Functional studies and in silico analyses to evaluate non-coding variants in inherited cardiomyopathies

Giulia Frisso, Nicola Detta, Pamela Coppola, Cristina Mazzaccara, Maria Rosaria Pricolo, Antonio D’Onofrio, Giuseppe Limongelli, Raffaele Calabrò, Francesco Salvatore

Research output: Contribution to journalArticle


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>C, MYBPC3-c.906-7G>T, MYBPC3-c.2308+3G>C, SCN5A-c.393-5C>A, and ACTC1-c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the MYBPC3-c.506-2A>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>C and SCN5A-c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either MYBPC3-c.906-7G>T or ACTC1-c.617-7T>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.

Original languageEnglish
Article number1883
JournalInternational Journal of Molecular Sciences
Issue number11
Publication statusPublished - Nov 1 2016


  • ACTC1
  • Inherited cardiomyopathies
  • Intronic mutations
  • Minigene
  • MYBPC3
  • Non-coding variations
  • SCN5A
  • Splicing analysis

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Computer Science Applications
  • Spectroscopy
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Fingerprint Dive into the research topics of 'Functional studies and in silico analyses to evaluate non-coding variants in inherited cardiomyopathies'. Together they form a unique fingerprint.

  • Cite this

    Frisso, G., Detta, N., Coppola, P., Mazzaccara, C., Pricolo, M. R., D’Onofrio, A., Limongelli, G., Calabrò, R., & Salvatore, F. (2016). Functional studies and in silico analyses to evaluate non-coding variants in inherited cardiomyopathies. International Journal of Molecular Sciences, 17(11), [1883].