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N. Borràs et al.
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 sequenc- ing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable infor- mation. Our data bring to light the importance of studying the effect of synonymous and missense muta- tions on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.
Introduction
Von Willebrand disease (VWD), the most common con- genital bleeding disorder, is caused by a genetic defect in the von Willebrand factor gene (VWF).1 VWF mutational analysis can be valuable for diagnosing and investigating the molecular etiology of VWD, as was seen in the Spanish (PCM-EVW-ES project) and Portuguese cohort of VWD patients.2-4 One interesting challenge in this condi- tion is to elucidate the pathogenic mechanism of VWF mutations. In silico analysis is considered a suitable sup- porting tool to predict the pathogenicity of the variants identified.5,6 However, functional studies remain essential to unequivocally determine their deleterious effect.7
Functional studies can be performed by analyzing the potential effect of splice site mutations (PSSM) in RNA. In addition to splice site consensus sequence mutations, deep intronic, missense, and synonymous mutations can also disturb splicing. Along this line, 25% of synonymous mutations positioned at exon-intron boundaries result in altered splicing, which, in itself, can cause disease, modify the severity of the disease phenotype, or be linked with disease susceptibility.8 In VWF, heterozygotes for PSSM may be associated with mild forms of VWD type 1 or be phenotypically silent, but when two such mutations are found in different alleles, the phenotype is associated with VWD type 3.9
Almost all related studies of splicing effects have exam- ined peripheral blood platelets, as VWF is exclusively expressed in these cells and endothelial cells.10,11 Platelets are anucleated, but they contain small amounts of transla- tionally active megakaryocytic mRNA.12,13 In contrast, the amount of mRNA obtained from leukocytes is higher and contains mRNA transcripts for genes that are not normal- ly expressed in these cells, known as ‘‘ectopic transcripts’’. Their analysis has been used to investigate mutations in several inherited disorders,14,15 as they facilitate the study of mRNA of those genes expressed in hard to reach tis- sues.16 PSSM can affect mRNA reorganization and intro- duce premature termination codons (PTCs) into open reading frames, a common cause of genetic disorders. Most nonsense transcripts are recognized and degraded by nonsense-mediated mRNA decay (NMD),17 a degrada- tion pathway to control synthesis of truncated proteins.18 The efficiency of NMD varies between cell types; hence, the use of RNA from platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, partic- ularly when NMD occurs in the allele carrying the muta- tion in platelets, as we reported.7
Since its development, next-generation sequencing (NGS) has been increasingly used in molecular genetics to identify mutations causing disease. However, few groups
have explored its potential for analyzing splicing variants following RT-PCR.19,20 In this new scenario, the procedure we previously described to analyze the effects of PSSM in VWF7 has been optimized and adapted to an NGS-based technique to investigate its value in this field. Our main objective was to elucidate the true effects of 18 selected mutations (intronic, synonymous, delins, and missense) on mRNA processing and their genotype/phenotype cor- respondence by analysis of leukocytes and platelets from clinical samples. Finally, the in vivo effects of the muta- tions were compared with the in silico predictions.
Methods
Patients
We studied 15 patients diagnosed with different types of VWD, 5 from Complexo Hospitalario Universitario A Coruña, 8 from Hospital Universitari Vall d’Hebron (HUVH), and 2 from Centro Hospitalar e Universitário de Coimbra. Samples from 4 healthy individuals were used as controls. The study was performed according to the guidelines of the Declaration of Helsinki and was approved by the local Research Ethics Committee. All partici- pants provided written informed consent.
Splice site prediction software
The predicted impact of potential splice site mutations was analyzed with NetGene221 and the splicing prediction module of Alamut Visual v.2.6.1 software (Interactive Biosoftware, Rouen, France), which integrates data from three methods: Splice Site Prediction by Neural Network (NNSplice), MaxEntScan, and Human Splicing Finder (HSF).
Platelet and leukocyte separation and RNA isolation
Leukocyte and platelet RNA from patients and controls was isolated from 10 mL of peripheral blood collected in EDTA tubes, as previously described.7
VWF mRNA amplification
After RNA isolation, cDNA was synthesized using the High- Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. The region including the mutation was ampli- fied by Platinum Taq DNA Polymerase (Thermo Fisher Scientific) in leukocyte and platelet cDNA (Online Supplementary Methods and Table S1). PCR products were separated on 1% agarose gel and visualized by SYBR Safe DNA Gel Stain (Thermo Fisher Scientific).
Sanger sequencing and analysis
PCR products were sequenced as previously described.22 However, multiple-band PCR products were previously agarose-
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haematologica | 2019; 104(3)