Identification of p.W246L As a Novel Mutation in the GP1BA Gene Responsible for Platelet-Type von Willebrand Disease

Abstract

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) < 0.2, normal VWF propeptide/VWF:Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, in his mother, and in 100 healthy control subjects. We identified a heterozygous substitution G > T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient.

Progress in understanding the diagnosis and molecular genetics of macrothrombocytopenias

“…Other mutations in the GP1BA gene have been described so far, M239V (Russell & Roth, ), G233S (Matsubara et al , ), a 27‐bp in‐frame deletion (Othman et al , ), D235Y (Enayat et al , ) and W246L (Woods et al , ). All of these mutations induce a gain of function resulting in an enhanced affinity for VWF and enhanced platelet aggregation to a low dose of ristocetin (0·5 mg/ml), which normally does not induce platelet agglutination.…”

Platelet-Type von Willebrand Disease: Toward an Improved Understanding of the “Sticky Situation”

“…A shorter survival indicated by an increased VWFpp: VWF ratio in patients with type 2B VWD was reported, where an enhanced affinity of the abnormal VWF to normal platelet GPIbα is observed. 19 In their report, 1 Woods et al showed that the VWFpp and VWFpp:VWF ratio were unexpectedly normal in PT-VWD Try246Leu mutation. This may indicate that the clearance and survival of VWF may be two different mechanisms in patients with PT-VWD.…”

“…We are pleased to highlight in this issue of Seminars in Thrombosis & Hemostasis, the study by Wood et al, 1 wherein the authors describe a novel mutation in the platelet GP1BA gene creating a hyperresponsive GPIbα protein-a receptor for von Willebrand factor (VWF)-and causing platelet-type von Willebrand disease (PT-VWD). This is a new naturally occurring mutation in a 23-year-old male patient and is considered the sixth reported mutation thus far in patients described with this disease worldwide.…”

“…Third, for the first time, the level of VWF propeptide (VWFpp) and VWFpp/VWF:Ag ratio was reported in a patient with PT-VWD. 1 VWFpp is a 741 amino acid portion that gets cleaved from mature VWF by proteolysis. After cleavage, the VWFpp remains in noncovalent association with the VWF multimers, and both are stored together in the α-granules (megakaryocytes/platelets) or Weibel-Palade bodies (endothelial cells).…”

“…25 Consistent with this hypothesis are site-directed mutagenesis studies involving a change of Lys at 237 to Val, which also resulted in a gain of function phenotype characterized in the CHO cell system. 30 Using the GPIbα crystal structure, we examined the newly reported mutation Trp230Leu (Trp246Leu) by Woods et al 1 ►Fig. 1A shows the position of Trp230 within the unliganded GPIbα R-loop structure illustrating that it forms a hydrogen bond to the Gln232 side chain and also packs against the side chain of Met239.…”