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Molecular and clinical markers for diagnosis and management of type 1 von willebrands disease


The project was dependant upon recruitment of type 1 VWD families of a suitable size to allow the initial aims and objectives of the project to be addressed. 154 families were recruited and this number represents by far the largest number of such families studied to date. None were shown subsequently clearly shown to have VWD that would be classified as type 2 or 3. A detailed questionnaire was used to assess the clinical picture of all affected family members and the subsequent assessment of this by the use of a bleeding score devised by the partners, has resulted in a very comprehensive and measurable clinical assessment. Central confirmation testing of VWF parameters showed no significant differences between these results and those obtained locally at each recruiting centre This we believe strongly supports the local use of WHO International reference materials in such assays. VWF multimeric analysis was performed centrally. 2 levels of sensitivity were used (low and medium resolution) and overall 55% of index cases showed normal multimeric patterns. This rose to 66% when only the low-resolution technique was analysed. This analysis will identify the precise relationship between VWF mutimeric profiles and the pathology of the disease as an aid to diagnosis and classification. PFA-100 analysis was performed locally. The closure times for both cartridges (EPI and ADP) were normal for VWF:RCo levels above 60 IU/DL but below that there was a sharp increase. VWF binding to collagen (VWF:CB) was assessed centrally. Analysis of VWF:CB in index cases, affected family members and non-affected family members. In the cohort of type 1 VWD families there was no functional discrimination in assaying VWF:CB as compared to VWF:RCo. VWF/FVIII binding assays were performed centrally. 468 samples were tested, from 154 recruited families from whom samples were available for this test. Individuals from 12 families showed abnormal results.. It was concluded that reduced FVIII binding to VWF (type 2N VWD) is not common in patients recruited as type 1 VWD. Platelet VWF was assessed centrally .A total of 134 samples from index cases were available. Results are defined as Normal, Low and Dysfunctional. Results showed normal platelet VWF in 98 cases, low platelet VWF levels in 24 cases and dysfunctional platelet VWF in 7 cases. DDAVP was administered locally to 71 index cases (46%). This represents a large cohort of well-characterised type 1 VWD patients. Overall the majority of index cases responded well to DDAVP infusions. Linkage analysis was performed centrally. Of 153 families available for study 64 (42%) showed complete association (linkage) of diagnosed type 1 VWD with a specific VWF gene. 44 (29%) did not show the association and 45 (29%) were non-informative. Overall of the informative families, 59% showed linkage between the diagnosis of VWD within the family and a specific VWF gene and 41% did not. Therefore the majority of families within this type1 VWD cohort showed linkage. Haplotype analysis was performed centrally. Three SNPs in the VWF gene were analysed.. All individuals entered in the study were analysed, where DNA quantity and quality were sufficient; 153 index cases, 278 affected family members, 312 unaffected family members and 1,128 normal controls. Analysis of each of the 3 markers was by TaqMan allelic discrimination using the 5¿ nuclease activity. Overall frequencies of each SNP in index cases compared with normal controls was the same. ABO blood group analysis was performed centrally. Overall 38% of controls were ABO group O, 47% group A, 11% group B and 4% group AB. In the index cases 67% were group O (p <0.0001), 28% group A, 3% group B and 2% group AB. The data confirms previous reports of increased levels of ABO group O in individuals with VWD. VWF gene mutation analysis formed a major part of the project. Prior to the project only a few mutations had been reported/detected in type 1 VWD patients and to date only 14 are recorded on the ISTH VWD database, which is maintained from Sheffield UK. In all, affected individuals from 153 families were analysed. Overall 124 candidate mutations in 103 patients have been identified and confirmed. 42 of the 146 cases (29%) have no mutation identified. This important aspect of the project has clearly shown that mutations in the VWF gene are the major cause of VWD in type 1 disease and that VWF gene analysis should be performed in all cases to assist in the diagnosis and to help understand the inheritance of the disease. VWF in vitro gene expression studies have been commenced on 29 of the mutations identified in the project. To date it would appear that most of the mutations result in reduced secretion of mutant alone or mutant plus co-transfected wild type VWF from the cells in vitro.