Deciphering the importance of heterogeneity in Myotonic Dystrophy type 1

Abstract

[eng] Myotonic dystrophy type 1 (DM1) is a genetic and multisystemic muscular dystrophy with autosomal dominant inheritance. It is caused by a CTG expansion in the 3’ end of DMPK gene. Symptoms are mainly caused by the accumulation of toxic RNA aggregates, called RNA foci, which sequester MBNL1 protein, a splicing regulator. The symptoms can appear at any age and they are highly heterogeneous between patients. This heterogeneity is partially derived from the CTG repeat length, which due to both somatic and intergenerational instability, differs both between patients and between different tissues of a single patient. In this thesis the main focus is put on DM1 heterogeneity, which is often ignored in studies. Heterogeneity is an important feature to consider because it could have an influence in the molecular DM1 alterations at cellular level and in the efficacy of a therapy, meaning that DM1 models representing said heterogeneity would be useful. The thesis is divided in three chapters, each of which, explores DM1 heterogeneity from a different perspective. Chapter 1 is focused on the study of heterogeneity from a molecular point of view. The study analyses differences in RNA foci and MBNL1 aggregates expression between different cell types or DM1 subtypes. On the one hand, the analysis of four different cell types (lymphoblasts, fibroblasts, myoblasts and myotubes) revealed significant differences between them, being myotubes the cell type with a higher number of RNA foci and myoblasts the cell type with a higher MBNL1 sequestration. Myoblasts were also used to perform a single cell study to determine heterogeneity in expression levels and whether it correlates with the number of RNA foci expressed in the same cell. Results revealed no correlation. On the other hand, the analysis of RNA foci in the 5 subtypes of DM1 (congenital, infantile, juvenile, adult and late-onset) revealed a higher accumulation of RNA foci in patients with an earlier age of onset. In this chapter the existence of molecular heterogeneity both between and within patients is confirmed. Chapter 2 is focused on how DM1 heterogeneity impacts the degree of efficacy of a treatment. The study assesses the therapeutic potential of antagomiRs 23b and 218, two antisense oligonucleotides that block MBNL1 repressors miRNAs 23b and 218. A previous study proved that these antagomiRs have a positive impact in DM1 pathogenesis, however DM1 heterogeneity was not

assessed. In this study eight independent human DM1 primary myoblast lines with CTG repeats ranging from 117 to 1054 were treated with both antagomiRs. It was found that miR-23b and 218 are upregulated in patient myoblasts and that miRNAs overexpression correlated with CTG expansion size. The treatment reduced the miRNAs activity, increasing MBNL1 protein levels and reducing DMPK expression and foci number. Moreover, MBNL1- dependent splicing events were significantly rescued and the degree of MBNL1 enhancement correlated with splicing rescue. In this chapter it is concluded that antagomiRs 23b and 218 have therapeutic potential across different genetic backgrounds. Chapter 3 is focused on the need for in vitro models to study DM1 and to validate therapies that represent the clinical and genetic heterogeneity observed in DM1 patients. In the study, three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds were immortalized and characterized at the genetic, epigenetic, and molecular levels. Results showed that the three immortalized cell lines displayed all the expected DM1 hallmarks with significant differences between the cell lines for several of the studied alterations. Moreover, the response of the immortalized cell lines to the previously tested therapeutics was also analysed with positive results. In this chapter it is concluded that the three immortalized DM1 cell lines developed in this study are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.