Flash-frozen neuromuscular disease patient muscle biopsies were taken for diagnostic purposes. Normal control biopsies were obtained from baseline samples from exercise experiments in young adult volunteers.
Two datasets were analyzed. The first (test set) has been previously published (Bakay et al., 2006 (link)). This set contained Affymetrix mRNA profiles from 117 patient muscle biopsies using both HG-U133A and HG-U133B microarrays (n = 234 microarrays total; GEO accession no.GSE3307 ). The muscle disease groups were DMD (n = 10), amyotrophic lateral sclerosis (ALS; n = 9), acute quadriplegic myopathy (n = 5), BMD (n = 5), CAL (calpain III gene mutations; LGMD2A; n = 10), DYSF (LGMD2B; n = 10), ED-R (Emery–Dreifuss muscular dystrophy X linked; n = 4), ED-D (n = 4), FKRP (LGMD2I; n = 7), facioscapulohumeral dystrophy (FSH; n = 14), JDM (n = 21), and normal human skeletal muscle (NHM; n = 18). Biopsies were taken generally at the time of diagnosis and were typically from the vastus lateralis. The original published dataset analyzed 13 groups, but we removed the hereditary spastic paraplegia group from analyses reported here because of a high proportion of outlier transcripts and also removed four JDM samples that were not made public. To generate the mRNA profiles, frozen muscle biopsies were solubilized, total RNA was isolated, RNA was converted into biotinylated cRNA probes, hybridized to Affymetrix microarrays, and detected using fluorescent streptavidin, and microarrays were scanned. Quality control metrics used were as previously reported (Tumor Analysis Best Practices Working Group, 2004 (link)).
A second set of 49 human patient mRNA profiles was generated using HG-U133 Plus 2.0 microarrays, and this dataset is new to this study. These datasets contained profiles from 6 normal controls, 17 DMD (absence of dystrophin), 11 BMD (present but abnormal dystrophin), 7 LGMD2I (FKRP deficiency, a glycosylation defect), and 8 LGMD2B (DYSF). Patients had a broad range of ages, clinical severity of their disease, and histopathological findings, although all neuromuscular disease patients showed evidence of a dystrophic process (degeneration/regeneration of muscle fibers). Hematoxylin and eosin stains were performed on frozen sections, and the amount of fibrotic replacement (fibrosis) visually approximated for by the same evaluator (E.P. Hoffman) into normal, mild, moderate, or severe fibrosis categories.
Two datasets were analyzed. The first (test set) has been previously published (Bakay et al., 2006 (link)). This set contained Affymetrix mRNA profiles from 117 patient muscle biopsies using both HG-U133A and HG-U133B microarrays (n = 234 microarrays total; GEO accession no.
A second set of 49 human patient mRNA profiles was generated using HG-U133 Plus 2.0 microarrays, and this dataset is new to this study. These datasets contained profiles from 6 normal controls, 17 DMD (absence of dystrophin), 11 BMD (present but abnormal dystrophin), 7 LGMD2I (FKRP deficiency, a glycosylation defect), and 8 LGMD2B (DYSF). Patients had a broad range of ages, clinical severity of their disease, and histopathological findings, although all neuromuscular disease patients showed evidence of a dystrophic process (degeneration/regeneration of muscle fibers). Hematoxylin and eosin stains were performed on frozen sections, and the amount of fibrotic replacement (fibrosis) visually approximated for by the same evaluator (E.P. Hoffman) into normal, mild, moderate, or severe fibrosis categories.
