Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method (original) (raw)
Related papers
2012
Type 2B von Willebrand disease (vWD) and platelet-type von Willebrand disease (pt-vWD) are two rare inherited bleeding disorders that remain under-diagnosed and incorrectly treated due to their shared clinical and laboratory features. Proper diagnosis of vWD requires a careful patient and family history and a panel of vWD assays. Acquired vWD (AvWD) may also complicate diagnosis of type 2 vWD, as it may present similar laboratory findings. DNA analysis of von Willebrand factor (vWF) exon 28 and platelet glycoprotein \ba(GPIba) gene can potentially aid in diagnosis when samples lack higher molecular weight multimers. To incorporate molecular testing, DNA was extracted from 63 deidentified patients' plasma samples that closely resembled type 2B vWD/pt-vWD by Western blot multimer analysis and from two positive plasma controls used for identification of type 2A and type 2B vWD by vWF multimer analysis. DNA analyses were performed on exon 28 of vWF and the GPIba gene. ABO blood typing was also determined, as it is a genetic modifier of vWF. The two positive plasma controls were identified as type 2A with the n.4517C>T (p.Serl506Leu) mutation and as type 2B with the n.3916C>T (p.Argl306Trp) mutation. From the 63 study plasma samples, none of the samples tested positively for 2B or pt-vWD mutations, two samples were type 1 vWD with n.3686T>G (p.Vall229Gly) and n. 4751A>G (p.Tyll584Cys), and two samples were found to be Type 2A vWD with n.4517C>T (p.Serl506Leu) and n.4789C>T (p.Argl597Trp) mutations. One specimen analysis was inconclusive. Three new silent polymorphisms were identified. The low mutation detection rate in this study highlights the importance of expanding the vWD coagulation panel to include assays such as collagen-binding assay (vWF:CB), Ristocetin Induced Platelet Aggregation (RIPA) and DNA analysis, as the routine coagulation assays lack sensitivity and reproducibility. The RIPA assay is more specific for diagnosis of type 2B and pt-vWD. Preanalytical sample handling is of concern in vWD diagnosis because cold storage of whole blood induces the loss of large and intermediate vWF multimers in healthy controls. AvWD laboratory findings are similar to those of vWD type 2, and require careful evaluation and analysis of the patient's bleeding history to exclude any role of vWD mutations. Due to the large size of the vWF gene and the heterogeneity of vWD presentation, diagnosis of vWD is very complex and no single assay or diagnosis approach is suitable for all patients.
Blood, 1996
Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Va1902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Va190-ON WILLEBRAND FACTOR (vWF) is a multimeric plasma glycoprotein that plays several important roles in hemostasis.' vWF binds platelets to the subendothelium at sites of vascular injury. In addition, vWF carries and stabilizes factor VI11 in the circulation. The vWF gene, located on chromosome 12, spans 178 kb and is interrupted by 51 introns.',' vWF is synthesized from an 8.7-kb mRNA and is expressed in endothelial cells and megakaryocytes. Synthesis of vWF is a complex, multistep process that results in assembly of multimers of up to 100 subunits.4 Studies in endothelial cells indicate that dimerization by disulfide bonding at carboxyl terminal domains occurs in the endoplasmic reticulum, whereas further multimerization takes place in Golgi or post-Golgi compartments through disulfide linkages at amino-terminal domains. In the blood vessel, vWF is constitutively secreted by endothelial cells. vWF is also stored within intracellular granules in both endothelial cells (Weibel-Palade bodies) and platelets (a-granules). These specialized granules release vWF in response to a variety of stimuli including vascular damage. The vWF stored within these granules contains larger multimers than those that are constitutively secreted by endothelial cells. These high molecular weight (HMW) multimers appear to be most effective in platelet binding,'.' therefore, rapid release of stored vWF into the circulation may be particularly useful at times of vessel injury. Defects in vWF result in von Willebrand disease (vWD), V
American Journal of Hematology, 2000
Willebrand factor (VWF) in which there is an increased affinity for the platelet glycoprotein Ib-IX-V receptor complex. Spontaneous binding of type 2B VWF to platelets and subsequent clearance from the plasma is thought to account for the characteristic phenotype of type 2B VWD. These gain-of-function mutations are due to single amino substitutions that are clustered within the functionally important A1 domain of VWF. We describe 13 individuals from five unrelated families in Australia with type 2B VWD, report their phenotypic abnormalities, and delineate their causative mutations. We confirm that the mutation Arg543Trp is also particularly common among families with type
American Journal of Hematology, 1998
von Willebrand Disease (vWD) is the most frequently inherited bleeding disorder in humans, and is caused by a qualitative and/or quantitative abnormality of the von Willebrand factor (vWF). A large number of defects that cause qualitative variants have been located in the A1 domain of the vWF, which contains sites for interaction with platelet glycoprotein Ib (GPIb). We have developed a new approach to detect mutations based on DdeI digestion and single-strand conformation polymorphism analysis. A segment of 487 nucleotides, extending from intron 27 to codon 1368 of the pre-pro vWF was amplified from genomic DNA. The cleavage with DdeI yields two fragments of appropriate size for this kind of analysis and confirms that the gene, rather than the pseudogene, is being investigated. Six families with type 2B vWD, one type 2M vWD family, and one another type 2A vWD family were studied. After sequencing the fragments with an altered electrophoretic pattern, we found four mutations previously described-R1308C, V1316M, P1337L, and R1306W-in patients with 2B vWD. The last one arose de novo in the patient. In addition, two new candidate mutations were observed: R1315C and R1341W. The first one was associated to type 2M vWD, whereas the one second cosegregated with type 2B vWD. The fact that these new mutations were not found in 100 normal alleles screened further supports their causal relationship with the disease. These mutations, which induce either a gain or a loss of function, further show an important regulatory role of this region in the binding of vWF to GPIb and its implications in causing disease. Am.
Laboratory Diagnosis and Molecular Classification of von Willebrand Disease
Acta Haematologica, 2009
CB, with only poor response for VWF:RCo in 2M and 2U, poor in 2A group I, 2B, 2C and 2D, and very poor or non-responsive in severe recessive VWD type 1 and 3. Homozygosity or double heterozygosity for nonsense (null) mutations in the VWF gene result in recessive VWD type 3. The combination of a nonsense and missense mutation or of two missense mutations (homozygous or double heterozygous) may cause recessive severe VWD type 1. Recessive VWD type 2A subtype IIC (2C) is caused by homozygous or double heterozygous gene defects in the D1-D2 domain. Homozygosity or double heterozygosity for a FVIII binding defect of the VWF is the cause of recessive VWD type 2N (Normandy) characterized by low FVIII:C, mild or moderate VWF deficiency and normal VWF multimers. Dominant VWD type 1/2E is a mixed quantitative and qualitative multimerization defect caused by a heterozygous cysteine mutation in the D3 domain resulting in abnormal multimerization with a secretion and clearance defect of VWF not due to increased proteolysis. Dominant VWD type 1 Vicenza is a qualitative defect with normal secretion but rapid clearance with equally low levels of FVIII:C, VWF:Ag, VWF:RCo, VWF:CB and the presence of unusually large VWF multimers in plasma due to a specific mutation (R1205H) in the D3 domain. Dominant VWD type 2M and 2U are caused by loss-of-function mutations in the A1 domain Abstract A complete set of laboratory investigations, including bleeding time, PFA-100 closure times, factor VIII (FVIII) coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), antigen (VWF: Ag) and propeptide (VWFpp), ristocetin-induced platelet aggregation (RIPA), multimeric analysis of VWF and the response of FVIII:C and VWF parameters to desmopressin (DDAVP), is necessary to fully diagnose all variants of von Willebrand disease (VWD) and to discriminate between type 1 and type 2 and between severe VWD type 1 and type 3. The response to DDAVP of VWF parameters is normal in pseudo VWD (mild VWF deficiency due to blood group O), in mild VWD type 1 and in carriers of recessive severe VWD type 1 and 3. The response to DDAVP is rather good but restricted followed by increased clearance in dominant type 1/2E, good but transient in mild type 2A group II, good for VWF: Published onlineActa Haematol 2009;121:71-84 72
International Journal of Clinical and Experimental Medical Sciences, 2019
A complete set of rapid activity and classical von Willebrand factor (VWF) assays for Willebrand disease (VWD) diagnosis was used in the present study to characterize VWD type 1, 2A, 2B and 2M patients due to mutations in the A1, A2 and A3 domains. The VWF:RCo/VWF:Ag, VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag ratios at cuttt off value of 0.7 separated VWD type 1 and LowVWF from VWD type 2. The results from the Brno cohort of VWD 2A patients with the G1579R mutation in the A2 domain in sixteen affected member from five families and in one case with the G1609R in the A2 domain revealed that the VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF: Ag ratios are marked decreased (range 0.03-0.27) to a similar degree as compared to VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios (range 0.03-0.27) due to the proteolytic loss of large and intermediate VWF multimers. The results in VWD 2B patients due to gain of ristocetin induced platelet agglutination (RIPA) function mutations R1306W, R1308C and R1341 in the A1 domain demonstrated that the ratios for VWF:GPIbM/VWF:Ag and VWF:GPIbR/VWF:Ag as compared to VWF:RCo/VWF:Ag ratio were markedly decreased in VWD 2B, whereas the VWF:GPIbM/VWF:Ag ratio was somewhat higher (range 0.32 to 0.36) in VWD 2M. VWD 2M patients due to loss of RIPA function mutation R1359K in the A1 domain are featured by decreased VWF ratios for WVF:RCo/Ag and VWF:GPIbR/Ag, but less decreased for the VWF:GPIbM/Ag ratio and normal VWF ratio for VWF:CB/Ag ratio the need to retain the VWF:CB assay to make a correct diagnosis of VWD 2M for its differentiation from VWD type 1. The G1415D mutation in the A1 domain is featured by decreased RIPA and decreased VWF:RCo/VWF:Ag ratio (VWD 2M) but normal values for VWF:CB/VWF:Ag, VWF:GPIbM/VWF:Ag and VWFGPIbR/VWF:Ag ratios consistent with VWD 2M. Double heterozygous P1266L/V1278I mutation in two patients and heterozygous E1292D/WT mutation in three patients in the A1 domain were diagnosed as VWD 2M or 1M associated with a secretion defect (SD). The Platelet Function Analyzer Closure Times (PFA-CT) are strongly prolonged in VWD 2A, 2B and 2M. and moderately prolonged between the upper limit of normal to 300 seconds in heterozygous mutated VWD type 1 patients.
World Journal of Hematology, 2016
products, resulting in loss of large VWF multimers with triplet structure of each individual VWF band. Dominant VWD type 2B due to a gain of function mutation in the A1 domain is featured by spontaneous interaction between platelet glycoprotein Ib (GPIb) and mutated VWF A1 followed by increased proteolysis with loss of large VWF multimers and presence of each VWF band. A new category of dominant VWD type 1 secretion or clearance defect due to mutations in the D3 domain or D4-C1-C5 domains consists of two groups� Those with normal or smeary pattern of VWF multimers.