Search for mutations in a segment of the exon 28 of the human von Willebrand factor gene: New mutations, R1315C and R1341W, associated with type 2M and 2B variants

Abstract

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.

Investigation of von Willebrand Factor Gene Mutations in Korean von Willebrand Disease Patients

“…were located in domain A1 and all previously reported in association with type 2B VWD [13][14][15][16][17][18]. Another was V1-279I (3835 G>A) reported in type 1 VWD [3].…”

“…Methods : In 40 known vWD patients mutations of vWF gene were sought by direct sequencing of PCR products targeting exons 18,19,20,26,28 and 52 frequently implicated as the locations of mutation. For factors other than VWF gene contributing to VWD phenotype, we tested ABO blood group and measured ADAMTS13 activity in VWD patients.…”

The Montreal platelet syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation

“…14,[18][19][20] As anticipated, the mutant VWF displays a high affinity to platelets, 19 and many patients have persistent or transient thrombocytopenia, [21][22][23] large platelets, and spontaneous platelet aggregation. 4,6 One illustrative example is a 5-year-old boy with the heterozygous V1316M VWF mutation who presented with macrothrombocytopenia (platelet count ϳ 20 ϫ 10 9 /L), platelet clumping on blood film, and loss of plasma high and intermediate molecular weight VWF multimers.…”

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

“…The other group includes six structures, of which, one, labeled as "WT-NC," was a WT A1 reconstructed through taking the body of A1 from the crystal structure of the complex WT-A1/GPIb␣ (Protein Data Bank code 1SQ0) (20) and adding the N-terminal arm with the same protocol used in R543Q and I546V, and others, including a GOF mutant E501A (32) and four type 2B mutants (R578Q, R578W, R578P, and R578L) (33)(34)(35), were modeled per homology by replacing the corresponding side chain in WT A1 and subsequently performing 1000 steps of minimization in vacuo while all atoms except the mutated residue were fixed. The WT-NC A1 was used for a negative test to verify whether the reconstructed R543Q and I546V mutants were rational or not, and each GOF mutation (E501A, R578Q, R578W, R578P, and R578L) was selected to further examine whether it would cause a change of the dynamic properties of A1.…”

“…To verify this hypothesis, we further examined the dynamic properties of other five mutants (E501A, R578Q, R578W, R578P, and R578L) via MD simulations (see "Experimental Procedures"). The mutant E501A has gain of function phenotype (32), and R578Q, R578W, R578P, and R578L are known as the type 2B mutations (33)(34)(35).…”