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Haematologica. 2019 Mar;104(3):587-598. doi: 10.3324/haematol.2018.203166. Epub 2018 Oct 25.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Author information

1
Banc de Sang i Teixits, Barcelona, Spain.
2
Institut de Recerca Vall d'Hebron -Universitat Autònoma de Barcelona (VHIR-UAB), Spain.
3
Complexo Hospitalario Universitario A Coruña, INIBIC, Spain.
4
Centro Hospitalar e Universitário de Coimbra, Portugal.
5
Hospital Universitario y Politécnico La Fe, Valencia, Spain.
6
Hospital Regional Universitario de Málaga, Spain.
7
Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain.
8
Hospital Universitario Marqués de Valdecilla, Santander, Spain.
9
Salud Castilla y León, Segovia, Spain.
10
Hospital Universitario Cruces, Barakaldo, Spain.
11
Hospital Universitario de Guadalajara, Spain.
12
Hospital Universitario Central de Asturias, Oviedo, Spain.
13
Hospital Universitario Miguel Servet, Zaragoza, Spain.
14
Hospital Universitario La Paz, Madrid, Spain.
15
Hospital Infanta Cristina, Badajoz, Spain.
16
Complexo Hospitalario Universitario Santiago de Compostela, Spain.
17
Hospital Universitario Lucus Augusti, Lugo, Spain.
18
Hospital Jerez de la Frontera, Cádiz, Spain.
19
Hospital Virgen del Camino, Pamplona, Spain.
20
Hospital San Pedro de Alcántara, Cáceres, Spain.
21
Hospital Sant Joan de Deu, Barcelona, Spain.
22
Hospital Sta Creu i St Pau, Barcelona, Spain.
23
Hospital Universitario Fundación de Alcorcón, Madrid, Spain.
24
Hospital General de Alicante, Spain.
25
Hospital Regional Universitario Carlos Haya, Málaga, Spain.
26
Hospital Universitario 12 de Octubre, Madrid, Spain.
27
Hospital Clínico San Carlos, Madrid, Spain.
28
Complejo Hospitalario de Jaén, Spain.
29
Fundación Jiménez Díaz, Madrid, Spain.
30
Hospital Nuestra Sra. de Sonsoles de Ávila, Spain.
31
Hospital Joan XXIII, Tarragona, Spain.
32
Hospital Montecelo, Pontevedra, Spain.
33
Hospital Río Hortega, Valladolid, Spain.
34
Hospital Gregorio Marañón, Madrid, Spain.
35
Hospital Virgen de la Salud, Toledo, Spain.
36
Hospital Severo Ochoa, Madrid, Spain.
37
Hospital Universitario Virgen Arrixaca, Murcia, Spain.
38
Hospital Lozano Blesa, Zaragoza, Spain.
39
Hospital Santa Bárbara, Soria, Spain.
40
Banc de Sang i Teixits, Barcelona, Spain icorrales@bst.cat fvidal@bst.cat.
41
CIBER de Enfermedades Cardiovasculares, Madrid, Spain.

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 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.

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