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Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1

Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis i...

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Autores principales: Valero, Rebeca, de Castro-Miró, Marta, Jiménez-Ochoa, Sofía, Rodríguez-Ezcurra, Juan José, Marfany, Gemma, Gonzàlez-Duarte, Roser
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826400/
https://www.ncbi.nlm.nih.gov/pubmed/31546658
http://dx.doi.org/10.3390/genes10100732
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author Valero, Rebeca
de Castro-Miró, Marta
Jiménez-Ochoa, Sofía
Rodríguez-Ezcurra, Juan José
Marfany, Gemma
Gonzàlez-Duarte, Roser
author_facet Valero, Rebeca
de Castro-Miró, Marta
Jiménez-Ochoa, Sofía
Rodríguez-Ezcurra, Juan José
Marfany, Gemma
Gonzàlez-Duarte, Roser
author_sort Valero, Rebeca
collection PubMed
description Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. Results: Two intronic variants were identified in intron 45 of CDH23—one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation—with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. Conclusions: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome.
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spelling pubmed-68264002019-11-18 Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1 Valero, Rebeca de Castro-Miró, Marta Jiménez-Ochoa, Sofía Rodríguez-Ezcurra, Juan José Marfany, Gemma Gonzàlez-Duarte, Roser Genes (Basel) Article Aims: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype–genotype correlation. Methods: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. Results: Two intronic variants were identified in intron 45 of CDH23—one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation—with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. Conclusions: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome. MDPI 2019-09-21 /pmc/articles/PMC6826400/ /pubmed/31546658 http://dx.doi.org/10.3390/genes10100732 Text en © 2019 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
Valero, Rebeca
de Castro-Miró, Marta
Jiménez-Ochoa, Sofía
Rodríguez-Ezcurra, Juan José
Marfany, Gemma
Gonzàlez-Duarte, Roser
Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title_full Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title_fullStr Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title_full_unstemmed Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title_short Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1
title_sort aberrant splicing events associated to cdh23 noncanonical splice site mutations in a proband with atypical usher syndrome 1
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826400/
https://www.ncbi.nlm.nih.gov/pubmed/31546658
http://dx.doi.org/10.3390/genes10100732
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