Browsing by Author "Cid, AR"
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- Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative studyPublication . Sánchez-Guiu, I; Antón, AI; Padilla, J; Velasco, F; Lucia, JF; Lozano, Ml; Cid, AR; Sevivas, T; Lopez-Fernandez, MF; Vicente, V; González-Manchón, C; Rivera, J; Lozano, MLBACKGROUND: The diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project "Functional and Molecular Characterization of Patients with Inherited Platelet Disorders" under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis. PATIENTS/METHODS: Subjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest. RESULTS: Of the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed. CONCLUSIONS: Our study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.
- Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNAPublication . Borràs, N; Orriols, G; Batlle, J; Pérez-Rodríguez, A; Fidalgo, T; Martinho, P; López-Fernández, MF; Rodríguez-Trillo, Á; Lourés, E; Parra, R; Altisent, C; Cid, AR; Bonanad, S; Cabrera, N; Moret, A; Mingot-Castellano, ME; Navarro, N; Pérez-Montes, R; Marcellini, S; Moreto, A; Herrero, S; Soto, I; Fernández-Mosteirín, N; Jiménez-Yuste, V; Alonso, N; de Andrés-Jacob, A; Fontanes, E; Campos, R; Paloma, MJ; Bermejo, N; Berrueco, R; Mateo, J; Arribalzaga, K; Marco, P; Palomo, Á; Castro Quismondo, N; Iñigo, B; Nieto, MM; Vidal, R; Martínez, MP; Aguinaco, R; Tenorio, JM; Ferreiro, M; García-Frade, J; Rodríguez-Huerta, AM; Cuesta, J; Rodríguez-González, R; García-Candel, F; Dobón, M; Aguilar, C; Vidal, F; Corrales, ILarge studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to 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.7473G>A (p.=) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of 3 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 4 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.