Introduction

Muscular dystrophy is the term given to a group of hereditary muscular disorders which cause wasting and weakening of the muscles. The disorders typically arise due to defects in muscle proteins which lead to the untimely death of multiple muscle cells causing progressive muscle weakness. There are over 100 different types of muscular dystrophy, two of interest are; Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease which occurs at a rate of around 1 in 3,500 male births, making it one of the most prevalent muscular dystrophies. The unequal gender distribution indicated that this is a recessive X-linked disease i.e. the defective gene is located on the X chromosome.

The disease causes delays in the development of the infant, for example, the late onset of walking. The rapid rate of muscle degeneration leads to complaints by the infant from the ages of 3-5, these include leg weaknesses and difficulties in movement. The complete loss of ambulation (walking ability) is lost by around 12 years of age.

Continued muscle wasting frequently results in the death of DMD patients, death arises due to failure of the respiratory system as diaphragm muscles fail during the late teen years to the early twenties.

Becker Muscular Dystrophy

Becker muscular dystrophy (BMD) is a similar X-linked recessive muscular disorder to DMD. However, it affects fewer infants (1 in 30,000 male births) and the course of the disease is much more variable than DMD.

The variability of BMD often means, many sufferers of the disease can live well into adult life without being affected too greatly by muscle wasting. Most adults remain ambulatory for the majority of their lives.

Both of these muscular dystrophies involve a loss of the individual muscle fibres, essentially they represent different severities of the same disease – DMD being the more severe disorder. The standard arrangement of muscle fibres is disrupted in dystrophy patients, there is also an observed increase in degeneration, regeneration and fibrosis in the muscle tissues.

Dystrophin

Dystrophin Protein

Dystrophin is a rod shaped, cytoplasmic protein, around 3,500 amino acids in length, which plays an important part in muscle contraction. It consist of four distinct structural domains, including:

• A small actin binding region at the N-terminus
• A large triple helical domain, making up the majority of the protein. The three helices are made up from 24 repeats of a 109 amino acid sequence, the repeating sequence is interrupted by proline-rich ‘hinge regions’ which add flexibility to the molecule.
• A calcium binding region
• The carboxyl terminus which may possibly interact with other glycoproteins

It is expressed upon the inner face of the plasma membrane of both smooth cardiac and striated muscles. It makes up part of a protein complex (a costamere) along with a multitude of other proteins.

It appears that dystrophin is able to interact with a variety of membrane proteins due to its localization within the cell and proximity to the cytoskeleton. The role of dystrophin is to act as a molecular ‘shock absorber’ which reduces stress placed on the plasma membrane during muscle contractions.

Dystrophin-Like Proteins

The dystrophin gene codes for several protein products including dystrophin and dystrophin-like proteins. Dystrophin-like proteins (DLPs) are comparatively small to the original dystrophin protein but are able to act as a substitute for dystrophin, as they express similar behaviour.

DLPs may also be encoded for by different genes other than the dystrophin gene, despite this, they still express similar behaviour. Typically, DLPs vary in length, often smaller than the standard dystrophin protein – because of this and other variations, their efficacy is reduced.

Dystrophin Gene

The dystrophin gene is X-linked i.e. located on the X-chromosome, this is deducible from the uneven distribution of the disease between males and females. Because males only have a single X chromosome, if they inherit a recessive X-linked allele, there is no possibility of a dominant allele being expressed (therefore the recessive phenotype is observed).

The dystrophin gene is one of the largest genes of the human genome, spanning around 2.5 million base pairs of DNA. Within this are at least 70 exons, now believed to be much closer to 80. The large size of the gene probably contributes to the high mutation rate – one thought is that it is much more likely for spontaneous mutations to occur i.e. not just inherited, again due to the size of the gene.

Diagnosis

Muscular dystrophies can be confirmed by analysising the genetic sequence of the dystrophin gene. If deletions are present in the sequence, then it is highly likely the observed dystrophies are due to the mutations of the dystrophin gene.

Methods used to detect deletions include Southern blotting (a process involving separation of DNA by electrophoresis and subsequent fragment detection by probe hybridization) and PCR. PCR is able to detect mutations (deletions or duplications) in one or more exons of dystrophy patients with an accuracy of around 65% – the remaining patients have more subtle alterations such as point mutations which are easier to miss.

If there is a complete lack of dystrophin within the body then DMD can be predicted with 99% accuracy, whilst an altered size or prevalence of dystrophin predicts BMD with 95% accuracy (It is likely that the altered dystrophin will still have some efficacy and thus it is unlikely DMD will arise).

It is also possible to carry out prenatal testing on a foetus of around 11-14 weeks old, as with all prenatal testing, there are ethical issues which arise – such as the increased likelihood of miscarriage and how to respond to a positive test.

Mutations of the Dystrophin Gene

The dystrophin gene consists of the four domains described earlier. Mutations which occur within the different domains can give rise to different levels of dystrophy, for example, a deleterious mutation in the first domain may have a completely different result to the same mutation in the fourth domain.

Example Mutations

Deletions within domain 1 (actin binding region) result in greatly reduced levels of dystrophin and as such, this type of mutation is associated with the most severe phenotypes of muscular dystrophy. Because this domain is responsible for binding the protein to actin, deletions can be expected to reduce overall protein stability by disrupting interactions with other components of the cytoskeleton.

The largest of the domains, domain 2, consists of the triple helical structure. Any mutations within this domain give rise to highly variable levels of dystrophies. Deletions to the centre of domain 2 cause very mild dystrophy phenotypes which supports the suggestion that this large domain is there merely to provide size (which would increase the level of absorption).

Mutations to domains 3 and 4 almost certainly give rise to DMD, this suggests their role is essential as any mutation in these domains leads to the more severe phenotypes. However, loss of the terminal portion of domain 4 is associated with the less severe phenotype – BMD.

Prevention and Treatment

Because the disorder is an inherited disease, essentially the only way to prevent it is to identify adult female carriers of the disorder. However, even this is not enough to completely prevent prevalence of the disease due to the high spontaneous mutation rate of the dystrophin gene.

It may be that an infant is born with either BMD or DMD and the disease goes undetected until late infancy. Whilst the disease cannot be cured, early treatment can help prolong the patients life and ease suffering. Early detection of the disease is thus quite important – screening infants for increased serum levels of creatine kinase (an indicator of damage to muscle tissue) could allow for such early detection.

Some treatments designed to control the onset of symptoms and improve quality of life are:

• Corticosteroids – Can increase energy and strength of the patient and defer the severity of some of the symptoms
• β₂ -agonists may increase muscle strength of the patient, but onset of the disease is not altered
• Physical therapies such as; motility aids, mild physical activity and respiratory support are important, especially as the disease progresses

Gene Therapy

One promising area of research into alleviating the symptoms of DMD is gene therapy. As of 2007, standard viral-mediated gene therapies (which allow for the transfer of normal function genes to the patient), have been undergoing clinical research. Recently (April 2010) it has been suggested that meganuclease enzymes may be of use in repairing defective genes associated with DMD.

However, there are a number of obstacles to overcome if successful gene therapy is to be carried out. Firstly, the sheer total mass of human muscle makes it difficult to administer any form of treatment across the whole area. Any vector which could administer its payload to the entire muscle mass would also need to be selective i.e. only cause expression of dystrophin in skeletal muscle. Another problem may be autoimmune responses mounted against the dystrophin proteins, although the use of viruses usually reduces the threat of such immunological problems.

Dystrophin-Like Protein Up-Regulation

As an alternative to gene therapy would be to identify dystrophin-like proteins and cause their up-regulation within the body. Hopefully this would allow the dystrophin-like proteins to compensate for the lack of actual dystrophin, although this may not cure the disease it may reduce the severity of it, for example, elevate the disease from DMD to a mild case of BMD.

One candidate for such a task is the protein utrophin (ubiquitous dystrophin). Utrophin is the closest endogenous analogue of dystrophin within the human genome. It is encoded for by a different gene, much smaller in length (less prone to mutation) and the up-regulation of this gene by pharmaceutical products could compensate for the muscle cells lacking dystrophin expression.

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