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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 23, 2005 as doi:10.1096/fj.05-4678fje. |
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* Leiden University Medical Center, Center for Human and Clinical Genetics, Leiden, The Netherlands;
Howard Hughes Medical Institute, Department of Physiology and Biophysics, Iowa City, Iowa, USA; and
National Institute of Neuroscience, Department of Neuromuscular Research, Tokyo, Japan
1Correspondence: Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, Leiden 2333 AL, Nederland. E-mail: p.a.c.hoen{at}lumc.nl
SPECIFIC AIMS
The aims of this study are to 1) find common and distinct molecular mechanisms that underlie different forms of Duchenne/Becker (DMD/BMD) and limb-girdle muscular dystrophies (LGMD) and 2) explore the feasibility of the use of microarrays for differential diagnosis of muscular dystrophies by establishing expression signatures in a large number of mouse models for muscular dystrophy.
PRINCIPAL FINDINGS
1. Expression profiling distinguishes severely affected dystrophin- and sarcoglyan-deficient mice from mildly affected dysferlin-deficient mice and control mice
We have carried out a comparative gene expression profiling of hind limb muscles of the following mouse models: dystrophin-deficient (mdx, mdx3cv, models for DMD), sarcoglycan-deficient (Sgca null, Sgcb null, Sgcg null, Sgcd null; models for LGMD-2C-F), dysferlin-deficient (Dysf null, SJLDysf; models for LGMD-2B), and sarcospan-deficient (Sspn null; no human disease known) mice. Two wild-type strains (C57Bl/6, C57Bl/10) were included as controls. All mice were 8 wk of age. A total of 2171 genes demonstrated differential expression between the 11 models (P<0.05, Benjamini and Hochberg correction for multiple testing). Hierarchical clustering of the expression profiles clearly separates two groups of mice. The first group contains the models for dystrophinopathy and sarcoglycanopathy, which have a severe dystrophic phenotype at the age of 8 wk and whose expression profiles are remarkably similar. The second group consists of models with a mild or unaffected phenotype.
2. Muscular dystrophies share many molecular and cellular responses
Analysis of the lists of 535 up-regulated and 493 down-regulated genes most discriminative for severely affected mice identified several major biological processes that are commonly altered in these muscular dystrophies (Fig. 1
): increased cell adhesion (up-regulation of Icam1, P-selectin, and several integrins, laminins, and thrombospondins), inflammation (increased levels of many chemokines, cytokines, cytokine receptors, lymphocyte antigens), and regulation of muscle contraction (Atpa2, Calsequestrin 1 and 2, and Troponin C, I, T1 and T2). Many of the up-regulated genes localized either in the extracellular matrix (including 12 different collagens) or the lysosome. The most striking feature of the list of down-regulated genes is the participation of 53% of the genes (259 genes) in highly diverse metabolic processes, indicative of an overall decline in metabolic activity in dystrophic tissue.
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3. Inflammation and remodeling of the extracellular matrix, sarcomere, and cytoskeleton are also evident in dysferlin-deficient mice, though present at lower levels, in agreement with the later age of onset and the earlier stage of the disease analyzed
The majority of up-regulated (101/154) and down-regulated (88/167) genes in dysferlin-deficient compared with wild-type strains were also differentially expressed between severely affected and mildly or nonaffected mouse models. Since the latter group includes the dysferlin-deficient mice, these are genes with subtle changes in dysferlin-deficient mice and higher fold-changes in the more severe models. The major pathogenic mechanisms in which these genes participate are depicted in the schematic diagram (Fig. 3
). It appears that the molecular and cellular details of the pathological processes secondary to the different primary defects are highly similar for the muscular dystrophies analyzed, and are likely to contribute to the progression of these diseases.
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4. Based on the differences in severity of the animal models analyzed, we identified biomarker genes for disease progression
Since the pathology is more progressive in sarcoglycan- and dystrophin-deficient animals than in dysferlin-deficient animals, we statistically identified a set of 33 biomarker genes whose expression levels correlate with disease progression and severity (Fig. 2
).
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5. For the first time, we show a molecular phenotype for sarcospan-deficient animals
Two genes up-regulated in both mildly and severely affected animal models, S100a9, coding for a phagocytic protein known as calgranulin B, and Spp1, coding for a cytokine known as osteopontin, were also significantly up-regulated in sarcospan-deficient mice. This observation suggests for the first time a subtle molecular phenotype for these mice. With quantitative RT-PCR, we confirmed the much higher expression of Spp1 and S100a9 in sarcospan-deficient mice (29- and 14-fold increase over controls, respectively), as well as in the other muscular dystrophy models.
6. Based on identified differences in expression signatures between the muscular dystrophies and on the correlation with studies in human muscular dystrophy patients, we think that it would be possible to build a diagnostic classifier
A comparison was made between sarcoglycan-deficient and dystrophin-deficient mice, revealing 46 differen- tially expressed genes. This indicates there are subtle differences between the different muscular dystrophies. However, given the observed significant inter-individual variability in affected mice and the expected even greater variability in humans, construction of a diagnostic classifier would necessitate the analysis a large number of samples per disease. The analyzed expression patterns in mouse models may speed up the selection of genes relevant for classification.
CONCLUSIONS AND SIGNIFICANCE
We have found remarkable similarity in the expression patterns of dystrophin-, sarcoglycan-, and dysferlin-deficient mice. Genes functioning in the inflammatory response and structural organization are significantly up-regulated compared with wild-type mice, whereas metabolism genes are down-regulated. We conclude that common pathogenic mechanisms underlie the onset and progression of different forms of muscular dystrophy in mice (Fig. 3
). Given the similarity with published human studies on specific forms of muscular dystrophy, these pathogenic mechanisms may also contribute to the different forms of muscular dystrophy in humans. Moreover, we have identified sets of biomarker genes that can be used to monitor disease progression in muscular dystrophies. Finally, by recognition of disease-specific expression signatures, we took the first step toward the development of expression profiling-based classification as a powerful diagnostic approach for muscular dystrophies.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4678fje;
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