Heterogeneity of dystrophin expression in patients with Duchenne and Becker muscular dystrophy

Abstract

This report documents the results of an integrated biochemical and immunocytochemical investigation into the expression of dystrophin (the protein product of the Duchenne muscular dystrophy gene) in muscle biopsies from 226 patients. It is the first study in which dystrophin has been analysed on blots and on tissue sections in such a large number of patients using the same (monoclonal) antibody. The 140 patients with Xp21 muscular dystrophy who were included in this study represent a continuous spectrum of disease severity and this range was reflected in the heterogeneity of dystrophin expression which was observed with respect to abundance, size and the pattern of tissue localisation. Approximately 40% of biopsies obtained from patients diagnosed as having Duchenne muscular dystrophy (DMD) contained isolated clearly positive fibres and a further 20% had very weak labelling on a large number of fibres. Biopsies from patients with Becker muscular dystrophy (BMD) showed labelling patterns which varied from weak labelling on the majority of fibres to clear labelling on all fibres. Typically, however, there was inter- and intra-fibre variation in labelling intensity. Approximately 85% of the 52 BMD and 54 DMD patients who had unequivocal labelling on blots demonstrated a protein of abnormal size. The remaining 15% had a protein of normal size but reduced abundance. Overall, the estimated abundance of dystrophin correlated well with clinical assessments of the disease severity expressed in patients. We conclude that dystrophin analysis is an essential and dependable technique for the differential diagnosis of patients with Xp21 muscular dystrophy.

Protein defects in neuromuscular diseases

“…A correlation between the amount of dystrophin and the severity of the phenotype has been suggested (2,29). However, in our experience, patients with an intermediate clinical phenotype between DMD and BMD (outlier patients) usually show the same dystrophin-deficient pattern as classical DMD (30,31).…”

“…However, a variable proportion (4-30%) of dystrophin-positive isolated or grouped fibers (called revertant fibers) are observed in most of them, mainly with the N-terminal antibody ( Figure 2). This small amount of dystrophin can be observed as faint bands by Western blotting, but is not correlated with the clinical course (29)(30)(31). This observation should be taken into account before assessing the effect of any therapeutic trial with the replacement of dystrophin through gene or cell therapy.…”

Multiplex Western Blotting System for the Analysis of Muscular Dystrophy Proteins

“…The addition of 4 mol/L urea to a treatment buffer already containing 4% SDS aided the dissociation and solubilization of large protein complexes in skeletal muscle, particularly those of myosin and laminin. The addition of 0.01% SDS to the blotting buffer encouraged large proteins out from the gel matrix (but did not appear to inhibit binding to the nitrocellulose, as judged by the detection of dystrophin in DMD patients 11 ), whereas the 20% methanol helped to fix the proteins to the nitrocellulose sheets.…”

“…The density of the myosin heavy chain band on the dried, post-blotted gel was used to indicate how much muscle protein (as opposed to fat and fibrous connective tissue) had been loaded in each sample lane. 11 …”

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice

“…In Becker dystrophy, dystrophin expression is heterogeneous: low to strong expression of an often truncated dystrophin is observed in the muscle fibers. 54 Dystrophin isoforms which are often constitutively expressed by DMD patients may at least partially protect against immune rejection of an exogenous dystrophin. A phase I clinical trial for dystrophin gene transfer into DMD patients should therefore address the anti-dystrophin rejection issue (see also Ref.…”