Summary Myotonic dystrophy (dystrophia myotonica; DM) is an inherited disorder that causes progressive muscle weakness and wasting. No effective treatments are available to slow or reverse symptoms. The mutation responsible for DM type 1 (DM1) is an expanded CTG repeat in the DM protein kinase (DMPK) gene. Clinical symptoms result from pathogenic effects of the DMPK mRNA that lead to mis-regulation of alternative pre- mRNA splicing of at least several dozen transcripts. Monitoring of a panel of RNA splicing outcomes in repeated muscle tissue biopsies has been proposed to measure DM1 disease activity during future clinical trials. However, the muscle biopsy procedure is invasive, painful, impractical for long-term monitoring, and in pediatric patients requires general anesthesia, which increases risk. Extracellular RNA (exRNA) refers to mRNAs and noncoding RNAs that are released from and taken up by cells as a novel form of cell-to-cell communication. exRNAs in blood and urine serve as genetic biomarkers of cancers and other disease states. Recent work has suggested that splicing outcomes in urine exRNA may have the capacity to serve as robust monitoring biomarkers of DM1. This prospective multi-center study will test the hypothesis that exRNA contains a representative transcriptome that is altered in DM and is quantifiable in urine. Splicing outcomes in urine exRNA are expected to correlate with DM1 disease severity using clinical measures of muscle function. R61 phase (Years 1 - 3): Aim 1 will identify and characterize the urine exRNA profile in DM1 patients and unaffected control subjects. Droplet digital PCR (ddPCR), a state-of-the-art technology, will be used to quantitate a panel of urine exRNA splice events previously reported as altered in DM1. Go/no-go milestone: candidate exRNA biomarkers show differential splicing in DM1 vs. unaffected controls with a P value < 0.01. Aim 2 will determine the feasibility and reliability of the exRNA splicing profile measured by ddPCR as useful biomarkers of disease severity in DM1 patients using repeated measurements over two years. Go/no-go milestone: candidate exRNA biomarkers demonstrate correlation of at least 0.45 with one or more measures of muscle function and a false discovery rate of 5%. R33 phase (Years 4 - 5): Aim 3 will evaluate the optimal exRNA biomarker candidate identified in the R61 phase as a predictor of DM1 disease status by longitudinal monitoring of ddPCR splice events and clinical measurements of muscle function in a prospective cohort of DM1 patients, and comparison of splice events in exRNA with those in muscle biopsy tissue. Proposed context of use (COU): monitoring biomarker for serial assessment of disease status or burden in patients with DM1. The approach used in this proposal also will be applicable to DM type 2 (DM2), a range of muscular dystrophies, and other genetic disorders.