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Georgiana Laura Cioanca Ákos Bogó Esther Hazane Leroyer Molecular Genetics Class Presentations JPEMS 2015, Szeged DUCHENNE MUSCULAR DYSTROPHY (DMD) INTRODUCTION Duchenne Muscular Dystrophy (DMD) is the most common muscular dystrophy and the most severe. It takes its name from the French neurologist Guillaume Duchenne, who was the one to describe the condition. DMD is a single-gene type of disorder, caused by loss-of-function mutations (usually deletions) affecting the dystrophin gene. This gene is located on the X chromosome, thus the disorder shows X-linked recessive inheritance pattern, with an incidence of 1 in 3500 males. 1 Female mutation carriers can also show mild signs in 20°% of the cases.2 Mutations in the DMD gene can also result in Becker Muscular Dystrophy (BMD), similar condition associated with milder symptoms and better prognosis. DMD is characterized by a childhood onset, as the first clinical signs are seen in males between the ages of 3 and 5 years. As dystrophin is found in the skeletal, heart and smooth muscle, as well as in the brain, these are the affected organs in DMD. Loss of function of dystrophin leads to muscle cell degeneration and as a consequence to an increased serum level of creatine kinase (CK). The progressive proximal muscle weakness, accompanied by calf muscle pseudohypertrophy is the most distinctive sign. Young affected boys show difficulties in rising from the floor (positive Gower’s sign, figure 1). Subsequently, patients develop increased lumbar lordosis and scoliosis, leading to a waddling gait (figure 2). Independent ambulation soon becomes impossible to perform, thus in most of the cases patients are wheelchair bound by the age of 11 years.1 Patients show breathing shortness, due to the respiratory muscles wasting. The myocardium is also affected and DMD patients develop dilative cardiomyopathy. Regarding the brain damage, a mild to moderate intellectual impairment can usually be observed. In spite of this fact, about 20% of patients present severe mental retardation. 2 Figure 1. Gower’s sign: patients have difficulties in rising from the floor, pushing on their hands in order to stand up Figure 2. Musculo-skeletal abnormalities in DMD leading to waddling gait JPEMS 2015, Szeged Unlike BMD, in which the life expectancy is only slightly reduced, Duchenne Muscular Dystrophy is associated with a bad prognosis. Without steroid medication, the lifespan of DMD patients has a mean of 18 years. The most common cause of death is represented by cardiorespiratory failure. 1 GENETIC DEFECT DYSTROPHIN GENE The dimer protein causing Duchenne Muscular Dystrophy (DMD) is called the dystrophin. Dystrophin is encoded by dystrophin gene, the larger gene in human genome. Indeed, it is composed of 2,4 Mb of genomic DNA. It contains 79 exons, corresponding to 14kb, transcripted into mRNA. The large size of the gene could possibly explain the high mutation rate. The transcription occurs in all muscle cells (striated, smooth and cardiac cells) as well as in the brain, which explains the mental disorders patients can sometimes endure. The gene is located on the X-chromosome, which is why DMD is an X-linked disease. Moreover, it is an X-recessive disease, so mostly males are supposed to be affected and females are called carriers (figure 3). However, the first detection of the gene and of its location was guessed because of female affected by DMD. In fact, those females bore an Xautosome translocation, with a common breakpoint at Xp21. In this case, due to the skewedinactivation of the X chromosome, only cells containing the X-autosome survive, because the X chromosome involved in the translocation survives preferentially so as to maintain functional disomy of the autosomal genes. Figure 3. Family tree of a Duchenne Muscular Dystrophy family with the disorder being transmitted by carrier females and affecting males. TYPE OF MUTATIONS About 2/3 of all dystrophin gene mutations are deletions. There are hot spots for deletion, namely, the first 21 exons, as well as around exon 45 to 53, leading to disturbances of the translational reading frame. Appearance of stop codons, frameshift mutations, altered splicing signals or promoter mutations can occur, mainly drafting to premature translational termination. A shorter protein, or often no protein at all is transcripted. 2 October 1, 2015 JPEMS 2015, Szeged About 30% of mutations in the dystrophin gene arise as de novo mutations (ie not present in the mother), which is important to take into account for genetic counselling. Less frequently, mutations can take the form of duplications or point-mutations. If deletions arise almost exclusively from maternal meiosis, point-mutations often arise from paternal-meiosis. PHENOTYPE correlation The absence or abnormality of dystrophin protein causes the muscle cell degeneration, which is the onset of DMD. Mutated dystrophin cannot undergo muscle strength anymore. Dystrophin usually binds to a glycoprotein complex (β- dystroglycan) in the muscle membrane through its Cterminal domain, which makes the link with extracellular laminin, and to intracellular actin through its N-terminal domain (figure 4). 1 Dystrophin provides mechanical reinforcement of the sarcolemma. It protects it from the membrane stresses developed during muscle contraction. The sarcolemma can usually undergo both radial and longitudinal tensions, and since myofilaments are connected to actin, then to dystrophin, and then to the sarcolemma, dystrophin helps to serve this function as well. Muscle cells cannot withstand the tension anymore, which leads to an impaired muscle cell function. 3 Figure 4. Dystrophin protein molecule, depicted as a dimer linking intracellular actin and extracellular laminin. DIAGNOSIS As mentioned above, there are some distinctive clinical signs in DMD which may indicate the diagnosis. However, these features are not enough and further investigations should always be performed in order to confirm the diagnosis. Before DNA analysis developed, the diagnosis was based on pedigree information and evaluation of creatine kinase levels.1 Nowaday, it is possible to have a precise diagnosis by investigating the dystrophin gene, since the majority of DNA mutations can be detected by DNA testing. The identification of larger duplications or deletions is obtained by tests such as arrays, amplification and hybridization. If these large mutations are not to be found, then further tests are coming for smaller changes, like point mutations. For example, Sanger Gene sequencing or Resequencing arrays are used for this purpose. 4 3 October 1, 2015 JPEMS 2015, Szeged Performing a biopsy might also provide additional information besides the previously mentioned methods. A sample is taken from the skeletal muscle and examined after the application of stains or specific antibodies for dystrophin or dystrophin associated molecules (immunofluorescence). Gamma-sarcoglycan is also a useful marker, since in immunofluorescence and western blotting in skeletal muscle from the DMD patients showed complete absence of it.5 Since female carriers are usually asymptomatic, it is of an utmost importance to detect the carriers of the mutant allele. Especially in pregnant female carriers, since their child might inherit the mutation, thus developing DMD. Prenatal diagnosis is also available by performing amniocentesis.6 TREATMENT OPTIONS No treatment to cure Duchenne Muscular Dystrophy have been discovered so far, thus for now the only possibility is to attempt to improve the life quality of these patients. Physotherapy and steroids show to be helpful to maintain the mobility for a longer period, but it do not have curable effects. It is only the gene therapy that might offer a real therapeutic solution for DMD.1 At the moment, several experiments are conducted in different animal models. The most commonly employed models are mdx mice and canine X-linked muscular dystrophy (CXMD). Studying the mdx mice model allows us to understand the mechanisms behind the muscle degeneration and regeneration in DMD. However, it does not provide a precise model of the human disease: mice show severe muscle weakness, but the lifespan is only reduced with 25%, whereas in humans it is reduced with 75%. CXMD dogs represent a better alternative, as the pathological expression of the diseases is more related to the human DMD: progressive skeletal muscle weakness, associated with degeneration of the myocardium and bad prognosis.7 Currently, several gene therapy strategies are under development. One approach is to modify the abnormal dystrophin gene (by gene replacement or gene repair) in order to encode a protein with some residual function. As a consequence, the expression of the disease might be characterized by less severe manifestations (obtaining Becker Muscular Dystrophy phenotype). In some mice with dystrophin-negative dystrophy a spontaneous muscle repair can occur and this is due to the on switching of the gene coding utrophin (protein expressed in the fetus, similar to dystrophin). Finding a way to reactivate the utrophin gene represents another possible treatment for DMD disease. The technologies used to accomplish these goals include recombinant DNA, viral vectors and myoblast implantation.1 In conclusion, finding a cure for Duchenne Muscular Dystrophy represents an important field of interest for researchers nowadays. Until an effective treatment will be found, the only available options are genetic counselling for the families with DMD history and offering support for DMD patients, in order to improve their life quality. REFERENCES 1 Peter D. Turnpenny, Sian Ellard. Emery’s Elements of Medical Genetics, Philadelphia, PA: Churchill Livingstone/Elsevier , 2012, 14th Edition 2 http://www.omim.org/entry/310200 Muscular Dystrophy, Duchenne type; DMD, 2014 3 Petrof and Al. 1993, Dystrophin protects the sarcolema from stresses developed during muscle contraction 4 October 1, 2015 JPEMS 2015, Szeged 4 http://research.duchenneconnect.org/index.php?option=com_content&view=article&id=155&Itemi d=241 5 Jung, D., Leturcq, F., Sunada, Y., Duclos, F., Tome, F. M. S., Moomaw, C., Merlini, L., Azibi, K., Chaouch, M., Slaughter, C., Fardeau, M., Kaplan, J.-C., Campbell, K. P. Absence of gamma-sarcoglycan (35 DAG) in autosomal recessive muscular dystrophy linked to chromosome 13q12. FEBS Lett. 381: 15-20, 1996 6 E.J Annexstad, I. Lund-Petersen, M. Rasmussen; “Duchenne muscular dystrophy”; Tidsskr Nor Legeforen Nr. 14; 5. august 2014 7 http://www.parentprojectmd.org/site/PageServer?pagename=Advance_research_strategies_animal models 5 October 1, 2015