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

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 .

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

“…After applying in silico predictive tools to 41 variants previously classified as missense variants, the authors demonstrated that over 45% of them were shown to affect splicing and claimed that such variants have been frequently underscored if not analysed in depth. These observations were further supported by studies using next-generation sequencing technology [32] and highthroughput splicing reporter assays [35] to unravel the effect on splicing of single nucleotide variants, including missense, in the von Willebrand factor gene and POU1F1, respectively. Altogether, there is a need for improvement of in silico tools to predict cryptic splicing, and for the development of guidelines for formal analysis of splicing defects induced by SNVs, particularly synonymous and missense variants.…”

“…Regarding the impact that complete or incomplete downregulation of the mutant allele may have for disease phenotype and severity, very little is known. However, it has been documented that certain genetic modifiers, and interindividual variability in NMD efficiency between patients carrying identical mutations, may lead to differences in the disease severity and clinical phenotype [32].…”

A deep exon cryptic splice site promotes aberrant intron retention in a von Willebrand disease patient

“…Von Willebrand disease is one of the most common inherited bleeding disorders, caused by any one of hundreds of mutations in the VWF gene [23, 24]. The mutation studied here, although present at low frequency among the populations surveyed (dbSNP: rs900907976 ; minor allele frequency=0.000026 (7/264690, TOPMED), has an unusual impact on pre-mRNA splicing by acting at a distance to promote retention of the downstream intron [16].…”

A Combination of Two Variants p. (Val510 =) and p. (Pro2145Thrfs * 5), Responsible for von Willebrand Disease Type 3 in a Caribbean Patient

“…In conclusion, VWF mutational analysis can be valuable for diagnosing and investigating the molecular etiology of VWD. 4 The prediction softwares used (SpliceSiteFinder-like, MaxEntScan, GeneSplicer, NSPLICE, ESEFinder, RESCUE-ESE, and EX-SKIP) regarding c.1530G > A (p.(Val510 = )) interpreted the appearance of a donor site at 6 base pairs at the end of exon 13 which could potentially alter splicing. This synonymous mutation could therefore have an effect on VWF messenger ribonucleic acid processing, causing a shift in the reading frame and the appearance of a termination codon deletion of two codons.…”