Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signalling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Dystrophin abnormalities in polymyositis and dermatomyositis.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

Characterisation of dystrophin in carriers of Duchenne muscular dystrophy.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in needle biopsy samples taken from the quadriceps muscle of 15 asymptomatic carriers of DMD (13 adults and 2 young girls) and one symptomatic adult carrier. Antibodies to N- and C-terminal regions of dystrophin were used for both Western blot analysis and immunocytochemistry and a monoclonal antibody to beta-spectrin used to assess membrane integrity. All asymptomatic adult carriers showed some abnormality in dystrophin immunostaining but very few negative fibres were present. A clear mosaic of dystrophin positive and negative fibres was seen only in the adult symptomatic carrier and the two young girls. On a Western blot, all carriers studied had dystrophin of normal molecular weight, but most had reduced abundance. In adult carriers, the amount of dystrophin relative to normal controls varied, but it was unrelated to age, serum creatine kinase (CK) levels or to the degree of pathology. Carriers with normal CK showed abnormalities in dystrophin expression. The dystrophin immunoblotting profile of the 2 young girls was very similar to that of their mothers, but the mosaic pattern of immunostaining was not apparent in the older carriers. In conclusion, dystrophin immunostaining and Western blot analysis of biopsy samples from asymptomatic carriers is often abnormal and they may be useful additional aids for establishing carrier status, particularly in younger girls.

A dystrophin-immunoreactive protein in mammalian brain.

Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of approximately 230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.