title: Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.
abstract: In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate 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 sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By 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 2 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 by using a novel in vitro assay, we found that the nucleotide exchange activity was 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.
authors.affiliation: Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Instituto Murciano de Investigaciones Biomédicas-Arrixaca, Murcia, Spain;
authors.forename: María Luisa
authors.initials: ML
authors.lastname: Lozano
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Aaron
authors.initials: A
authors.lastname: Cook
authors.affiliation: Department of Hematology, Instituto de Investigación Biomédica de Salamanca-Hospital Universitario de Salamanca, Salamanca, Spain;
authors.forename: José María
authors.initials: JM
authors.lastname: Bastida
authors.affiliation: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: David S
authors.initials: DS
authors.lastname: Paul
authors.affiliation: Servicios de Hematología, Pediatría y Oncología Infantil, Hospital de Cruces, Bilbao, Spain;
authors.forename: Gemma
authors.initials: G
authors.lastname: Iruin
authors.affiliation: Unidad de Hemostasia y Trombosis, Servicio de Hematología y Hemostasia, Hospital Universitario Politécnico de la Fe, Valencia, Spain;
authors.forename: Ana Rosa
authors.initials: AR
authors.lastname: Cid
authors.affiliation: Servicios de Hematología, Pediatría y Oncología Infantil, Hospital de Cruces, Bilbao, Spain;
authors.forename: Rosa
authors.initials: R
authors.lastname: Adan-Pedroso
authors.affiliation: Department of Hematology, Instituto de Investigación Biomédica de Salamanca-Hospital Universitario de Salamanca, Salamanca, Spain;
authors.forename: José
authors.initials: J
authors.lastname: Ramón González-Porras
authors.affiliation: Department of Hematology, Instituto de Investigación Biomédica de Salamanca-Hospital Universitario de Salamanca, Salamanca, Spain;
authors.forename: Jesús María
authors.initials: JM
authors.lastname: Hernández-Rivas
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Fletcher
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Ben
authors.initials: B
authors.lastname: Johnson
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Neil
authors.initials: N
authors.lastname: Morgan
authors.affiliation: Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Instituto Murciano de Investigaciones Biomédicas-Arrixaca, Murcia, Spain;
authors.forename: Francisca
authors.initials: F
authors.lastname: Ferrer-Marin
authors.affiliation: Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Instituto Murciano de Investigaciones Biomédicas-Arrixaca, Murcia, Spain;
authors.forename: Vicente
authors.initials: V
authors.lastname: Vicente
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC.
authors.forename: John
authors.initials: J
authors.lastname: Sondek
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; and.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC; McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Bergmeier
authors.affiliation: Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Instituto Murciano de Investigaciones Biomédicas-Arrixaca, Murcia, Spain;
authors.forename: José
authors.identifier: 0000-0003-4225-6840
authors.initials: J
authors.lastname: Rivera
mesh_terms: D019943:Amino Acid Substitution; D001792:Blood Platelets / Q000378:metabolism* / Q000473:pathology; D002648:Child; D004789:Enzyme Activation / Q000235:genetics; D005091:Exons; D005260:Female; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D015539:Platelet Activation / Q000235:genetics*; D040201:Platelet Membrane Glycoprotein IIb / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism; D013915:Thrombasthenia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C061248:ITGB3 protein, human; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; C415483:RASGRP2 protein, human; D020827:rap1 GTP-Binding Proteins