Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies

• Published in: International journal of molecular sciences 2016-11, Vol.17 (11), p.1883-1883
• Main Authors: Frisso, Giulia, Detta, Nicola, Coppola, Pamela, Mazzaccara, Cristina, Pricolo, Maria Rosaria, D'Onofrio, Antonio, Limongelli, Giuseppe, Calabrò, Raffaele, Salvatore, Francesco
• Format: Article
• Language: English
• Subjects: Actins - genetics; Base Sequence; Bioinformatics; Cardiomyopathies - genetics; Cardiomyopathy; Cardiomyopathy, Hypertrophic - genetics; Carrier Proteins - genetics; Computer Simulation; Family Health; Female; Genetic disorders; Genetic Predisposition to Disease - genetics; Genomics; HEK293 Cells; Humans; Introns - genetics; Male; Models, Genetic; Mutation; NAV1.5 Voltage-Gated Sodium Channel - genetics; Pedigree; Point Mutation; RNA Splice Sites - genetics; RNA Splicing
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms17111883

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 ( -c.506-2A>C, -c.906-7G>T, -c.2308+3G>C, -c.393-5C>A, and -c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the -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 ( -G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed that -c.2308+3G>C and -c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either -c.906-7G>T or -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.