Associations of multiple genetic variations with plasma levels of Von Willebrand Factor and clinical phenotype in Iranian patients with Von Willebrand disease type 1.

BACKGROUND: Genetic variations influence the Von Willebrand Factor plasma level and function. This study aims to evaluate the frequency and clinical phenotype effects of eight single nucleotide polymorphism candidates in four genes (VWF, STXBP5, CLEC4M, and ABO) in Iranian patients with VWD type 1. METHOD: The study recruited 50 patients with VWD type 1 and 100 healthy individuals. The demographic data from all participants were collected, and the High-Resolution Melting technique was used to determine the frequency of specific single nucleotide polymorphisms. Bleeding scores were also obtained from all patients to assess how these genetic variations might affect the severity of their bleeding symptoms. RESULTS: The study found notable variations in the occurrence of certain SNPs (rs7853989 and rs8176743 for ABO gene and rs1063856 and rs1063857 for VWF gene) between the control group and the patients. Additionally, the study discovered that two SNPs (rs868875 for CLEC4M gene and rs9390459 for STXBP5 gene) were significantly linked to the severity of bleeding, and two others (rs868875 for CLEC4M gene and rs8176746 for ABO gene) were associated with reduced levels of VWF antigen in the patients. CONCLUSION: According to this study, the above-selected SNPs can cause variations in VWF plasma levels in patients with VWD type 1. Furthermore, the effects of SNPs on bleeding phenotype prove the role of these SNPs in the severity of bleeding manifestations in patients.

Genetic determinants of enhanced von Willebrand factor clearance from plasma.

BACKGROUND: Enhanced von Willebrand factor (VWF) clearance from plasma is associated with von Willebrand disease (VWD). However, the genetic background of this disease mechanism is not well defined. OBJECTIVE: To determine VWF variants that are associated with reduced VWF survival. METHODS: Two hundred fifty-four patients with VWD (type 1 = 50 and type 2 = 204) were investigated, and the results were compared with 120 healthy controls. The patients were comprehensively characterized for phenotypic and genetic features. The ratio of VWF propeptide (VWFpp)/VWF antigen (VWFpp ratio) was used to establish in each patient the VWF clearance state. RESULTS: Out of 92 variants associated with type 1 (7 were novel) and type 2 VWD, 19 had a VWFpp ratio ranging from 1.7 to 2.2, 24 had a VWFpp ratio between 2.3 and 2.9, and 24 variants had a ratio of ≥3. The VWFpp median ratio in healthy controls was 0.98 (0.55-1.6) so that a cut-off value of >1.6 was considered an indicator of accelerated VWF clearance from plasma. An enhanced VWF clearance was observed in 34% of type 1 cases, 100% of type 1 Vicenza cases, 81% of 2A cases, 77% of 2B cases, 88% of 2M cases, and 36% of 2N cases. CONCLUSIONS: An accelerated VWF clearance was found in most patients with type 2A, 2B, and 2M VWD, with a lower proportion of type 1 and 2N. Sixty-seven different variants alone or in combination with other variants were associated with an increased VWFpp ratio. The variants with the highest VWFpp ratio were mostly located in the D3-A1 VWF domains.

Type 1 von Willebrand Disease in a Pediatric Patient Caused by a Novel Heterozygous Deletion of Exons 1 to 6 of the von Willebrand Factor Gene: A Case Report.

A 6-year-old boy was referred to a hematologist due to excessive mucocutaneous bleeding. Diagnostic assessment for von Willebrand disease (VWD) was indicated and included both coagulation and genetic testing. Laboratory testing revealed proportionally decreased von Willebrand factor (VWF) glycoprotein Ib-binding activity (23.6%) compared to VWF antigen (24.7%), similarly decreased VWF collagen-binding activity (24.2%), and normally distributed VWF multimers, with decreased intensity of all fractions. Diagnosis of type 1 VWD was established. Genetic analysis by means of next-generation sequencing (NGS) of VWF and coagulation factor VIII genes did not identify any causative mutations. Additionally, multiplex ligation-dependent probe amplification (MLPA) of VWF gene exons revealed a heterozygous deletion of exons 1 to 6, which is reported in type 1 VWD for the first time. Application of MLPA was crucial for revealing the genetic basis of type 1 VWD in this case, which would have remained undetected if only NGS was used.

A transcriptome analysis of basal and stimulated VWF release from endothelial cells derived from patients with type 1 VWD.

Type 1 von Willebrand disease (VWD) is associated with a reduction in qualitatively normal von Willebrand factor (VWF). Current diagnostic guidelines only take into consideration the contribution of basal VWF levels, despite a lack of correlation with bleeding severity. Defects in stimulated VWF release, which occurs after hemostatic challenge, may contribute to bleeding in type 1 VWD, but the pathogenic mechanisms are poorly defined. In this study, a layered multiomic approach including messenger RNA (mRNA) and microRNA (miRNA) sequencing was used to evaluate transcriptome-wide differences between type 1 VWD- and control-derived endothelial colony forming cells (ECFCs) during basal and stimulated VWF release. ECFCs from 8 patients with type 1 VWD and 4 other patients were included in this study as controls. VWF protein analysis revealed heterogenous responses to stimulation among type 1 VWD and control ECFCs. During basal VWF release, 64 mRNAs and 7 miRNAs were differentially regulated between type 1 VWD and control ECFCs, and 65 putatively pathogenic miRNA-mRNA interactions were identified. During stimulated VWF release, 190 mRNAs and 5 mRNAs were differentially regulated between type 1 VWD and control ECFCs, and 110 putatively pathogenic miRNA-mRNA interactions were identified. Five gene ontology terms including coagulation, regulation of cell shape, and regulation of cell signaling were also differentially regulated in type 1 VWD ECFCs during stimulated release. To our knowledge, we have shown for the first time that transcriptome-wide differences exist between type 1 VWD and control ECFCs. These differences may contribute to bleeding in type 1 VWD, and further investigation may reveal novel biomarkers and therapeutic targets.

von Willebrand factor antigen levels are associated with burden of rare nonsynonymous variants in the VWF gene.

Approximately 35% of patients with type 1 von Willebrand disease (VWD) do not have a known pathogenic variant in the von Willebrand factor (VWF) gene. We aimed to understand the impact of VWF coding variants on VWD risk and VWF antigen (VWF:Ag) levels, studying 527 patients with low VWF and VWD and 210 healthy controls. VWF sequencing was performed and VWF:Ag levels assayed. A combined annotation-dependent depletion (CADD) score >20 was used as a predicted pathogenicity measure. The number of rare nonsynonymous VWF variants significantly predicted VWF:Ag levels (P = 1.62 × 10-21). There was an association between average number of rare nonsynonymous VWF variants with VWD type 1 (P = 2.4 × 10-13) and low VWF (P = 1.6 × 10-27) compared with healthy subjects: type 1 subjects possessed on average >2 times as many rare variants as those with low VWF and 8 times as many as healthy subjects. The number of rare nonsynonymous variants significantly predicts VWF:Ag levels even after controlling for presence of a variant with a CADD score >20 or a known pathogenic variant in VWF (P = 2.7 × 10-14). The number of rare nonsynonymous variants in VWF as well as the presence of a variant with CADD >20 are both significantly associated with VWF levels. The association with rare nonsynonymous variants holds even when controlling for known pathogenic variants, suggesting that additional variants, in VWF or elsewhere, are associated with VWF:Ag levels. Patients with higher VWF:Ag levels with fewer rare nonsynonymous VWF gene variants could benefit from next-generation sequencing to find the cause of their bleeding.

Clinical significance of slightly reduced von Willebrand factor activity.

Von Willebrand disease (VWD) is the most common congenital bleeding disorder, with a clinical presentation of mucocutaneous and surgical bleeding varying from mild to severe. It is inherited in an autosomal dominant or autosomal recessive manner. The disease is caused by quantitative or qualitative deficiency of the von Willebrand factor (VWF) and is classified as type 1, 2 (2A, 2B, 2M, 2N), and 3. Although type 1 VWD is the most common form of VWD, the f ormal cutoff for diagnosis remains a subject of debate. In our paper, we present results of studies regarding the clinical and laboratory importance of a new type of bleeding disorder called low VWF. The new guidelines for VWD diagnosis and management suggested that patients with historically type 1 VWD should be divided into 2 subsets: type 1 VWD with a VWF antigen level (VWF:Ag) of less than 30 IU/dl or less than 40 IU/dl, in which about 80% of patients exhibited VWF gene mutations, and low VWF with a VWF:Ag level of 30 to 50 IU/dl or 40 to 50 IU/dl, in which the causative mutation is detected in merely 40% of patients and in most families, inheritance is not dependent on the locus of VWF on chromosome 12. Previously, moderately reduced VWF levels (30-50 IU/dl) were considered a risk factor for bleeding, but not a true bleeding disorder, and this condition was named low VWF. Recently, it was documented in a large group of patients with type 1 VWD and low VWF that bleeding score does not correlate with VWF:Ag and bleeding symptoms in type 1 VWD (<30 IU/dl) and low VWF can change from infrequent and moderate to severe bleeds. Because the plasma concentration of VWF depends on many physiological and pathological factors that may mask the diagnosis of VWD, separation of the group of patients with low VWF (30-50 IU/dl) from those with type 1 VWD may delay or prevent them from receiving appropriate treatment. Diagnosis of VWD in each case, particularly those with a slight decrease in VWF (30-50 IU/dl), should be based primarily on the clinical manifestations and family history of hemorrhagic diathesis.

The role of genetics in the pathogenesis and diagnosis of type 1 Von Willebrand disease.

PURPOSE OF REVIEW: Von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis of VWD is challenging, particularly with type 1 VWD. Although most clinicians use specific tests of von Willebrand factor (VWF) activity to classify patients with VWD, genetic testing for VWF defects is another potential method of diagnosis. RECENT FINDINGS: Studies of patients with type 1 VWD report consistently that many, but not all, study participants have VWF gene defects. Certain populations, including those with VWF levels less than 30 IU/dl and those with clearance defects, are more likely to have a VWF sequence variant. In addition, a number of loci outside the VWF gene have been shown to affect VWF levels, including ABO, CLEC4M, STXBP5, and STAB2. SUMMARY: Genetic defects in VWF are common, but not all defects lead to disease. Type 1 VWD in particular does not always have an associated VWF sequence variant. New data stemming from genome-wide association studies on modifier genes suggest that the etiology of type 1 VWD is multifactorial.

Diagnostic challenges of inherited mild bleeding disorders: a bait for poorly explored clinical and basic research.

The best-known inherited mild bleeding disorders (MBDs), i.e. type 1 von Willebrand disease (VWD), platelet function disorders (PFDs), and mild to moderate clotting factor deficiencies, are characterized clinically by mucocutaneous bleeding, and, although they are highly prevalent, still pose difficult diagnostic problems. These include establishing the pathological nature of bleeding, and the uncertainties surrounding the clinical relevance of laboratory results. Furthermore, the high frequency of bleeding symptoms in the normal population and the subjective appraisal of symptoms by patients or parents makes elucidating the pathological nature of bleeding difficult. Standardized bleeding assessment tools and semiquantitative bleeding scores (BSs) help to discriminate normal from abnormal bleeding. However, as most MBDs have similar bleeding patterns, for example, bleeding sites, frequency, and severity, BSs are of little help for diagnosing specific diseases. Global tests of primary hemostasis (bleeding time; PFA-100/200) lack sensitivity and, like BSs, are not disease-specific. Problems with the diagnosis of type 1 VWD and PFD include assay standardization, uncertain definition of von Willebrand factor cut-off levels, and the lack of universal diagnostic criteria for PFD. Regarding clotting factor deficiencies, the bleeding thresholds of some coagulation factors, such as factor VII and FXI, are highly variable, and may lead to misinterpretation of the clinical relevance of mild to moderate deficiencies. Remarkably, a large proportion of MBDs remain undiagnosed even after comprehensive and repeated laboratory testing. These are tentatively considered to represent bleeding of undefined cause, with clinical features indistinguishable from those of classical MBD; the pathogenesis of this is probably multifactorial, and unveiling these mechanisms should constitute a fertile source of translational research.

Genetic Variation in the Syntaxin-Binding Protein STXBP5 in Type 1 von Willebrand Disease Patients.

von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10-4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.

A case of inherited type 1 and type 2A von Willebrand disease confirmed by diagnostic exome sequencing.

A 10-year-old male and his family members visited a pediatric hematology clinic due to coagulopathy. Laboratory tests indicated von Willebrand disease (vWD) in all the family members. We conducted diagnostic exome sequencing for confirmation. The patient was confirmed to be a compound heterozygote for vWD: c.2574C > G (p.Cys858Trp) from his father (known variant of vWD type 1) and c.3390C > T (p.Pro1127_Gly1180delinsArg) from his mother (variant known to result in exon 26 skipping in vWD type 2A). He was managed with factor VIII and von Willebrand factor complex concentrate during palatoplasty due to bleeding despite pre-operative desmopressin injection. The operation was completed successfully.

Genetic variation in the C-type lectin receptor CLEC4M in type 1 von Willebrand Disease patients.

von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, e.g. VWF, ABO, STXBP5 and CLEC4M. This study aims to screen comprehensively for CLEC4M variants and investigate their association with type 1 VWD in the Swedish population. In order to screen for CLEC4M variants, the CLEC4M gene region was re-sequenced and the polymorphic neck region was genotyped in 106 type 1 VWD patients from unrelated type 1 VWD families. Single nucleotide variants (SNV) and variable number tandem repeat (VNTR) allele and genotype frequencies were then compared with 294 individuals from the 1000Genomes project and 436 Swedish control individuals. Re-sequencing identified a total of 42 SNVs. Rare variants showed no accumulation in type 1 VWD patients and are not thought to contribute substantially to type 1 VWD. The only missense mutation (rs2277998, NP_001138379.1:p.Asp224Asn) had a higher frequency in type 1 VWD patients than in controls (4.9%). The VNTR genotypes 57 and 67 were observed at higher frequencies than expected in type 1 VWD patients (6.4% and 6.2%) and showed an increase in patients compared with controls (7.4% and 3.1%). Strong linkage disequilibrium in the CLEC4M region makes it difficult to distinguish between the effect of the missense mutation and the VNTR genotypes. In conclusion, heterozygous VNTR genotypes 57 and 67 of CLEC4M were highly enriched and are the most likely mechanism through which CLEC4M contributes to disease in the Swedish type 1 VWD population.

Advances in the diagnosis and treatment of Von Willebrand disease.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder, yet diagnosis and management remain challenging. Development and use of bleeding assessment tools allows for improved stratification of which patients may require further assessment and which patients are most likely to require treatment of their VWD. New options for laboratory assessment of von Willebrand factor (VWF) activity include a new platelet-binding assay, the VWF:GPIbM, which is subject to less variability than the ristocetin cofactor activity assay, and collagen-binding assays that provide insight into a different function of VWF. Genetic testing may be helpful in some cases where a type 2 VWD variant is suspected but is usually not helpful in type 1 VWD. Finally, treatment options for VWD are reviewed, including the use of recombinant VWF. Despite these advances, still more work is required to improve diagnosis, treatment, and quality of life for affected patients.

Controversies in the diagnosis of Type 1 von Willebrand disease.

von Willebrand disease (VWD) is the most common bleeding disorder. Type 1 VWD represents the majority of cases and results from a partial quantitative deficiency of von Willebrand factor (VWF). The diagnosis of Type 1 VWD presents many challenges, despite there being three established diagnostic criteria: a personal history of mucocutaneous bleeding, a family history and low VWF levels. These criteria do not always coexist, and there is great overlap of clinical phenotypes and laboratory parameters between healthy individuals and those with VWD. Mild bleeding symptoms can have any number of causes and bleeding is commonly reported in the general population. VWF levels do not always correlate with bleeding symptoms and can be variable between affected family members. Additionally, VWF levels vary widely as a result of both genetic and non-genetic influences. Perhaps the greatest current controversy in the diagnosis of Type 1 VWD is that there is no consensus laboratory cut-off for the diagnosis, raising concern about both over- and under-diagnosis. Ongoing studies are addressing these issues by clarifying the underlying pathogenesis of the disease, as well as the natural history and the risk of future bleeding in those with the diagnosis.

Haemostatic patterns and bleeding scores of a genetically characterised Italian family with combined haemophilia A and type 1 von Willebrand disease.

: We report on the coinheritance of mild haemophilia A and type 1 von Willebrand disease (VWD) in a genetically characterized Italian family. The proband is a 56-year-old man carrying both the c.2167G>A mutation in the factor VIII (FVIII) gene (responsible for p.A723T substitution) and the c.4751A>G mutation (p.Y1584C) in the von Willebrand factor (VWF) gene. His FVIII and VWF levels were 9.8 and 43.2 IU/dl, respectively. His bleeding symptoms included mucocutaneous bleeding, haemarthrosis, and muscle haematomas. Using the bleeding assessment tool, a questionnaire currently employed in diagnosing VWD, the patient had a bleeding score of 27 as compared with the 10.2 ±â€Š3.4 found in patients with mild-to-moderate haemophilia A, and 0-3 in normal men. One of the proband's two daughters (both obligate carriers of haemophilia A) also harboured the VWF p.Y1584C mutation. Her FVIII and VWF levels were 45.9 and 54 IU/dl, respectively, and her bleeding score was slightly higher than normal for women (6 vs. 0-5). The other daughter had a normal bleeding score, and so did the proband's father (with type 1 VWD) and mother (haemophilia A carrier). Discrepancies between haemostatic patterns and bleeding symptoms in cases of haemophilia A, as seen in our patient, suggest the need to search for other coagulation defects, especially involving VWF, which is the carrier of FVIII. Although the presence of a VWF mutation significantly exacerbates the haemorrhagic complications in patients with mild haemophilia A, it has only mild effects on haemophilia A carriers.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

BACKGROUND: Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. MATERIALS AND METHODS: The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. RESULTS: As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of ß-sheet secondary structure. DISCUSSION: The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

von Willebrand disease type 1 mutation p.Arg1379Cys and the variant p.Ala1377Val synergistically determine a 2M phenotype in four Italian patients.

INTRODUCTION: We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also have the variant p.Ala1377Val in cis with p.Arg1379Cys. AIM: To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype. METHODS: Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1-VWF-WT and mutant (A1377V, R1379C and A1377V-R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild-type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL-1 ) and the other with a gain-of-function mutant rGpIbα (VWF:GPIbM). RESULTS: The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL-1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12-fold increased binding to rGpIbα, respectively. A1377V-R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3-fold increased, at 1.50 mg mL-1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V-R1379C rVWF and the 2M patients' plasma. CONCLUSION: Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype.

Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site.

Disease-associated silent mutations are considered to affect the accurate pre-messenger RNA (mRNA) splicing either by influencing regulatory elements, leading to exon skipping, or by creating a new cryptic splice site. This study describes a new molecular pathological mechanism by which a silent mutation inhibits splicing and leads to intron retention. We identified a heterozygous silent mutation, c.7464C>T, in exon 44 of the von Willebrand factor (VWF) gene in a family with type 1 von Willebrand disease. In vivo and ex vivo transcript analysis revealed an aberrantly spliced transcript, with intron 44 retained in the mRNA, implying disruption of the first catalytic step of splicing at the 5' splice site (5'ss). The abnormal transcript with the retained intronic region coded a truncated protein that lacked the carboxy-terminal end of the VWF protein. Confocal immunofluorescence characterizations of blood outgrowth endothelial cells derived from the patient confirmed the presence of the truncated protein by demonstrating accumulation of VWF in the endoplasmic reticulum. In silico pre-mRNA secondary and tertiary structure analysis revealed that this substitution, despite its distal position from the 5'ss (85 bp downstream), induces cis alterations in pre-mRNA structure that result in the formation of a stable hairpin at the 5'ss. This hairpin sequesters the 5'ss residues involved in U1 small nuclear RNA interactions, thereby inhibiting excision of the pre-mRNA intronic region. This study is the first to show the allosteric-like/far-reaching effect of an exonic variation on pre-mRNA splicing that is mediated by structural changes in the pre-mRNA.

Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.

Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.

Severe, recessive type 1 is a discrete form of von Willebrand disease: the lesson learned from the c.1534-3C>A von Willebrand factor mutation.

Type 1 von Willebrand disease (VWD) is transmitted mainly as a dominant trait - especially in forms involving von Willebrand factor (VWF) levels below 20 U/dL - and less frequently as a recessive trait. In the latter case, mutations at heterozygous level may be associated with type 3 carrier status, while mutations at homozygous or compound heterozygous level often coincide with type 3 VWD. Here we present a recessive, severe type 1 form as a distinct type of VWD. Eight patients with severe type 1 VWD belonging to 7 unrelated families were studied. They had VWF levels below 10 U/dL, FVIII higher than 10 U/dL, and a significantly lower than normal platelet VWF content. All patients were homozygous or compound heterozygous for the c.1534-3C>A VWF mutation, that simultaneously induces the skipping of exon 14, the activation of a cryptic splice site, and a normal VWF gene transcription. This means that one of the three different mRNA generated assures the synthesis of normal VWF. The probands' relatives who were heterozygous for the c.1534-3C>A mutation always had low platelet VWF levels, sometimes with circulating VWF levels within normal range. This finding confirms the utility of measuring platelet VWF content to identify an abnormal VWF synthesis. Because the c.1534-3C>A mutation impairs, but does not abolish normal mRNA processing, it may never cause type 3 VWD. We propose a model of severe recessive type 1 VWF defect associated with mutations that sporadically go undetected by the cells' molecular machinery, as the c.1534-3C>A VWF mutation. BULLET POINTS: What is known about this topic? - Type 1 VWD is transmitted mainly as a dominant trait. - Recessive type 1 mutations at homozygous or compound heterozygous level are often associated with type 3 VWD, and at heterozygous level with type 3 VWD carrier status. What does this paper add? - There are quantitative VWF mutations, such as c.1534-3C>A, that impair, but do not abolish normal mRNA processing. - The c.1534-3C>A VWF mutation simultaneously induces the skipping of exon 14, the activation of a cryptic splice site, and a normal VWF gene transcription. - The c.1534-3C>A mutation is the archetype of mutations that cause severe recessive type 1 VWD, but never type 3 VWD. - Recessive, severe type 1 appears to be a distinct form of VWD.