Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA
Por:
Borras, N, Orriols, G, Batlle, J, Perez-Rodriguez, A, Fidalgo, T, Martinho, P, Lopez-Fernandez, MF, Rodriguez-Trillo, A, Loures, E, Parra, R, Altisent, C, Cid, AR, Bonanad, S, Cabrera, N, Moret, A, Mingot-Castellano, ME, Navarro, N, Perez-Montes, R, Marcellin, S, Moreto, A, Herrero, S, Soto, I, Fernandez-Mosteirin, N, Jimenez-Yuste, V, Alonso, N, de Andres-Jacob, A, Fontanes, E, Campos, R, Paloma, MJ, Bermejo, N, Berrueco, R, Mateo, J, Arribalzaga, K, Marco, P, Palomo, A, Quismondo, NC, Inigo, B, Nieto, MD, Vidal, R, Martinez, MP, Aguinaco, R, Tenorio, JM, Ferreiro, M, Garcia-Frade, J, Rodriguez-Huerta, AM, Cuesta, J, Rodriguez-Gonzalez, R, Garcia-Candel, F, Dobon, M, Aguilar, C, Vidal, F, Corrales, I
Publicada:
1 mar 2019
Resumen:
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 MT mRNA processing, investigate the contribution of next -generation sequencing to in V1.1'0 mRNA study in von Willebrand disease, and compare the findings with in silk 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 VIVE 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 'dent( kr:02869074.
Filiaciones:
Borras, N:
Banc Sang & Teixits, Barcelona, Spain
UAB, Inst Recerca Vall dHebron, VHIR, Barcelona, Spain
Orriols, G:
Banc Sang & Teixits, Barcelona, Spain
Batlle, J:
CHU A Coruna, INIBIC, La Coruna, Spain
Perez-Rodriguez, A:
CHU A Coruna, INIBIC, La Coruna, Spain
Fidalgo, T:
CHU Coimbra, Coimbra, Portugal
Martinho, P:
CHU Coimbra, Coimbra, Portugal
Lopez-Fernandez, MF:
CHU A Coruna, INIBIC, La Coruna, Spain
Rodriguez-Trillo, A:
CHU A Coruna, INIBIC, La Coruna, Spain
Loures, E:
CHU A Coruna, INIBIC, La Coruna, Spain
Parra, R:
Banc Sang & Teixits, Barcelona, Spain
UAB, Inst Recerca Vall dHebron, VHIR, Barcelona, Spain
Altisent, C:
UAB, Inst Recerca Vall dHebron, VHIR, Barcelona, Spain
Cid, AR:
Hosp Univ & Politecn La Fe, Valencia, Spain
Bonanad, S:
Hosp Univ & Politecn La Fe, Valencia, Spain
Cabrera, N:
Hosp Univ & Politecn La Fe, Valencia, Spain
Moret, A:
Hosp Univ & Politecn La Fe, Valencia, Spain
Mingot-Castellano, ME:
Hosp Reg Univ Malaga, Malaga, Spain
Navarro, N:
Hosp Univ Dr Negrin, Las Palmas Gran Canaria, Spain
Perez-Montes, R:
Hosp Univ Marques Valdecilla, Santander, Spain
Marcellin, S:
Salud Castilla & Leon, Segovia, Spain
Moreto, A:
Hosp Univ Cruces, Baracaldo, Spain
Herrero, S:
Hosp Univ Guadalajara, Guadalajara, Spain
Soto, I:
Hosp Univ Cent Asturias, Oviedo, Spain
Fernandez-Mosteirin, N:
Hosp Univ Miguel Servet, Zaragoza, Spain
Jimenez-Yuste, V:
Hosp Univ La Paz, Madrid, Spain
Alonso, N:
Hosp Infanta Cristina, Badajoz, Spain
de Andres-Jacob, A:
CHU Santiago De Compostela, Santiago De Compostela, Spain
Fontanes, E:
Hosp Univ Lucus Augusti, Lugo, Spain
Campos, R:
Hosp Jerez Frontera, Cadiz, Spain
Paloma, MJ:
Hosp Virgen Camino, Pamplona, Spain
Bermejo, N:
Hosp San Pedro Alcantara, Caceres, Spain
Berrueco, R:
Hosp San Juan Dios, Barcelona, Spain
Mateo, J:
Hosp Sta Creu i St Pau, Barcelona, Spain
Arribalzaga, K:
Hosp Univ Fdn Alcorcon, Madrid, Spain
Marco, P:
Hosp Gen Alicante, Alicante, Spain
Palomo, A:
Hosp Reg Univ Carlos Haya, Malaga, Spain
Quismondo, NC:
Hosp Univ 12 Octubre, Madrid, Spain
Inigo, B:
Hosp Clin San Carlos, Madrid, Spain
Nieto, MD:
Complejo Hosp Jaen, Jaen, Spain
Vidal, R:
Fdn Jimenez Diaz, Madrid, Spain
Martinez, MP:
Hosp Nuestra Sra Sonsoles Avila, Avila, Spain
Garcia-Frade, J:
Hosp Montecelo, Pontevedra, Spain
Hosp Rio Hortega, Valladolid, Spain
Rodriguez-Huerta, AM:
Hosp Gregorio Maranon, Madrid, Spain
Cuesta, J:
Hosp Virgen Salud, Toledo, Spain
Rodriguez-Gonzalez, R:
Hosp Severo Ochoa, Madrid, Spain
Garcia-Candel, F:
Hosp Univ Virgen Arrixaca, Murcia, Spain
Dobon, M:
Hosp Lozano Blesa, Zaragoza, Spain
Aguilar, C:
Hosp Santa Barbara, Santa Barbara, CA, Spain
Vidal, F:
Banc Sang & Teixits, Barcelona, Spain
UAB, Inst Recerca Vall dHebron, VHIR, Barcelona, Spain
CIBER Enfermedades Cardiovasc, Madrid, Spain
Corrales, I:
Banc Sang & Teixits, Barcelona, Spain
UAB, Inst Recerca Vall dHebron, VHIR, Barcelona, Spain
Hosp Joan 23, Tarragona, Spain.
Hosp Ramon & Cajal, Madrid, Spain.
Gold, Green Published
|