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

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. identifier:02869074

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project - Fig 2

<p><b>Comparison between the diagnostic definition contribution of VWF:CB (in step “screening tests” [a]) and the multimeric analysis (MA) instead of VWF:CB first step, (step “screening tests” [b]).</b> A greater degree of efficiency was observed for MA (50.4% <i>versus</i> 33.1% for VWF:CB).</p

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

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. identifier:02869074

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project

<div><p>The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study ‘Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)’ has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.</p></div

Multimeric analysis of von Willebrand factor (VWF) in low-resolution SDS-agarose gels in patients with type 2A VWD and some discrepancy.

<p>VWF from platelet lysate (NPt), plasmas of a normal subject (NP), patients with type 2A VWD and a patient with VWD type 2A (IIA) used as a control 2A are shown. <b>(a-b):</b> Patients with discrepancy between ratios and multimeric analysis; <b>(c-d):</b> Patients re-classified as type 2A on the basis of the genetic study.</p

Evaluation of the contribution of different laboratory steps in the diagnosis definition.

<p>Four different successive laboratory assessment steps are considered. The progress in the diagnostic definition of the patients according to each step is shown. After the first and second steps, the multimeric analysis (MA) was of great significance in the diagnosis definition of 83 additional patients. Moreover, MA was in agreement with the diagnostic definition accomplished in steps 1 and 2 (179 patients). Finally, MA was also in agreement with the diagnostic definition achieved by molecular analysis in 186 additional patients. Thus, MA contributed to the diagnosis definition in a total of 448 (93.3%) patients.</p

Distribution of patients according to their coincidence between ratios and multimeric analysis before genetic study and between multimeric analysis and mutation after genetic study.

<p>Of 110 patients with some type of discrepancy, in 76 (48 + 28) the MA was in line with the molecular study while in 18 (14+4) patients there was not concordance. In the remaining 16, the similarity could not be demonstrated because the mutation found has not been described previously.</p

Patients with consistency between VWF:RCo/VWF:Ag-VWF:CB/VWF:Ag-multimeric pattern but not with genetic analysis.

<p>Patients with consistency between VWF:RCo/VWF:Ag-VWF:CB/VWF:Ag-multimeric pattern but not with genetic analysis.</p