Muscular Dystrophy

University Degree Medicine and Dentistry

Muscular Dystrophy Essay

Discuss: (a) the way in which a mutation in the gene encoding the protein dystrophin leads to defects in the muscle cell membrane of patients with Duchenne muscular dystrophy; and, (b) the practical issues involved in potential treatments and cures.

The dystrophin protein is a large rod shaped protein with a molecular weight of 427 kDa. The gene itself is 2.4 million bp long making it one of the largest in the human genome, the mRNA transcribed from it is reduced to 14 000 bp due to the many exons it is encoded by. The gene is located on the short arm of the X chromosome at position Xp21.2 bestowing upon it sex linked heredity. Muscular dystrophies in general are rarely manifested in females and this is the case with Duchenne Muscular Dystrophy (DMD). In two thirds of all cases a mutated form of the dystrophin gene is passed on to sons from the mother, however, in the remaining third the deficiency is due to a novel mutation. The high mutatation rate (possibly 1 in 10 000) is thought to be a product of the genes extraordinary length and this can cause problems with genetic counselling.

Dystrophin is made up of (3700 amino acids (aa’s) and has four structural domains:

∙ N-terminal: associates with F-actin in the sub-sarcolemmal cytoskeleton

∙ Rod domain: 25 repeats of 109 aa’s in a triple helix

∙ Cysteine rich domain: binds to sarcolemmal proteins (approx. 300 aa’s)

∙ C-terminal: leucine rich containing 420 aa’s

Dystrophin is located on the cytoplasmic face of skeletal and cardiac sarcolemma, providing a stabilising link between the subsarcolemmal cytoskeleton (probably F-actin) and the extracellular matrix (bound to laminin). Dystrophin does not have a transmembraneous region but forms a complex at its C-terminal end with dystrophin associated proteins (DAP’s), and dystrophin associated glycoproteins (DAG’s). Thus the dystrophin-glycoprotein complex (DGC) is thought to confer structural support to the sarcolemma during muscle contraction and stretch, and helps prevent tearing of the tissues that could lead to muscle cell necrosis. The sarcolemma is also thought to aid in signal transduction and help regulate the intracellular Ca2+ concentration. Other areas that dystrophin has been isolated from include cells surrounding neuromuscular junctions and transverse tubules (T-tubules).

DMD is the most frequent and severe of all muscular dystrophies with an incidence of around 3 in every 10 000. Most boys are wheelchair bound by their early teens and many die before reaching the age of 20, often through resulting complications e.g. respiratory/cardiovascular failure. Proximal muscles such as the shoulders and upper arms, dorsal muscles of the lower limbs are affected first which leads to hypertrophy in other muscles (esp. calf’s) in order to compensate for the lack of function.

A number of mutations may be responsible for genetic defects in Muscular dystrophies, where additions and/or deletions alter the reading frame. DMD occurs as a result of an out-of-frame deletionleading to almost complete loss of the dystrophin protein. In-frame deletions lead to a shorter or less abundant gene product causing the milder phenotype of Becker muscular dystrophy (BMD). DMD and BMD are thought to be alleles of the same gene due to their high homology with each other and location at the same area of the X chromosome. BMD is less common and severe than DMD and therefore the focus of this essay will be on the latter.

The various mutations the dystrophin gene is prone to have different pathological effects depending on the type of mutation, but not its size or position. As described earlier, dystrophin and its associated proteins provides a necessary link between the sarcolemma and the extracellular matrix. The complications involved with DMD arise from loss of the dystrophin molecule due to an out of frame mutation. This causes an alteration in the reading frame of the gene leading to a premature stop codon being generated. Thus synthesis of the mRNA transcript ceases early and the subsequent protein is degraded rapidly in the cytoplasm. The rod domain consists of 25 repeats and mutations tend to occur in this region (the other domains appear to be highly conserved). Removal of one or more of these repeats will not have as serious an effect as, say, a loss of the N-terminal domain. Other mutations that reduce the size or abundance of dystrophin (as in BMD) have less severe consequences.

The sarcolemma is a continuous plasma membrane that forms an intricate system of tubules (including T-tubules) spanning the whole of skeletal muscles. It is attached to these muscles via the DGC, which in turn binds to cytoskeleton filaments (probably F-actin) and provides a flexible framework for the muscles to pull against during contraction and stretching. The sarcolemma membrane is also able to bind Ca2+ ions and then release them after appropriate stimulation and this is thought to aid signal transduction throughout the muscle fibre. In DMD patients, dystrophin is absent from the subsarcolemmal cytoskeleton leading to a lack of DAP/DAG ...

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Stress on the sarcolemma from the repeated contraction and stretching of muscle fibres causes it to tear and Ca2+ ions are released on to the muscle fibre, Ca2+ ions enter the muscle cells and activate proteases which begin to break down cellular constituents leading to necrosis of the tissue. A wedge shaped infarct forms on affected cells and phagocytes arrive to digest and remove the resulting cellular debris. After this stage one of two things occurs: either satellite cells divide giving rise to myoblasts (if the necrosis is contained) which may eventually repair the damaged cell(s), or, the infarct spreads along the fibre ending in its eventual death. Continued muscle cell necrosis from persistent tearing of the sarcolemma results in loss of muscle fibres probably from exhaustion of its regenerative properties and also due to fibrous and fatty infiltration.

When a muscle cell or fibre becomes damaged some of the cellular contents leak out into the extracelular fluid and find their way into the bloodstream. In this way macrophages and other phagocytic cells are attracted to the damaged area. One of the proteins released from necrotic cells is Creatine Kinase (CK) found in high quantities in muscle cells, thus, analysis of blood samples for high levels of CK is a useful diagnostic tool for doctors when considering possible DMD cases. In DMD the serum CK levels may be up to 300 times higher than normal during the first 5 years, dropping in later years but remaining at abnormal levels. Other diagnostic techniques are used to confirm DMD rather than some other myopathy. Genetic counselling using pedigree analysis and muscle biopsy are essential, whereas electromyography (EMG) and electrocardiography (ECG) are less important for diagnosis but may highlight associated heart problems (present in 80% of DMD cases).

There are currently no cures available for DMD and treatment is largely centred on prolonging patients’ lives and making them as comfortable as possible. It is interesting, and indeed distressing, to note that despite recent advances in technology and molecular biology there has been no significant decrease in the number of cases of DMD and similarly little enhancement to patients’ quality of life.

Maintaining a patients ability to use as many healthy muscles as possible is the main priority in most cases. Physiotherapy and occupational therapy must be tailored to the individuals needs as exercising muscles will help keep them healthy but repeated strenuous work may accelerate breakdown of tissue. Joints tend to become restricted in their range of movements as the disease progresses (contracture) and occurs in ankles first then hips, knees and finally shoulders and elbows. Surgery may be attempted to rectify contractures but again will depend upon the individuals’ status, age and stage of disease.

Surgery is increasingly used to correct contractures causing curvature of the spine, known as scoliosis; this involves inserting a metal rod to hold the spine straight. In scoliosis the spine becomes curved to one side accompanied by rotation which causes prominence of the chest wall on one side. In severe cases this can become painful and limit lung function especially during puberty when growth can be most rapid. Before surgery is undertaken the status of the individual needs to be evaluated as patients become increasingly weak with age.

The respiratory muscles become affected in the latter stages of DMD and lung function becomes inadequate. Assistance is given through a facemask used during sleep or even 24 hours a day if normal function can no longer support life. Cardiac muscles generally cause less problems than those of the respiratory system.

Many recent technological advances in the fields of molecular biology and genetics have opened up possibilities for potential treatments and cures for Duchenne muscular dystrophy. The most recent and topical of these is the culture of stem cells for introduction into damaged muscle fibres in the hope that normal function will be restored. Other methods involve the introduction of modified genes via viral vectors and the inducement of revertant muscle fibres back to normal phenotype. Many potential therapies being considered are still in the developmental stage and require extensive testing and support from trials on humans before widespread application is considered.

Ideal gene therapy would be applicable at any stage in the disease, although an associated screening program to give early warning of new mutations would speed up the treatment process. Such a therapy would provide an alternative or replacement gene for the production of dystrophin, which should be expressed throughout the body for an extended period before complications such as contracture arose. Provided no other complications were experienced from increasing the life expectancy of patients they could be expected to live normally with few restrictions.

Such ideal therapies have frequently eluded scientists and doctors and as yet no such ‘miracle cure’ is even on the horizon. There have been a number of recent investigations and discoveries, however, that may yet offer potential new treatments for DMD but so far a cure for the disease is a long way off and limited treatments are the only therapy available at the moment. Elucidating a feasible gene therapy applicable to humans will not simply be a case of ‘switching on’ the dystrophin gene or introducing a working replacement. Lack of dystrophin leads to a lack of the DGC in the sarcolemma membrane; the reasons for this are still unclear but it is not thought to be a result of muscle cell necrosis, yet this may prove just as difficult an obstacle to overcome.

Unless there are improvements in the screening process, many cases of DMD still go unnoticed for a number of years, after this time some of the clinical manifestations of the disease may have caused irreparable damage to some muscle groups. Any therapy used to ease DMD in these patients may be extremely different from that given to one whose disorder is detected early on.

As with any new treatment the potential benefits must be carefully evaluated. Certain complications arising from DMD such as effects on the respiratory and cardiac muscles must be taken into account as these groups must ultimately be preserved to render a treatment worthwhile. The stage at which the treatment is administered could be decisive in terms of benefits gained, generally the sooner therapy could be undertaken the better, provided the child is healthy enough.

A major obstacle for gene therapy is the need to transfer genetic material directly into muscle cells. A variety of techniques exist for this purpose but are by no means without their drawbacks. The conventional model at the moment is the adenoviral vector (Ad); which is capable of carrying inserts of up to 8kbp, it can be modified to carry the mini-dystrophin gene (6.3 kbp, found in some BMD patients) and is then injected into muscle tissue. The Ad then infects muscle cells via its normal route and they can begin to synthesize the encoded protein. This and similar methods have proved successful in a number of studies using mdx mice (a homologue of the human disorder), with expression of mini-dystrophin and an associated accumulation of DAP/DAG’s. Studies such as these are encouraging in respect of their applications for humans but the prospects as yet appear a long way off. Accumulation of the DGC at the sarcolemma appears not to be sufficient for restoration of dystrophin function and long term expression may be necessary to prove the applicability of this technique.

One of the inherent problems with using viral vectors is their ability to bring about an immune response. Viral proteins can be toxic to human and mouse models and must be removed or rendered harmless as they could damage adjacent cells, their ability to replicate must also be removed to avoid viral proliferation. Work is currently underway to engineer such vectors to have reduced immunogenecity as it may be possible to incorporate muscle specific regulatory elements whilst at the same time removing viral genes in order to circumvent the immune response. The ideal Ad would also have increased cloning capacity, enabling the insertion of a full-length dystrophin gene into the viral genome. Other uncertainties such as how best to transfect cells with viral DNA still remain unanswered: is it possible to direct a virus straight to muscle cells from an intravenous injection? what amounts of Ad would be needed to bring about a widespread and persistent expression of dystrophin? These questions and more continue to perplex investigators across the world, however, gene therapy involving viral vectors is by no means the only avenue open to current research.

Some of the most recent advances in cytology have taken place with stem cells and they are raising ideas about possible solutions to DMD, and objections to the research, largely based around the issue of using unborn embryos to provide the cells. Stem cells tend to be defined as undifferentiated cells capable of becoming any type of cell, however, the cells best suited to DMD therapy may be satellite cells isolated from the patients themselves. Satellite cells provide a natural means of generating the muscle mass necessary for growth and are activated (in adults) in response to stress from exercise or trauma. Their daughter cells - myogenic precursor cells - undergo multiple rounds of division and then fuse with existing or new myofibrils. Satellite cells remain at relatively constant levels and persist after continued division throughout adult life, however in DMD their levels become greatly reduced presumably from repeated regeneration. Modified satellite cells from the donors muscle tissue would not be rejected in an immune response and could perform a dual role of regenerating muscle fibres and introducing the normal dystrophin gene. In a recent study, again using the mdx mouse, satellite cells were introduced using similar procedures to bone marrow transplants. The results showed partial expression of dystrophin in affected muscles and may provide an unanticipated avenue for treating DMD. A problem with this technique is the lack of stem cells in cardiac muscle, as maintenance of heart function will be vital to any successful therapy. Muscle fibres generated from regular satellite cells would become tired quickly in a cardiac environment and are thus not readily transferable.

Despite the mutations manifested in DMD patients some 50% show a small number of dystrophin positive fibres, indicating that somewhere in the transcriptional process the premature stop codon is ‘skipped’ with the production of a truncated but functional gene product. Muscle tissues exhibiting this condition are said to have ‘revertant fibres’ and their expression appears as yet to be random and possibly widespread across several species. Thus there appears to be a certain degree of flexibility in the expression of dystrophin. The presence of a premature stop codon should terminate transcription yet if this is not always the case there must be some inbuilt (or consequent) mechanism for overriding a stop. The proposed phenomenon is called exon skipping which can bypass these frameshift mutations with the loss of a small portion of the gene. It could simply be that a further mutation corrects the original defect which could account for clusters of ‘revertant fibres’. Prospects for therapy would involve inducing fibres to revert back to the normal phenotype and/or encouraging proliferation of existing revertants in patients already exhibiting the trait.

Recently, Takeshima et al (2001) reported that oligonucleotides against a splicing enhancer sequence led to dystrophin production in muscle cells from a DMD patient. In revertant fibres of mdx mice a splicing sequence causes exon skipping with the result of an internally deleted dystrophin protein. The introduction of a complementary oligonucleotide to this splicing sequence has also been shown to induce exon skipping with a truncated gene product being formed. In the investigation only cells transfected with the olignucleotide produced revertant fibres and the production of truncated dystrophin was confirmed at the mRNA level. This method too represents a novel way of treating DMD although in some cases the best that could be hoped for is a transformation of the DMD to a BMD phenotype. Further research is necessary to produce other oligonucleotides that could induce exon skipping in other sections of the DMD gene that have mutated.

CONCLUSION- It is worrying to think that despite the extensive documentation of the disease and its alarming fatality rate, an appropriate treatment or cure still eludes us. Doctors trying to diagnose and treat the disease still have no screening program and are at best only able to alleviate the symptoms of DMD.

There are a number of techniques currently in development aimed at providing gene therapy for DMD and are being added to at an ever-increasing rate. They may be aimed at one or a number of the complications it causes but it is likely that future treatments will involve a combined approach to restoring muscle function. Many of the present techniques have been proven and well documented in mouse models but still await clinical trials for them to become accepted treatments.

Utrophin as a replacement for Dystrophin?