Phenotypic and genotypic (exon 28) characterization of patients diagnosed with von Willebrand disease type 1 in Eastern Saudi Arabia.

Von Willebrand factor (VWF) is a plasma glycoprotein that plays a key role in hemostasis. Mutations in this protein can result in von Willebrand disease (VWD), the most common form of bleeding disorder in humans. Patients with type 1 VWD have a quantitative plasmatic deficiency of normal structural and functional VWF. Our study aimed to investigate the phenotypic and genotypic characteristics of VWD type 1 patients in eastern Saudi Arabia, focusing on exon 28. We included patients previously diagnosed with WWD type 1 at the King Fahad teaching hospital in Al Khobar and their family members. The correlations between various phenotypic data and genotypic (exon 28) were analyzed using statistical software (SPSS) version 21. While these variants were generally considered benign with minor clinical effects, our analysis did identify two pathogenic variants that could lead to severe VWD symptoms. Specifically, we found these two pathogenic variants in three VWD patients from Saudi Arabia, providing essential insights into pathogenic VWD mutations in this population. Our study, therefore, sheds light on the prevalence of VWF variants in the eastern province of the Kingdom and highlights the need for continued research into the genetic causes of VWD in this region.

Phenotypic and Genotypic Signatures of VWF Exon 18 in Eastern Saudi Patients Previously Diagnosed with Type 1 von Willebrand Disease.

Introduction: von Willebrand disease (VWD) is the most prevalent bleeding disease, which is associated with either low levels of von Willebrand factor (VWF) or abnormality in its structure. Three types of the disease have been described; type 1 (VWD1) and 3 (VWD3) are caused by deficiency of VWF and type 2 (VWD2) is caused by production of defective VWF. The aim of the current study was to characterize gene variants of VWF gene; exon 18 in particular, in a cohort of Saudi families as well as healthy control subjects. Methods: A total of 19 families comprising 60 subjects of type 1 VWD were enrolled in the study. Participants were divided into 22 index cases, 21 affected family members and 17 unaffected family members ranging in age from 6 to 70 years. Blood samples were collected from all participants to measure activated partial thromboplastin time test (APTT), von Willebrand antigen level (VWF:Ag), Factor VIII activity (FVIII:C) and ristocetin cofactor activity (VWF:RCo), platelet count, determining the ABO blood group and for genetic analysis by Sanger sequencing. Results: The results indicated that VWD1 patients have lower levels of VWF and factor VIII than the non-affected family members and the control subjects. In addition, five gene variants were reported in VWF exon 18; of these, c.2365A>G and c.2385T>C were more common in the control group and might be protective from VWD. Discussion: In conclusion, VWF levels are influenced by blood group, and there was no association between variants in exon 18 of VWF gene reported in all groups and the disease status; however, blood group analysis and genome-wide genotyping could help to highlight high-risk groups and improve clinical management of VWD.

Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

Type 3 von Willebrand disease (VWD3) is a rare and severe bleeding disorder characterized by often undetectable von Willebrand factor (VWF) plasma levels, a recessive inheritance pattern, and heterogeneous genotype. The objective of this study was to identify the VWF defects in 265 European and Iranian patients with VWD3 enrolled in 3WINTERS-IPS (Type 3 Von Willebrand International Registries Inhibitor Prospective Study). All analyses were performed in centralized laboratories. The VWF genotype was studied in 231 patients with available DNA (121 [115 families] from Europe [EU], and 110 [91 families] from Iran [IR]). Among 206 unrelated patients, 134 were homozygous (EU/IR = 57/77) and 50 were compound heterozygous (EU/IR = 43/7) for VWF variants. In 22 patients, no or only one variant was found. A total of 154 different VWF variants (EU/IR = 101/58 [5 shared]) were identified among the 379 affected alleles (EU/IR = 210/169), of which 48 (EU/IR = 18/30) were novel. The variants p.Arg1659*, p.Arg1853*, p.Arg2535*, p.Cys275Ser, and delEx1_Ex5 were found in both European and Iranian VWD3 patients. Sixty variants were identified only in a single allele (EU/IR = 50/10), whereas 18 were recurrent (≥3 patients) within 144 affected alleles. Nine large deletions and one large insertion were found. Although most variants predicted null alleles, 21% of patients carried at least 1 missense variant. VWD3 genotype was more heterogeneous in the European population than in the Iranian population, with nearly twice as many different variants. A higher number of novel variants were found in the Iranian VWD3 patients.

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms.

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.

Bleeding symptoms in patients diagnosed as type 3 von Willebrand disease: Results from 3WINTERS-IPS, an international and collaborative cross-sectional study.

BACKGROUND: Type 3 von Willebrand's disease (VWD) patients present markedly reduced levels of von Willebrand factor and factor VIII. Because of its rarity, the bleeding phenotype of type 3 VWD is poorly described, as compared to type 1 VWD. AIMS: To evaluate the frequency and the severity of bleeding symptoms across age and sex groups in type 3 patients and to compare these with those observed in type 1 VWD patients to investigate any possible clustering of bleeding symptoms within type 3 patients. METHODS: We compared the bleeding phenotype and computed the bleeding score (BS) using the MCMDM-1VWD bleeding questionnaire in patients enrolled in the 3WINTERS-IPS and MCMDM-1VWD studies. RESULTS: In 223 unrelated type 3 VWD patients, both the BS and the number of clinically relevant bleeding symptoms were increased in type 3 as compared to type 1 VWD patients (15 versus 6 and 5 versus 3). Intracranial bleeding, oral cavity, hemarthroses, and deep hematomas were at least five-fold over-represented in type 3 VWD. A more severe bleeding phenotype was evident in patients having von Willebrand factor antigen levels < 20 IU/dL at diagnosis in the two merged cohorts. In type 3 patients, there was an apparent clustering of hemarthrosis with gastrointestinal bleeding and epistaxis, whereas bleeding after surgery or tooth extraction clusters with oral bleeding and menorrhagia. CONCLUSIONS: In the largest cohort of type 3 VWD patients, we were able to describe a distinct clinical phenotype that is associated with the presence of a more severe hemostatic defect.

Next Generation Sequencing in Newborn Screening in the United Kingdom National Health Service.

Next generation DNA sequencing (NGS) has the potential to improve the diagnostic and prognostic utility of newborn screening programmes. This study assesses the feasibility of automating NGS on dried blood spot (DBS) DNA in a United Kingdom National Health Service (UK NHS) laboratory. An NGS panel targeting the entire coding sequence of five genes relevant to disorders currently screened for in newborns in the UK was validated on DBS DNA. An automated process for DNA extraction, NGS and bioinformatics analysis was developed. The process was tested on DBS to determine feasibility, turnaround time and cost. The analytical sensitivity of the assay was 100% and analytical specificity was 99.96%, with a mean 99.5% concordance of variant calls between DBS and venous blood samples in regions with ≥30× coverage (96.8% across all regions; all variant calls were single nucleotide variants (SNVs), with indel performance not assessed). The pipeline enabled processing of up to 1000 samples a week with a turnaround time of four days from receipt of sample to reporting. This study concluded that it is feasible to automate targeted NGS on routine DBS samples in a UK NHS laboratory setting, but it may not currently be cost effective as a first line test.

Correction to: Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

[This corrects the article DOI: 10.1186/s12959-017-0143-3.].

Evaluation of a semi-automated von Willebrand factor multimer assay, the Hydragel 5 von Willebrand multimer, by two European Centers.

BACKGROUND: The phenotypic diagnosis of von Willebrand disease (VWD) is a multistep process with classification dependent on the quantification of von Willebrand factor (VWF) multimeric structure. VWF multimer analysis is a technically challenging, lengthy and non-standardised assay, usually performed in specialist laboratories. Recently, a new semi-automated multimer assay, the Hydragel 5 von Willebrand multimers (H5VWM) has become available. OBJECTIVES: This study, performed in two European centres, compared existing in-house multimer assays to the H5VWM in individuals with and without VWD. RESULTS: Overall agreement of 91.1% was observed in 74 individuals with normal VWF levels, 57 patients grouped as type 1 VWD, 33 type 2A, 16 type 2B, 28 type 2M, 11 type 2N. Patients tested following Desmopressin or VWF concentrate, with thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome were also evaluated. Many of the discrepancies between methods were in patients with genetic mutations linked to more than one type of VWD including p.R1374C/H and p.R1315C. Quantifiable multimer results were available within one working day. Densitometry improved the interpretation of the multimers with slight structural variations that were not apparent by visual inspection of the in-house method. CONCLUSIONS: 5VWM was a rapid, sensitive, standardised assay which used existing technology and could be included as an initial screen of VWF multimers in a VWD diagnostic algorithm in conjunction with traditional multimer analysis.

The common VWF single nucleotide variants c.2365A>G and c.2385T>C modify VWF biosynthesis and clearance.

Plasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in VWF, rs1063856 (c.2365A>G) and rs1063857 (c.2385T>C), although to date the mechanistic basis for their association with VWF level is unknown. Using genotypic/phenotypic information from a European healthy control population, in vitro analyses of recombinant VWF expressing both SNVs, and in vivo murine models, this study determined the precise nature of their association with VWF level and investigated the mechanism(s) involved. Possession of either SNV corresponded with a significant increase in plasma VWF in healthy controls (P < .0001). In vitro expression confirmed this observation and highlighted an independent effect for each SNV (P < .0001 and P < .01, respectively), despite close proximity and strong linkage disequilibrium between them both. The influence of c.2365A>G on VWF levels was also confirmed in vivo. This increase in VWF protein corresponded to an increase in VWF messenger RNA (mRNA) resulting, in part, from prolonged mRNA half-life. In addition, coinheritance of both SNVs was associated with a lower VWF propeptide-to-VWF antigen ratio in healthy controls (P < .05) and a longer VWF half-life in VWF knockout mice (P < .0001). Both SNVs therefore directly increase VWF plasma levels through a combined influence on VWF biosynthesis and clearance, and may have an impact on disease phenotype in both hemostatic and thrombotic disorders.

Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

BACKGROUND: Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients. METHODS: This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study. RESULTS: Ten patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein. CONCLUSIONS: Congenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients.

Diagnosing von Willebrand disease: genetic analysis.

Investigation of a patient with possible von Willebrand disease (VWD) includes a range of phenotypic analyses. Often, this is sufficient to discern disease type, and this will suggest relevant treatment. However, for some patients, phenotypic analysis does not sufficiently explain the patient's disorder, and for this group, genetic analysis can aid diagnosis of disease type. Polymerase chain reaction and Sanger sequencing have been mainstays of genetic analysis for several years. More recently, next-generation sequencing has become available, with the advantage that several genes can be simultaneously analyzed where necessary, eg, for discrimination of possible type 2N VWD or mild hemophilia A. Additionally, several techniques can now identify deletions/duplications of an exon or more that result in VWD including multiplex ligation-dependent probe amplification and microarray analysis. Algorithms based on next-generation sequencing data can also identify missing or duplicated regions. These newer techniques enable causative von Willebrand factor defects to be identified in more patients than previously, aiding in a specific VWD diagnosis. Genetic analysis can also be helpful in the discrimination between type 2B and platelet-type VWD and in prenatal diagnosis for families with type 3.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Clinical and laboratory variability in a cohort of patients diagnosed with type 1 VWD in the United States.

von Willebrand disease (VWD) is the most common inherited bleeding disorder, and type 1 VWD is the most common VWD variant. Despite its frequency, diagnosis of type 1 VWD remains the subject of debate. In order to study the spectrum of type 1 VWD in the United States, the Zimmerman Program enrolled 482 subjects with a previous diagnosis of type 1 VWD without stringent laboratory diagnostic criteria. von Willebrand factor (VWF) laboratory testing and full-length VWF gene sequencing was performed for all index cases and healthy control subjects in a central laboratory. Bleeding phenotype was characterized using the International Society on Thrombosis and Haemostasis bleeding assessment tool. At study entry, 64% of subjects had VWF antigen (VWF:Ag) or VWF ristocetin cofactor activity below the lower limit of normal, whereas 36% had normal VWF levels. VWF sequence variations were most frequent in subjects with VWF:Ag <30 IU/dL (82%), whereas subjects with type 1 VWD and VWF:Ag ≥30 IU/dL had an intermediate frequency of variants (44%). Subjects whose VWF testing was normal at study entry had a similar rate of sequence variations as the healthy controls (14%). All subjects with severe type 1 VWD and VWF:Ag ≤5 IU/dL had an abnormal bleeding score (BS), but otherwise BS did not correlate with VWF:Ag. Subjects with a historical diagnosis of type 1 VWD had similar rates of abnormal BS compared with subjects with low VWF levels at study entry. Type 1 VWD in the United States is highly variable, and bleeding symptoms are frequent in this population.

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

The UK National External Quality Assessment Scheme for heritable bleeding disorders.

Molecular genetic analysis of families with hemophilia and other heritable bleeding disorders is a frequently requested laboratory investigation. In the United Kingdom, laboratories undertaking genetic testing must participate in a recognized external quality assessment scheme for formal accreditation. The UK National External Quality Assessment Scheme (UK NEQAS) for heritable bleeding disorders was established in its current format in 2003, and currently has 27 registered participants in the United Kingdom, the European Union (EU), and the non-EU countries. Two exercises per annum are circulated to participants comprising either whole blood or DNA isolated from cell lines, and laboratories are allowed 6 weeks to analyze the samples and generate a report. Reports are assessed by a panel comprising clinicians and scientists with expertise in this area. Samples to date have involved analysis of the F8 gene (10 exercises), the F9 gene (4 exercises), and the VWF gene (3 exercises) and have comprised a wide spectrum of mutations representing the routine workload encountered in the molecular genetics laboratory. The majority of laboratories in each exercise passed, but a small number did not and reasons for failing included clerical errors, genotyping inaccuracies, and a failure to correctly interpret data. Overall we have seen an improvement in quality of reports submitted for assessment, with a more concise format that will be of value to referring clinicians and counsellors. Informal feedback from participants has been very positive.

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A.

The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.