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Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies
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 vari...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133883/ https://www.ncbi.nlm.nih.gov/pubmed/27834932 http://dx.doi.org/10.3390/ijms17111883 |
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author | Frisso, Giulia Detta, Nicola Coppola, Pamela Mazzaccara, Cristina Pricolo, Maria Rosaria D’Onofrio, Antonio Limongelli, Giuseppe Calabrò, Raffaele Salvatore, Francesco |
author_facet | Frisso, Giulia Detta, Nicola Coppola, Pamela Mazzaccara, Cristina Pricolo, Maria Rosaria D’Onofrio, Antonio Limongelli, Giuseppe Calabrò, Raffaele Salvatore, Francesco |
author_sort | Frisso, Giulia |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5133883 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-51338832016-12-12 Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies Frisso, Giulia Detta, Nicola Coppola, Pamela Mazzaccara, Cristina Pricolo, Maria Rosaria D’Onofrio, Antonio Limongelli, Giuseppe Calabrò, Raffaele Salvatore, Francesco Int J Mol Sci Article 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. MDPI 2016-11-10 /pmc/articles/PMC5133883/ /pubmed/27834932 http://dx.doi.org/10.3390/ijms17111883 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Frisso, Giulia Detta, Nicola Coppola, Pamela Mazzaccara, Cristina Pricolo, Maria Rosaria D’Onofrio, Antonio Limongelli, Giuseppe Calabrò, Raffaele Salvatore, Francesco Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title | Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title_full | Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title_fullStr | Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title_full_unstemmed | Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title_short | Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies |
title_sort | functional studies and in silico analyses to evaluate non-coding variants in inherited cardiomyopathies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133883/ https://www.ncbi.nlm.nih.gov/pubmed/27834932 http://dx.doi.org/10.3390/ijms17111883 |
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