Novel mutations in RASGRP2 encoding for CalDAG-GEFI abrogate Rap1 activation causing platelet dysfunction

In addition to mutations in ITG2B or ITGB3 genes causing defective αIIbβ3 expression/function in Glanzmann's thrombasthenia patients, platelet dysfunction can be due to genetic variability in proteins mediating inside-out activation of αIIbβ3. The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation (NGS) and whole exome sequencing (WES) to identify two novel function-disrupting mutations in RASGRP2 accounting for the bleeding diathesis and platelet dysfunction in two unrelated families. Using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year old child of Chinese origin (Family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In two Spanish siblings from Family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients and, using a novel in vitro assay, the nucleotide exchange activity was found to be dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.