Diagnostic Tools For The Detection Of DMD Biology Essay


Locomotion, the fundamental characteristic of mammals, is due to the presence of skeletal muscle tissues that convert Adenosine Tri-Phosphate into mechanical energy. Degeneration of these muscles would result in impaired locomotion and movement. The genetic disorders that result in progressive muscle weakness, degeneration and wasting are referred to as muscular dystrophies (MD). MDs are a single-gene, hereditary disorders that are transmitted according to the Mendelian Inheritance patterns.

Duchenne Muscular Dystrophy (DMD), x-linked, is the most severe type of muscular dystrophy and affects ~1 in 3500 live born males. (Scott et al., 1998) Males are more prone to this disease because it is caused by the mutation in the dystrophin gene located at Xp21 transmitted from mother to son. It has been found that 30% of individuals with DMD have mothers who are not carriers of the defective gene. In these instances, a spontaneous genetic mutation and not genetic inheritance results in muscular dystrophy (Bullock, 1992). The mutations cause an absence of active dystrophin leading to decreased levels of the Dystrophin associated proteins. This disrupts the localization of dystrophin and its associated proteins making the sarcolemma more susceptible to contraction mediated ruptures and degeneration.


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DMD associated progressive muscle wastage results in gradual loss of motor function. Difficulty in walking is seen in the initial stages and includes abnormalities in stepping gait. The patient will eventually require crutches and ultimately be confined to a wheelchair. The enlargement of calf muscles (pseudo-hypertrophy) results from macrophage infiltration. Deformities of the skeletal structure include scoliosis, osteoporosis and lumbar lordosis (inward curvature of the spine). There are also deformities of the muscle where the function of the achilles tendon and hamstrings are impaired due to shortening of the muscle fibers and fibrosis occuring in connective tissue. Individuals have an increased likelihood of suffering from behavioural and learning disabilities, and suffer notable fatigue.

Diagnostic tests

There are several diagnostic tools for the detection of DMD. However, the co

Blood tests

The levels of serum creatine kinase are an important marker in the determination and detection of MDs.

This test is used to evaluate neuromuscular diseases in three basic ways:

To determine whether symptoms of muscle weakness is caused by a nerve or muscle problem

To differentiate between types of disorders i.e. Dystophies vs congenital myopathies

To detect carriers of neuromuscular disorders particularly in DMD.

Venous blood samples should be taken from individuals before and after the completion of exercise. The serum creatine kinase levels will be significantly greater in DMD carriers before and after exercise than in normal individuals. Creatine kinase is an enzyme necessary in the conversion of ADP to ATP. Larger than normal quantities within in the bloodstream indicates a large-scale destruction of muscle cells. DMD causes muscle cell membranes to break down, allowing creatine kinase to spill out into the bloodstream resulting in these characteristic high levels.


In sufferers of DMD, cardiomyocytes and the Purkinje system are progressively replaced by connective tissue or fat leading to cardiac abnormalities (Finsterer and Stollberger, 2003). Absence of dystrophin in Purkinje fibres alters conduction leading to atrioventricular blockage. In DMD patients, cardiac autonomic nervous system disturbances are present especially reduced vagal activity and enhanced tone. (Vita et al., 2001) Other abnormalities include tachycardia.

Usually, the ECG readings reveal tall R waves in the right precordial leads. An increase in the R/S ratio and W waves are shown in lead augmented vector left (aVL), V5 and V6 (precordial leads) due to posterior wall involvement. The posterobasal and the inferior wall of the left ventricle show abnormalities due to which the QRS wave is shifted to the right causing an increase of R/S ratio in ECG readings. (Sanyal et al., 1978)

Muscle biopsy

Immunohistochemistry methods are used to analyse muscle biopsies for the presence of dystrophin. This involves using antibodies against the N-terminal, C- terminal and - rod-like domain of the protein. No dystrophin is shown in the biopsies of DMD patients except that which is confined to a small proportion of the muscle fibre. The samples are further analysed by Western blotting.

Musculoskeletal examination

This diagnostic test is one of the most complex features of the physical examination. This examination focuses on observation of the functions of the musculoskeletal system. An inspection of the patient's active range of motion is assessed at the joint junctions. Palpations are undertaken to check for swelling, tenderness and bony enlargement on the joints. (Bushby et al., 2009)

Molecular genetic analysis

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This test involves DNA extraction from the patient and testing it for the gene mutations characteristic of DMD. In most cases, multiplex PCR combined with southern blotting proves to be helpful in detecting DMD. This is because almost 66% of the DMD patients show large deletions or amplifications in parts of the dystrophin gene (Prior and Bridgeman, 2005). For the remaining 33%, the diagnosis by this method faces challenges due to point mutations. However, this technique proves to be an important tool in pre-natal screening there by enabling early management.

Pathophysiology of DMD

The dystrophin-glycoprotein complex (DGC) encompasses several proteins like dystrophin, dystroglycans (α and β), sarcoglycans (α,β,γ and δ), sarcospan, syntrophins (α1,β1 and β2) and dytstrobrevin. This entire complex of membrane-associated proteins functions to connect the F-actin in the sub-sarcoplasm and the DGC in the extracellular matrix in the skeletal and cardiac muscles of the human body (Bunnell et al, 2008; Petrof et al, 1993). Hence, it provides protection against stress which is frequently imposed during the course of muscle contractions along with regulation of the cell-signalling cascades (Bunnell et al, 2008; Evans et al, 2009). Several alterations like mutations in any component of the DGC are held responsible for a variety of pathological conditions in humans such as cardiomyopathy, vasospasm and muscular dystrophy demonstrated in both animal models and humans (Kim et al, 2009).

Of all the proteins that are involved in the formation of the DGC, the dystrophin protein is one of the best characterised DGC proteins. Despite the wide spectrum of Pathophysiology of DMD, the primary defect identified is the absence of dystrophin in humans and several animal models like the mdx mouse. Usually out-of-frame mutations in the dystrophin gene lead to reduced dystrophin protein expression in the myofibers (Kimura et al, 2008). These mutations results in destabilisation of the protein complex, myofiber degeneration, fragility of the sarcoplasm and severe muscular weakness (Tidball et al, 2007; Blake et al, 2002).

Dystrophin protein: The rod-shaped, membrane-associated dystrophin protein is a 470 kDa product of the dystrophin gene, whose defect is characteristic of one the types of progressive muscular dystrophy, DMD. The dystrophin gene has been mapped to the short (p) arm of the X chromosome at position 21.2 (http://ghr.nlm.nih.gov/gene=dmd). It spans a region >2,200 kb, which roughly means about 0.1% of the entire genome (Hegde et al, 2008). The protein is primarily found in the skeletal muscle, however even there it is found at very low concentrations (Tidball et al, 2007). Structurally it is composed of four essential domains, which include the actin-binding domains, cysteine-rich domains and a carboxyl terminal domain (Ervasti et al, 2007) (Fig 1).

Fig 1: Dystrophin protein and its interaction with the other proteins of the dystrophin-glycoprotein complex. (Davies et al, 2006)

The absence of the entire dystrophin protein or reduction of the protein levels below that of the threshold level, results in increased myofiber sensitivity to injury caused by muscle contraction (Li et al, 2008). These contraction-induced injuries lead to a ripped sarcoplasm, thus activating a series of pathological conditions beginning from muscular degeneration, inflammation and subsequent cell death (Li et al, 2008). Muscles examined from specimens of mdx mice indicate that these dystrophin-absent fibres are susceptible to rupture of the sarcolemma during contraction. Upon contraction, (primarily eccentric contractions) the compression of the myofilaments transmits a force to the sarcolemma thus putting a heavy amount of pressure upon this membrane. Contraction therefore can rupture membranes, damaging the fibres, and limiting or inhibiting their usage. The fibres however are not more susceptible to rupture by quantity of contractions, but rather by the stress exerted upon the fibre i.e. the percentage the muscle is contracted (Petrof et al, 1993). Hence, consequent degeneration of the muscle cells through this contraction-induced process, accompanied by gradual accumulation of adipose and fibrous tissues results in reduced functional muscle mass and loss in contractility (Gregorevic, 2008). Several studies have also indicated that absence of the dystrophin protein results in an elevated intracellular calcium level, in addition to an altered calcium-mediated muscle contractility. Numerous studies propose abnormality in the regulatory mechanism of calcium ions in the body is linked to necrotic cell death and excitotoxic injury (Kim et al, 2009)

Prior to the identification of the deficient dystrophin protein as a causative factor in DMD, oxidative stress was proposed as the chief cause of the disease condition. However, current research supports the notion that interactions occurring between the primary genetic defect and disturbed free radical production in the body participate in the pathophysiology of DMD. Currently, three broad routes are known through which the disrupted free radical production can contribute to the developing pathology. Firstly, significant variations in the production of free radicals disrupt the default signalling processes, hence aggravating the disease pathology. Secondly, tissue-specific responses to the presence of pathology lead to a significant disruption in free radical production. Lastly, certain behavioural changes in an affected individual result in additional alterations in the production and stoichiometry of free radicals, thus contributing to the disease pathology (Tidball et al, 2007). Also, an immunological response to a dystrophin-deficient muscle, demonstrated on the mdx mouse model, has been found responsible for the promotion of DMD. The release of major basic protein (MBP) by the eosinophils results in the lysis of muscle cells in vitro, indicating it's prominence in DMD (Wehling-Henricks et al, 2008).

Complications and Prognosis

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No drug therapies or occupational therapeutics cannot prevent the gradual and progressive degradation of muscle. Decreased mobility and gradual inability to care for oneself is the result. Weight loss will occur due to this wastage, though most often in the later stages. However, motion manoeuvres, passive and active, as well as a walking regime can delay the speed of deterioration, though not prevent it.

Of those who become wheelchair bound, 90% develop scoliosis which will worsen over time (Galasko, 1995). Osteoporosis, seen in 20% of sufferers, can result from glucocorticoid therapy (Bachrach, 2005). Grip strength will also decline. Joint contractures; a stiffening of joints preventing contraction to the full extent are often seen.

Respiratory muscle strength will decline over time, resulting in progressive respiratory failure. Early, non specific respiratory symptoms will be witnessed eventually leading to hypoventilation and an inability to cough. Additionally, respiratory tract infections such as pneumonia are likely. This is likely to be the cause of death (McConnell, 2005).

In some instances, smooth muscle can be affected; GI symptoms such as constipation, gastric dilation and pseudo-obstruction can occur.

All voluntary muscles are affected in the later stages. Towards the end of life, the cardiac and respiratory muscles will be affected. As a result, cardiac arrhythmias, dilated cardiomyopahty and congestive heart failure can be seen. Life expectancy ranges from late teens to mid thirties and is usually the result of lung disorders/infection, heart failure, or airway obstruction.


Disease management

Although the exact mechanism of DMD deficiency is known, the conclusive treatment for the same remains elusive. Hence, the most preferred strategy towards DMD is management of the disease symptoms. The current scenario can be categorised in several care management areas including:

Pharmacotherapy- Glucocorticoid corticosteroids are currently the most effective medication that successfully reduces the declining of the muscle strength and its function in DMD (Bushby et al, 2009). Consequently, this reduces the risk of unstabilised pulmonary function while cardiac impairment shows a gradual improvement. Prednisone and Deflazacort remain the most commonly used glucocorticoid corticosteroids available for DMD management. (Bushby et al, 2009) Additionally, several supplements like coenzyme Q10 and anti-oxidants are endorsed for the management of DMD (Bushby et al, 2009).

Physiotherapy- Categorized as rehabilitation medicine, physiotherapy promotes frequent walking and prevents joint deformities (Manzur et al, 2008). Exercises like swimming are highly recommended (Kinali et al, 2008).

Management of cardiac and respiratory impairments- Studies by Dubec at al, show that perindopril medication in the initial stages of the disease leads to a reduction in the onset and progression of left ventricular dysfunction (Kinali et al, 2007). The nascent stages of DMD are often characterised by decreased respiratory reserve and sleep hypoventilation. These symptoms are best managed by domiciliary non-invasive ventilation (NIV) (Manzur et al, 2008).

As discussed earlier, DMD is a fatal muscular dystrophy with no effective therapy against it. But now there is a hope for the treatment of this deadly genetic disorder and it is thought that it will be curable in one decade or so. There are numerous novel approaches for the treatment of DMD that have entered or are ready to enter the clinical trials. The three basic strategies that can be adopted include: cell therapy, gene therapy and drug therapy. Owing to the fact that the nuclei of muscle fibers cannot divide, any cell or gene replacement therapy should restore the proper gene expression and thereby restoring the proper function. Correspondingly, the drug therapy approaches try to interfere with the complex biochemical mechanism of fiber degeneration. The above approaches are discussed, in some details, with their current status as a tool for the treatment.

Gene Therapy:

The gene therapy involves either replacing the defective gene with functionally active gene or repairing the defective gene. Repairing the dystrophin gene appears to be more promising because the viral vectors are not capable of integrating the full-size cDNA of dystrophin (14 kb).

Exon skipping, one of the approach to repair the endogenous gene, involves epigenetic correction of the gene by eliminating the mutation containing exons. This skipping allows the restoration of the reading frame there by generating a functionally active dystrophin though it is internally deleted. These specific mutated exons can be targeted by small nuclear ribonucleoproteins (snRNP) expressed by adeno-associated vectors or by antisense oligonucleotides (AONs). Both the methods involve targeting exonic sequence (that defines an exon), acceptor splice site or donor splice site. These sites are essential for proper pre-mRNA splicing of specific exons and upon binding to these sites, the specific exon gets spliced out along with its flanking introns thus restoring the reading frame. The UK MDEX Consortium started a clinical trial, in collaboration with AVI BioPharma, of intramuscular injection of morpholino AONs targeting exon 51 in DMD patients.

Mutation in the dystrophin that result in the premature translation termination, typically known as nonsense mutation, occurs in approximately 15% of the DMD patients. PTC Therapeutics discovered a new bio-available drug called PTC124 (or Ataluren) that makes ribosome less sensitive to stop codons there by ignoring the non-sense mutation. By doing this, Ataluren promotes the translation machinery to continue the translation till the end there by producing a functional dystrophin. PTC Therapeutics, Inc. and Genzyme Corporation declared the introductory results of the clinical trial phase 2b of Ataluren on 3rd March, 2010 indicating well tolerability of the drug. The drug will now be investigated further for it efficacy, and on satisfactory results, the drug will be commercialised, first in USA and Canada, and then in rest of the world.


Cell Therapy:

The cell therapy strategy dates back to 1989 when Partridge, T.A. et al. showed that dystrophin-expressing myofibers can be generated by transplanting myoblasts into the dystrophic muscles. Based on this experiment, several clinical trials were initiated in early 1990s indicating poor efficacy. The main reasons behind this were the immune response to donor myoblasts and non-efficient delivery. Several adult stem cells including Bone marrow-derived stem cells and Mesoangioblasts are now further investigated for the optimisation of the therapy.

Drug Therapy:

There are several drugs that can prove to be promising in the treatment of DMD and are thus under clinical investigations. There are several pathways in which these drugs can intervene and treat the disease. Some of the major drugs under clinical trials are discussed below.

SMT C1100:

It is now a well known fact that Utrophin (similar to dystrophin) upregulation by a certain extent can restore the normal function and prevent the further development of muscular dystrophy. Based on this, Summit plc. analyzed the utrophin promoter and after a high-throughput screening, SMT C110 (also known as BMN-195) was selected as it is capable of upregulating the utrophin. SMT C110 is now under phase I clinical trial by BioMarin Pharmaceuticals Inc. and the results are expected in the third quarter of 2010.

HCT 1026:

HCT 1026 is an NO-releasing, non-steroidal anti-inflammatory drug derived from flurbiprofen. After the oral administration, the drug helped in reducing the inflammation and preventing muscle damage there by slowing down the disease progression. Moreover, HCT 1026 has been shown to complement the efficacy of arterially delivered donor stem cells by reducing the immune response against them.

Histone deacetylase inhibitors:

Histone acetylation/deacetylation cycles play an important role in the regulation of gene expression. By inhibiting histone deacetylases, the regeneration-activated genes (for e.g. follistatin gene) can be upregulated thereby promoting muscle regeneration and development. Trichostatin A or MS27 are examples of class I deacetylases that are currently under investigation.

Pros and Cons:

Though all the above discussed therapeutic strategies display potential, they have their own advantages and disadvantages. The cell and gene therapies are able to treat the disease completely, but at the same time they are limited by their availability and cost. On the other hand, drug therapies carry a disadvantage of their side effects. The strategies applied with an intention to repair the endogenous gene, is limited only to certain amount of patients because there are several different mutations in dystrophin gene causing DMD. The other major hindrance lies in the delivery of the therapeutic agent, especially the systemic delivery. Once these limitations are overcome, may be by combinatorial therapies, the treatment of DMD would be much easier and may be treatable

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