Abstract
Objective: Identify minimally invasive biomarkers of disease activity in muscular dystrophies.
Background: DM1 results from expression of a pathogenic RNA that contains an expanded CUG repeat (CUGexp) and leads to abnormal regulation of alternative pre-mRNA splicing. A previous study (Nakamori, 2013) identified DM1-specific slice outcomes in muscle biopsies as biomarkers of disease severity. Some gene deletions causing DMD are amenable to therapeutic modulation of RNA splicing to restore the open reading frame and produce a partially functional dystrophin protein. In clinical trials for DM1 and DMD, serial muscle biopsies are used to monitor therapeutic antisense oligonucleotide (ASO) drug effects on splicing outcomes. Extracellular RNA (exRNA) in serum and urine includes mRNA and non-coding RNA that can serve as genetic biomarkers of glioblastoma, prostate cancer, and other disease states.
Design/Methods: We isolated exRNA from urine and serum samples of individuals with DM1 (N=18), DMD due to deletion mutations (N=3), and unaffected (UA) controls (N=16), examined RNA integrity by capillary gel electrophoresis, gene expression by qPCR, splicing by RT-PCR, and DMD deletion transcripts by sequencing.
Results: In the urine, 11 of 30 transcripts examined show differential splicing in DM1 as compared to DMD and UA subjects, and display the characteristic pattern found muscle biopsies. By contrast, alternative splicing of these transcripts in serum is similar in DM1, DMD, and UA subjects. In DMD subjects, sequencing of urine exRNA confirms the presence of dystrophin gene deletions amenable to therapeutic exon-skipping ASOs.
Conclusions: Human urine contains genetic markers of DM1 and DMD. The surprising ability to monitor mRNA splicing outcomes in urine may reduce or eliminate the need for muscle biopsies to follow therapeutic ASO effects in DM1 and DMD.
Study Supported by: Slye family fund and NIH (NCIP01CA069246).
Disclosure: Dr. Antoury has nothing to disclose. Dr. Hu has nothing to disclose. Dr. Balaj has nothing to disclose. Dr. Breakefield has nothing to disclose. Dr. Wheeler has received (royalty or license fee or contractual rights) payments from Ionis Pharmaceuticals. Dr. Wheeler has received research support from Sanofi Innovative Awards Program.
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