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

International Journal of Molecular Sciences

Pamela Coppola¹  Giulia Frisso¹  Cristina Mazzaccara¹  Nicola Detta¹  Francesco Salvatore¹  Giuseppe Limongelli²  Antonio D’Onofrio²  Raffaele Calabrò²  Maria Rosaria Pricolo³ 

[1] CEINGE-Biotecnologie Avanzate s.c.a r.l., 80145 Napoli, Italy;Dipartimento di Cardiologia, Seconda Università degli Studi di Napoli, A.O. Monaldi, Azienda dei Colli, 80131 Napoli, Italy;Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy;

关键词: minigene;    splicing analysis;    inherited cardiomyopathies;    non-coding variations;    intronic mutations;    SCN5A;    MYBPC3;    ACTC1;   

DOI  :  10.3390/ijms17111883

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown