The nemaline myopathies (NMs) are a clinically and genetically heterogeneous group of disorders characterized by nemaline rods and skeletal muscle weakness. Mutations in five sarcomeric thin filament genes have been identified. However, the molecular consequences of these mutations are unknown. Using Affymetrix oligonucleotide microarrays, we have analyzed the expression patterns of >21,000 genes and expressed sequence tags in skeletal muscles of 12 NM patients and 21 controls. Multiple complementary approaches were used for data analysis, including geometric fold analysis, two-tailed unequal variance t test, hierarchical clustering, relevance network, and nearest-neighbor analysis. We report the identification of high satellite cell populations in NM and the significant down-regulation of transcripts for key enzymes of glucose and glycogen metabolism as well as a possible regulator of fatty acid metabolism, UCP3. Interestingly, transcript level changes of multiple genes suggest possible changes in Ca(2+) homeostasis. The increased expression of multiple structural proteins was consistent with increased fibrosis. This comprehensive study of downstream molecular consequences of NM gene mutations provides insights in the cellular events leading to the NM phenotype.

OBJECTIVE: To report pathologic findings in 124 Australian and North American cases of primary nemaline myopathy. METHODS: Results of 164 muscle biopsies from 124 Australian and North American patients with primary nemaline myopathy were reviewed, including biopsies from 19 patients with nemaline myopathy due to alpha-actin (ACTA1) mutations and three with mutations in alpha-tropomyosin(SLOW) (TPM3). For each biopsy rod number per fiber, percentage of fibers with rods, fiber-type distribution of rods, and presence or absence of intranuclear rods were documented. RESULTS: Rods were present in all skeletal muscles and diagnosis was possible at all ages. Most biopsies contained nemaline bodies in more than 50% of fibers, although rods were seen only on electron microscopy in 10 patients. Rod numbers and localization correlated poorly with clinical severity. Frequent findings included internal nuclei and increased fiber size variation, type 1 fiber predominance and atrophy, and altered expression of fiber type specific proteins. Marked sarcomeric disruption, increased glycogen deposition, and intranuclear rods were associated with more severe clinical phenotypes. Serial biopsies showed progressive fiber size variation and increasing numbers of rods with time. Pathologic findings varied widely in families with multiple affected members. CONCLUSIONS: Very numerous nemaline bodies, glycogen accumulation, and marked sarcomeric disruption were common in nemaline myopathy associated with mutations in skeletal alpha-actin. Nemaline myopathy due to mutations in alpha-tropomyosin(SLOW) was characterized by preferential rod formation in, and atrophy of, type 1 fibers. Light microscopic features of nemaline myopathy correlate poorly with disease course. Electron microscopy may correlate better with disease severity and genotype.

BACKGROUND: The development of large-scale gene expression profiling technologies is rapidly changing the norms of biological investigation. But the rapid pace of change itself presents challenges. Commercial microarrays are regularly modified to incorporate new genes and improved target sequences. Although the ability to compare datasets across generations is crucial for any long-term research project, to date no means to allow such comparisons have been developed. In this study the reproducibility of gene expression levels across two generations of Affymetrix GeneChips (HuGeneFL and HG-U95A) was measured. RESULTS: Correlation coefficients were computed for gene expression values across chip generations based on different measures of similarity. Comparing the absolute calls assigned to the individual probe sets across the generations found them to be largely unchanged. CONCLUSION: We show that experimental replicates are highly reproducible, but that reproducibility across generations depends on the degree of similarity of the probe sets and the expression level of the corresponding transcript.

Myxopapillary ependymomas (MPEs) are low-grade neuroepithelial tumors typically occurring in the conus-cauda equina-filum terminale region. Limited molecular and cytogenetic analysis of MPEs has not demonstrated consistent abnormalities. In an attempt to clarify the chromosomal status of these tumors and identify commonly aberrant regions in the genome we have combined 3 molecular/cyto/genetic methods to study 17 MPEs. Comparative genomic hybridization of 7/17 tumors identified concurrent gain on chromosomes 9 and 18 as the most frequent finding. The majority of the 17 tumors were also studied using microsatellite analysis with marker spanning the whole chromosomes 9 and 18 and interphase-FISH with centromeric probes for both chromosomes. Our combined results were consistent with concurrent gain in both chromosomes 9 and 18 in 11/17 cases, gain of either chromosome 9 or 18 and imbalance in the other chromosome in 3/17 tumors and allelic imbalances of chromosomes 9 or 18 in 3/17 and 1/17 tumors, respectively. Other abnormalities observed included gain of chromosomes 3, 4, 7, 8, 11, 13, 17q, 20, and X and loss of chromosomes 10 and 22. Our findings represent some steps towards understanding the molecular mechanisms involved in the development of MPE.

The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene, leading to absence of the corresponding protein, disruption of the dystrophin-associated protein complex, and substantial changes in skeletal muscle pathology. Although the primary defect is known and the histological pathology well documented, the underlying molecular pathways remain in question. To clarify these pathways, we used expression microarrays to compare individual gene expression profiles for skeletal muscle biopsies from DMD patients and unaffected controls. We have previously published expression data for the 12,500 known genes and full-length expressed sequence tags (ESTs) on the Affymetrix HG-U95Av2 chips. Here we present comparative expression analysis of the 50,000 EST clusters represented on the remainder of the Affymetrix HG-U95 set. Individual expression profiles were generated for biopsies from 10 DMD patients and 10 unaffected control patients. Two methods of statistical analysis were used to interpret the resulting data (t-test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression). These analyses identified 183 probe sets (59 of which represent known genes) that differ significantly in expression level between unaffected and disease muscle. This study adds to our knowledge of the molecular pathways that are altered in the dystrophic state. In particular, it suggests that signaling pathways might be substantially involved in the disease process. It also highlights a large number of unknown genes whose expression is altered and whose identity therefore becomes important in understanding the pathogenesis of muscular dystrophy.