Identification of two novel mutations in RASGRP2 affecting platelet CalDAG-GEFI expression and function in patients with bleeding diathesis

Abstract

The RASGRP2 gene encodes the Ca²⁺ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn²⁺-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.

Keywords:

• Bleeding
• dysfunction
• platelets
• RASGRP2
• signaling

Acknowledgments

This study was conducted according to the aims of the Project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” (approved by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis). We thank the families for providing samples. We also thank Constantino Martínez and José Padilla for their help in some platelet studies and Sanger sequencing.

Declaration of interest

The authors declare no competing financial interests.

Funding

JMB group is supported by Gerencia Regional de Salud (GRS 1370/A/16). Research by the group of J.R. is supported by grants from Instituto de Salud Carlos III and Feder (PI14/01956 and CB15/00055). Research by the group of S.P.W is supported by the British Heart Foundation (RG/PG/13/36/30275; RG/09/007). W.B. is supported by grants from the National Institutes of Health (R01 HL130404 and R01 HL121650) and the American Heart Association (14EIA18910004).

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Additional information

Funding

Research by the group of J.R. is supported by grants from Instituto de Salud Carlos III and Feder (PI14/01956 and CB15/00055). Research by the group of S.P.W is supported by the British Heart Foundation (RG/PG/13/36/30275; RG/09/007). W.B. is supported by grants from the National Institutes of Health (R01 HL130404 and R01 HL121650) and the American Heart Association (14EIA18910004).