"Frisso","year":"2009","journal-title":"Clin. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.gene.2014.02.012","article-title":"Position of premature termination codons determines susceptibility of herg mutations to nonsense-mediated mrna decay in long qt syndrome","volume":"539","author":"Gong","year":"2014","journal-title":"Gene"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.jmoldx.2011.09.001","article-title":"Extensive molecular analysis of patients bearing cftr-related disorders","volume":"14","author":"Amato","year":"2012","journal-title":"J. Mol. Diagn."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1016\/j.jcf.2010.12.008","article-title":"Splicing defects in the CFTR gene: Minigene analysis of two mutations, 1811+1G>C and 1898+3A>G","volume":"10","author":"Dujardin","year":"2011","journal-title":"J. Cyst. Fibros."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1136\/jmg.2007.056895","article-title":"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","volume":"45","author":"Bonnet","year":"2008","journal-title":"J. Med. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1362","DOI":"10.1038\/ejhg.2014.40","article-title":"Functional characterization of BRCA1 gene variants by mini-gene splicing assay","volume":"22","author":"Steffensen","year":"2014","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1486","DOI":"10.1002\/humu.21106","article-title":"The genetic basis of long QT and short QT syndromes: A mutation update","volume":"30","author":"Hedley","year":"2009","journal-title":"Hum. Mutat."},{"key":"ref_16","unstructured":"National Center for Biotechnology Information, Available online: http:\/\/www.ncbi.nlm.nih.gov\/snp\/."},{"key":"ref_17","unstructured":"Exome Aggregatium Consortium. Available online: http:\/\/exac.broadinstitute.org\/."},{"key":"ref_18","unstructured":"Exome Variant Server. Available online: http:\/\/evs.gs.washington.edu\/evs\/."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1006\/jmbi.1999.3276","article-title":"COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and\/or incorporation in fetal rat cardiomyocytes","volume":"294","author":"Flavigny","year":"1999","journal-title":"J. Mol. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e6","DOI":"10.4081\/cardiogenetics.2012.e6","article-title":"Combined use of in silico and in vitro splicing assays for interpretation of genomic variants of unknown significance in cardiomyopathies and channelopathies","volume":"2","author":"Crehalet","year":"2012","journal-title":"Cardiogenetics"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1161\/CIRCGENETICS.113.000448","article-title":"Sarcomere mutation-specific expression patterns in human hypertrophic cardiomyopathy","volume":"7","author":"Helms","year":"2014","journal-title":"Circ. Cardiovasc. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1016\/j.cca.2014.05.026","article-title":"Evaluation of damaging effects of splicing mutations: Validation of an in vitro method for diagnostic laboratories","volume":"436","author":"Manzoni","year":"2014","journal-title":"Clin. Chim. Acta Int. J. Clin. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"E5383","DOI":"10.1073\/pnas.1419553111","article-title":"Recessive cardiac phenotypes in induced pluripotent stem cell models of jervell and lange-nielsen syndrome: Disease mechanisms and pharmacological rescue","volume":"111","author":"Zhang","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.abb.2016.03.027","article-title":"Mutations in troponin T associated with hypertrophic cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation","volume":"601","author":"Messer","year":"2016","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1502","DOI":"10.1016\/j.bbapap.2015.07.009","article-title":"The multi-faceted aspects of the complex cardiac nav1.5 protein in membrane function and pathophysiology","volume":"1854","author":"Detta","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_26","unstructured":"Human Gene Mutation Database. Available online: http:\/\/www.hgmd.cf.ac.uk\/ac\/index.php."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1038\/ejhg.2013.81","article-title":"Pre-symptomatic genetic testing for inherited cardiac conditions: A qualitative exploration of psychosocial and ethical implications","volume":"22","author":"Ormondroyd","year":"2014","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_28","unstructured":"Wallis, Y., Payne, S., McAnulty, C., Bodmer, D., Sistermans, E., Robertson, K., Moore, D., Abbs, S., Deans, Z., and Devereau, A. Practice guidelines for the evaluation of pathogenicity and the reporting of sequence variants in clinical molecular genetics. Available online: http:\/\/www.ukcgg.org\/media\/774853\/evaluation_and_reporting_of_sequence_variants_bpgs_june_2013_-_finalpdf.pdf."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9","DOI":"10.3389\/fcvm.2016.00009","article-title":"The brugada syndrome: A rare arrhythmia disorder with complex inheritance","volume":"3","author":"Gourraud","year":"2016","journal-title":"Front. Cardiovasc. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1136\/jmedgenet-2014-102923","article-title":"A founder MYBPC3 mutation results in HCM with a high risk of sudden death after the fourth decade of life","volume":"52","author":"Calore","year":"2015","journal-title":"J. Med. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1085\/jgp.201411304","article-title":"Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy","volume":"145","author":"Moreau","year":"2015","journal-title":"J. Gen. Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1016\/j.amjcard.2014.05.065","article-title":"Significance of sarcomere gene mutations analysis in the end-stage phase of hypertrophic cardiomyopathy","volume":"114","author":"Biagini","year":"2014","journal-title":"Am. J. Cardiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.jmoldx.2013.07.008","article-title":"DNA sequence capture and next-generation sequencing for the molecular diagnosis of genetic cardiomyopathies","volume":"16","author":"Frisso","year":"2014","journal-title":"J. Mol. Diagn."},{"key":"ref_34","unstructured":"(2013). WMA Declaration of Helsinki\u2014Ethical Principles for Medical Research Involving Human Subjects. JAMA, 310, 2191\u20132194."},{"key":"ref_35","unstructured":"Alamut Interactive Biosciences. Available online: http:\/\/www.interactive-biosoftware.com\/."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/17\/11\/1883\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T19:35:16Z","timestamp":1760211316000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/17\/11\/1883"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,11,10]]},"references-count":35,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2016,11]]}},"alternative-id":["ijms17111883"],"URL":"https:\/\/doi.org\/10.3390\/ijms17111883","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2016,11,10]]}}}