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Severe type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14-15, 80,830bp (-41510_657+7928A*del) and 2,231bp [1534-2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

Background: Von Willebrand disease type 3 VWD is an autosomal-recessively inherited severe bleeding disorder with a homogeneous phenotype on the basis of very heterogeneous genotypes. Many different molecular defects have been reported to date. We tried to assess the molecular background of Indian and Greek patients with VWD type 3 by doing a complete VWF gene screen in all index patients., Materials and Methods: We investigated 21 unrelated Indian and six Greek patients with type 3 VWD. Mutation screening was done by PCR and direct sequencing of the coding VWF exons 2-52 including flanking intron sequences., Results: The diagnosis of VWD type 3 could be confirmed by the detection of null alleles or two mutations each in 22 patients. In one patient only one heterozygous mutation was identified. In four patients no mutations were identified for unknown reasons. Most of the defects cause null alleles. Eight patients had homozygous nonsense mutations - R1659X (6 patients), W553X (1 patients) and L1267X (1 patient); 2 patients were compound heterozygous - R324X/R373X and N318K/Q565X; 3 patients had small insertions - 3259insT, 3737insCC and 7173insT; 2 patients had small deletions - 3938delG and 1381delG; 2 patients had a duplication of 8 bp (duplAGTGTGGA) in exon 28 and a missense mutation (R273W) in exon 7; one patient had a heterozygous mutation K1794E (second mutation not identified); 5 patients had gene conversions between VWF and its pseudogene (117 bp to 335 bp in length corresponding to the 5' end of exon 28). The mutations as part of the gene conversion were - S1263P, P1266L, V1279I, Q1311X, A1317, I1343V, V1360A, and F1369I., Conclusion: VWD type 3 is caused by a broad variety of mutations distributed over the entire VWF sequence. As expected most mutations cause null alleles (16/23). The most common molecular defects found were gene conversions and R1659X in exon 28.

von Willebrand disease (VWD), the most common inherited bleeding disorder, is very heterogeneous, both in its phenotype and genotype. One particular molecular mechanism of VWD is due to recombination events between the true gene and its pseudogene on chromosome 22. We assessed the frequency and extension of such events in 50 multi-ethnic index patients with severe VWD type 3 and in five index patients with VWD type 2M Vicenza. One additional unclassified patient had been diagnosed with possible VWD in Russia solely on a clinical basis. Gene conversions, previously thought to be rare events, were identified in >10% of our study population: in six multi-ethnic patients with severe VWD type 3, in one patient with VWD type 2M Vicenza and the Russian patient was finally diagnosed with VWD type 2B New York/Malmoe. Our results suggest a significant contribution of this particular molecular mechanism to the manifestation of VWD. The location of the gene conversions, their extension and their occurrence as homozygous, compound heterozygous or heterozygous mutations determines the resulting phenotype.