**AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy**

Ingrid E. C. Verhaart and Annemieke Aartsma-Rus

*Department of Human Genetics, Leiden University Medical Center The Netherlands* 

#### **1. Introduction**

54 Neuromuscular Disorders

Zeng, W., de Greef, J.C., Chen, Y.Y., Chien, R., Kong, X., Gregson, H.C., Winokur, S.T., Pyle,

with facioscapulohumeral dystrophy (FSHD). *PLoS Genet* 5, e1000559. Zouvelou, V., Manta, P., Kalfakis, N., Evdokimidis, I., & Vassilopoulos, D. (2009).

facioscapulohumeral muscular dystrophy. *J Clin Neurosci* 16, 1218-1219.

A., Robertson, K.D., Schmiesing, J.A., *et al.* (2009). Specific loss of histone H3 lysine 9 trimethylation and HP1gamma/cohesin binding at D4Z4 repeats is associated

Asymptomatic elevation of serum creatine kinase leading to the diagnosis of 4q35

Duchenne muscular dystrophy (DMD) is a genetic, X-chromosome recessive, severe and progressive muscle wasting disorder, affecting around 1 in 3500 newborn boys. The onset of the disease is in early childhood and, nowadays, most children are diagnosed before the age of 5. The first signs of muscular weakness become apparent around the age of 2 or 3 years. In most patients the age at which the child starts to walk is delayed (retarded motor development). The children have less endurance and difficulties with running and climbing stairs (Moser, 1984). Gower's sign is a reflection of the weakness of the muscles of the lower extremities (knee and hip extensors): the child helps himself to get upright from sitting position by using his upper extremities: first by rising to stand on his arms and knees, and then "walking" his hands up his legs to stand upright (Gowers, 1895). Muscle wasting is often symmetrical, however not all muscles are affected to the same extent. A prominent feature of the disease is enlargement of the calve muscle, caused by replacement of muscle fibres by connective and adipose tissue. Furthermore, the pelvic girdle, trunk and abdomen are severely affected and to a lesser extent the shoulder girdle and proximal muscles of the upper extremities. Progressive weakness and contractures of the leg muscles lead to wheelchair-dependency around the age of 10. Thereafter the muscle contractions increase rapidly leading to spinal deformities and scoliosis, often with an asymmetric distribution pattern. Involvement of the intercostal muscles and distortion of the thorax lead to respiratory failure and patients often require assisted ventilation in the mid to late teens. Thereafter dilated cardiomyopathy becomes apparent and most patients die before the age of 30. Another common feature is mental retardation (IQ less than 70) in around 20-30% of the patients (Emery, 2002).

Becker Muscular Dystrophy (BMD) is a related, but much milder, form of muscular weakness, affecting around 1 in 20 000 men. The phenotype varies between individual patients, from very mild to moderately severe, but the course of the disease is more benign compared to DMD. On average, the age of onset is around 12 years; however some patients remain asymptomatic until much higher ages. The age of wheelchair-dependency also shows more variability, but in general is in their second or third decade of life. The most severely affected patients die between 40 and 50 years of age, whereas patients with a mild

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 57

The dystrophin-associated glycoprotein complex (DGC) is composed of α- and β-dystroglycan, a sarcoglycan-sarcospan complex and the dystrophin containing cytoplasmic complex. Dystrophin (purple) forms the link between the actin cytoskeleton with its N-terminal domain and extracellular matrix component laminin-2 (lilac) via α- and β-dystroglycan (dark blue) with its C-terminal domain. Bdystroglycan is also bound to the sarcoglycan-sarcospan complex (light blue/black) and to caveolin-3 (orange), a scaffolding protein of skeletal muscle caveolae. Furthermore, the C-terminal domain of dystrophin is connected to α-dystrobrevin (green) and syntrophin (salmon pink), which recruits nNOS (yellow), a vasodilator, to the membrane. Α-dystrobrevin, in turn, is linked to syncoilin (brown), forming a bridge between the DGC and the desmin intermediate filament protein network (brown).

In addition to its mechanical linker function, dystrophin is involved in the organisation of the DGC as well as many other proteins, the maintenance of the calcium homeostasis and control of the growth of the muscle cells (Hoffman et al., 1987). In the DGC, β-dystroglycan is connected to a complex of α-, β-, γ- and δ-sarcoglycans and sarcospan. This complex functions in maintaining membrane stability (Miller et al., 2007). B-dystroglycan is also bound to caveolin-3, a structural protein of skeletal muscle caveolae, small invaginations of the plasma membrane playing a role in, among others, signal transduction. Caveolins act as scaffolding proteins to compartmentalise and functionally regulate signalling molecules (Hezel et al., 2010). Furthermore, the C-terminal domain of dystrophin is connected to αdystrobrevin and syntrophin. nNOS is recruited to the membrane by binding to dystrophin and syntrophin. In contracting muscles, nNOS produces NO to induce vasodilatation in order to increase the local blood flow necessary for the increased mechanical load. The absence of nNOS in DMD causes abnormal vasoconstriction and ischemic stress, which contributes to the muscle degeneration (Brenman et al., 1995). Syntrophin is also connected to sodium channels, which are involved in regulating the Na+ distribution. In DMD, defects in cardiac conduction systems are thought to be caused by disturbances in Na+ distribution (Gee et al., 1998). A-dystrobrevin is linked to syncoilin too, thereby forming a bridge between the DGC and the desmin intermediate filament protein network at the

Fig. 1. The dystrophin-associated glycoprotein complex

neuromuscular junction (Newey et al., 2001).

phenotype have (nearly) normal life expectancies. Around 50% of patients also develops cardiomyopathy (Emery, 1993).

The majority of female carriers shows no signs of disease. Only in 5 to 10% some degree of skeletal muscular weakness and enlarged calves are reported, but this is generally very mild and often does not affect daily activities. A small part of these carriers develops cardiomyopathy later in life; however most of the women with cardiac abnormalities on echocardiogram or ECG (left ventricular dilatation and decreased shortening fraction), are asymptomatic. There is no relation between the presence of skeletal muscle weakness and the development of cardiomyopathy (Grain et al., 2001).

At present there is no cure for DMD. However, during the past decades pharmacological interventions and improved care (e.g. physiotherapy and assisted ventilation) have led to increased function and quality of life and prolonged life expectancy for currently diagnosed patients into their forties. The current standard of care also consists of corticosteroids (mainly predniso(lo)ne or deflazacourt). These are anti-inflammatory/immunosuppressive drugs that have shown to improve muscle function, prolong ambulation for around 3 years and to have a positive effect on cardiac function (Bushby et al., 2010).
