Myotonic Dystrophy Type 1: the heterogeneity of a complex disease in a global research approach

Abstract

Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion. In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor Resumen de la Tesis: Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion. In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor CTG expansion found in muscle correlates with the age of disease onset of these patients. We studied as well the presence of interruptions in the CTG expansion and how these variant repeats can affect the genetic transmission in between generations and the phenotype of the patients. After analyzing a cohort of 49 DM1 patients we have seen that 10% of the patients carried CCG interruptions. Moreover, the presence of interruptions resulted, in one case, in a contraction of the CTG expansion in between generations, but also resulted in an expansion of the CTG track in the other case, linked to anticipation. As it has been previously described, we have seen that the presence of interruptions can be linked to non-typical phenotypic traits of DM1, such as proximal weakness. However, contrary of what is believed, we have seen that patients carrying interruptions can develop a clinical phenotype with a high severity. We also studied the pathomolecular mechanism of RNA gain-of function, trough 3D imaging. We studied myoblasts cells derived from DM1 patients, analyzing the presence of RNA toxic aggregates. 3D imaging allowed us to have a full characterization of the cell, quantifying the number of the RNA toxic aggregates and studying the relationship between the main molecular players. We have seen that the CTG expansion leads the number of RNA foci that is accumulated in the cell and the number of the RNA foci correlates with the DMPK expression. We have seen as well that the RNA foci is not only present in the nucleus, but also in the cytoplasm, and the area of these toxic aggregates is related with its presence in the cytoplasm. Finally, we studied the efficacy (reduction of RNA foci number) and toxicity (cell mortality) of a promising therapeutic approach for DM1, using antisense oligonucleotides with BNANC modifications, in cells derived from DM1 patients. We have studied the treatment response in three different subtypes, since due to the multisystemic nature of DM1, it is necessary to know how the different cells would react to a specific treatment. We have seen that the RNA foci reduction and the cell mortality is different in fibroblasts, lymphoblasts and myoblasts of Dm1 patients. Myoblasts, derived from one of the most affected tissues in DM1 patients, are the cells responding less to the treatment, which highlights the importance to perform experiments in order to improve the delivery in these cells. Moreover, we have seen that the treatment response seems to not be related with the CTG expansion size.