A protocol for single nucleus RNA-seq from frozen skeletal muscle.

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

Transcriptome analysis from muscle biopsy tissues in late-onset myopathies identifies potential biomarkers correlating to muscle pathology.

The diagnosis of adult-onset genetic muscle diseases is challenging because of the diversity of clinical phenotypes, findings on muscle biopsy that may be nonspecific, and the large number of genetic causes. Even with thorough investigation, the diagnostic yield for genetic testing in these populations is very low, and the distinction from acquired conditions such as sporadic inclusion body myositis [sIBM] can also prove difficult. In this study, we analysed whole transcriptome data generated from RNA isolated from muscle biopsy tissues, from a cohort of 16 participants with sIBM and other histologic diagnoses. Our objective was to identify candidate RNA biomarkers that could be an adjunctive tool in differentiating these conditions. Principal component analysis was able to delineate the groups based on their histologic diagnoses. Gene ontology and pathway analyses demonstrated dysregulation of immune pathways in sIBM. In mitochondrial myopathy we observed upregulation of FGF21, GDF15, ASNS and TRIB3, which are known candidate biomarkers for mitochondrial myopathy. Novel findings included the identification of transcripts of unknown function that were dysregulated in myofibrillar myopathy [JPX], dystrophic changes [MEG3], and mitochondrial myopathy [GAS5]. We suggest future investigations with larger cohorts of participants to confirm the findings of this study, with further directed experiments to determine the role of novel transcripts in disease pathogenesis.

Highly Elevated Prevalence of Spinobulbar Muscular Atrophy in Indigenous Communities in Canada Due to a Founder Effect.

OBJECTIVE: Spinobulbar muscular atrophy (SBMA) is an X-linked adult-onset neuromuscular disorder that causes progressive weakness and androgen insensitivity in hemizygous males. This condition is reported to be extremely rare, but has higher prevalence in certain populations due to multiple founder effects. Anecdotal observations of a higher prevalence of SBMA in patients of Indigenous descent in Saskatchewan led us to perform this study, to estimate the disease prevalence, and to attempt to identify a founder effect. METHODS: For our prevalence estimation, we identified patients with confirmed SBMA diagnosis from the Saskatoon neuromuscular clinic database for comparison with population data available from Statistics Canada. For our haplotype analysis, participants with SBMA were recruited from 2 neuromuscular clinics, as well as 5 control participants. Clinical data were collected, as well as a DNA sample using saliva kits. We performed targeted quantification of DXS1194, DXS1111, DXS135, and DXS1125 microsatellite repeats and the AR GGC repeat to attempt to identify a disease haplotype and compare it with prior studies. RESULTS: We estimate the prevalence of SBMA among persons of Indigenous descent in Saskatchewan as 14.7 per 100,000 population. Although we believe that this is an underestimate, this still appears to be the highest population prevalence for SBMA in the world. A total of 21 participants were recruited for the haplotype study, and we identified a unique haplotype that was shared among 13 participants with Indigenous ancestry. A second shared haplotype was identified in 2 participants, which may represent a second founder haplotype, but this would need to be confirmed with future studies. CONCLUSIONS: We describe a very high prevalence of SBMA in western Canadians of Indigenous descent, which appears to predominantly be due to a founder effect. This necessitates further studies of SBMA in these populations to comprehensively ascertain the disease prevalence and allow appropriate allocation of resources to support individuals living with this chronic disease.