Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA
| Autor: | Borràs, Nina, Orriols, Gerard, Batlle, Javier, Pérez-Rodríguez, Almudena, Fidalgo, Teresa, Martinho, Patricia, López-Fernández, María Fernanda, Rodríguez-Trillo, Ángela, Lourés, Esther, Parra, Rafael, Altisent, Carme, Cid, Ana Rosa, Bonanad, Santiago, Cabrera, Noelia, Moret, Andrés, Mingot-Castellano, María Eva, Navarro, Nira, Pérez-Montes, Rocío, Marcellin, Sally, Moreto, Ana, Herrero, Sonia, Soto, Inmaculada, Fernández-Mosteirín, Núria, Jiménez-Yuste, Víctor, Alonso, Nieves, de Andrés-Jacob, Aurora, Fontanes, Emilia, Campos, Rosa, Paloma, María José, Bermejo, Nuria, Berrueco, Ruben, Mateo, José, Arribalzaga, Karmele, Marco, Pascual, Palomo, Ángeles, Quismondo, Nerea Castro, Iñigo, Belén, Nieto, María del Mar, Vidal, Rosa, Martínez, María Paz, Aguinaco, Reyes, Tenorio, Jesús María, Ferreiro, María, García-Frade, Javier, Rodríguez-Huerta, Ana María, Cuesta, Jorge, Rodríguez-González, Ramón, García-Candel, Faustino, Dobón, Manuela, Aguilar, Carlos, Vidal, Francisco, Corrales, Irene, Universitat Autònoma de Barcelona |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Blood Platelets
Male Genotype RNA Splicing Mutation Missense Biology medicine.disease_cause Article DNA sequencing Processamento de RNA Exon Gene Frequency Von Willebrand factor von Willebrand Factor Leukocytes medicine Humans Missense mutation Gene Silencing RNA Messenger Mutação Alleles Genetics Hemostasis Mutation Base Sequence Intron Computational Biology High-Throughput Nucleotide Sequencing Exons Hematology Introns Exon skipping von Willebrand Diseases RNA splicing biology.protein Female RNA Splice Sites |
| Zdroj: | HAEMATOLOGICA r-IIB SANT PAU. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica Sant Pau instname Dipòsit Digital de Documents de la UAB Universitat Autònoma de Barcelona r-FISABIO: Repositorio Institucional de Producción Científica Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunitat Valenciana (FISABIO) Repositorio Institucional de la Consejería de Sanidad de la Comunidad de Madrid Consejería de Sanidad de la Comunidad de Madrid HAEMATOLOGICA-THE HEMATOLOGY JOURNAL r-FISABIO. Repositorio Institucional de Producción Científica r-IIS La Fe. Repositorio Institucional de Producción Científica del Instituto de Investigación Sanitaria La Fe r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu Haematologica r-ISABIAL. Repositorio Institucional de Producción Científica del Instituto de Investigación Biomédica y Sanitaria de Alicante r-FSJD: Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu Fundació Sant Joan de Déu |
| ISSN: | 0390-6078 |
| Popis: | Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|