The Molecular Basis of Muscular Dystrophy

1. The Molecular Basis of Muscular Dystrophy 肌肉萎缩症的分子基础 AUTHER：刘少伟 刘寅 王青 吕潇

2. 肌肉萎缩症（假性肥大肌营养不良） Connective tissue结缔组织 A Baby of seven month

3. What Are Muscular Dystrophies? The muscular dystrophies are a group of Muscle diseases which have three features in common: • muscle-fiber degeneration and muscle weakness • Hereditary: It passes from parent to child • Progressive: It gets worse over time

4. Different Types of MD There are nine major types of MD affecting people of all ages, from infancy（幼年）to middle age or later. The two most common types of MD affect children: • Duchenne muscular dystrophy (DMD) • Becker muscular dystrophy (BMD)

5. Guillaume Duchenne • Guillaume Benjamin Amand Duchenne is the man credited with identifying the symptoms and naming the disease known as Duchenne Muscular Dystrophy.  • He was a medical doctor that became very interested in the relationship between electric stimulation and muscles.  This interest caused him to travel from hospital to hospital in an attempt to experiment with and treat those with muscular diseases.  Along his journey, he encountered and recorded details of many boys who had the condition that he identified as Duchenne Muscular Dystrophy.

6. Why are DMD and BMD discussed together? DMD and BMD cause similar patterns of weakness and disability and are inherited in the same way. Because they are due to defects of the same gene on the X chromosome which encodes a protein ——“dystrophin”. They are inherited as X-linked recessive diseases. ( X染色体隐性基因 ）

7. The gene of Dystrophin(抗肌肉萎缩蛋白) The gene responsible for DMD and BMD is nameddystrophin。It’s the largest gene so far identified in the mammalian(哺乳动物)genome . The gene stretches for more than 2.3 million bases along the X chromosome , a length that requires 16 hours to be transcribed into a single mRNA! Only about 0.6 % of the gene actually codes for amino acids(exons); the remaining 99.4% consist of noncoding introns .

8. The exons are put together for code assembly during a process called splicing. Due to its length the Dystrophingene is relatively vulnerable for rearrangements (mutations)

9. X-linked recessive diseases

10. The protein dystrophin reside in the membrane skeleton of striatedmuscle(横纹肌) cells. It is important for the strength and flexibility of the muscle fiber membranes.

11. Dystrophin are rod-shaped dimers（杆状二聚物） beneath the plasma membrane

12. laminin DAP (dystophin-associated proteins) dystrophin F-actin

13. On their cytoplasmic side ,dystrophin molecules are attached to actin filaments, and on their membrane side, they are attached to a cluster of dystophin-associated proteins (DAPs), in turn, are linked on their extracellular surface to components of the basement membrane that surround these contractile(有收缩性的) cells. Taken together, these various proteins form a functional pathway linking the extracellular matrix with the internal cytoskeleton. In addition, dystrophin provides structural support for the plasma membrane as the muscle fiber repeatedly contracts and relaxes. 抗肌萎缩蛋白的C端连接着一系列与抗肌萎缩蛋白相关的糖蛋白(DAPs)，进而与收缩细胞的细胞外基质成分连接.内部端（氨基酸端）连接肌动蛋白（肌动蛋白是肌细胞的骨架），所以说抗肌萎缩蛋白是连接肌细胞内部骨架和细胞膜外的重要桥梁，当肌肉收缩时，它起到稳定肌肉细胞的作用。

14. How mutations cause Duchenne and Becker dystrophy? There are three types of mutations or damage of the dystrophin gene: • Deletions：if one or more entire exons of the gene are missing; • Duplications：if parts of the gene are repeated; • Point mutations：if single base pairs are exchanged.

15. DMD——Many mutations in the gene disrupt the reading frame—“out of frame”， and thus cause the premature abortion（流产） of the synthesis of the dystrophin, BMD——Numerous mutations are “in-frame”, and conserve the reading frame. Despite a large internal deletion, a truncated（删节的） but mostly functional dystrophin is produced. In these BMD patients, the extent and progression of the muscle weakness is less severe.

16. 如果基因的突变没有完全打乱 “三子一窝”的基因阅读框， 也就说基因字母（核酣酸）缺 失或增加的数量能被3整除， 没有余数。这种情况下抗肌萎 缩蛋白被缩短或者拉长，同时 基因的改变发生在非关键区 域，例如抗肌萎缩蛋白的中 段，使得抗肌萎缩蛋白仍然能 发挥部分的功能，这就形成了 良性的假肥大型进行性肌营养 不良。 假如抗肌萎缩蛋白的基因突变 完全打乱了基因阅读框，也就 是缺失或增加的核酣酸数量不 能被3整除，那么从基因突变的 位点直到抗肌萎缩蛋白的终 点，所有合成的氨基酸都是错 误的，那么不完全的抗肌萎缩 蛋白无法发挥它的正常功能， 这样就形成了杜氏进行性肌营 养不良。 BMD in-frame out of frame DMD

17. The comparison between Becker and Duchenne

19. Duchenne Muscular Dystrophy: Dystrophin staining（染色） Normal dystrophin Staining around the rim of muscle fibers Absent dystrophin No staining around the rim of any muscle fibers

20. Western blot of dystrophin Lane 1: Becker dystrophy; Dystrophin has reduced abundance but normal size. Lane 2: Becker dystrophy; Dystrophin has reduced size and abundance. Lane 3: Normal; Dystrophin has normal size and amount. Lane 4: Duchenne dystrophy; Almost no protein is present. Lane 5: Duchenne dystrophy; Dystrophin has severely reduced abundance. BMD BMD Normal DMD DMD

21. DMD & BMD • BMD is a less severe form ,in which the same protein dystrophin is present but is abnormal or greatly reduced in amount . • An arbitary means of distinguishing the two disorders depends on whether the affected person can still walk at age 16 years.

22. Symptoms症状 Duchenne muscular dystrophy is the most common form of MD and primarily affects boys. Onset is between 3 and 5 years and the disorder progresses rapidly. Most boys are unable to walk by age 12. DMD occurs in about 1 in 3,500 male births, and affects approximately 8,000 boys and young men in the United States.

23. WHAT HAPPENS TO THE VOLUNTARY MUSCLES OF SOMEONE WITH DMD ? Difficulty walking, such as being late in learning how to walk (more than 18 months old), having a waddling gait or walking on toes or balls of the feet. • Difficulty running or jumping caused by weakness in leg muscles. • Frequent falls, stumbling and difficulty climbing stairs. • Difficulty standing from a lying or sitting position. • Reduced endurance. • Enlarged calf muscles. • Mild mental retardation, in some cases.

25. Because of weakened leg muscles, boys with DMD have a distinctive way of rising from the floor, calledthe Gowers' maneuver. They first get on hands andknees, then elevate the posterior, then "walk" their hands up the legs to raise their upper body.

26. The Gower's Maneuver

27. Other symptoms • Respiratory Function After a boy with DMD is about 10 years old, the diaphragm and other muscles that operate the lungs may weaken, making them less effective in moving air in and out. • Heart diseasesThemuscle layer(myocardium) of the heart deteriorates, just as the skeletal muscles do, putting the person at risk of fatal heart failure. • Learning disabilitydystrophin abnormalities in the brain may cause subtle cognitive and behavioral deficits.

28. Patients with DMD showdramatic abnormalities in both cardiac and skeletal muscle tissue . At the light microscopic level, some of the muscle fibers are seen to be degenerating Necrotic（and often infiltrated by macrophages of the immune system. 患DMD的病人在心肌和骨骼肌系统中表现得相当反常。在显微水平，我们看到一些肌纤维会逐渐坏死，并且被免疫系统的巨噬细胞所浸润。 横切面上见核从周边移入纤维中央，一些肌纤维萎缩。 在萎缩的肌纤维间有脂肪浸润和纤维组织形成。

29. 在电子显微水平,可以看到肌肉收缩时所产生的机械压力造成质膜的破裂,质膜片断从细胞表面流失， 导致细胞内部结构的显著变化，包括：原生质网状结构的扩张，线粒体的膨胀，肌酶从胞浆中大量“漏出”并使血清中有关酶相应增加；肌酶的外溢导致核糖体代偿性合成更多的肌酶，由于这种代偿作用相当有限，一定时间后肌细胞即遭受破坏，为增生的结缔组织取代，出现肥大症状。

30. Symptoms 症状 Becker muscular dystrophy is basically a milder form of Duchenne. Boys with Becker MD (very similar to but less severe than Duchenne MD) have faulty or not enough dystrophin.The illness is about 10 times rarer than Duchenne. BMD affects older boys and young men, following a milder course than DMD. BMD occurs in about 1 in 30,000 male births.As with Duchenne, the pattern of muscle loss in BMD usually begins with the hips and pelvic area, the thighs and the shoulders. But in BMD, the rate of muscle degeneration varies a great deal from one person to another.

31. Symptoms症状 The disease progresses with increasing muscular weakness and debilitation(衰弱) , finally taking the patient’s life as the result of respiratory (呼吸)or cardiac failure（心脏衰竭）

32. What types of muscular dystrophy are there? • DMD;BMD • Facioscapulohumeral muscular dystrophy (FSH) • Emery-Dreifuss muscular dystrophy (EDMD) • Limb-girdle muscular dystrophy (LGMD) • Oculopharyngeal muscular dystrophy (OPMD) • Distal muscular dystrophy (DD) • Congenital muscular dystrophy (CMD)

33. Other Types Of muscular dystrophy Congenital muscular dystrophy(CMD)——先天肌肉萎缩症 is a rare form of the disease that develops in early infancy（婴儿时期），and strikes boys and girls with equal frequency . The disease has been traced to a deficiency in one of the subunits of the laminin molecule that forms a key component of the muscle cell basement membrane。

34. Congenital muscular dystrophy (CMD) ——先天肌肉萎缩症 由层粘连蛋白（laminin）缺陷引起

35. Other Types Of muscular dystrophy SCARMD--severe childhood autosomal recessive 儿童常染色体隐性肌肉萎缩症 。A deficiency of a dystrophin-associated protein could be the cause of severe childhood autosomal recessive mus-cular dystrophy (SCARMD)由DAG上的一个单位缺陷引起的

36. SCARMD的分子病理学 • Dystrophin是DMD基因的蛋白质产物，近年来人们发现事实上Dystrophin只是连接肌细胞骨架与细胞外基质的蛋白复合体的一部分，即Dystrophin要与糖蛋白，如adhalin，β-，γ-，δ-sarcoglycan等结合形成Dystrophin-糖蛋白复合物［Dystrophin－associated-glycoprotein(DAG)complex］才能维持肌肉收缩时的稳定性，Dystrophin缺乏则不能形成此复合物，使细胞膜不稳定，最终造成肌细胞的坏死，导致DMD的发生。同理，构成DAG复合物的其它糖蛋白的异常或缺失，也可影响DAG复合物的形成，这可能是SCARMD的分子基础。已有很多研究发现了adhalin和γ-sarcoglycan在SCARMD患者骨骼肌中特异性缺失.

37. 综上，DMD, BMD，CMD,和SCARMD都表现出该病的严格症状，这表明分子链上的所有三种组分dystrophin，DAG和Laminin都是肌肉功能所必需的。

38. WHAT TESTS ARE USED TO DIAGNOSE ? 1/Taking a patient and family history 2/performing a physical examination 3/Have genetic testing 4/Have a muscle biopsy

39. Thank you!