Caracterización funcional y molecular de nuevas variantes genéticas y desarrollo de una nueva aproximación de terapia génica en trastornos plaquetarios congénitos

Abstract

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