Phenotype Analysis and Clinical Management in a Large Family with a Novel Truncating Mutation in RASGRP2, the Caldag-GEFI Encoding Gene
Authors: Desai Amrita , Jy Wenche , Bergmeier Wolfgang , Canault Mathias , Alessi Marie-Christine , Nurden Paquita , Ahn Yeon-Soong , Nurden Alan T. 
Published: November 2019

Abstract
The use of new generation sequencing technology has led to the recent identification of disease-causing mutations in the RASGRP2gene that encodes CalDAG-GEFI, a small GTPase-activating nucleotide exchange factor essential for integrin activation in platelets and megakaryocytes. Abrogation of CalDAG-GEFI function leads to a Glanzmann thrombasthenia (GT)-like platelet defect with a major loss in the platelet aggregation response. It is important to know the clinical profiles of patients with CalDAG-GEFI deficiency. Here we present variable phenotypes encountered in a large family with Jamaican ancestry associated with a truncating CalDAG-GEFI mutation.
The initial propositus (case 1) was a 51 yr-old Jamaican man with a GT-like platelet function phenotype and absent platelet aggregation to a range of physiologic agonists (including ADP and collagen) but a normal response to ristocetin. His platelets contained a full complement of the αIIbβ3 integrin and Sanger sequencing failed to reveal any mutations in the ITGA2B and ITGB3genes. His sister presented a similar profile and flow cytometry showed that platelets of both patients bound 5-30% of the normal levels of PAC-1 after platelet stimulation with ADP thereby confirming a much decreased αIIbβ3 function. Yet western blotting confirmed normal levels of αIIb and β3 in their platelets that also contained an abundant storage pool of fibrinogen (Fg). Clot retraction occurred but was reduced and platelets bound to a Fg-covered surface (result provided by the late Professor JG White, Minneapolis, MN). DNA from case 1 was analyzed by whole exome sequencing within the BRIDGE - Bleeding and Platelet Disorders consortium (Cambridge, UK) and a homozygous c.1490delT predicted to give rise to a p.F497fs*22 truncating mutation near to the C-terminal domain of CalDAG-GEFI was highlighted in Bordeaux. Consanguinity is present within the family. Sanger sequencing performed in Marseille showed that case 1 and his sister were homozygous for the c.1490delT and that 3 (mother, aunt and niece) out of 4 close family members were heterozygous for the mutation. Transfection of human embryonic kidney (HEK) cells with the mutated CalDAG-GEFI confirmed the expression of the truncated protein.
The propositus and his sister have experienced spontaneous bleeding and both have had excessive bleeding after surgery. In particular, the sister had episodes of severe epistaxis and heavy menstruation requiring frequent hospitalizations as a child with no benefit from platelet transfusions; she underwent hysterectomy and cholecystectomy under platelet transfusions and rFVIIa infusion. The propositus had episodes of gingival bleeding as a child and needed hospitalization for a tooth extraction. In 2006 he presented with headaches and was found to have a meningioma that was removed surgically (6 hours) under the cover first of platelet transfusions that again failed to prevent bleeding. This led to the use of rFVIIa that reduced bleeding despite a transient hypoxemia that led to a reduced dose and the adjunction of aminocaproic acid. The patient made a full recovery. Of interest, he has previously reported problems with wound repair namely abnormal scarring with excessive keloid. Also of note is the occurrence of ITP within 2 family members (aunt and her son who is otherwise asymptomatic) and his father died from bleeding complications following a major accident. We recommend the assembly of as much data as possible on the range of phenotypes and treatment of bleeding in families with inherited RASGRP2 defects.

Disclosures
No relevant conflicts of interest to declare.