Muscular Dystrophy. Biol 405 Molecular Medicine

Transcription

1 Muscular Dystrophy Biol 405 Molecular Medicine

2 Duchenne muscular dystrophy Duchenne muscular dystrophy is a neuromuscular disease that occurs in ~ 1/3,500 male births. The disease causes developmental delays e.g. late onset of walking. By the age of 3-5 years complaints include leg weakness i.e. running and climbing difficulties. Subsequently, gradual muscle wasting occurs leading to loss of ambulation by age 12. Death frequently occurs in the late teens or early twenties as a consequence of respiratory failure (through weakening of the diaphragm).

3 Becker muscular dystrophy The usual arrangement of fibres is disrupted and there is marked degeneration, regeneration and fibrosis in the muscles. Becker muscular dystrophy affects ~ 1/30,000 male births. Course is much more variable and less severe than Duchenne muscular dystrophy. Consequently, many Becker patients remain ambulatory into adulthood and live full and minimally restricted lives. Both dystrophies involve loss of individual muscle fibres and represent different degrees of severity of essentially the same disease.

4 The dystrophin gene Unequal gender distribution indicates that the gene is on the X chromosome. Analysis of the gene proved to be difficult because there was no prior knowledge of the nature of the gene product. Initial strategies depended upon mapping the large numbers of mutations causing the disease. The gene spans ~ 2.5 million base pairs of genomic DNA. It consists of at least 70 exons and gives rise to a 14kb transcript that encodes dystrophin (Mr ~ 427,000). The large size of the gene probably accounts for its high mutation rate.

5 The dystrophin protein The dystrophin gene codes for several protein products. Three different promoters control the expression of different products in muscle, brain and other non-muscle tissues respectively. Alternative splicing also occurs. The dystrophin protein found in muscle is composed of four structural domains. Dystrophin is stained brown in normal muscle (left), but is absent in muscle from a boy with DMD (right).

6 Severe BMD Cramps and/or weakness BMD DMD BMD Hinge region 1 - actin binding N-terminus 2 - triple helical domain 3 - calcium binding region? 4 - carboxyl terminus - interaction with glycoprotein? The Dystrophin Protein 1 shows homology to the actin-binding regions of -actinin and -spectrin. 2 consists of a series of amino acid repeats, which form a triple helical structure. The repeats are interrupted by proline-rich hinge regions that add flexibility to the molecule. 3 is similar to the Ca 2+ binding region of -actinin. 4 is similar to the carboxyl terminus of the chromosome 6- encoded dystrophin-related protein (utropin).

7 Dystropin-like proteins A 71 kda protein - expressed by internal promoter between exons 62 and 63 of the dystrophin gene. Contains 583 out of the 610 amino acids of the cys-rich and carboxyl terminus domains of the dystrophin protein, this core is flanked by unique amino- and carboxyl terminus sequences. An 87 kda protein encoded by a separate gene. This is a component of the acetylcholine receptor-rich postsynaptic membranes from electric tissues. Its core is modestly homologous to the dystrophin cys-rich and carboxyl terminus domains.

8 Immunocytochemical mapping has shown that dystrophin is expressed on the inner face of plasma membranes of smooth, cardiac, and striated muscle as part of a glycoprotein complex (it is also expressed in specifc neurones). The protein appears to be part of the cytoskeleton and, as such, interacts with a variety membrane proteins. Overall the function, of dystrophin is to act as a molecular 'shock absorber' protecting the plasma membrane from the stresses developed during muscle contraction.

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10 Diagnosis Southern blotting can be used to test for deletions in the coding sequence of the dystrophin gene. With the aid of PCR technology, deletions, or duplications, of one or more exons can be detected in approximately 65% of dystrophic patients. The remaining 35% of patients have more subtle alterations such as splicing mutations or point mutations in the coding sequence. Some of these mutations are detectable by mrna RT-PCR.

11 Immunoblotting of muscle proteins from tissue biopsies allows the identification of altered size dystrophin mutants. The total absence of dystrophin predicts Duchenne muscular dystrophy with 99% accuracy. The presence of larger or smaller sized dystrophins and/or reduced abundance of dystrophin predicts Becker muscular dystrophy with 95% accuracy.

12 Severe BMD Cramps and/or weakness BMD DMD BMD Hinge region 1 - actin binding N-terminus 2 - triple helical domain 3 - calcium binding region? 4 - carboxyl terminus - interaction with glycoprotein? The Dystrophin Protein Deletions within domain 1 result in low levels of dystrophin and the more severe phenotypes. This domain binds actin and deletions might be expected to reduce protein stability by disrupting interactions with other components of the cytoskeleton. The phenotypes of patients with deletions or duplications within domain 2 are more variable.

13 Severe BMD Cramps and/or weakness BMD DMD BMD Hinge region 1 - actin binding N-terminus 2 - triple helical domain 3 - calcium binding region? 4 - carboxyl terminus - interaction with glycoprotein? The Dystrophin Protein Loss of the middle section of domain 2 causes a very mild phenotype. If domain 2 only provides size then deletions may be predicted to have minimal impact. Deletions around exons cause Becker muscular dystrophy. Phenotypic variability suggests that environmental factors may play important roles in clinical progression. Domain 3 and the proximal region of domain 4 are apparently essential - loss leads to Duchenne muscular dystrophy. Loss of the terminal portion of domain 4 is associated with mild Becker muscular dystrophy.

14 Prevention and possible treatment Prevention relies on the identification of (female) carriers with demonstrable mutations and on prenatal diagnosis from foetal cells. Unfortunately the high spontaneous mutation rate of the dystrophin gene limits the effectiveness of these techniques. Infant screening for serum creatine kinase may allow identification of affected children.

15 Subcellular structures and pathways of a skeletal muscle fibre targeted in treatment strategies.

16 Gene Therapy There are a number of obstacles to successful gene therapy: Large size of the human skeletal muscle volume. Vectors needed that can deliver and appropriately express dystrophin only in skeletal muscle. Immunological problems - if the immune system has never seen a protein (dystrophin) it may mount an autoimmune response to it. Adenoviral vectors, gutted of almost the entire viral genome, are potentially useful as there should be few immunological problems with viral proteins. Use of antisense oligonucleotides to force alternative splicing around mutations - thus restoring reading frame and converting a Duchene phenotype to the milder Becker phenotype may be a useful approach.

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18 Gene therapy and cell (myoblast) transplantation may be combined i.e. transplantation of genetically modified myoblasts. e.g. derive a primary culture from a patient s muscle biopsy, then carry out adenovirus-mediated dystrophin gene transfer into these cultures with subsequent expression of the dystrophin transgene. The transduced cultures can then, hopefully, be transplanted back into the patient. An alternative approach may lie in the identification of proteins similar to dystrophin. The expression of such proteins (e.g. the chromosome 6 encoded dystrophinrelated protein) may be up-regulated, by appropriate pharmacological agents, to compensate for the absence of dystrophin. Adult stem cells producing dystrophin in dystrophin-deficient mice.

19 Summary Duchenne muscular dystrophy is a neuromuscular disease that occurs in ~ 1/3,500 male births. The milder Becker muscular dystrophy affects ~ 1/30,000 male births. The dystrophin gene consists of at least 70 exons and encodes the dystrophin protein (Mr ~ 427,000). Dystrophin acts as a molecular 'shock absorber' protecting the plasma membrane from the stresses developed during muscle contraction. The absence of dystrophin predicts Duchenne muscular dystrophy with 99% accuracy. Larger or smaller sized dystrophins and/or reduced abundance predicts Becker muscular dystrophy with 95% accuracy. The high spontaneous mutation rate of the dystrophin gene prevents reliable identification of carriers or prenatal diagnosis. There are considerable obstacles to successful gene therapy.

20 References Le Rumeur, E. et al., (2010) Biochim. Biophys. Acta 1804, dystrophin structure. Vilquin, J.-T. et al., (2011) Curr. Opin. Organ Transplant. 16, Cell therapy for muscular dystrophies. Hoffman, E. P. et al., (2011) Am. J. Pathol. 179, Exon skipping and stop codon read through. Goyenvalle, A. et al., (2011) Hum. Molec. Gen. 20, R69-R78. Therapeutic approaches to muscular dystrophy. Sahenk, Z. & Mendell, J. R. (2011) Curr. Rheumatol. Rep. 13, Distinct pathogenic mechanisms and novel therapeutic strategies.