Phenotype analysis and clinical management in a large family with a novel truncating mutation in RASGRP2, the CalDAG‐GEFI encoding gene

Abstract Essentials Mutations in the RASGRP2 gene represent a new inherited platelet function disorder. Report a five generation family with a novel frameshift mutation in RASGRP2 (p.F497Sfs*22). Partial platelet activation defect and serious bleeding complications in homozygous patients. Patients respond to recombinant Factor VIIa infusion but not platelet transfusions. Background Genetic variants in the RASGRP2 gene encoding calcium and diacylglycerol‐regulated guanine nucleotide exchange factor I (CalDAG‐GEFI) represent a new inherited bleeding disorder linked to major defects of platelet aggregation and activation of αIIbβ3 integrin. They are of major interest as CalDAG‐GEFI is receiving attention as a potential target for antiplatelet therapy for prevention and treatment of cardiovascular disorders including arterial thrombosis and atherosclerosis. Objectives To better understand the phenotypical and clinical profiles of patients with CalDAG‐GEFI deficiency. Patients We report a five‐generation family with a novel truncating CalDAG‐GEFI mutation detailing clinical management and phenotypic variability. Results Patients IV.6 & IV.4 manifested with episodes of serious mucocutanous bleeding or bleeding after surgery not responding to platelet transfusion but responding well to recombinant Factor VIIa infusions. Their blood counts and coagulation parameters were normal but platelet aggregation to ADP and collagen was defective. Further work‐up confirmed normal levels of αIIb and β3 in their platelets but decreased αIIbβ3 function. DNA analysis by whole exome sequencing within the BRIDGE‐BPD consortium (Cambridge, UK), allowed us to highlight a homozygous c.1490delT predicted to give rise to a p.F497Sfs*22 truncating mutation near to the C‐terminal domain of CalDAG‐GEFI. Sanger sequencing confirmed that both patients were homozygous for the c.1490delT and 3 out of 4 close family members were heterozygous. Conclusions A long‐term prospective study is warranted for full clinical exploration of CalDAG‐GEFI to understand the bleeding phenotyes and their management.


| INTRODUCTION
Platelet adhesion and aggregation to form a primary hemostatic plug is an essential step in stopping bleeding. Inherited disorders of platelet production and/or function lead to increased spontaneous or surgical mucocutaneous bleeding and greater mortality in patients harboring these defects. 1,2 Integrin αIIbβ3, formerly known as GPIIb/IIIa, is the adhesive protein receptor that in an activated state mediates platelet aggregation. 3 αIIbβ3 undergoes conformational changes from a bent to an extended and open form in stimulated platelets; changes brought about by "inside-out" signaling through GTPase Ras-proximate-1 (Rap1) promoted talin and kindlin interactions with β3. 4 In a major pathway, activated phospholipase C causes increased intracellular Ca 2+ that in turn activates calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) which then activates Rap1. 5,6 Significantly, the role of CalDAG-GEFI extends to other integrins including those in white blood cells. The patho-physiological importance of CalDAG-GEFI is highlighted by studies on knockout mice, which exhibit marked defects in hemostasis, arterial thrombosis and atherosclerotic plaque formation; studies that have suggested CalDAG-GEFI as a therapeutic target for thrombosis and atherosclerosis. [7][8][9][10] Abrogation of CalDAG-GEFI function in man leads to a Glanzmann thrombasthenia (GT)-like bleeding disorder and a major loss of platelet aggregation. 11 Next generation sequencing technology has facilitated the identification of disease-causing mutations in the RASGRP2 gene that encodes CalDAG-GEFI; genetic variants that have segregated with a severe bleeding diathesis in families from France, Spain and Japan. [12][13][14] However, the number of cases studied remains small and the description of clinical profiles within families having genetically altered CalDAG-GEFI function is incomplete. Here we present the clinical management of a large family with Jamaican ancestry with unexpectedly variable phenotypes associated with a novel truncating CalDAG-GEFI mutation. Figure 1A gives the pedigree of a five-generation consanguineous family of Jamaican origin, genotyped members are highlighted (+/+, +/− or −/−) with + representing presence of the mutation. Essential laboratory parameters are given in Table 1.

| Patient IV.6
During childhood, a Jamaican man, the index case IV.6 (+/+), now middle-aged, experienced excessive gum bleeding and easy bruising leading to a suspected diagnosis of GT (see George et al. 15 , for the GT phenotype). His first major hemorrhage at age 11 after a dental extraction needed hospitalization, where he received platelet transfusions with no apparent benefit. He was then given fresh frozen plasma (FFP) and whole blood products. Excessive bleeding during elective hernia repair was controlled by surgical glue although he again received FFP

Essentials
• Mutations in the RASGRP2 gene represent a new inherited platelet function disorder.
• Report a five generation family with a novel frameshift mutation in RASGRP2 (p.F497Sfs*22).
• Partial platelet activation defect and serious bleeding complications in homozygous patients.
• Patients respond to recombinant Factor VIIa infusion but not platelet transfusions.

| Patient IV.4
The affected sister of the index case (+/+), now aged 55, manifested extensive petechial rash at birth and early work-up also led to a misdiagnosis of GT. She had severe epistaxis during childhood requiring frequent hospitalizations, nasal packing, and whole blood and platelet transfusions. Epistaxis stopped during adolescence but she experienced menorrhagia at menarche. Surgical bleeding during cholecystectomy and hysterectomy led to platelet transfusions but like her brother there was little benefit. During a recent appendectomy, she initially received platelets without benefit but abnormal bleeding stopped after the administration of rFVIIa. She currently has occasional gum/nose bleeding but is otherwise healthy with no history of abnormal wound healing, infections or cardiac problems.

| Other genotyped family members
The

| GENETIC AND BIOLOGICAL STUDIES (RESULTS)
The and III.2 in Figure 1C). This segregation fits with the mutation being the cause of the platelet defect. The localisation of this novel mutation is compared to that of other reported variants [12][13][14] for CalDAG-GEFI in Figure 1D. , wild-type mice and Caldaggef1 knock-out (Cdg1−/−) mice; technical details were as described. 13 The intact CalDGAG-GEFI protein is missing for the index case and his sister but present in mother and WT mice

| DISCUSSION
CalDAG-GEFI-mediated Rap1 activation is rapid and reversible and is necessary for full platelet activation and stable thrombus formation. Similar to other patients with mutations in RASGRP2 [12][13][14] or animals with altered CalDAG-GEFI-Rap1 signaling, 7,18 platelet aggregation in our patients was partially impaired, with the strongest defect seen in response to activation with ADP or collagen.
Since the first report from Canault et al., 12 two other groups have described mutations in RASGRP2 associated with an absence of CalDAG-GEFI expression or function; 3 cases in 2 Spanish families and a young Japanese woman. 13,14 Severe bleeding is a consistent finding recapitulating the findings in knock-out mouse models for CalDAG-GEFI. 7,8 For the family described here, the index case and his sister, both with homozygous expression, have experienced spontaneous bleeding and excessive bleeding after surgery.
We were surprised by the severity of the hemostatic defects in our patients, as mice expressing CalDAG-GEFIΔC1 in blood cells exhibited a less severe bleeding phenotype than CalDAG-GEFI knockout mice. 8 However, the CalDAG-GEFIΔC1 mice were generated by transplantation of retrovirally transduced bone marrow cells into lethally irradiated mice, i.e., an approach that may have led to chimeric mice with residual amounts of circulating platelets expressing intact CalDAG-GEFI. It is also important to remember that defective function of CalDAG-GEFI in endothelial cells and/or extravascular cells may contribute to the more severe hemostasis defect in our patients when compared to the chimeric mice. Lastly, our Western blotting studies suggest that the frameshift mutation in our patients affects both the size and the expression level of CalDAG-GEFI in platelets. Less protein with impaired function would explain markedly impaired platelet function and moderate to severe bleeding in the patients. Intriguingly, both homozygous patients gained no observable benefit from platelet transfusions; but when rFVIIa was used hemostasis was restored. This was probably life-saving for the index case (IV.6) who developed a meningioma that required major surgery (6 hours). While the development of a mengioma may be coincidental, it is of interest that CalDAG-GEFI is highly expressed in the brain. 19 The index case and his father experienced problems with wound repair namely abnormal scarring with excessive keloid. Pasvolsky et al. 20 reported a case of Leukocyte Adhesion Deficiency III (LAD III) with defective expression of CalDAG-GEFI manifesting poor wound healing; however, this was later corrected to a kindlin-3 deficiency. 21 Kindlin-3 is normally expressed in the platelets of IV.6 and IV.4 ( Figure 1F). Although mildly altered neutrophil function associated with decreased activity of β1 or β2 integrins has been reported for the Spanish cases of inherited CalDAG-GEFI deficiency it was not so for the Japanese and French patients or for the present family. [12][13][14] We did not perform in vitro studies to assess leukocyte function as none of our patients show increased suspectibity to infections or infectious complications.
Clearly, the phenotype of CalDAG-GEFI deficiency differs from Leukocyte Adhesion Deficiency type III where the loss of kindlin-3, also essential for integrin activation, combines bleeding with infections. 21,22 Transient ITP occurred in one heterozygous family member (III.2), but thrombocytopenia in two other family members was less well defined and did not always segregate with the RASGRP2 genotype. In summary, we have reported the largest kindred so far associated with RASGRP2 mutations and the variety of associated phenotypes together with the observed lack of efficacity of platelet transfusions recommend the assembly of as much data as possible to define the phenotypes and treatment of bleeding in families with inherited CalDAG-GEFI expression and/or function. This is particularly so as CalDAG-GEFI is receiving increasing attention as a new target for antiplatelet and antithrombotic therapy for prevention and treatment of cardiovascular disorders.