SRC-RELATED THROMBOCYTOPENIA - A FINE LINE BETWEEN A MEGAKARYOCYTE DYSFUNCTION AND AN IMMUNE MEDIATED DISEASE

Autores:

VERÓNICA PALMA BARQUEROS1, Nuria Revilla Calvo1, CARLO ZANINETTI2, ANA Mª GALERA MIÑARRO3, ANA SANCHEZ FUENTES4, ANA ZAMORA CANOVAS5, NATALIYA BOHDAN 1, José Padilla Ruiz1, ANA MARIN QUILEZ6, AGUSTIN RODRIGUEZ ALEN7, José Luis Fuster Soler3, ANDREAS GREINACHER2, Vicente Vicente García1, JOSE MARIA BASTIDA BERMEJO8, José Rivera Pozo1, Mª Luisa LOZANO ALMELA1

Afiliaciones:

(1) HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL, IMIB-Arrixaca, España
(2) Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald Hospital,, Alemania
(3) TRASPLANTE HEMATOPOYÉTICO / TERAPIA CELULAR, IMIB-Arrixaca, España
(4) Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, España (Región de Murcia)
(5)
(6) department of Hematology, Complejo Asistencial Universitario de Salamanca (CAUSA), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca, España (Castilla y León)
(7) Hospital Virgen de la Salud, Complejo Hospitalario de Toledo, España (Castilla la Mancha)
(8) Department of Hematology, Complejo Asistencial Universitario de Salamanca (CAUSA), Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca, España (Castilla y León)

Comunicación:

Antecedentes:

Src-related thrombocytopenia (SRC-RT) is a rare autosomal dominant inherited thrombocytopenia due to the p.E527K heterozygous germline gain-of-function variant of Src. To date, genetic diagnosis of the disease has only been reported in seven patients from three unrelated families. We report a new three-generation pedigree with SRC-RT, exhibiting a syndrome that, from the therapeutic perspective, is difficult to differentiate from immune thrombocytopenia (ITP).

Métodos:

The members of this family were recruited through the Spanish multicenter project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders”. All participants gave written informed consent. Clinical records were reviewed. Blood samples were drawn (EDTA, sodium citrate, serum) for the different studies. DNA from two index cases was analyzed by a predesigned high throughput sequencing (HTS) gene panel using an Ion Torrent PGM platform. Sequences were annotated and candidate genetic variants were confirmed in these probands, and segregated in the family by and Sanger sequencing. Extensive platelet phenotyping (full blood counts, immature platelet fraction, blood smears, aggregation activation and secretion, electron microscopy, immunofluorescence staining of α-granule proteins ..) was performed. Plasma levels of various cytokines were measured using Luminex xMAP® technology. Protein content and tyrosine phosphorylation of specific targets (Src, BTK, phospholipase Cγ2) were evaluated in platelet lysates by standard immunoblotting using specific antibodies .Human peripheral blood mononuclear cells (PBMCs) were isolated stained with anti CD19 and stimulated with 20 μg/mL soluble anti-IgM antibody. Cells were fixed permeabilized using the Intrasure kit (BD), and evaluated by flow cytometry for P-Syk, P-BTK and P-ERK content.

Resultados:

Patients with the Src p.E527K variant presented with variable thrombocytopenia, increased immature platelet fraction, and with a significant proportion of large-giant platelets. Remarkably, four adults from the family were diagnosed with ITP and underwent splenectomy, achieving sustained platelet counts above 75x109/L for several years; increases in platelet counts were also observed after corticosteroid therapy. Patients suffered from frequent infections and autoimmune manifestations. In addition, a range of neurological symptoms, from specific language impairment to epilepsy was seen in some family members. Patient platelets exhibited constitutive Src, BTK, and PLC2 activation, and after stimulating CD19 cells by crosslinking surface IgM, the levels of phosphorylated ERK were above those seen in healthy control samples. Platelet functional studies revealed platelet hypo-responsiveness toward several agonists, and reduced expression of GPVI.

Conclusiones:

Here we report the largest pedigree with SRC-RT, the four family worldwide. These patients display an autosomal dominant disorder of megakaryopoiesis, with an apparent disturbed B cell receptor (BCR) signaling, which may be central to the clinical presentation, including a myriad of autoimmune features. A sensitive recognition of SRC-RT should be attempted in families with atypical inherited thrombocytopenia who have ITP-like features. Tyrosine kinase inhibitors may be investigated for their potential role as targeted therapies to treat the autoimmune manifestations associated to this disorder.


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Dirección

Campus de Ciencias de la Salud
Carretera Buenavista s/n, 30120 El Palmar
Murcia, España

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