Transcriptomic analysis of paired healthy human skeletal muscles to identify modulators of disease severity in DMD.

Muscle damage and fibro-fatty replacement of skeletal muscles is a main pathologic feature of Duchenne muscular dystrophy (DMD) with more proximal muscles affected earlier and more distal affected later in the disease course, suggesting that different skeletal muscle groups possess distinctive characteristics that influence their susceptibility to disease. To explore transcriptomic factors driving differential gene expression and modulating DMD skeletal muscle severity, we characterized the transcriptome of vastus lateralis (VL), a more proximal and susceptible muscle, relative to tibialis anterior (TA), a more distal and protected muscle, in 15 healthy individuals using bulk RNA sequencing to identify gene expression differences that may mediate their relative susceptibility to damage with loss of dystrophin. Matching single nuclei RNA sequencing data was generated for 3 of the healthy individuals, to infer cell composition in the bulk RNA sequencing dataset and to improve mapping of differentially expressed genes to their cell source of expression. A total of 3,410 differentially expressed genes were identified and mapped to cell type using single nuclei RNA sequencing of muscle, including long non-coding RNAs and protein coding genes. There was an enrichment of genes involved in calcium release from the sarcoplasmic reticulum, particularly in the myofibers and these myofiber genes were higher in the VL. There was an enrichment of genes in "Collagen-Containing Extracellular Matrix" expressed by fibroblasts, endothelial, smooth muscle and pericytes, with most genes higher in the TA, as well as genes in "Regulation Of Apoptotic Process" expressed across all cell types. Previously reported genetic modifiers were also enriched within the differentially expressed genes. We also identify 6 genes with differential isoform usage between the VL and TA. Lastly, we integrate our findings with DMD RNA sequencing data from the TA, and identify "Collagen-Containing Extracellular Matrix" and "Negative Regulation Of Apoptotic Process" as differentially expressed between DMD compared to healthy. Collectively, these findings propose novel candidate mechanisms that may mediate differential muscle susceptibility in muscular dystrophies and provide new insight into potential therapeutic targets.

Single nuclei transcriptomics of muscle reveals intra-muscular cell dynamics linked to dystrophin loss and rescue.

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.

Complex Patterns of Association between Pleiotropy and Transcription Factor Evolution.

Pleiotropy has been claimed to constrain gene evolution but specific mechanisms and extent of these constraints have been difficult to demonstrate. The expansion of molecular data makes it possible to investigate these pleiotropic effects. Few classes of genes have been characterized as intensely as human transcription factors (TFs). We therefore analyzed the evolutionary rates of full TF proteins, along with their DNA binding domains and protein-protein interacting domains (PID) in light of the degree of pleiotropy, measured by the number of TF-TF interactions, or the number of DNA-binding targets. Data were extracted from the ENCODE Chip-Seq dataset, the String v 9.2 database, and the NHGRI GWAS catalog. Evolutionary rates of proteins and domains were calculated using the PAML CodeML package. Our analysis shows that the numbers of TF-TF interactions and DNA binding targets associated with constrained gene evolution; however, the constraint caused by the number of DNA binding targets was restricted to the DNA binding domains, whereas the number of TF-TF interactions constrained the full protein and did so more strongly. Additionally, we found a positive correlation between the number of protein-PIDs and the evolutionary rates of the protein-PIDs. These findings show that not only does pleiotropy associate with constrained protein evolution but the constraint differs by domain function. Finally, we show that GWAS associated TF genes are more highly pleiotropic : The GWAS data illustrates that mutations in highly pleiotropic genes are more likely to be associated with disease phenotypes.

Identification of putative circadian clock genes in the American horseshoe crab, Limulus polyphemus.

While the American horseshoe crab, Limulus polyphemus, has robust circadian and circatidal rhythms, virtually nothing is known about the molecular basis of these rhythms in this species or any other chelicerate. In this study, next generation sequencing was used to assemble transcriptomic reads and then putative homologs of known core and accessory circadian genes were identified in these databases. Homologous transcripts were discovered for one circadian clock input gene, five core genes, 22 accessory genes, and two possible output pathways. Alignments and functional domain analyses showed generally high conservation between the putative L. polyphemus clock genes and homologs from Drosophila melanogaster and Daphnia pulex. The presence of both cry1 and cry2 in the L. polyphemus transcriptome would classify its system as an "ancestral", type 2 clock system. In addition, a novel duplication of CYCLE, and a novel triplication of PERIOD were found. Investigations are currently underway to determine if any of these "circadian" genes also participate in the molecular processes that drive the Limulus circatidal clock.