RT Journal Article
T1 Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.
A1 Borràs, Nina
A1 Orriols, Gerard
A1 Batlle, Javier
A1 Pérez-Rodríguez, Almudena
A1 Fidalgo, Teresa
A1 Martinho, Patricia
A1 López-Fernández, María Fernanda
A1 Rodríguez-Trillo, Ángela
A1 Lourés, Esther
A1 Parra, Rafael
A1 Altisent, Carme
A1 Cid, Ana Rosa
A1 Bonanad, Santiago
A1 Cabrera, Noelia
A1 Moret, Andrés
A1 Mingot-Castellano, María Eva
A1 Navarro, Nira
A1 Pérez-Montes, Rocío
A1 Marcellin, Sally
A1 Moreto, Ana
A1 Herrero, Sonia
A1 Soto, Inmaculada
A1 Fernández-Mosteirín, Núria
A1 Jiménez-Yuste, Víctor
A1 Alonso, Nieves
A1 de Andrés-Jacob, Aurora
A1 Fontanes, Emilia
A1 Campos, Rosa
A1 Paloma, María José
A1 Bermejo, Nuria
A1 Berrueco, Ruben
A1 Mateo, José
A1 Arribalzaga, Karmele
A1 Marco, Pascual
A1 Palomo, Ángeles
A1 Quismondo, Nerea Castro
A1 Iñigo, Belén
A1 Nieto, María Del Mar
A1 Vidal, Rosa
A1 Martínez, María Paz
A1 Aguinaco, Reyes
A1 Tenorio, Jesús María
A1 Ferreiro, María
A1 García-Frade, Javier
A1 Rodríguez-Huerta, Ana María
A1 Cuesta, Jorge
A1 Rodríguez-González, Ramón
A1 García-Candel, Faustino
A1 Dobón, Manuela
A1 Aguilar, Carlos
A1 Vidal, Francisco
A1 Corrales, Irene
AB 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.
YR 2018
FD 2018-10-25
LK http://hdl.handle.net/10668/13131
UL http://hdl.handle.net/10668/13131
LA en
DS RISalud
RD Apr 7, 2025