Second Report of A GPIbα LRR7 Variant (P.Leu194Phe) Supporting the Pathogenicity of Mutations Outside The C-Terminal Domain In Platelet-Type Von Willebrand Disease

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant platelet bleeding disorder, with fewer than 60 patients reported worldwide so far.[ 1 ] While type 2B von Willebrand disease (type 2B VWD) results from a functionally abnormal von Willebrand factor (VWF) molecule arising from mutations in the VWF gene, PT-VWD is caused by hyperresponsive platelets resulting from mutations in the platelet GP1BA gene (GPIα).[ 2 ] [ 3 ] PT-VWD may be misdiagnosed in approximately 15% of those with type 2B VWD.[ 4 ] GPIbα is the major ligand-binding subunit of the GPIb-IX-V complex that binds VWF.[ 5 ] The N-terminal domain of GPIbα consists of eight leucine-rich repeats (LRR) that forms a concave shape.[ 6 ] The interaction between VWF A1 domain and GPIbα occurs in the presence of high shear stress.[ 7 ] Affected patients with PT-VWD suffer from mild to moderate mucocutaneous bleeding, low VWF activity compared with antigen, decreased high-molecular-weight (HMW) VWF multimers, variable degree of thrombocytopenia and typically platelet agglutination in response to low concentrations of ristocetin.[ 8 ] To date, only eight variants causing PT-VWD have been described, five in the C-terminal disulfide loop of the VWF-binding domain of GPIbα, two in the LRR domain, and one deletion in the macroglycopeptide.[ 9 ] [ 10 ] The C-terminal disulfide loop, known as the β-switch, is the region crucial for the binding to the A1 domain of VWF, and it harbors five of the eight gain-of-function (GoF) variants of GPIbα causing PT-VWD described so far.[ 9 ] Recently, Bury et al and Monteiro et al reported the case of two patients with moderate bleeding symptoms, increased ristocetin induced platelet agglutination (RIPA) and GoF GP1BA variants in the LRR domain (c.G380A/p.Arg127Gln and c.580C > T/p. Leu194Phe), outside the C-terminal loop, showing that a single substitution in this LRR structure may also cause a conformational change in the GPIbα binding site, directly affecting the VWF ligation.[ 9 ] [ 10 ] Bury et al identified the GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD.[ 9 ] Hydrogen-deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. The (p.Leu194Phe) variant affecting the LRR7 domain of GPIbα with moderate bleeding phenotype described by Monteiro et al was in a 17-year-old boy adopted patient; thus, there were no other available family members to assess, and segregation studies were not performed.[ 10 ] Moreover, study of HMW VWF multimers or functional studies confirming the potential impact of this variant on the structure and/or function of GPIbα were not realized, leaving some uncertainty regarding the pathogenicity of this variant. Supporting these findings, we herein describe another family with moderate bleeding symptoms, normal platelet counts, and increased RIPA harboring as yet reported p.Leu194Phe GP1BA GoF variant in LRR7. A 1-year-old boy was referred to us for a possible finding of VWD before a cranioplasty for trigonocephaly. There was no family history of bleeding disorders in his parents, uncles and aunts, first cousin, or grandparents. He was born by cesarean section with vacuum extraction; he did not develop hematomas after intramuscular vaccinations or blood sampling. He has not presented with ecchymosis so far, nor prolonged bleeding from scratches. He has no history of gingival bleeding, no erupted teeth, and no bleeding at umbilical cord detachment. Standard laboratory evaluation showed that platelet counts ranged from 194 × 10 9 /L to 415 × 10 9 /L with normal Mean Platelet Volume (MPV; 8.9 fL). Closure times (platelet function assay–PFA-200) performed using collagen/adenosine diphosphate (153 second) and collagen/epinephrine cartridges (182 second) were moderately increased while FVIII:C (43%), VWF:Ag (immunologic quantification by HemosIL AcuStar VWF Antigen from Werfen; 55%) and VWF:GPIbR (functional activity of VWF measured by ristocetin induced binding to recombinant GPIb fragment; HemosIL VWF Ristocetin Cofactor Activity reagent from Werfen; 36%) were moderately decreased ([ Table 1 ]). His blood group was A + . Platelet glycoprotein levels, assessed by flow cytometry, were normal (35,847 sites/plt), whereas RIPA was increased (70% of normal) at a low ristocetin concentration (0.625 mg/mL). VWF multimer pattern was also analyzed by SDS-agarose electrophoresis, showing a normal result ([ Fig. 1 ]). Table 1 ISTH-BAT bleeding scores and laboratory results of the different reported mild forms of PT-VWD Bleeding scores and laboratory parameters Normal values Our patient Mother Patient from Monteiro et al[ 10 ] Patient from Bury et al[ 9 ] GP1BA variant – c.580C > T/p. Leu194Phe c.580C > T/p. Leu194Phe c.580C > T/p. Leu194Phe c.G380A/p.Arg127Gln GPIbα domain – LRR7 LRR7 LRR7 LRR5 Sex, age (y) – Male, 1 y Female, 39 y Male, 17 y Male, 14 y Bleeding score (International Society of Thrombosis and Haemostasis-Bleeding Assessment Tool)