Source code of paper "A central role for disordered renal metabolism in myotonic dystrophy type 1"
Chronic kidney disease (CKD) is associated with progressive muscle wasting, whole-body insulin resistance, and impaired systemic metabolism. Reduced renal function has been described in the genetic muscle wasting disorder myotonic dystrophy type 1 (DM1). However, the mechanism for kidney involvement in DM1 is unknown and its relationship to clinical features uncertain. Here we use urinary extracellular vesicles (EVs), RNA sequencing, droplet digital PCR, and predictive modeling to identify downregulation of metabolism transcripts PCK1, HPD, DPYS, GSTA1, ACY1, and ETFB in DM1. Expression of these genes localizes to the kidney, especially the proximal tubule, and correlates with muscle strength and function. In DM1 autopsy kidney tissue, characteristic ribonuclear inclusions are evident throughout the nephron. We show that urinary organic acids and acylglycines are elevated in DM1, and correspond to enzyme deficits of downregulated genes. Our study identifies a previously unrecognized site of DM1 molecular pathogenesis, highlights the potential of urinary EVs as biomarkers, and supports longitudinal investigation of the relationship between urinary EVs and renal function in this population.
We generated libraries from subjects with DM1 and DMD (N = 4 each group) using 10 ng urine exRNA from each sample and the Takara SMARTer Stranded Total RNA Sequencing kit V2 - pico input mammalian (Takara product number 634412). Sequencing was performed in two separate batches using an Illumina NextSeq 500 platform at the George Washington University Genomics Core (https://www.gwgenomics.org) and a high output 150-cycle kit to generate more than 83 million paired end reads per sample. Reads were aligned to the human GRCh38 genome and quantified using Salmon (v. 1.10.2)67 (see pipeline). Genes that have average expression of greater than 5 transcripts per million (TPM) in at least one group (DM1 and/or DMD) were considered as expressed genes and included in the down-stream analysis. One DM1 and one DMD sample from batch 1 were excluded because they failed quality control and had too few reads. Differential expression analysis was done using the edgeR (v.3.30.3) packages in R20 with a false discovery rate Q < 0.05 (see script).