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Hemostasis
Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA
Ferrata Storti Foundation
Haematologica 2019 Volume 104(1):587-598
Nina Borràs,1,2 Gerard Orriols,1 Javier Batlle,3 Almudena Pérez-Rodríguez,3 Teresa Fidalgo,4 Patricia Martinho,4 María Fernanda López-Fernández,3 Ángela Rodríguez-Trillo,3 Esther Lourés,3 Rafael Parra,1,2 Carme Altisent,2 Ana Rosa Cid,5 Santiago Bonanad,5 Noelia Cabrera,5 Andrés Moret,5
María Eva Mingot-Castellano,6 Nira Navarro,7 Rocío Pérez-Montes,8
Sally Marcellin,9 Ana Moreto,10 Sonia Herrero,11 Inmaculada Soto,12 Núria Fernández-Mosteirín,13 Víctor Jiménez-Yuste,14 Nieves Alonso,15 Aurora de Andrés-Jacob,16 Emilia Fontanes,17 Rosa Campos,18
María José Paloma,19 Nuria Bermejo,20 Ruben Berrueco,21 José Mateo,22 Karmele Arribalzaga,23 Pascual Marco,24 Ángeles Palomo,25
Nerea Castro Quismondo,26 Belén Iñigo,27 María del Mar Nieto,28 Rosa Vidal,29 María Paz Martínez,30 Reyes Aguinaco,31 Jesús María Tenorio,33
María Ferreiro,33 Javier García-Frade,34 Ana María Rodríguez-Huerta,35
Jorge Cuesta,36 Ramón Rodríguez-González,37 Faustino García-Candel,38 Manuela Dobón,39 Carlos Aguilar,40 Francisco Vidal1,2,41 and Irene Corrales1,2
1Banc de Sang i Teixits, Barcelona, Spain; 2Institut de Recerca Vall d’Hebron - Universitat Autònoma de Barcelona (VHIR-UAB), Spain; 3Complexo Hospitalario Universitario A Coruña, INIBIC, Spain; 4Centro Hospitalar e Universitário de Coimbra, Portugal; 5Hospital Universitario y Politécnico La Fe, Valencia, Spain; 6Hospital Regional Universitario de Málaga, Spain; 7Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain; 8Hospital Universitario Marqués de Valdecilla, Santander, Spain; 9Salud Castilla y León, Segovia, Spain; 10Hospital Universitario Cruces, Barakaldo, Spain; 11Hospital Universitario de Guadalajara, Spain; 12Hospital Universitario Central de Asturias, Oviedo, Spain; 13Hospital Universitario Miguel Servet, Zaragoza, Spain; 14Hospital Universitario La Paz, Madrid, Spain; 15Hospital Infanta Cristina, Badajoz, Spain; 16Complexo Hospitalario Universitario Santiago de Compostela, Spain; 17Hospital Universitario Lucus Augusti, Lugo, Spain; 18Hospital Jerez de la Frontera, Cádiz, Spain; 19Hospital Virgen del Camino, Pamplona, Spain; 20Hospital San Pedro de Alcántara, Cáceres, Spain; 21Hospital Sant Joan de Deu, Barcelona, Spain; 22Hospital Sta Creu i St Pau, Barcelona, Spain; 23Hospital Universitario Fundación de Alcorcón, Madrid, Spain; 24Hospital General de Alicante, Spain; 25Hospital Regional Universitario Carlos Haya, Málaga, Spain; 26Hospital Universitario 12 de Octubre, Madrid, Spain; 27Hospital Clínico San Carlos, Madrid, Spain; 28Complejo Hospitalario de Jaén, Spain; 29Fundación Jiménez Díaz, Madrid, Spain; 30Hospital Nuestra Sra. de Sonsoles de Ávila, Spain; 32Hospital Joan XXIII, Tarragona, Spain; 32Hospital Ramón y Cajal, Madrid, Spain; 33Hospital Montecelo, Pontevedra, Spain; 34Hospital Río Hortega, Valladolid, Spain; 35Hospital Gregorio Marañón, Madrid, Spain; 36Hospital Virgen de la Salud, Toledo, Spain; 37Hospital Severo Ochoa, Madrid, Spain; 38Hospital Universitario Virgen Arrixaca, Murcia, Spain; 39Hospital Lozano Blesa, Zaragoza, Spain; 40Hospital Santa Bárbara, Soria, Spain and 41CIBER de Enfermedades Cardiovasculares, Madrid, Spain
Correspondence:
IRENE CORRALES
icorrales@bst.cat/fvidal@bst.cat
Received: August 3, 2018. Accepted: October 19, 2018. Pre-published: October 25, 2018.
doi:10.3324/haematol.2018.203166
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/3/587
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ABSTRACT
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 elu- cidate the true effects of 18 mutations on VWF mRNA processing, inves- tigate 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 skip- ping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryp- tic 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
haematologica | 2019; 104(3)
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ARTICLE