Duchenne muscular dystrophy: Still an incurable disease
Muscular dystrophies (MDs) are heterogeneous, genetic muscle disorders.,,,,, Genetic mapping techniques have helped to identify more than 50 diseases, caused by specific gene mutations, which constitute MDs.,, Muscle histology has identified changes in the fiber size, the presence of necrotic areas in muscles, and subsequently, the fatty infiltration in MDs.,, MDs are classified according to their phenotypic features, inheritance patterns, age of onset, and the rate of disease progression., They present clinically with progressive muscle degeneration and weakness, which affects different muscles in different variations.,,,, DMD is the most common form of MDs among children.,
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by out-of-frame mutations in the dystrophin gene, leading to an absence of the dystrophin protein.,,, It has an incidence of 1 in 3500–5000 live male births.,,,,, It mostly affects males, who are diagnosed between the ages of 3 and 5 years when their motor milestones are delayed, compared with those of their peers.,, Females are non-symptomatic carriers most of the time, but in 5%–22% of the cases, they exhibit clinical symptoms ranging from mild muscle weakness to severe cardiac involvement.,, DMD is diagnosed by increased serum creatine kinase (CK) levels, muscle biopsy, genetic testing, and observed clinical features.,,, The purpose of this article is to focus on the causes, pathogenesis, and potential treatments of this disease, globally as well as specifically in the Indian population. The literature search was done through PubMed and Google Scholar for selecting the articles titled with DMD.
DMD is inherited in a X-linked recessive fashion. Since females have two X chromosomes, in the event that there is a mutation in one of them, they still have the second X chromosome to produce a normal dystrophin gene. Therefore, female carriers are usually unaffected, barring rare cases of abnormal X chromosome inactivation. Symptomatic female carriers show similar DMD features of proximal muscle weakness, increased serum creatine kinase (CK) levels, and hypertrophy of calf muscles, as reported by Padma et al. (1996). Males, on the other hand, have only one X chromosome. Thus, mutation in their X chromosome would always result in DMD. Therefore, two-thirds of the DMD cases are the result of an X-linked recessive pattern and one-third of the cases are due to spontaneous mutations.
The dystrophin gene, the largest gene known, is present on the Xp21 chromosome.,,,, It is approximately 2.2–2.3 megabases in size.,, Various mutations can occur on this gene, such as deletions (60%–65%), duplications (5%–10%), and point mutations (remaining cases).,, The “reading-frame hypothesis” states that in DMD, there are out-of-frame mutations that cause an early stoppage of protein translation and lead to an absence of dystrophin. This characteristic of DMD differentiates it from Becker muscular dystrophy (BMD), where in-frame mutations occur and form an abnormal but partially functional dystrophin protein.,,, In DMD cases in India, the reading-frame rule is observed and also there has been an increase in new mutations in North Indian DMD/BMD patients. However, it has been observed that there have been no ethnic differences in deletion mutations across different parts of India. Genetic studies have shown to be valuable markers for diagnosing DMD, as reported by a study done in South India, and deletion mutations have been more prevalent in Northern, Southern, Western, and Eastern India.,, The prenatal diagnosis can be helpful, so parents can make an informed choice.
The dystrophin protein is normally present in the inner surface of sarcolemma.,, The main functions of dystrophin are the stabilizing of muscle cell membranes and the linking of internal cytoskeleton to the extracellular matrix.,,, Dystrophin acts like a shock absorber during contraction of the muscle. In DMD, due to the absence of dystrophin, there is an abnormal permeability and fragility of cell membranes,, which increases the chance of muscle damage at any given stress level and is particularly pronounced in high force muscle contractions.
The various mechanisms involved in muscle damage are mechanical damage, an increase in intracellular calcium level, impairment of signaling molecules, and an increase in immune cells. They are described as below:
Mechanical damage: Dystrophin helps in the transmission of force between the intracellular cytoskeleton and the extracellular matrix. A lack of dystrophin causes increased stress on the cell membrane, even with normal muscle contractions.,,,, It also leads to an increase in the permeability of the muscle membrane.,
Increase in intracellular calcium level: Damage of a muscle membrane causes an increase in the intracellular calcium concentration.,,,,,, This leads to the protease activation, especially of calpains, which further causes necrosis of fibers.,,,
Impairment of signaling molecules: Dystrophin supports the signaling molecule, neuronal nitric oxide synthase (nNOS), through its dystrophin–glycoprotein complex (DGC)., nNOS forms nitric oxide (NO), whose primary function is to maintain vascular tone by reacting with free radicals. Absence of dystrophin causes downregulation of nNOS and its displacement into the cytoplasm from the plasma membrane.,, This leads to the reduced production of NO and causes muscle damage associated with free radicals.,, It also leads to vasoconstriction, and ultimately, ischemia of the muscle.,,
Increase in immune cells: Increased inflammation is also considered to play a role in the pathogenesis of DMD. There are increased levels of cluster of differentiation (CD)4+ and CD8+ T cells found in the dystrophic muscles, causing more muscle damage and fibrosis.,,,
Boys with DMD are born with a normal muscle function. As they age, there is a decrease in functional mass and a degeneration of the muscle fibers, followed by fibrofatty tissue replacement. There is appearance of fibers with central nuclei, the marker of regeneration. By the time a boy reaches his teenage years, reduced muscle fibers are present and fibrofatty tissue is highly prevalent.,
Patients with DMD exhibit progressive muscle weakness, from the proximal to distal direction.,,,,,, Boys with DMD exhibit a waddling gait, clumsiness, flat feet, and an inability to walk until 18 months of age., When they start walking around 18–24 months of age, boys have a wide-based gait, have a difficulty in running, and present with an increased lordosis of the spine due to weakness in the hip extensors.,,, Moreover, they struggle with getting up from the floor, jumping, walking on their toes, and they fall frequently. They also often exhibit head lag when they sit up from a lying position due to weakness of neck flexor muscles., When these boys stand up from a supine position, they tend to place their hands on their knees, exhibiting what is referred to as the “Gowers' sign.”,, They also exhibit enlargement of calf muscles, referred to as pseudohypertrophy, when the fatty tissue replaces degenerated muscle. A “Valley sign” has been described to characterize patients with DMD where there was depression of the posterior axillary fold, with hypertrophied or preserved muscles on its two sides, as measured from the back. The clinical features and the pattern of losing major milestones are independent of the ethnicity and have been shown to be similar between the Indian and the Western populations.
Boys with DMD have increased difficulty in walking and lose the ability to both rise from the floor and climb stairs. Boys typically lose the ability to walk by the age of 10–12 years,, without steroid treatment. However, they can still sustain their posture. Scoliosis is usually seen, which affects their respiration and worsens as a boy becomes increasingly non-ambulatory.,, Upper limb functions decline at a later stage which leads to difficulty in performing activities of daily living such as eating, bathing, and brushing teeth. At this point, they also find it difficult to sustain their posture.
Other clinical features, such as joint contractures, are usually first seen in the ankles and then in the hips, knees, elbows, and wrists. Long bone fractures due to falls, and osteoporosis are other orthopedic complications present in boys with DMD., Impaired cognition, attention deficit hyperactive disorder, autism spectrum disorder, and obsessive compulsive disorder have also been reported in some cases., As the disease progresses, boys become increasingly dependent on others for assistance with their activities of daily living. Dilated cardiomyopathy is usually seen after the age of 10 years and is evident in all patients of 18 years or older., As boys with DMD age, respiratory and cardiac problems continue to worsen, ultimately leading to their death.,,,,
DMD is currently incurable, but many experimental therapeutic strategies are currently in clinical trials., The current management of DMD focuses on the multidisciplinary approach for treating symptoms and improving the quality of life and function.,,,
Contractures are commonly seen in these patients, so appropriate physiotherapy is essential to maintain the range of motion. In physiotherapy, stretching, proper positioning of the joints, and range of motion exercises are recommended to prevent contractures., Orthoses can be used either to prevent or minimize contractures. A study reported in India showed that the use of ankle foot orthosis has the potential of prolonging ambulatory status. Spinal corset can be used to reduce scoliosis. In a few cases, surgery can be considered as an option to correct contractures or scoliosis.,, There is not enough evidence to suggest the type of exercise that may be beneficial in this population., Swimming is recommended for aerobic conditioning of the muscles., Since fractures are commonly seen in these patients, splints or casts can be used for fracture healing. Vitamin D and calcium supplements can be considered for maintaining bone health.,
Airway clearance techniques, including manual and mechanical assisted cough, can be used to prevent respiratory complications. Respiratory infections can be treated with antibiotics and chest physiotherapy.,,,, Noninvasive ventilation is suggested when patients start experiencing hypoventilation.,,,,
Echocardiography is used for detecting cardiac dysfunction. Angiotensin-converting enzyme (ACE) inhibitors are considered to be the first-line drugs in cardiac management.,,,,,, Beta blockers can be used with ACE inhibitors for even more beneficial results.,
Glucocorticoids have been shown to delay the loss of ambulation in patients with DMD by 1–2 years.,, They also reduce the chance of scoliosis. Currently, prednisone with a daily dose of 0.75 mg/kg or deflazacort with a daily dose of 0.9 mg/kg are the recommended drugs.,,,,, Weight gain, behavioral problems, vertebral and long bone fractures, stunted growth, puberty delay, hypertension, and cataracts are some of the common side effects seen in glucocorticoid-treated boys.,,,,, In a recent study, it has been observed that treatment with deflazacort would result in delayed deterioration of ambulation and maintenance of key motor functions, when compared with prednisone. However, a study reported in India showed that prednisolone has more effects than deflazacort. Thus, the use of steroids and prednisolone versus deflazacort still remains controversial.
There are many experimental therapeutic strategies in preclinical and clinical trials that look promising for slowing down disease progression in DMD in the future. Below is a review of some of the major strategies.
Stem cell therapy
Muscle stem cells (myoblasts) have a capacity for differentiation into muscle cells. Promising results were seen when myoblasts were transplanted into immunosuppressed mdx mice, a mouse model for DMD.,,, Unfortunately, these results could not be replicated in boys with DMD because of their immune responses., Another limitation of myoblasts is that they do not travel long distances in muscles and die soon after injection. A study performed in various hospitals of India showed that the administration of umbilical cord mesenchymal stem cells resulted in the stability of muscle power and could be used as a potential treatment in DMD.
Gene replacement therapy
The large size of the dystrophin gene and the relatively limited packaging capacity of viral vectors necessitate the creation of micro- and mini-dystrophin genes for delivery.,,,, Recombinant adeno-associated viral (rAAV) vectors are used to carry micro- and mini-dystrophins.,, This therapy showed successful results in mdx mice, but failed to replicate the results in boys with DMD. The reason behind its failure is the immune responses shown by boys with DMD., Recently, a study showed that delivery of micro-dystrophin using AAV vector could reduce inflammation and fibrosis in skeletal muscles of DBA/2J-mdx mice, a severe mouse model for DMD; though this therapy has not been tested clinically as yet.
Stop codon read through drugs
Aminoglycoside antibiotics like gentamicin help in reading through premature stop codons. Gentamicin has its application in identifying nonsense mutations in the dystrophin gene,,, but its major disadvantage is that it causes toxicity in patients with DMD.,, PTC124 (ataluren) is another synthetic drug that can read through premature stop codons, thus allowing full translation and formation of the functional protein.,,,, It is a well-tolerated drug and is also used for nonsense mutations., Unfortunately, in phase 2b of its clinical trial, ataluren failed to show a significant effect in its primary outcome measure – the six-minute walk test (6 MWT), which caused the clinical trial to be terminated.,, A significant effect was seen in patients with DMD who received a low dose of the drug, after subsequent post hoc analysis. Based on this, ataluren has been conditionally approved in Europe for patients with DMD and has started its phase 3 clinical trial., Recently, phase 3 clinical trial results demonstrated that ataluren showed a 15 meter improvement in the 6 MWT from baseline to 48 weeks but with no statistical significance.,, The Food and Drug Administration (FDA) had initially refused to review the drug's application, stating that the drug does not show efficacy;, however, they recently conducted a review and did not approve ataluren.
Exon skipping drugs
Exon skipping drugs use antisense oligonucleotides to skip exons in the pre-mRNA phase, restoring the reading frame of dystrophin. These drugs form a partially functional dystrophin, thus converting DMD to BMD.,,, Successful results have been observed in mdx mice. Drisapersen and eteplirsen are two of the drugs in clinical trials for patients with DMD. Both of them skip exon 51 and can be used in 13% of the DMD population., Drisapersen failed to show a significant improvement in the 6 MWT in its phase 3 clinical trial over a 48-week treatment,,,, which has led to the discontinuation of further clinical trials. Eteplirsen showed promising results when tested on 12 patients in the phase 2 trial and it is currently completing a phase 3 clinical trial. Recently, the FDA has granted conditional approval to eteplirsen.,,,
Utrophin is structurally very similar to dystrophin.,,,,, It is thought that utrophin can substitute for dystrophin deficiency, when it is upregulated.,,, Utrophin upregulation has shown effective results in the mdx mice and the golden retriever MD, a dog model for DMD. Clinical trials for the drug SMT C1100 are ongoing to upregulate utrophin expression in patients with DMD.,,, A phase 1 clinical trial showed that this drug is well tolerated by boys with DMD. The drug was in phase 2 of its clinical trial., Recent results of its phase 2 proof-of-concept trial showed that the drug did not show efficacy at its primary endpoint and the researchers decided to discontinue their study.
Phosphodiesterase 5A inhibitors
Absence of dystrophin leads to the displacement of neuronal nitric oxide synthase (nNOS) from the dystrophin glycoprotein complex (DGC), resulting in decreased nitric oxide (NO) production. Reduction in NO leads to muscle ischemia. Phosphodiesterase 5A (PDE5A) inhibitor drugs like sildenafil and tadalafil have a vasodilatory effect that could help to reduce muscle ischemia. After showing promising results in mdx mice,, a clinical trial was initiated. Unfortunately, sildenafil showed no improvement in cardiomyopathy in a phase 2, double-blind, randomized study of patients with DMD.
Activation of nuclear factor (NF)-κB results in the degradation of muscle proteins, which produces pro-inflammatory mediators like cytokines and chemokines. Activated NF-κB is seen in patients with DMD. The drug edasalonexent (CAT-1004) has been developed to inhibit the NF-κB pathway and is currently in a phase 1/2 clinical trial. This drug was shown to be safe and well-tolerated in patients with DMD in phase 1 of its clinical trial. However, it was recently reported that the drug did not show significant improvement in boys with DMD after 12 weeks of treatment. Recently, the phase 2 clinical trial was completed. Positive results were found and phase 3 clinical trial was being started.
Myostatin and insulin growth factor-1
Myostatin inhibits muscle growth., Myostatin antibodies have been used in reducing muscle pathology in mdx mice, but similar results were not found in patients with DMD. In a recent phase 2 clinical trial, myostatin inhibitors showed a trend of maintaining performance on the 6-minute walk test in the treatment group, but no statistical difference could be found between the placebo and treated groups. Insulin growth factor-1 helps in muscle growth. Its use in increasing the size of muscle fibers is currently being investigated in a clinical trial.
Histone deacetylase inhibitors
Histone deacetylase (HDAC) inhibitors help in the regeneration of muscles and reduce fibrosis and fatty infiltration in mdx mice. Givinostat, a HDAC inhibitor, has shown to improve muscle growth in mdx mice. This drug is starting a phase 2 clinical trial., It is assumed that this drug delays the progression of DMD in patients at an early stage of the disease.
CRISPR/Cas9 is used for gene editing, where Cas9 nuclease is used to cleave DNA sequences with the help of a single-guide RNA. It has already been shown that this method can re-establish expression of dystrophin in patient-derived induced pluripotent stem cells (iPSCs) and in murine iPSCs. It can restore partial dystrophin expression and enhance muscle function in the mdx mice.,,, However, its efficacy and safety have not been tested in patients with DMD so far, though it is projected that this method can treat about 80% of the patients. CRISPR/Cas9 gene editing has several challenges such as the unknown efficacy, the specificity of delivery vehicles, and the possibility of off-target effects. Translatability of the majority of in vitro research to in vivo delivery methods in the clinic is another challenge. Immune responses to delivery vehicles and Cas9 peptides may also be a hurdle, as with any gene therapy. These challenges need to be overcome in the future.
MDs are a heterogeneous group of disorders, characterized by progressive muscle weakness. DMD is one of the most common forms of MD and mostly affects boys. DMD is caused by the absence of the dystrophin protein, which leads to abnormal membrane permeability and fragility of the cell membrane. This permeability and fragility make the cell membrane more susceptible to damage. In the early stages, the affected muscles undergo numerous cycles of damage and repair, but in later stages, there is a progressive replacement by fat and fibrotic tissue. Boys with DMD exhibit progressive muscle weakness in the proximal to distal direction, which leads to gradual functional decline. There is no known cure for DMD, and glucocorticoids are the only drugs which have been shown to delay the loss of ambulation by 1–2 years. Many drugs in clinical trials have shown success preclinically in animals but have shown minimal success in humans. There are significant barriers in the treatment of DMD. It is worth noting that the use of a single drug may not be successful in treating DMD and it may be helpful to administer multiple drugs, targeting different pathways, for successful treatment of DMD. Moreover, most of the clinical trials target ambulatory patients with DMD, thus excluding roughly two-thirds of patients with DMD who are no longer able to walk.
DMD is not an easy disease to live with, especially in the traditional Indian setting, where there are social taboos and absence of much-needed infrastructure to help these patients. It becomes difficult for the patients and their families to get counseling support because of psychosocial issues. In a recent MD conference, I was made aware that there were not many clinical trials going on in India, unlike the practice in the United States or Europe. It is disheartening for the patients and their families that they would not be able to participate in the potential therapies. Thus, there is an urgent need for creating more awareness about this disease, and pharmaceutical companies need to be encouraged to conduct clinical trials in India. There has been stem cell research going on in India, which is promising, but more is needed to fight this incurable disease.