Characterization of novel genes and variants involved in Congenital Platelet Disordersfrom the genomic data to the functional studies

  1. Marín Quílez, Ana
Dirigida per:
  1. José María Bastida Bermejo Director
  2. J. R. González Porras Codirector
  3. Ignacio García Codirector/a

Universitat de defensa: Universidad de Salamanca

Fecha de defensa: 16 de de setembre de 2022

  1. María Luisa Lozano Almela President/a
  2. Miguel Pericacho Bustos Secretari
  3. Josefa Verónica Alonso Pérez Vocal

Tipus: Tesi


Congenital Platelet Disorders [CPDs] comprise a wide and heterogeneous group of rare diseases that predispose patients to thrombocytopenia and/or bleeding but also, in some cases, to other complications, such as hematological malignancies or syndromic manifestations. Diagnosis is challenging and requires multiple laboratory tests as well as a final molecular confirmation. The introduction of High Throughput Sequencing [HTS] tools into the clinical practice has significantly improved and streamlined patients' diagnosis. However, despite the substantial improvement that HTS tools have brought, two major challenges still persist. Firstly, the interpretation of the variants found by HTS and their classification. For the curation of the variants, especially for novel or uncertain significance, it is encouraging the development of functional models to formally demonstrate its role in the pathogenesis and the mechanism underlying the disease. The second challenge to be faced is the fact that, regardless of the success in the diagnosis and phenotypic characterization by an integrative approach using targeted sequencing panels and functional studies, there are still patients and families without a final diagnosis. Considering the unknown prevalence and the underdiagnosis of the CPDs, in addition to the fact that several physiological processes of the megakaryopoiesis and platelet function are not well understood, it is highly possible that a substantial number of disorders are caused by variants in genes that have not been described or characterized so far. The aim of this thesis dissertation was to characterize the clinical and platelet phenotype associated with molecular alterations found by the high throughput sequencing tools in genes involved in Congenital Platelet Disorders, and to demonstrate the pathogenicity-related mechanism using in vitro and ex vivo megakaryocyte cultures, and in vivo disease models generated by the CRISPR/Cas9 technology. Specifically, to characterize the RUNX1 p.Leu56Ser variant in a knock-in murine model to establish its involvement in platelet disorder, and to investigate the signaling pathways and biological determinants responsible for progression to myeloid leukemia. Next, applying the whole exome sequencing for the identification of novel genes involved in Congenital Platelet Disorders, and the evaluation of the mechanisms of pathogenicity: evaluation of the clinical and platelet phenotype of a family with thrombocytopenia carrying a variant in TPM4, but also to determine the functional effect of four genetic variants identified in GALE and their implication in platelet function, megakaryopoiesis and thrombopoiesis, and their role in megakaryocyte and platelet glycosylation.