Role of CCG Interruptions within CTG Triplet Expansion in Myotonic Dystrophy Type 1

Short tandem DNA repeats make a significant contribution to genetic diversity and phenotypic variation, with some unstable repeat expansions associated with more than sixty neurological and neuromuscular diseases. One of them is Myotonic dystrophy type 1 (DM1). DM1 is an autosomal dominant progressive neuromuscular disorder, caused by an unstable expansion of CTG repeats in the 3’-untranslated region of the DMPK gene. It is an incurable and variable disorder resulting in different symptoms’ severity, age of onset and CTG repeats instability. DM1 is associated with anticipation phenomenon characterized as a worsening of symptoms and earlier age of the disease onset through every successive generation. Anticipation is explained at the molecular level by intergenerational instability biased towards expansion which, in turn, results in an increase of CTG repeat number across successive generations. In general, patients tend to display positive correlation between clinical severity and the number of CTG repeat tracts. Apart from intergenerational instability, there is somatic mosaicism characterized as the tendency of CTG repeats to demonstrate genetic variations between and within tissues. This results in the repeats’ length variability in different body parts of the patients. Around 10% of the DM1 patient population carries an interrupted expanded allele, where CTG repeat expansions are interrupted by other types of triplets (CCG, CAG, CTC, CGG). Apart from the type, there is also difference in its frequency and the position within the expansions. The presence of interruptions is associated with stabilization and a decrease in the repeat number (contraction) which imposes the direct influence of repeat interruptions on repeat instability. Nevertheless, the exact mechanism and the pathway, by which the stabilization is happening, is not explored. Taken into account the fact that the repeat interruptions are associated with milder and less severe symptoms, there is an important question: what is the role of the interruptions in CTG repeat expansion in DM1 patients? The goal of my thesis is to determine how CCG interruptions affect the repeat instability by investigating two significant processes - DNA methylation and DNA repair, known to impact pure CTG repeat instability across different models. To do so, I focus on four lines of immortalized fibroblasts with and without interruptions to check if the methylation pattern is affected between interrupted and interrupted cell lines using Oxford Nanopore long read sequencing. Moreover, to investigate interruption's role on DNA repair, I have initiated experiment collaboration with Veronica Brito from University of Barcelona. In addition, I participated in the generation of isogenic cell lines to create new cell lines with interruptions by using system CRISPR-Cas9 system for future research steps. My thesis project aims to enhance our understanding of the role of interruptions in repeat instability, particularly in the stabilization associated with less severe symptoms. The data generated will contribute to advancing our knowledge and, ultimately, help develop new therapeutic approaches for DM1.