Publication: Diagnóstico integrado en trastornos plaquetarios congénitos : caracterización de nuevas variantes y aplicación de estrategias innovadoras de diagnóstico molecular
Authors
Zamora Cánovas, Ana
item.page.secondaryauthor
Escuela Internacional de Doctorado
item.page.director
Rivera Pozo, José ; Lozano Almela, María Luisa
Publisher
Universidad de Murcia
publication.page.editor
publication.page.department
DOI
item.page.type
info:eu-repo/semantics/doctoralThesis
Description
Abstract
Los trastornos plaquetarios congénitos (TPC) constituyen un grupo heterogéneo de enfermedades raras causadas por alteraciones genéticas que afectan a la producción, estructura o función de las plaquetas. Su espectro clínico abarca desde manifestaciones mucocutáneas leves hasta hemorragias graves potencialmente mortales, así como formas sindrómicas que pueden comprometer distintos órganos o asociarse a riesgo de neoplasias hematológicas. Las técnicas de secuenciación masiva (high-throughput sequencing, HTS) han facilitado el diagnóstico molecular de estas entidades, pero aún existen pacientes sin diagnóstico genético definitivo. Además, los mecanismos patogénicos de los TPC permanecen insuficientemente caracterizados. Esta tesis doctoral se centra en la caracterización clínica, funcional y molecular de pacientes con sospecha de TPC, y ampliaa el conocimiento del espectro genético y fisiopatológico de estas enfermedades, profundizando especialmente en los mecanismos implicados en la señalización plaquetaria, la biogénesis de gránulos y la organización del citoesqueleto. El Capítulo I describe nuevas variantes en los genes P2RY12, GP6 y TBXAS1, implicados en la reactividad plaquetaria. Se identificaron mutaciones nonsense, missense y frameshift en pacientes con historia hemorrágica desde la infancia. Los estudios funcionales demostraron que las variantes en P2RY12 alteraban selectivamente la respuesta al ADP, con defectos en la inhibición de la adenilato ciclasa y en la fosforilación de VASP; la variante en GP6 reducía la expresión del receptor GPVI y de la cadena FcRgamma, comprometiendo la respuesta a colágeno al altera señalización aguas debajo de GPVI dependiente de ITAM; la variante en TBXAS1 disminuía la producción de tromboxano A2 como se demostró en modelos celulares. Estos hallazgos amplían el espectro mutacional conocido y evidencian la necesidad de integrar fenotipo plaquetario detallado con estudios funcionales para establecer la patogenicidad de variantes raras. El Capítulo II amplía el conocimiento del Síndrome de Plaqueta Gris, causado por variantes en NBEAL2. Se describen cuatro nuevos pacientes portadores de tres variantes, dos de ellas nuevas. Algunos de estos pacientes mostraban manifestaciones extrahematológicas como esplenomegalia y alteraciones medulares. El análisis plaquetario confirmó la reducción de marcadores de gránulos alfa y, curiosamente, de gránulos densos en dos casos. La integración de datos clínicos, morfológicos y genéticos permitió clasificar las nuevas variantes como probablemente patogénicas y reforzar el papel de NBEAL2 en la biogénesis de gránulos alfa y en procesos inflamatorios e inmunitarios. El Capítulo III aborda la variante p.Arg1213* en DIAPH1, asociada a macrotrombocitopenia autosómica dominante con sordera neurosensorial y neutropenia variable. Aunque la función plaquetaria estaba relativamente conservada, se identificaron alteraciones significativas en la organización del citoesqueleto, con desestructuración del anillo marginal de microtúbulos y polimerización aberrante de F-actina en megacariocitos derivados de células CD34+. Se observó un defecto severo en la formación de proplaquetas, lo que sugiere que la activación constitutiva de DIAPH1 altera el equilibrio dinámico entre microtúbulos y actina durante la trombopoyesis. Por último, el Capítulo IV demuestra la utilidad de la secuenciación de lectura larga mediante tecnología Nanopore en pacientes sin diagnóstico tras HTS convencional, concretamente en dos casos de Trombastenia de Glanzmann y en tres pacientes con Síndrome de Hermansky-Pudlak. En estos pacientes, Nanopore identificó variantes estructurales complejas, lo que hizo posible establecer el diagnóstico genético definitivo. En conjunto, esta tesis amplía el conocimiento de la genética de los TPC, profundiza en sus mecanismos moleculares y refuerza la importancia de un enfoque multidisciplinar para optimizar su diagnóstico y comprensión fisiopatológica.
Inherited platelet disorders (IPDs) constitute a heterogeneous group of rare diseases caused by genetic alterations affecting platelet production, structure, or function. The clinical spectrum of these conditions ranges from mild mucocutaneous manifestations to severe, potentially life-threatening haemorrhage, as well as syndromic forms that may involve multiple organs or be associated with an increased risk of haematological malignancies. High-throughput sequencing (HTS) technologies have facilitated the molecular diagnosis of these conditions; however, a subset of patients still lacks a definitive genetic diagnosis. Furthermore, the pathogenic mechanisms underlying IPDs remain incompletely characterised. The present doctoral thesis focuses on the clinical, functional and molecular characterisation of patients suspected of having IPDs, with a view to expanding current knowledge of the genetic and pathophysiological spectrum of these disorders. The thesis makes particular emphasis on mechanisms involved in platelet signalling, granule biogenesis and cytoskeletal organisation. Chapter I provides a comprehensive overview of the novel variants observed in the P2RY12, GP6, and TBXAS1 genes, which play a pivotal role in platelet reactivity. Nonsense, missense, and frameshift mutations were identified in patients with a lifelong history of bleeding. A series of platelet investigations have demonstrated that specific P2RY12 variants result in a selective impairment of the response to ADP, characterised by defects in adenylate cyclase inhibition and VASP phosphorylation. The GP6 variant has been demonstrated to reduce the expression of both the GPVI receptor and the FcRgamma chain, thereby compromising collagen-induced responses by disrupting downstream ITAM-dependent signalling. The TBXAS1 variant has been demonstrated to decrease thromboxane A2 production in cellular models. These findings expand the known mutational spectrum and highlight the importance of integrating detailed platelet phenotyping with functional studies to establish the pathogenicity of rare variants. Chapter II provides a comprehensive overview of the scientific literature pertaining to Gray Platelet Syndrome, a condition that arises from genetic variations in the NBEAL2 gene. The following report details four new patients carrying three variants, two of which are novel. Certain patients demonstrated the presence of extrahematological manifestations, which included splenomegaly and bone marrow abnormalities. The platelet analysis revealed a decrease in the levels of alfa-granule markers and, notably, a reduction in the number of dense granules in two cases. The integration of clinical, morphological, and genetic data enabled the classification of the newly identified variants as likely pathogenic, thereby reinforcing the role of NBEAL2 in alfa-granule biogenesis, as well as in inflammatory and immune processes. Chapter III is concerned with the DIAPH1 p.Arg1213* variant, which has been linked to autosomal dominant macrothrombocytopenia, accompanied by sensorineural hearing loss and variable neutropenia. Despite the relatively preserved platelet function, significant alterations in cytoskeletal organisation were identified, including disruption of the marginal microtubule band and aberrant F-actin polymerisation in CD34+ derived megakaryocytes. A severe defect in proplatelet formation was observed, suggesting that constitutive activation of DIAPH1 disrupts the dynamic balance between microtubules and actin during thrombopoiesis. Chapter IV demonstrates the utility of long-read sequencing using Nanopore technology in patients lacking a molecular diagnosis after conventional HTS, specifically in two cases of Glanzmann thrombasthenia and three patients with Hermansky-Pudlak syndrome. In these patients, Nanopore sequencing identified complex structural variants, enabling definitive genetic diagnosis. Overall, this thesis contributes to the existing body of knowledge on the genetic basis of IPDs, deepens our understanding of their molecular mechanisms, and emphasises the importance of a multidisciplinary approach to optimise diagnosis and pathophysiological insight.
Inherited platelet disorders (IPDs) constitute a heterogeneous group of rare diseases caused by genetic alterations affecting platelet production, structure, or function. The clinical spectrum of these conditions ranges from mild mucocutaneous manifestations to severe, potentially life-threatening haemorrhage, as well as syndromic forms that may involve multiple organs or be associated with an increased risk of haematological malignancies. High-throughput sequencing (HTS) technologies have facilitated the molecular diagnosis of these conditions; however, a subset of patients still lacks a definitive genetic diagnosis. Furthermore, the pathogenic mechanisms underlying IPDs remain incompletely characterised. The present doctoral thesis focuses on the clinical, functional and molecular characterisation of patients suspected of having IPDs, with a view to expanding current knowledge of the genetic and pathophysiological spectrum of these disorders. The thesis makes particular emphasis on mechanisms involved in platelet signalling, granule biogenesis and cytoskeletal organisation. Chapter I provides a comprehensive overview of the novel variants observed in the P2RY12, GP6, and TBXAS1 genes, which play a pivotal role in platelet reactivity. Nonsense, missense, and frameshift mutations were identified in patients with a lifelong history of bleeding. A series of platelet investigations have demonstrated that specific P2RY12 variants result in a selective impairment of the response to ADP, characterised by defects in adenylate cyclase inhibition and VASP phosphorylation. The GP6 variant has been demonstrated to reduce the expression of both the GPVI receptor and the FcRgamma chain, thereby compromising collagen-induced responses by disrupting downstream ITAM-dependent signalling. The TBXAS1 variant has been demonstrated to decrease thromboxane A2 production in cellular models. These findings expand the known mutational spectrum and highlight the importance of integrating detailed platelet phenotyping with functional studies to establish the pathogenicity of rare variants. Chapter II provides a comprehensive overview of the scientific literature pertaining to Gray Platelet Syndrome, a condition that arises from genetic variations in the NBEAL2 gene. The following report details four new patients carrying three variants, two of which are novel. Certain patients demonstrated the presence of extrahematological manifestations, which included splenomegaly and bone marrow abnormalities. The platelet analysis revealed a decrease in the levels of alfa-granule markers and, notably, a reduction in the number of dense granules in two cases. The integration of clinical, morphological, and genetic data enabled the classification of the newly identified variants as likely pathogenic, thereby reinforcing the role of NBEAL2 in alfa-granule biogenesis, as well as in inflammatory and immune processes. Chapter III is concerned with the DIAPH1 p.Arg1213* variant, which has been linked to autosomal dominant macrothrombocytopenia, accompanied by sensorineural hearing loss and variable neutropenia. Despite the relatively preserved platelet function, significant alterations in cytoskeletal organisation were identified, including disruption of the marginal microtubule band and aberrant F-actin polymerisation in CD34+ derived megakaryocytes. A severe defect in proplatelet formation was observed, suggesting that constitutive activation of DIAPH1 disrupts the dynamic balance between microtubules and actin during thrombopoiesis. Chapter IV demonstrates the utility of long-read sequencing using Nanopore technology in patients lacking a molecular diagnosis after conventional HTS, specifically in two cases of Glanzmann thrombasthenia and three patients with Hermansky-Pudlak syndrome. In these patients, Nanopore sequencing identified complex structural variants, enabling definitive genetic diagnosis. Overall, this thesis contributes to the existing body of knowledge on the genetic basis of IPDs, deepens our understanding of their molecular mechanisms, and emphasises the importance of a multidisciplinary approach to optimise diagnosis and pathophysiological insight.
publication.page.subject
Citation
item.page.embargo
Collections
Ir a Estadísticas
Este ítem está sujeto a una licencia Creative Commons. http://creativecommons.org/licenses/by-nc-nd/4.0/