Hemostasis ARTICLE Unraveling the effect of silent, intronic and Ferrata Storti Foundation missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA Nina Borràs,1,2 Gerard Orriols,1 Javier Batlle,3 Almudena Pérez-Rodríguez,3 Teresa Fidalgo,4 Patricia Martinho,4 María Fernanda López-Fernández,3 Ángela Rodríguez-Trillo,3 Esther Lourés,3 Rafael Parra,1,2 Carme Altisent,2 Ana Rosa Cid,5 Santiago Bonanad,5 Noelia Cabrera,5 Andrés Moret,5 María Eva Mingot-Castellano,6 Nira Navarro,7 Rocío Pérez-Montes,8 Haematologica 2019 Sally Marcellin,9 Ana Moreto,10 Sonia Herrero,11 Inmaculada Soto,12 Volume 104(1):587-598 Núria Fernández-Mosteirín,13 Víctor Jiménez-Yuste,14 Nieves Alonso,15 Aurora de Andrés-Jacob,16 Emilia Fontanes,17 Rosa Campos,18 María José Paloma,19 Nuria Bermejo,20 Ruben Berrueco,21 José Mateo,22 Karmele Arribalzaga,23 Pascual Marco,24 Ángeles Palomo,25 Nerea Castro Quismondo,26 Belén Iñigo,27 María del Mar Nieto,28 Rosa Vidal,29 María Paz Martínez,30 Reyes Aguinaco,31 Jesús María Tenorio,33 María Ferreiro,33 Javier García-Frade,34 Ana María Rodríguez-Huerta,35 Jorge Cuesta,36 Ramón Rodríguez-González,37 Faustino García-Candel,38 Manuela Dobón,39 Carlos Aguilar,40 Francisco Vidal1,2,41 and Irene Corrales1,2 1 Banc de Sang i Teixits, Barcelona, Spain; 2Institut de Recerca Vall d’Hebron - Universitat Autònoma de Barcelona (VHIR-UAB), Spain; 3Complexo Hospitalario Universitario A Coruña, INIBIC, Spain; 4Centro Hospitalar e Universitário de Coimbra, Portugal; 5Hospital Universitario y Politécnico La Fe, Valencia, Spain; 6Hospital Regional Universitario de Málaga, Spain; 7Hospital Universitario Dr. Negrín, Las Palmas de Gran Canaria, Spain; 8Hospital Universitario Marqués de Valdecilla, Santander, Spain; 9Salud Castilla y León, Segovia, Spain; 10Hospital Universitario Cruces, Barakaldo, Spain; 11 Hospital Universitario de Guadalajara, Spain; 12Hospital Universitario Central de Asturias, Oviedo, Spain; 13Hospital Universitario Miguel Servet, Zaragoza, Spain; 14 Hospital Universitario La Paz, Madrid, Spain; 15Hospital Infanta Cristina, Badajoz, Spain; 16Complexo Hospitalario Universitario Santiago de Compostela, Spain; 17Hospital Universitario Lucus Augusti, Lugo, Spain; 18Hospital Jerez de la Frontera, Cádiz, Spain; Hospital Virgen del Camino, Pamplona, Spain; 20Hospital San Pedro de Alcántara, 19 Correspondence: Cáceres, Spain; 21Hospital Sant Joan de Deu, Barcelona, Spain; 22Hospital Sta Creu i St Pau, Barcelona, Spain; 23Hospital Universitario Fundación de Alcorcón, Madrid, Spain; IRENE CORRALES Hospital General de Alicante, Spain; 25Hospital Regional Universitario Carlos Haya, 24 icorrales@bst.cat/fvidal@bst.cat Málaga, Spain; 26Hospital Universitario 12 de Octubre, Madrid, Spain; 27Hospital Clínico San Carlos, Madrid, Spain; 28Complejo Hospitalario de Jaén, Spain; 29Fundación Jiménez Díaz, Madrid, Spain; 30Hospital Nuestra Sra. de Sonsoles de Ávila, Spain; 32Hospital Joan Received: August 3, 2018. XXIII, Tarragona, Spain; 32Hospital Ramón y Cajal, Madrid, Spain; 33Hospital Montecelo, Pontevedra, Spain; 34Hospital Río Hortega, Valladolid, Spain; 35Hospital Gregorio Accepted: October 19, 2018. Marañón, Madrid, Spain; 36Hospital Virgen de la Salud, Toledo, Spain; 37Hospital Severo Pre-published: October 25, 2018. Ochoa, Madrid, Spain; 38Hospital Universitario Virgen Arrixaca, Murcia, Spain; 39Hospital Lozano Blesa, Zaragoza, Spain; 40Hospital Santa Bárbara, Soria, Spain and 41CIBER de Enfermedades Cardiovasculares, Madrid, Spain doi:10.3324/haematol.2018.203166 Check the online version for the most updated ABSTRACT information on this article, online supplements, and information on authorship & disclosures: L arge studies in von Willebrand disease patients, including Spanish www.haematologica.org/content/104/3/587 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 elu- ©2019 Ferrata Storti Foundation cidate the true effects of 18 mutations on VWF mRNA processing, inves- Material published in Haematologica is covered by copyright. All rights are reserved to the Ferrata Storti Foundation. Use of tigate the contribution of next-generation sequencing to in vivo mRNA published material is allowed under the following terms and study in von Willebrand disease, and compare the findings with in silico conditions: prediction. RNA extracted from patient platelets and leukocytes was https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- amplified by RT-PCR and sequenced using Sanger and next generation nal use. Sharing published material for non-commercial pur- sequencing techniques. Eight mutations affected VWF splicing: poses is subject to the following conditions: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skip- https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- ping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryp- mercial purposes is not allowed without permission in writing tic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular from the publisher. pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete haematologica | 2019; 104(3) 587 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 have explored its potential for analyzing splicing variants following RT-PCR.19,20 In this new scenario, the procedure Von Willebrand disease (VWD), the most common con- we previously described to analyze the effects of PSSM in genital bleeding disorder, is caused by a genetic defect in VWF7 has been optimized and adapted to an NGS-based the von Willebrand factor gene (VWF).1 VWF mutational technique to investigate its value in this field. Our main analysis can be valuable for diagnosing and investigating objective was to elucidate the true effects of 18 selected the molecular etiology of VWD, as was seen in the mutations (intronic, synonymous, delins, and missense) Spanish (PCM-EVW-ES project) and Portuguese cohort of on mRNA processing and their genotype/phenotype cor- VWD patients.2-4 One interesting challenge in this condi- respondence by analysis of leukocytes and platelets from tion is to elucidate the pathogenic mechanism of VWF clinical samples. Finally, the in vivo effects of the muta- mutations. In silico analysis is considered a suitable sup- tions were compared with the in silico predictions. porting tool to predict the pathogenicity of the variants identified.5,6 However, functional studies remain essential to unequivocally determine their deleterious effect.7 Methods Functional studies can be performed by analyzing the potential effect of splice site mutations (PSSM) in RNA. In Patients addition to splice site consensus sequence mutations, We studied 15 patients diagnosed with different types of VWD, deep intronic, missense, and synonymous mutations can 5 from Complexo Hospitalario Universitario A Coruña, 8 from also disturb splicing. Along this line, 25% of synonymous Hospital Universitari Vall d’Hebron (HUVH), and 2 from Centro mutations positioned at exon-intron boundaries result in Hospitalar e Universitário de Coimbra. Samples from 4 healthy altered splicing, which, in itself, can cause disease, modify individuals were used as controls. The study was performed the severity of the disease phenotype, or be linked with according to the guidelines of the Declaration of Helsinki and was disease susceptibility.8 In VWF, heterozygotes for PSSM approved by the local Research Ethics Committee. All partici- may be associated with mild forms of VWD type 1 or be pants provided written informed consent. phenotypically silent, but when two such mutations are found in different alleles, the phenotype is associated Splice site prediction software with VWD type 3.9 The predicted impact of potential splice site mutations was Almost all related studies of splicing effects have exam- analyzed with NetGene221 and the splicing prediction module of ined peripheral blood platelets, as VWF is exclusively Alamut Visual v.2.6.1 software (Interactive Biosoftware, Rouen, expressed in these cells and endothelial cells.10,11 Platelets France), which integrates data from three methods: Splice Site are anucleated, but they contain small amounts of transla- Prediction by Neural Network (NNSplice), MaxEntScan, and tionally active megakaryocytic mRNA.12,13 In contrast, the Human Splicing Finder (HSF). amount of mRNA obtained from leukocytes is higher and contains mRNA transcripts for genes that are not normal- Platelet and leukocyte separation and RNA isolation ly expressed in these cells, known as ‘‘ectopic transcripts’’. Leukocyte and platelet RNA from patients and controls was Their analysis has been used to investigate mutations in isolated from 10 mL of peripheral blood collected in EDTA tubes, several inherited disorders,14,15 as they facilitate the study as previously described.7 of mRNA of those genes expressed in hard to reach tis- sues.16 PSSM can affect mRNA reorganization and intro- VWF mRNA amplification duce premature termination codons (PTCs) into open After RNA isolation, cDNA was synthesized using the High- reading frames, a common cause of genetic disorders. Capacity cDNA Reverse Transcription Kit (Thermo Fisher Most nonsense transcripts are recognized and degraded Scientific, Waltham, MA, USA) according to the manufacturer’s by nonsense-mediated mRNA decay (NMD),17 a degrada- recommendations. The region including the mutation was ampli- tion pathway to control synthesis of truncated proteins.18 fied by Platinum Taq DNA Polymerase (Thermo Fisher Scientific) The efficiency of NMD varies between cell types; hence, in leukocyte and platelet cDNA (Online Supplementary Methods the use of RNA from platelets and leukocytes for in vivo and Table S1). PCR products were separated on 1% agarose gel study of VWF PSSM offers complementary results, partic- and visualized by SYBR Safe DNA Gel Stain (Thermo Fisher ularly when NMD occurs in the allele carrying the muta- Scientific). tion in platelets, as we reported.7 Since its development, next-generation sequencing Sanger sequencing and analysis (NGS) has been increasingly used in molecular genetics to PCR products were sequenced as previously described.22 identify mutations causing disease. However, few groups However, multiple-band PCR products were previously agarose- 588 haematologica | 2019; 104(3) Effect of VWF mutations on mRNA splicing Table 1. Laboratory and molecular data of VWD patients Patients code VWD VWF:Ag VWF:RCo FVIII:C NT change AA change Exon Intron Domain UMP01 3 carrier 70 70.5 111 c.1533+1G>A - - 13 intronic UMP02 1H 36 49 101 c.3379+1G>A * - - 25 intronic c.-2627C>T * - - - upstream UMP03 1 26 21.2 64.8 c.5664+2T>C * - - 33 intronic c.7220T>C * p.Leu2407Pro 42 - C2 UMP04 1 50 52 47 c.7081+6G>T - - 41 intronic UMP05 1 27 32 72 c.7730-56C>T - - 45 intronic UMP06 2A/2M 12 4 24 c.4120C>T * p.Arg1374Cys 28 A1 c.7730-4C>G * - - 45 intronic UMP07 1 37 28 60 c.8254-5T>G - - 51 intronic UMP08 1 42.4 41.7 73.4 c.546G>A† p.Ser182 6 - D1 c.4866C>T† p.Asp1622 28 - A1-A3 UMP09‡ 1 51 49.8 71 c.3291C>T p.Cys1097 25 - D3 UMP10 3 carrier 46.4 45 105 c.1109G>A p.Cys370Tyr 9 - D1 UMP11 1 31 24 57 c.3223-7_3236dup p.Pro1079_Tyr1080ins 25 - D3 LeuGlnValAspProGluPro UMP12 3 3.7 5 6.7 c.449T>C† p.Leu150Pro 5 - D1 c.7082-2A>G† - - 41 intronic UMP13 2A 117 6.5 90 c.3426T>C§ p.Cys1142 26 - D3 c.3485_3486delinsTG§ p.Pro1162Leu 26 - D3 c.8318G>C† p.Cys2773Ser 52 - CK UMP14 1 6 6 4,5 c.6699_6702dup† p.Cys2235ArgfsTer8 38 - D4 c.7437G>A† p.Ser2479 43 - C3 UMP15|| 3 2 <1 5 c.546G>A§ p.Ser182 6 - D1 c.7082-2A>G§ - - 41 intronic c.8155+3G>C† - - 50 intronic All mutations were identified in heterozygous state. In bold, mutations selected to study their effect on VWF mRNA. The subtype 1H (historical) refers to patients previously diag- nosed as type 1 VWD that, at the time of enrollment at the PCM-EVW-ES project, show a slight decrease or even a normal VWF plasma levels. AA:amino acid; NT: nucleotide;VWD: von Willebrand disease. *The allelic phase (cis/trans) of mutations could not be determined since no informative relatives are available. †Mutations in trans. ‡Patient with an addi- tional mutation in the F8 gene. §Mutations in cis. ||Patient previously studied at mRNA level by Corrales et al.7 purified using the MiniElute Gel Extraction kit (Qiagen). The ly undescribed VWF mutations and mutations with an sequences obtained were assembled and aligned against the con- unknown or controversial pathogenic mechanism. sensus wild-type (WT) VWF mRNA sequence (GenBank Eighteen mutations (15 patients) were selected: 8 intronic NM_000552) using SeqScape v2.7 software (Thermo Fisher (4 in canonical, GT and AG, splice site sequences), 5 syn- Scientific). onymous, 2 missense, and 3 delins. The patients’ pheno- typic and molecular data are summarized in Table 1 and Next-generation sequencing and analysis Online Supplementary Table S2. Total mRNA was obtained PCR amplicons obtained per patient were equimolarly mixed in from platelets and leukocytes of all patients, with the a single tube in a total amount of 250 ng. Subsequently, the exception of patients UMP08 and UMP14, in whom libraries were fragmented and the adapter and barcodes were lig- platelet RNA isolation failed due to blood lysis. All muta- ated using the NGSgo protocol (GenDX, Utrecht, Netherlands) tions were analyzed by both Sanger sequencing and NGS, following the manufacturer’s recommendations. Resulting and results were compared to the predictions generated libraries were combined and sequenced on a MiSeq platform by in silico analysis (Online Supplementary Table S3). (Illumina, San Diego, CA). After sequencing, barcoded sequences were demultiplexed and Mutations in canonical splice site sequences analyzed individually. The paired sequence files (fastq format) The c.1533+1G>A mutation (intron 13) was identified were used as input for analysis with the CLC Genomic in a type 3 VWD carrier (UMP01). The exon 11-15 region Workbench v.11 software (Qiagen, Aarhus, Denmark) (Online was analyzed with a specific primer pair to avoid amplifi- Supplementary Methods and Figure S1-S2). cation of a prevalent alternative-splicing product (skipping of exons 14 and 15) in VWF from leukocytes.7 Whereas only the expected PCR product was observed in platelets, Results leukocyte amplification resulted in 4 PCR bands. Sanger analysis of leukocytes showed 2 mRNA aberrant tran- An in-depth study was performed in PCM-EVW-ES,3 the scripts: 1) lacking exon 13; and 2) lacking exon 13 and 14 Portuguese cohort,4 and HUVH patients to select previous- (Online Supplementary Figure S3). By means of NGS, it was haematologica | 2019; 104(3) 589 N. Borràs et al. A B C D Figure 1. Analysis of the c.5664+2T>C mutation in patient UMP03. A) RT-PCR products amplified with primers located in exons 30 and 35 in leukocyte (L) and platelet (P) RNA, separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR products from patient leukocytes (L2) and platelets (P), and control leuko- cytes (L). Analysis of the band 2 from patient leukocyte on agarose gel demonstrates exon 33 skipping. In platelets, only the allele without c.5664+2T>C mutation could be amplified. C) NGS of PCR products from leukocytes showed exon 33 skipping, indicated by arrows depict aberrant transcripts. However, exon 33-34 skipping was also detected, but in a really low of transcripts. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indi- cates a 100-bp DNA ladder. determined that 19% were transcripts without exon 13, transcripts were detected: the major one, which lacked 45% transcripts without exons 13 and 14, and an addi- exon 25, was detected in 43% of all reads, and the minor tional aberrant transcript without exon 14 was detected in one, which resulted from activation of a cryptic donor 8% transcripts (Online Supplementary Table S4). By both splice site (DSS) 31 nucleotides upstream from the WT- techniques, no effect was visible in platelets due to NMD. DSS, was found in 1.4% of reads (p.Pro1117ValfsTer88) The c.3379+1G>A mutation (intron 25) was identified (Table 2 and Online Supplementary Table S4). In platelets, in a type 1H VWD patient (UMP02). We designed two however, the mutated allele had undergone NMD, which new primers to analyze the exon 22-26 region. precluded observation of aberrant transcripts. Amplification of leukocyte cDNA yielded two bands, one The c.5664+2T>C mutation was identified in a type 1 with the expected size and a smaller band, whereas in VWD patient (UMP03), in trans with the p.Leu2407Pro platelets only the expected PCR product was observed mutation. To analyze the PSSM in intron 33, the exon 30- (Online Supplementary Figure S4). Sanger analysis of leuko- 35 region was amplified. The PCR product had the expect- cytes showed that c.3379+1G>A caused exon 25 skipping, ed size in platelets, whereas an additional smaller one was leading to a frameshift at position 1075 and adding 88 observed in leukocytes (Figure 1). Sanger sequencing of aberrant amino acids before a PTC was encountered the leukocyte PCR product confirmed exon 33 skipping (p.Pro1075ValfsTer88). On NGS, however, two aberrant (p.Gly1874AlafsTer32) (Table 2). NGS detected 2 aberrant 590 haematologica | 2019; 104(3) Effect of VWF mutations on mRNA splicing A B C D Figure 2. Analysis of the c.546G>A mutation, located at nucleotide 14 from the beginning of exon 6, in patient UMP08. A) RT-PCR products amplified with primers located in exons 4 and 7 in leukocyte RNA (L), separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient L (1) showed the single nucleotide change. C) NGS of PCR products from leukocytes identified the single nucleotide variant, as well as exon 6 skipping. Arrows show aberrant transcripts. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100-bp DNA ladder. transcripts: the major one (37% of reads) showed exon 33 analyzed: rs216321 and rs1800380. Surprisingly, the skipping, and the minor (2% of reads) showed exons 33 sequence obtained in both cell types indicated that only and 34 skipping. Moreover, analysis of the p.Leu2407Pro one allele was expressed. To further explore these results, mutation confirmed that NMD of the allele carrying the we tested whether intron 45 was retained within the c.5664+2T>C mutation had occurred in platelets. mature mRNA (Online Supplementary Methods) and per- formed complete sequencing of VWF cDNA from leuko- Candidate intronic mutations cytes. Nonetheless, no changes were observed (data not The c.7081+6G>T mutation, identified in a type 1 VWD shown). patient (UMP04), generated a new GT dinucleotide in The c.7730-4C>G mutation (intron 45) was identified in intron 41 (Online Supplementary Table S3). The exon 38-43 a type 2A/2M patient (UMP06), combined with the region showed the expected PCR product in both cell types p.Arg1374Cys mutation. The HSF predicted activation of (772 bp). To confirm these results, two informative SNPs a cryptic intronic acceptor splice site (ASS), but in the exon (rs216321 in exon 20 and rs216902 in exon 35) were geno- 43-49 region, splicing was not affected by the mutation. typed. Both were in heterozygous state, indicating that the To confirm the presence of the allele carrying c.7730- allele carrying the mutation was unaffected by NMD. 4C>G, the p.Arg1374Cys mutation was analyzed, and The c.7730-56C>T mutation (intron 45) was identified both nucleotides were seen in platelets and leukocytes, in a type 1 VWD patient (UMP05). The exon 43-49 region, suggesting expression of both alleles. We then performed examined by Sanger and NGS, showed no visible effect on the same experiment as was done in patient UMP05 to mRNA splicing. To confirm the presence of the allele car- test intron 45 retention, but no changes in VWF cDNA rying the intronic mutation, two informative SNPs were were observed (data not shown). haematologica | 2019; 104(3) 591 N. Borràs et al. A B C D Figure 3. Analysis of the c.1109G>A (p.Cys370Tyr) mutation in patient UMP10, located in the last nucleotide of exon 9. A) RT-PCR products amplified with primers located in exons 6 and 12 in leukocyte (L) and platelet (P) RNA, separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient L (2) showed exon 9 skipping. The L (3) band could not be purified in the agarose gel due to low concentration, thus it was not analyzed by Sanger. In platelets, only the non-mutated allele could be amplified. C) NGS of PCR products from leukocytes identified transcripts lacking exon 9, as well as transcripts lacking exons 8 and 9. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100-bp DNA ladder. The c.8254-5T>G mutation (intron 51) was found in a slightly diffuse, smaller band. Sanger sequencing detected type 1 VWD patient (UMP07). The exon 49-52 region was the c.546G>A change (Figure 2). However, NGS not only amplified, and the expected 546-bp size was observed in detected the nucleotide change, but also recognized a loss both cell types. Sequence analysis by the two techniques of 125 nucleotides corresponding to exon 6 revealed no changes in VWF cDNA. Interestingly, this (p.Thr179ProfsTer31) in small 2.3% of reads (Online patient’s two children, heterozygous for the c.8254-5T>G Supplementary Table S4). The predicted impact of the mutation, had bleeding symptoms. Hence, we performed c.546G>A mutation was discrepant and only two in silico complete sequencing of VWF cDNA from leukocytes. algorithms predicted an effect on mRNA splicing (Online However, no changes were identified. Supplementary Table S3) that was confirmed in vivo even though with a slight effect. To study c.4866C>T, the exon Synonymous mutations 25-31 region was amplified and sequenced, but no effect The mutations c.546G>A (exon 6) and c.4866C>T (exon on mRNA processing was seen, as was predicted by in sil- 28) were identified in trans in a type 1 VWD patient ico tools. Both nucleotides were identified in heterozygous (UMP08). These mutations were studied only in leukocyte state, indicating the presence of both alleles. Only off- RNA. To investigate c.546G>A, the exon 4-7 region was spring carrying the c.546G>A mutation (1 of 3 children) amplified, which resulted in the expected band and a had lower levels of VWF:Ag and VWF:RCo compared to 592 haematologica | 2019; 104(3) Effect of VWF mutations on mRNA splicing Table 2. Effect of VWF mutations on mRNA from leukocytes and platelets. Mutation type NT change AA change Exon Intron Leukocyte effect Platelet effect Protein prediction Intronic c.1533+1G>A - - 13 Exon 13 skipping (r.1433_1533del) NMD p.Gly478AlafsTer138 c.3379+1G>A* - - 25 Exon 25 skipping (r.3223_3379del) NMD p.Pro1075ValfsTer88 Activation of a cryptic site at +126 nt NMD p.Pro1117ValfsTer88 in exon 25 (r.3349_3379del) ‡ c.5664+2T>C - - 33 Exon 33 skipping (r.5621_5664del) NMD p.Gly1874AlafsTer32 Exon 33+34 skipping (r.5621_5842del) ‡ NMD p.Phe1875_Cys1948del c.7081+6G>T - - 41 No visible effect No visible effect - c.7082-2A>G † - - 41 Activation of a cryptic site +7 nt NMD p.Ala2361GlyfsTer40 in exon 42 (r.7082_7088del) c.7730-4C>G - - 45 No visible effect No visible effect - c.7730-56C>T - - 45 No visible effect No visible effect - c.8254-5T>G - - 51 No visible effect No visible effect - Synonymous c.546G>A † p.Ser182 6 - Exon 6 skipping (r.533_657del) ‡ NA p.Thr179ProfsTer31 c.3291C>T p.Cys1097 25 - No visible effect No visible effect - c.3426T>C p.Cys1142 26 - No visible effect No visible effect - c.4866C>T p.Asp1622 28 - No visible effect NA - c.7437G>A p.Ser2479 43 - Exon 43 skipping (r.7288_7437) NA p.Val2430GlyfsTer335 Activation of a cryptic site at +146 nt NA p.Ser2479AlafsTer23 in exon 43 (r.7434_7437del) Missense c.449T>C p.Leu150Pro 5 - No visible effect No visible effect - c.1109G>A p.Cys370Tyr 9 - Exon 9 skipping (r.998_1109del) NMD p.Glu333AlafsTer87 Exon 8+9 skipping (r.875_1109del) NMD p.Ser292ThrfsTer87 DelIns c.3485_3486delinsTG p.Pro1162Leu 26 - No visible effect No visible effect - c.3223-7_3236dup p.Pro1079_Tyr1080insLeu GlnValAspProGluPro 25 - (r.3477_3478instttgcaggtggaccccgagcc) (r.3477_3478instttgcaggt p.Pro1079_Tyr1080insLeu ggaccccgagcc) GlnValAspProGluPro c.6699_6702dup p.Cys2235ArgfsTer8 38 - No visible effect NMD p.Cys2235ArgfsTer8 AA:amino acid; NA: not available; NMD: nonsense-mediated decay; NT:nucleotide. *Previous functional studies by Nesbitt et al.23 †Previous functional studies by Corrales et al.7 ‡Leukocyte effect observed in a really low % of reads by NGS. the others. This may be explained by the mutation and/or 12 region was investigated. Whereas the expected size resulted in 2 additional bands of ̴650 and ̴500 bp (Figure other factors not explored in the current study. was observed in platelets, amplification from leukocytes The c.3291C>T mutation (exon 25), was identified in patient UMP09 (female), as well as a novel mutation in F8 3). Sanger sequencing of leukocyte mRNA confirmed that (c.1346C>G, p.Ala430Gly). Of note, her brother, who was the 650-bp band corresponded to a transcript lacking exon not included in this study, also had these mutations. As 9. NGS of leukocyte mRNA revealed 3 different tran- both siblings experienced bleeding, and the HSF predic- scripts: the WT, a transcript skipping exon 9 tion regarding c.3291C>T interpreted creation of an exon- (p.Glu333AlafsTer87) (13% of reads), and a transcript ic splicing silencer site that could potentially alter splicing, skipping exons 8 and 9 (p.Ser292ThrfsTer87) (7% of we hypothesized that this synonymous mutation could reads)(Table 2). Sequencing of PCR products from have an effect on VWF mRNA processing. Hence, the platelets showed no changes, suggesting that the mutated exon 22-26 region was amplified, but no change in the allele had experienced NMD. VWF cDNA sequence was observed. In addition, we test- ed whether intron 25 was retained within mature VWF Duplication mutation mRNA (Online Supplementary Methods), but no differences The c.3223-7_3236dup mutation, which generated an compared to control leukocyte cDNA were found (data not in-frame tandem duplication of 21 nucleotides, including shown). Finally, as both siblings presented similar FVIII:C 7 nucleotides from intron 24 and the first 14 from exon levels, we investigated X chromosome inactivation in 25, was detected in a type 1 VWD patient (UMP11). patient UMP09. The results demonstrated skewed inacti- Study of this mutation by the two techniques demon- vation of the WT X chromosome in this patient (Online strated a change in the native ASS of intron 24 in both cell Supplementary Figure S5). types, which resulted in maintaining the 21-bp insert cor- responding to 7 aberrant amino acids in mature VWF Missense mutations mRNA (p.Pro1079_Tyr1080insLeuGlnValAspProGluPro) The mutation c.1109G>A (p.Cys370Tyr, exon 9) was (Table 2 and Online Supplementary Figure S6). Furthermore, identified in a type 3 VWD carrier (UMP10). The exon 6- NGS showed that the aberrant mRNA transcript was haematologica | 2019; 104(3) 593 N. Borràs et al. present in ̴ 14% of reads in both cell types (Online within exon 42. This led to deletion of the 7 initial Supplementary Table S4). nucleotides of exon 42 (r.7082_7088del), as was predicted by the 4 algorithms, and resulted in a frameshift and a Combined potential splice site mutations in individual PTC (p.Ala2361GlyfsTer40) (Figure 4). Additionally, NGS patients showed that the aberrant transcript was present in 24% of The c.7082-2A>G mutation (intron 41) was identified in reads. On sequencing of platelet amplicons, there were no a type 3 VWD patient (UMP12) combined in trans with the changes, a finding highly suggestive of NMD. As type 3 p.Leu150Pro (c.449C>T, exon 5) mutation. To study VWD is characterized by absent or non-functional expres- c.7082-2A>G, the exon 38-43 region was amplified in sion of both VWF alleles, we postulated that the platelets and leukocytes, which yielded a band of the p.Leu150Pro missense mutation could have an effect on expected size. However, Sanger and NGS sequencing of VWF mRNA. Nonetheless, no sequence change was iden- the PCR products revealed activation of a cryptic ASS tified. These results were confirmed by analysis of 2 Figure 4. Analysis of the A c.7082-2A>G mutation in patient UMP12. Agarose gel electrophoresis results of RT- PCR amplification of exon 38 to 43 using RNA from leuko- cytes and platelets were the same as those of healthy controls (data not shown). A) Traditional Sanger sequenc- ing of PCR product from patient leukocyte (L) demon- strate activation of a cryptic splice site 7 nucleotides downstream of the native splice site within exon 42. In patient platelet (P), only the allele carrying the p.Leu150Pro mutation could be amplified. B) NGS of PCR products from leukocytes showed deletion of the 7 ini- tial nucleotides of exon 42. Arrows show aberrant tran- scripts. C) Schematic repre- B sentation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. WO: without. C 594 haematologica | 2019; 104(3) Effect of VWF mutations on mRNA splicing informative SNPs, rs1800375 and rs1800376, which were niques revealed 2 aberrant transcripts: one lacked exon found in heterozygous state in leukocyte transcripts and 43 (p.Val2430GlyfsTer335) and the other showed activa- homozygous state in platelets. tion of a cryptic DSS within exon 43, leading to deletion The c.[3426T>C; 3485_3486delinsTG] mutations (exon of its 4 final nucleotides (p.Ser2479AlafsTer23) (Table 2). 26) were found in a type 2A VWD patient (UMP13), com- Study of c.6699_6702dup in leukocyte mRNA showed bined in trans with p.Cys2773Ser. In silico analysis predict- no splicing changes, but the duplication introduced 4 ed no impact on the splice site for c. 3485_3486delinsTG, nucleotides that changed the reading frame and generat- but 2 of the 4 algorithms predicted that c.3426T>C would ed a PTC (p.Cys2235ArgfsTer8). have an effect on splicing. The exon 25-28 region was The c.546G>A (p.=, exon 6) and c.7082-2A>G (intron investigated, but no effect was found on mRNA process- 41) mutations in trans with c.8155+3G>C (intron 50) were ing. Study of the p.Cys2773Ser mutation and 2 informa- detected in a type 3 VWD patient (UMP15). This patient tive SNPs (rs2228317 and rs4021576) confirmed that the 2 had been described in our previous article, but at that time alleles were present, suggesting that exon 26 mutations do we were only able to identify the effect of c.8155+3G>C not have an effect on VWF splicing. (p.Gly2706ValfsTer24) on splicing. On reanalysis of this The mutations (c.7437G>A exon 43), and case with NGS, we were able to characterize the effect of c.6699_6702dup (p.Cys2235ArgfsTer8, exon 38) were c.546G>A (p.Thr179ProfsTer31) and c.7082-2A>G identified in trans in a severe type 1 VWD patient (p.Ala2361GlyfsTer40) in cis in leukocyte mRNA. The (UMP14) and studied only in leukocyte RNA. For results are consistent with those observed in patients c.7437G>A, we amplified exons 41-45, and 2 bands UMP08 and UMP12, who harbored each mutation indi- of ̴ 350 bp (Figure 5). Sequencing analysis by both tech- emerged: one with the expected size, and a smaller band vidually. In addition, in leukocytes, transcripts resulting from c.546G>A were detected in 2% of reads, transcripts A B C D Figure 5. Analysis of the c.7437G>A (p.=) mutation in patient UMP14, located in the last nucleotide of exon 43. A) RT-PCR products amplified with primers located in exons 41 and 45 in leukocyte RNA (L), separated on 1% agarose gel. B) Traditional Sanger sequencing of PCR product from patient leukocyte (L) shows two aber- rant transcripts: activation of a cryptic splice site - 4 nucleotides upstream to WT-DSS - in exon 43 (1), and exon 43 skipping (2). C) NGS of PCR products gave the same results than Sanger sequencing. D) Schematic representation of the mutation in genomic DNA and its effect on the VWF mRNA sequence. M indicates a 100- bp DNA ladder. WO: without. haematologica | 2019; 104(3) 595 N. Borràs et al. from c.7082-2A>G in 6% and transcripts from leukocytes generated three aberrant transcripts. One of c.8155+3G>C in 95% of reads. In platelet analyses, NGS them, which leads to the inframe deletion of exons 13-14, detected transcripts without exon 6 resulting from the should have been detected in platelets since it would not c.546G>A mutation in 11% of the total reads but no aber- generate a PTC and would not be degraded. However, rant transcript derived from the mutation c.7082-2A>G because the mutation is located in a region with weak indicating that NMD rate could differ between 5' and 3' splicing signal sequences, as previously demonstrated,7,25,26 regions of mRNA. These results indicate the c.[546G>A; we suggest that the results obtained in leukocytes could 7082-2A>G] allele is under-represented compared to the be an artifact that would not occur in the natural cellular allele carrying the c.8155+3G>C mutation, showing that type of VWF expression. Based on these observations, we this allele underwent NMD in both cell types, as was pre- propose that the real effect of c.1533+1G>A is the skip- viously hypothesized.7 ping of exon 13, which predicts a frameshift at position 478 and addition of 138 aberrant amino acids before a PTC is encountered (p.Gly478AlafsTer138). Discussion Two synonymous mutations included in our study, c.546G>A and c.7437G>A, affect splicing of VWF mRNA. This study reports the results of in-depth analysis of 18 To our knowledge, only three synonymous mutations PSSM in samples from VWD patients. The novel and affecting VWF mRNA processing have been reported: robust procedure used, combining two-step RT-PCR and c.7056C>T,28 c.7464C>T,29 and c.3390C>T.26 Of note, the NGS sequencing, was faster and more sensitive than the results of the present study have almost doubled the num- method used in our previous article.7 This new approach ber of reported mutations of this type causing VWD. The provides several advantages, such as allele-specific indi- pathogenic effect of c.546G>A could only be documented vidual sequences, higher sensitivity to detect transcript by NGS; it eluded Sanger detection because of the low variants present at low copy numbers, and simplified sam- expression in leukocytes and platelets. The c.7437G>A ple preparation. Moreover, the NGS data on total reads mutation located at the last nucleotide of exon 43 was obtained for each transcript is additional information that found in a patient with severe type 1 VWD (UMP14) can provide an approximation of the expression levels of in trans with c.6699_6702dup (p.Cys2235ArgfsTer8). This each VWF mRNA. Studies are currently ongoing to con- synonymous mutation produced 2 splicing variants. First, firm that the relative expression of each transcript creation of a new DSS 4 nucleotides upstream of the WT- obtained by NGS is comparable to that provided by real- DSS in exon 43 that generated a PTC in exon 44 and time RT-PCR. would lead to NMD in platelets; and second, exon 43 skip- The proven usefulness of leukocyte analysis to interpret ping and generation of a PTC in exon 52. In this latter case, mutations masked by NMD in platelets was seen in rela- we would expect that NMD had been abolished in tion to the c.3379+1G>A mutation, which had been pre- platelets, since this cellular mechanism is not effective viously investigated by RT-PCR in platelets.23 In that study, when a PTC is encountered within 50 bp of the last exon- the true pathogenic effect of this mutation could not be exon junction.30 determined due to NMD. In the present study, using Study of the p.Cys370Tyr (c.1109G>A in exon 9) muta- leukocyte mRNA and NGS technology, we found that the tion, resulted in generation of 2 VWF transcripts, one of mutation induces two aberrant transcripts. them identified only by NGS because of their low expres- Similarly, the pathogenic mechanism of 7 PSSM sion. Of note, only one other missense mutation, (c.1533+1G>A, c.5664+2T>C, c.7082-2A>G, c.546G>A, p.Gly1108Arg, has been reported to affect VWF splicing. c.7437G>A, and p.Cys370Tyr) was determined in leuko- Thus, these two mutations support the concept that mis- cytes, and only the c.3223-7_3236dup mutation effect was sense mutations on the last exonic nucleotides can also observed in both cell types. Because this mutation is locat- have repercussions on the splicing process.31 Interestingly, ed in the D3 domain implicated in multimerization, it is the pathogenic effect of this mutation is the same as that possible that the pathogenic mechanism leading to type 1 described for c.1109+2T>C (intron 9).32 In addition to the VWD may be related to impaired VWF secretion due to patient reported here, the p.Cys370Tyr mutation was intracellular retention, as has been described for other D3 identified in 6 additional related patients included in the mutations such as p.Cys1130Phe.24 To confirm this PCM-EVW-ES: in homozygous state in a type 3 VWD hypothesis, expression studies by means of heterologous patient and in heterozygosis in 4 type 3 carriers and 1 cell lines or blood outgrowth endothelial cells (BOECs) patient with type 1 VWD, based on their phenotype lev- will remain the gold standard. els. Certain genetic modifiers of VWF levels33 and inter- As would be expected, mutations located in the consen- individual variability in NMD efficiency between patients sus splicing sequence affected mRNA processing. These carrying identical mutations may lead to differences in the included c.1533+1G>A, c.5664+2T>C, leading to exon disease severity and clinical phenotype.34 skipping, and c.7082-2A>G, activating a cryptic splice site. Lastly, study of c.7081+6G>T, c.7730-4C>G, c.7730- Both these molecular effects have been reported previous- 56C>T, c.8254-5T>G, c.3291C>T, c.4866C>T and ly in splicing mutations causing VWD.7,25,26 The c.7082- c.[3426T>C; 3485_3486delinsTG] showed no visible 2A>G in trans with p.Leu150Pro leads to the development effect on mRNA processing, although this does not neces- of type 3 VWD. The p.Leu150Pro mutation does not affect sarily mean that they have no effect on splicing. For splicing. However, other mutations located in the propep- instance, mRNA SNP analysis in patient UMP05 with tide, such as p.Asp141Tyr, have been identified in type 3 c.7730-56C>T showed an absence of 1 allele in leukocytes patients,27 and, as has been described in these cases, we and platelets, suggesting that the allele may have experi- suspect that p.Leu150Pro may compromise propeptide enced NMD in both cell types or lack expression due to a folding and affect intracellular survival and the capacity to mutation that was not detected by our sequencing proto- mediate multimerization. The c.1533+1G>A mutation in col. Of particular note, deep intronic mutations such as 596 haematologica | 2019; 104(3) Effect of VWF mutations on mRNA splicing c.6599-20A>T have been found to cause VWD.35 In patient onymous or missense mutations, such as c.546G>A and UMP07 carrying the c.8254-5T>G mutation, no informa- p.Cys370Tyr. Therefore, the results here should not be con- tive SNP was identified and we were unable to determine sidered definitive, and as with all analytical approaches, whether there was a lack of expression of 1 allele, which should form one aspect of a wider investigation.5 could explain cosegregation of the mutation in the family In conclusion, we present an extensive study reporting with bleeding symptoms. the effect of 18 candidate mutations on VWF mRNA pro- Study of c.[3426T>C; 3485_3486delinsTG] and c.7730- cessing. In vivo mRNA studies incorporating NGS technol- 4C>G showed no visible effect on splicing. However, ogy together with traditional sequencing enabled us to these mutations have been identified in homozygous state determine the pathogenic effect of 8 PSSM (44%). Our in a type 3 patient (c.[3426T>C; 3485_3486delinsTG])3 study emphasizes the importance of examining selected and in a severe type 1 VWD patient (c.7730-4C>G)4 with mutations, including synonymous and missense muta- VWF:Ag at 7% and VWF:RCo at 5%, combined with the tions, to determine their pathogenic role in splicing. Taken heterozygous p.Ala631Val (previously reported in a together, our results add to the current knowledge about healthy control).36 Based on these findings, there is sub- the molecular events leading to VWD. stantial evidence that these mutations would have an effect on VWF levels. Therefore, to unequivocally deter- Acknowledgments mine the potential deleterious effect of these variants, We are indebted to Baxalta US Inc., now a part of Shire, for functional studies remain essential. These studies are tra- support of the PCM-EVW-ES (Grant H13-000845). ditionally carried out by in vitro analyses performed using heterologous cell lines (COS7, AtT-20 and HEK293). Funding However, the advent of the possibility of obtaining This study was also supported by the Spanish Ministry of the Economiá y Competitividad), Instituto de Salud Carlos III (ISCI- BOECs from patients represents a valuable alternative, Economy and Competitiveness (MINECO, Ministerio de since this is the functional expression site of VWF.37 Moreover, BOECs allows protein expression and mRNA II) (PI12/01494, PI15/01643 and RD12/0042/0053). We are studies simultaneously. very grateful for the kind collaboration of the participating In silico algorithms used to assess the impact of mutations patients and their families. CIBERCV is an initiative of ISCIII, on splicing are more sensitive and accurate in determining co-financed by the European Regional Development Fund the putative effect of intronic mutations than that of syn- (ERDF), “A way to build Europe”. 7. Corrales I, Ramirez L, Altisent C, Parra R, Tuddenham EG, McVey JH. Analysis of the References Vidal F. The study of the effect of splicing consequences of premature termination mutations in von Willebrand factor using codons within factor VIII coding 1. Rodeghiero F, Castaman G, Dini E. RNA isolated from patients' platelets and sequences. J Thromb Haemost. 2003;1(1): Epidemiological investigation of the preva- leukocytes. J Thromb Haemost. 139-146. lence of von Willebrand's disease. Blood. 2011;9(4):679-688. 16. Roberts RG, Bentley DR, Bobrow M. 1987;69(2):454-459. 8. Wang GS, Cooper TA. Splicing in disease: Infidelity in the structure of ectopic tran- 2. Batlle J, Perez-Rodriguez A, Corrales I, et disruption of the splicing code and the scripts: a novel exon in lymphocyte dys- al. Molecular and clinical profile of von decoding machinery. Nat Re Genet. trophin transcripts. Hum Mutat. 1993;2 Willebrand disease in Spain (PCM-EVW- 2007;8(10):749-761. (4):293-299. ES): Proposal for a new diagnostic para- 9. Eikenboom JC, Reitsma PH, Peerlinck KM, 17. Byers PH. Killing the messenger: new digm. Thromb Haemost. 2016;115(1):40- Briët E. Recessive inheritance of von insights into nonsense-mediated mRNA 50. Willebrand's disease type I. Lancet. decay. J Clin Invest. 2002;109(1):3-6. 3. Borras N, Batlle J, Perez-Rodriguez A, et al. 1993;341(8851):982-986. 18. Lewis BP, Green RE, Brenner SE. Evidence Molecular and clinical profile of von 10. Jaffe EA, Hoyer LW, Nachman RL. for the widespread coupling of alternative Willebrand disease in Spain (PCM-EVW- Synthesis of von Willebrand factor by cul- splicing and nonsense-mediated mRNA ES): comprehensive genetic analysis by tured human endothelial cells. Proc Natl decay in humans. Proc Natl Acad Sci U S A. next-generation sequencing of 480 Acad Sci U S A. 1974;71(5):1906-1909. 2003;100(1):189-192. patients. Haematologica. 2017;102(12): 11. Sporn LA, Chavin SI, Marder VJ, Wagner 19. Martorell L, Luce E, Vazquez JL, et al. 2005-2014. DD. Biosynthesis of von Willebrand pro- Advanced cell-based modeling of the royal 4. Fidalgo T, Salvado R, Corrales I, et al. tein by human megakaryocytes. J Clin disease: characterization of the mutated F9 Genotype-phenotype correlation in a cohort Invest. 1985;76(3):1102-1106. mRNA. J Thromb Haemost. 2017;15(11): of Portuguese patients comprising the entire 12. Kieffer N, Guichard J, Farcet JP, 2188-2197. spectrum of VWD types: impact of NGS. Vainchenker W, Breton-Gorius J. 20. Hojny J, Zemankova P, Lhota F, et al. Thromb Haemost. 2016;116(1):17-31. Biosynthesis of major platelet proteins in Multiplex PCR and NGS-based identifica- 5. Richards S, Aziz N, Bale S, et al. Standards human blood platelets. Eur J Biochem. tion of mRNA splicing variants: Analysis of and guidelines for the interpretation of 1987;164(1):189-195. BRCA1 splicing pattern as a model. Gene. sequence variants: a joint consensus rec- 13. Fink L, Holschermann H, Kwapiszewska 2017;637:41-49. ommendation of the American College of G, et al. Characterization of platelet-specif- 21. Brunak S, Engelbrecht J, Knudsen S. Medical Genetics and Genomics and the ic mRNA by real-time PCR after laser- Prediction of human mRNA donor and Association for Molecular Pathology. assisted microdissection. Thromb acceptor sites from the DNA sequence. J Genet Med. 2015;17(5):405-424. Haemost. 2003;90(4):749-756. Mol Biol. 1991;220(1):49-65. 6. Wang QY, Song J, Gibbs RA, Boerwinkle E, 14. McVey JH, Boswell EJ, Takamiya O, et al. 22. Corrales I, Ramirez L, Altisent C, Parra R, Dong JF, Yu FL. Characterizing polymor- Exclusion of the first EGF domain of factor Vidal F. Rapid molecular diagnosis of von phisms and allelic diversity of von VII by a splice site mutation causes lethal Willebrand disease by direct sequencing. Willebrand factor gene in the 1000 factor VII deficiency. Blood. Detection of 12 novel putative mutations Genomes. J Thromb Haemost. 1998;92(3):920-926. in VWF gene. Thromb Haemost. 2013;11(2):261-269. 15. David D, Santos IM, Johnson K, 2009;101(3):570-576. haematologica | 2019; 104(3) 597 N. Borràs et al. 23. Nesbitt IM, Hampton KK, Preston FE, with type 3 von Willebrand disease. quantitative deficiencies. J Thromb Peake IR, Goodeve AC. A common splice Haemophilia. 2008;14(3):549-555. Haemost. 2010;8(12):2736-2742. site mutation is shared by two families 28. Daidone V, Gallinaro L, Grazia Cattini M, 33. James PD, Lillicrap D. von Willebrand dis- with different type 2N von Willebrand dis- et al. An apparently silent nucleotide sub- ease: clinical and laboratory lessons learned ease mutations. Thromb Haemost. stitution (c.7056C>T) in the von from the large von Willebrand disease stud- 1999;82(3):1061-1064. Willebrand factor gene is responsible for ies. Am J Hematol. 2012;87 Suppl 1:S4-11. 24. Tjernberg P, Vos HL, Castaman G, Bertina type 1 von Willebrand disease. 34. Miller JN, Pearce DA. Nonsense-mediated RM, Eikenboom JC. Dimerization and mul- Haematologica. 2011;96(6):881-887. decay in genetic disease: friend or foe? timerization defects of von Willebrand fac- 29. Yadegari H, Biswas A, Akhter MS, et al. Muta Res Rev Mutat Res. 2014;762:52-64. tor due to mutated cysteine residues. J Intron retention resulting from a silent 35. Hawke L, Bowman ML, Poon MC, Scully Thromb Haemost. 2004;2(2):257-265. mutation in the VWF gene that structurally MF, Rivard GE, James PD. Characterization 25. Gallinaro L, Sartorello F, Pontara E, et al. influences the 5' splice site. Blood. of aberrant splicing of von Willebrand fac- Combined partial exon skipping and cryp- 2016;128(17):2144-2152. tor in von Willebrand disease: an underrec- tic splice site activation as a new molecular 30. Maquat LE. Nonsense-mediated mRNA ognized mechanism. Blood. 2016;128(4): mechanism for recessive type 1 von decay: splicing, translation and mRNP 584-593. Willebrand disease. Thromb Haemost. dynamics. Nat Rev Mol Cell Biol. 2004;5(2): 36. Bellissimo DB, Christopherson PA, Flood 2006;96(6):711-716. 89-99. VH, et al. VWF mutations and new 26. Pagliari MT, Baronciani L, Garcia Oya I, et 31. James PD, O'Brien LA, Hegadorn CA, et al. sequence variations identified in healthy al. A synonymous (c.3390C>T) or a splice- A novel type 2A von Willebrand factor controls are more frequent in the African- site (c.3380-2A>G) mutation causes exon mutation located at the last nucleotide of American population. Blood. 2012;119(9): 26 skipping in four patients with von exon 26 (3538G>A) causes skipping of 2 2135-2140. Willebrand disease (2A/IIE). J Thromb nonadjacent exons. Blood. 2004;104(9): 37. Wang JW, Bouwens EA, Pintao MC, et al. Haemost. 2013;11(7):1251-1259. 2739-2745. Analysis of the storage and secretion of von 27. Baronciani L, Federici AB, Cozzi G, et al. 32. Castaman G, Plate M, Giacomelli SH, Willebrand factor in blood outgrowth Expression studies of missense mutations Rodeghiero F, Duga S. Alterations of endothelial cells derived from patients with p.D141Y, p.C275S located in the propep- mRNA processing and stability as a patho- von Willebrand disease. Blood. 2013;121 tide of von Willebrand factor in patients genic mechanism in von Willebrand factor (14):2762-2772. 598 haematologica | 2019; 104(3)