Abstract Congenital myotonic dystrophy (CDM), the most severe form of myotonic dystrophy, causes muscle weakness, breathing problems, and feeding difficulties at birth. During childhood affected individuals experience intellectual impairment and gastrointestinal issues while, in contrast, muscle strength and weakness improve. Muscle symptoms associated with adults with myotonic dystrophy, including myotonia and fatigue, are not observed until individuals reach adolescence. In the previous proposal period, the investigators clinically defined this triphasic pattern of motor involvement via enrollment of over 100 children in a comprehensive clinical study to evaluate measures of physical function over 12 months. In adults with myotonic dystrophy, a toxic RNA repeat expansion leads to global dysregulation of RNA splicing. Within the previous proposal period we performed RNA sequencing on 36 congenital myotonic dystrophy muscle biopsies from individuals 2 weeks to 16 years of age. We found that the severity of RNA mis- splicing mirrored the triphasic course of muscle symptoms captured clinically; children in early childhood showed improvement in RNA splicing dysregulation that regressed in adolescence. This result was bolstered by the inclusion of biopsies sampled from individuals at two ages across development. While these observations correlate with the clinical course of CDM, the mechanisms responsible for these dynamic shifts in mis-splicing remain unknown. This proposal is designed further clarify and define the molecular mechanisms responsible for the clinical and molecular progression of CDM. In Aim 1, we will enroll additional children with CDM that have acquired a previous muscle biopsy in a clinical follow-up study to acquire both longitudinal assessments of muscle function and a secondary muscle biopsy. RNA sequencing will be performed and mis-splicing and gene expression quantified to validate the course of CDM disease pathogenesis. In Aim 2 and 3, we will evaluate two potential molecular mechanisms based on our prior data that may contribute to the complex trajectory of CDM disease. In Aim 2, we will define the proliferative and regenerative capacity of CDM muscle stem cells across pediatric development and the contribution mediated via the IGF2 mitogenic signaling axis. In Aim 3, we will define how expression of core spliceosomal patterns contribute to the unique mis-splicing signatures observed within cohorts of CDM individuals. At the completion of this project, we will have validated the clinical and molecular course of CDM disease progression across pediatric development and performed experiments vital to understanding the mechanisms that contribute to this dynamic pattern. By better understanding the pathogenesis of CDM, we both allow access of these children to forthcoming disease modifying therapies in myotonic dystrophy, as well as, identify new therapeutic targets for drug development.