Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets

<div><p>Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. </p> </div

Biglycan immunolocalisation in UCMD muscle biopsies.

<p>Biglican (red) and perlecan (green) conventional immunofluorescence in muscle sections of controls (<b>A</b>-<b>C</b>), UCMD patient 9 (D-F) and patient 2 (G-I) showing a variable degree of biglican reduction. Scale bar: 50 µm.</p

Characterization of inflammation found in UCMD muscle.

<p>HLA immunostaining in healthy muscle sections located in the endothelial cells of capillaries (<b>A</b>). Sarcolemmal and cytoplasmatic HLA staining on UCMD muscle sections including strong staining of mononucleated cells (<b>B</b>). Immunohistochemistry for CD68 demonstrate macrophage infiltration (<b>C</b>). Immunohistochemistry for CD206 identified M2-type macrophages (<b>D</b>). Arrows point to mononucleated cells CD68+ and CD206+ whereas arrowheads point to only CD68+ cells. Scale bar: 50 µm.</p

Secondary reduction of biglycan at the basal lamina of UCMD patients.

<p>The integrity of the basal lamina and the reduction of biglycan was further studied using confocal microscopy. An example for UCMD patient 6 at two different magnifications is shown. Arrows point to representative areas where biglycan (A, D, G) appears reduced relative to perlecan (B, E, H). Scale bar: 50 µm.</p

GO BP categories in UCMD vs control muscle.

<p>Pie chart representing GO BP categories up-regulated in UCMD vs control muscle.</p