A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

[Background]: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. [Objectives]: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. [Patients/Methods]: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. [Results]: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. [Conclusions]: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01966, PI20/00926), Gerencia Regional de Salud (GRS2061A/19, GRS2135/A/2020, GRS2314/A/2021), Fundación Mutua Madrileña (FMM, AP172142019) and Sociedad Española de Trombosis y Hemostasia (SETHFETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2020 and 2021).Peer reviewe

Characterization of the platelet phenotype caused by a germline RUNX1 Variant in a CRISPR/Cas9-generated murine model

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.Research by the group of F.F.-M. and J.R. is supported by grants from Instituto de Salud Carlos III and Feder (PI14/ 01956) and Fundación Séneca (19873/GERM/15). V.P.-B. holds a research fellowship from CIBERER (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)

Expanding the genetic spectrum of TUBB1-related thrombocytopenia

β1-Tubulin plays a major role in proplatelet formation and platelet shape maintenance, and pathogenic variants in TUBB1 lead to thrombocytopenia and platelet anisocytosis (TUBB1-RT). To date, the reported number of pedigrees with TUBB1-RT and of rare TUBB1 variants with experimental demonstration of pathogenicity is limited. Here, we report 9 unrelated families presenting with thrombocytopenia carrying 6 β1-tubulin variants, p.Cys12LeufsTer12, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp, the last of which novel. Segregation studies showed incomplete penetrance of these variants for platelet traits. Indeed, most carriers showed macrothrombocytopenia, some only increased platelet size, and a minority had no abnormalities. Moreover, only homozygous carriers of the p.Gly109Glu variant displayed macrothrombocytopenia, highlighting the importance of allele burden in the phenotypic expression of TUBB1-RT. The p.Arg359Trp, p.Gly269Asp, and p.Gly109Glu variants deranged β1-tubulin incorporation into the microtubular marginal ring in platelets but had a negligible effect on platelet activation, secretion, or spreading, suggesting that β1-tubulin is dispensable for these processes. Transfection of TUBB1 missense variants in CHO cells altered β1-tubulin incorporation into the microtubular network. In addition, TUBB1 variants markedly impaired proplatelet formation from peripheral blood CD34+ cell-derived megakaryocytes. Our study, using in vitro modeling, molecular characterization, and clinical investigations provides a deeper insight into the pathogenicity of rare TUBB1 variants. These novel data expand the genetic spectrum of TUBB1-RT and highlight a remarkable heterogeneity in its clinical presentation, indicating that allelic burden or combination with other genetic or environmental factors modulate the phenotypic impact of rare TUBB1 variants.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, PI20/00926 and CB15/00055), Fundacion Séneca (19873/ GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundacion Mutua Madrile´ña (AP172142019), and ~ Sociedad Espanola de Trombosis y Hemostasia (Premio L ~ opez Borrasca 2019 and Ayuda a Grupos de Trabajo en Patologıa Hemorragica). The authors’ research on inherited platelet disorders is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, from Grupo Espanol de Alteraciones Plaqueta- ~ rias Congenitas, which is supported by the Spanish Society of Thrombosis and Haemostasis. V.P.-B. has a predoctoral contract from CIBERER. L.B. was supported by a fellowship from Fondazione Umberto Veronesi. M.E.d.l.M.-B. holds a postdoctoral fellowship from the University of Murcia. A.M.-Q. holds a predoctoral grant from the Junta de Castilla y Leon

Significant hypo-responsiveness to GPVI and CLEC-2 agonists in pre-term and full term neonatal platelets and following immune thrombocytopenia

AbstractNeonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.</jats:p

Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.Peer Reviewe

A novel genetic variant in PTGS1 affects N-glycosylation of cyclooxygenase-1 causing a dominant-negative effect on platelet function and bleeding diathesis.

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease.Fundación Mutua Madrileña, Grant/Award Number: AP172142019; Fundación Séneca, Grant/Award Number: 19873/GERM/15; Gerencia Regional de Salud, Grant/Award Numbers: 1647/A/17, 2061A/19; Instituto de Salud Carlos III (ISCIII) & Feder, Grant/Award Numbers: CB15/00055, PI17/01966, PI18/00598, PI20/00926, PI17/01311; Junta de Castilla y León; British Heart Foundation, Grant/Award Number: PG/17/40/33028; Ayuda a Grupos de Trabajo en Patología Hemorrágica; Premio López Borrasca 2019; Sociedad Española de Trombosis y Hemostasia

Functional and molecular characterization of new genetic variants and development of a new gene therapy approach in Congenital Platelet Disorders

Introducción: Los Trastornos plaquetarios congénitos [TPC] son un grupo heterogéneo de enfermedades raras que afectan al recuento de plaquetas o a sus funciones. Su severidad clínica es variable, desde complicaciones insignificantes a potencialmente mortales. Su diagnóstico genético se ha visto facilitado en los últimos años con la introducción de la secuenciación masiva. Por el contrario, no ha habido grandes avances en su manejo clínico. Objetivo: Caracterizar una serie de pacientes con sospecha de padecer un TPC, identificar su patología molecular subyacente, y demostrar la patogenicidad de sus variantes moleculares. Adicionalmente, explorar el potencial de la terapia génica en los TPC clínicamente severos Metodología: Reclutamos pacientes con diátesis hemorrágica, disfunción plaquetaria y/o trombocitopenia en el proyecto multicéntrico de “Caracterización Funcional y Molecular de pacientes con TPC”. Re-evaluamos su historial clínico y antecedentes familiares, estudiamos el fenotipo plaquetario, y abordamos el diagnóstico genético mediante secuenciación masiva de un panel de genes prediseñado. Se evaluó la patogenicidad de las variantes candidatas identificadas en los enfermos, aplicando los criterios de ACMG, y realizando ensayos específicos. Aplicando la tecnología CRISPR/Cas9, generamos un modelo de células CD34+ con fenotipo de Trombastenia de Glanzmann [TG] (CD34+ TG-like). Diseñamos un vector viral como potencial terapia génica para la TG. Resultados: Hemos caracterizado funcional y molecularmente 50 pacientes de 14 familias no emparentadas. i) Demostramos que la diátesis hemorrágica en una niña está causada por la presencia en heterocigosis de la variante p.Asn143Ser en PTGS1 (ciclooxigenasa -1). Se demostró un patrón de disfunción plaquetaria aspirina-like, con defecto de agregación plaquetaria selectivo para ácido araquidónico y reducida capacidad de síntesis plaquetaria de TXA2. Estudios en plaquetas y en células 293THEK, demostraron que la mutación provoca la pérdida de un N-glicano generando una proteína hipoglicosilada con un efecto dominante negativo en la función. ii) Caracterizamos un pedigrí con macrotrombocitopenia tipo SRC-RT, con 7 portadores de la variante p.E527K en Src. Confirmamos un defecto de agregación, activación y secreción plaquetaria, más marcado, pero no exclusivo, para agonistas de GPVI; un defecto de marcadores -granulares mediante inmunfluorescencia en frotis sanguíneos; también la ganancia de función de esta mutación causando fosforilación constitutiva en tirosinas de proteínas plaquetarias incluyendo Src, PLCy2 y BTK. Evidenciamos que en estos pacientes coexisten complicaciones de tipo inmune y neurológico. Su respuesta plaquetaria a la esplenectomía y los esteroides, así como el aparente estado inflamatorio en algunos de ellos, sugiere cierta similitud clínica con la trombocitopenia inmune [PTI]. iii) Describimos la serie más larga, descrita hasta la fecha, de pacientes con TUBB1-RT (9 familias), identificando en ellos 6 variantes en TUBB1 (p.Cys12Leufs12*, p.Thr107Pro, p.Gln423*, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp). Se observó en ellas una penetrancia incompleta, con una mayoría de pacientes portadores con macrotrombocitopena, otros sin trombocitopenia pero con plaquetas grandes y una minoría de portadores con plaquetas normales. Encontramos en un pedigrí, por primera vez en TUBB1-RT, que la mutación responsable, p.Gly109Glu, causa enfermedad solo en homocigosis, poniendo de manifiesto la importancia de la carga alélica. Los estudios plaquetarios (expresión de GPs, agregación, activación y secreción, spreading) demostraron un escaso efecto de estas mutaciones en la funcionalidad plaquetaria, acorde con la moderada o ausente clínica hemorrágica en pacientes con TUBB1-RT. La expresión de estas mutaciones (solo las misssense) en células CHO demostró que afectan significativamente la expresión y localización de la tubulina-1. Los ensayos de cultivo in vitro de células CD34+ de los enfermos, demostraron que las mutaciones alteran la diferenciación/maduración de los Mks y la formación de proplaquetas. Los datos obtenidos en esta serie de familias y mutaciones de TUBB1, nos ha permitido reclasificar la patogenicidad de las variantes identificadas y definir criterios de patogenicidad ACGM adaptados a mutaciones en TUBB1. iv) La caracterización del fenotipo plaquetario (agregación, activación, cuantificación de gránulos por microscopia electrónica e inmunofluorescencia), los estudios de segregación familiar y, de forma muy novedosa, el análisis del transcriptoma plaquetario, apoyan fuertemente la patogenicidad de las variantes de RUNX1 p.Gln268* y p.Thr196Ala, pero no así de p.Asn159Ser. Hasta el 70% de los genes previamente descritos como diana de RUNX1, (incluyendo entre otros MYL9, MYH9, ALOX12, TREML1 e ITGA2), mostraron en nuestro microarray una expresión alterada en los portadores de las variantes p.Gln268* y p.Thr196Ala, en comparación con controles sanos. Estos datos se confirmaron, para algunos genes, mediante RT-qPCR. En el caso de p.Asn159Ser solo el 7% de los genes mostraron expresión alterada. Clasificamos los 120 genes más infraexpresados en 5 grupos: genes implicados en el citoesqueleto (18%); genes que participan en la transducción de señales (40%); genes relacionados con la interacción con ADN; genes implicados en el ciclo celular; genes asociados, al menos ocasionalmente, con procesos tumorales. También, encontramos un gran número de genes sobreexpresados, siendo el primero CA1, el gen que codifica la anhidrasa carbónica I (Fold change 140.04 vs. controles). Entre los sobreexpresados, hay genes que codifican proteínas de origen eritroide, transportadores de membrana, proteínas ribosomales y otras implicadas en la síntesis, modificación y degradación de proteínas y LXN, gen que codifica la latexina que es una proteína reconocida recientemente como supresor tumoral en neoplasias hematológicas. v) Desarrollamos un modelo celular con fenotipo de TG en el que demostramos la eficacia de un vector viral para revertir el fenotipo patológico. Conclusiones: Presentamos el diagnóstico clínico, funcional y molecular de un amplia número de pacientes con sospecha de TPC, caracterizando 11 variantes genéticas en 4 genes distintos. Reportamos el tercer caso de disfunción plaquetaria y sangrado asociado a patología molecular de PTGS1. Esta paciente revela la importancia de la N-glicosilación en la funcionalidad de la COX-1. Caracterizamos la cuarta familia con la mutación p.E527K y SRC-RT, que pone de manifiesto la coexistencia de alteraciones plaquetarias, inmunes y neurológicas en esta patología. La aparente parcial similitud con la PTI, podrían ayudar a establecer futuras estrategias de tratamiento. El estudio de la serie más larga descrita hasta la fecha de TUBB1-RT, revelan que la penetrancia incompleta es un fenómeno común en esta enfermedad, resalta la importancia de la carga alélica en la enfermedad, consolida el defecto de maduración de Mks, y el escaso efecto deletéreo de estas mutaciones en la reactividad plaquetaria, acorde con la moderada/ausente clínica hemorrágica en pacientes con TUBB1-RT. El análisis de trascriptoma plaquetario en tres casos con sospecha de RUNX1-RT, demuestran que puede ser una herramienta útil para establecer la patogenicidad de mutaciones nuevas de RUNX1, identificadas en pacientes con o sin historial familiar de neoplasia. Por último, demostramos la utilidad de la tecnología CRISPR/Cas9 para diseñar modelos de TPC, como la Trombastenia de Glanzmann. También mostramos la eficacia de un vector viral para revertir in vitro el fenotipo de la TG. Estos datos pre-clínicos son un importante avance en el camino hacia el potencial uso clínico de la terapia génica como alternativa curativa en TPC graves.Introduction: Inherited platelet disorders [IPD] are a heterogeneous group of rare diseases that affect the platelet count or its functions. The clinical severity is variable, from insignificant to life-threatening. Its genetic diagnosis has been facilitated in recent years with the introduction of high throughput sequencing [HTS]. On the contrary, there have not been major advances in the clinical management of IPD. Objective: To characterize a series of patients with suspected TPC, identify the underlying molecular pathology, and demonstrate the pathogenicity of their molecular variants. Additionally, to explore the value of gene therapy in clinically severe IPD. Methodology: We recruited patients with bleeding diathesis, platelet dysfunction and/orthrombocytopenia to the multicenter project of "Functional and Molecular Characterization of patients with IPD". We re-evaluate their clinical history and family background, studied the platelet phenotype, and addressed their genetic diagnosis by means of HTS of pre-designed panel of genes. The pathogenicity of the candidate variants identified in the patients was evaluated applying the ACMG criteria, and carrying out specific tests in patient samples and cell models. Using CRISPR/Cas9 technology, we generated a model of CD34+ cells of Glanzmann Thrombasthenia [GT] (CD34+ GT-like] phenotype. We designed a viral vector to correct the defect in the CD34+ GT-like cells. Results: We have functionally and molecularly characterized 50 patients from 14 unrelated families. i) We show that the bleeding diathesis in a young girl is caused by the heterozygous presence of the p.Asn143Ser variant in PTGS1 (cyclooxygenase -1 or COX-1). An aspirin-like platelet dysfunction pattern was demonstrated, with a selective platelet aggregation defect for arachidonic acid and a reduced platelet capacity for TXA2 synthesis. Studies in platelets and 293 cells demonstrated that the mutation causes the loss of an N-glycan, generating a hypoglycosylated protein with a dominant negative effect on COX-1 function. ii) We characterize a large pedigree with SRC-RT macrothrombocytopenia, with 7 carriers of the p.E527K variant in Src. We confirmed a platelet aggregation, activation and secretion defect, more marked, but not exclusive, for GPVI agonists; we also found an -granule markers defect by immunfluorescence of blood smears; the gain of function of this mutation caused constitutive tyrosine phosphorylation of platelet proteins including Src, PLC and Btk. We show that in these patients coexist platelets, immune and neurological alterations. The platelet response to splenectomy and steroids, as well as the apparent inflammatory status in some of patients, suggested a certain clinical similarity to immune thrombocytopenia (ITP). iii) We describe the largest series, so far, of patients with TUBB1-RT (9 families), carrying 6 TUBB1 variants (p.Cys12Leufs12 *, p.Thr107Pro, p.Gln423 *, p.Arg359Trp, p.Gly109Glu, and p.Gly269Asp). Incomplete penetrance was observed in these pedigrees, with most carriers presenting with macrothrombocytopenia, few others without thrombocytopenia but with large platelets, and a minority of carriers with normal platelets. We found in a pedigree, for the first time in TUBB1-RT, that the TUBB1 mutation, p.Gly109Glu, causes disease only in homozygosity, highlighting the importance of allelic burden. Platelet studies (expression of GPs, aggregation, activation and secretion, spreading) showed little effect of these mutations on platelet function, consistently with the moderate or absent clinical hemorrhage in patients with TUBB1-RT. The expression of these mutations (only the misssense ones) in CHO cells was shown to significantly affect the expression and localization of1-tubulin. In vitro culture of CD34+ cells from these patients demonstrated that the mutations alter the differentiation/maturation of Mks and proplatelets formation. The data obtained in this series of families and mutations of TUBB1, has allowed us to reclassify the pathogenicity of the identified variants and to define ACGM pathogenicity criteria adapted to utations in TUBB1.iv) Characterization of the platelet phenotype (aggregation, activation, granule quantification by electron microscopy and immunofluorescence), family segregation studies and, most novelty, platelet transcriptome analysis, strongly support the pathogenicity of RUNX1 variants p.Gln268* and p.Thr196Ala, but not that of p.Asn159Ser. Up to 70% of the genes previously described as RUNX1 targets, (including among others MYL9, MYH9, ALOX12, TREML1 and ITGA2),), showed in our array a decreased expression in carriers of the p.Gln268* and p.Thr196Ala variants, compared to healthy controls. These data were confirmed, for some genes, by qPCR-RT. In the case of p.Asn159Ser, only 7% of the genes showed altered expression. We classified the 120 most downregulated genes into 5 groups: genes involved in the cytoskeleton (18%); genes involved in signal transduction (40%); genes related to interaction with DNA; genes involved in the cell cycle; genes associated, at least occasionally, with tumor processes. We also found a large number of overexpressed genes, the first being CA1, the gene that encodes carbonic anhydrase I. Among those overexpressed, there are genes that encode proteins of erythroid origin, membrane transporters, ribosomal proteins and others involved in the synthesis, modification and degradation of proteins. Also LXN, a gene that encodes latexin, a protein recently recognized as a tumor suppressor in hematological neoplasms. v) We developed a cell model with a TG phenotype in which we demonstrated the efficacy of our viral vector to reverse the pathological phenotype. Conclusions: We present the clinical, functional and molecular diagnosis of a large number of patients with suspected IPD, characterizing 11 genetic variants in 4 different genes. We report the third case of platelet dysfunction and bleeding associated with molecular pathology of PTGS1. This patient reveals the importance of N-glycosylation in the function activity of COX-1. We also have characterized the fourth family with the p.E527K mutation and SRC-RT, which reveals the coexistence of platelet, immune and neurological alterations in this pathology. The apparent partial similarity with ITP could help to establish future treatment strategies. The study of the largest series described to date of TUBB1-RT, reveals that incomplete penetrance is a common phenomenon in this disease, highlights the importance of the allelic burden, consolidates the defect in Mks maturation and proplatelets formation caused by TUBB1 mutations, and reflect an almost negligible effect of these mutations on platelet reactivity, consistent with the moderate or absent clinical bleeding in patients with TUBB1-RT. Platelet transcriptome analysis in three cases with suspected RUNX1-RT shows that it can be a useful tool to establish the pathogenicity of new RUNX1 mutations, identified in patients with or without a personal or family history of neoplasia. Finally, we demonstrate the usefulness of CRISPR/Cas9 technology to design IPD models, such as Glanzmann's Thrombasthenia. We also show the efficacy of a viral vector to reverse the GT phenotype in vitro. These pre-clinical data represent an important step forward to the potential clinical use of gene therapy as a curative alternative in severe IPD

RASGRP2 gene variations associated with platelet dysfunction and bleeding

This manuscript reviews pathogenic variants in RASGRP2, which are the cause of a relatively new autosomal recessive and nonsyndromic inherited platelet function disorder, referred to as platelet-type bleeding disorder-18 (BDPLT18)(OMIM:615888). To date, 18 unrelated BDPLT18 pedigrees have been reported, harboring 19 different homozygous or compound heterozygous RASGRP2 variants. Patients with this disease present with lifelong moderate to severe bleeding, with epistaxis as the most common and relevant bleeding symptom. Biologically, they exhibit normal platelet count and morphology, reduced aggregation responses to ADP, epinephrine and low-dose collagen, and impaired αIIbβ3 integrin activation (fibrinogen or PAC-1 binding) in response to most agonists except PMA. Diagnosis is confirmed by genetic analysis of RASGRP2