1. The Medical Genetics of Dystrophinopathies 张咸宁 zhangxianning@zju.edu.cn Tel ： 13105819271; 88208367 Office: C303, Teaching Building 2015/11

2. Learning Objectives 1. To appreciate the clinical presentations and courses of patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) 2. To understand the differences in the mutations and molecular pathogenesis between DMD and BMD 3. To appreciate the diagnostic approaches to individuals suspected of having DMD or BMD, and their family members

3. Required Reading ●Thompson &Thompson Genetics in Medicine, 7 th ed. （双语版， 2009 ） p. 284-288, Chapter 12, The Molecular and Biochemical Basis of Genetic Disease - Disorders of Structural Proteins - Duchenne and Becker Muscular Dystrophies: Defects in Dystrophin Case Study, 12. Duchenne Muscular Dystrophy ●Thompson &Thompson Genetics in Medicine, 8 th ed., 2016 p. 233-238, 270-271 Case 14. Duchenne Muscular Dystrophy

4. Muscular dystrophies (MD) are primary diseases of muscle characterized by muscle weakness and abnormalities of muscle fibers on histopathological examination. are clinically and genetically heterogeneous （临床异质性、 遗传异质性） show variable onset, ranging from prenatal/birth (congenital muscular dystrophies, CMDs), childhood (DMD), early adulthood (most common age of presentation of the limb girdle muscular dystrophies, LGMDs), or late adult life (myofibrillar myopathies, MFMs). Severe muscle disorder –Progressive clinical course –No effective treatment at this time www.mdausa.org 、 www.china-dmd.org

10. DMD & BMD Incidences of DMD and BMD are ~ 1:3500 males and 1:20 000 males Gene cloning in 1987 (Kunkel LM’s group) — Improved diagnosis and counseling — Provided insight into pathogenesis of DMD and BMD — Suggested therapeutic strategies: gene therapy

11. Clinical Phenotypes of DMD and BMD

12. Early History in Boys with DMD Appear normal for first 1-2 yrs of life In retrospect many have delayed walking beyond 18 mos of age Muscle weakness recognized at 3-5 yrs of age when difficulties noted with –Rising from seated position, especially when sitting on the floor –Climbing stairs

13. Muscle Pseudohypertrophy in DMD Pseudohypertrophy of calves in a 8-yo boy with DMD Muscles appear hypertrophied but are weak Muscle tissue replaced with connective tissue and fat

14. Gowers Maneuver (sign) （ Gowers 征） Classic maneuver that boys with DMD independently discover and most use by age 5 yrs Illustrates progressive myopathy –Begins with hip girdle muscles and neck flexors –Progresses to involve shoulder girdle and then muscles of distal limbs and trunk Uses limb muscles to “climb up legs” to standing position Common solution to weakness indicates that the pattern of progression is similar among boys affected with DMD

15. Early Clin Lab Finding in DMD In preclinical and early stages of DMD Serum creatine kinase (CK or CPK) –Dramatically increased: 50-100 times normal –Increased circulating CK due to release from breakdown of diseased muscle

17. Later History in Boys with DMD Usually confined to wheelchair by 12 yrs Survival beyond age 25 yrs is uncommon –Median age at death 18 yrs of age Patients die of skeletal or cardiac muscle involvement –Respiratory failure –Pneumonia –Cardiac failure

19. A male with DMD at 4-, 10-, and 29-yr-old showing the progression of the disease. He has deletion of exons 49–54 of the dystrophin gene.

20. Cardiac Involvement in DMD Cardiac and respiratory involvement is commonly observed in MDs and is often the leading death cause 95% DMD have cardiac compromise –Dilated cardiomyopathy and/or abnormal EKG 84% DMD have cardiac findings at autopsy  50% DMD have chronic cardiac failure May present rarely with heart failure

21. Mental retardation (MR) /developmental disability (DD) in Patients with DMD CNS involvement is not common in MDs and intelligence is usually preserved Statistics on MR/DD in boys with DMD somewhat misleading –Average IQ reduced 10-20 points –  30% have some degree of MR/DD Dystrophin expressed in the brain and therefore MR/DD attributed to reduced brain expression

22. Becker Muscular Dystrophy (BMD) Milder phenotype than DMD –BMD if still ambulatory at 16 yrs –Extreme variability in progression of the disease in patients with BMD, some with very mild myopathy BMD also due to mutations in DMD gene

23. DMD Mutations in DMD and BMD DMD:  85% of DMD mutations –Mutations result in absence or near absence of dystrophin BMD:  15% of DMD mutations –Mutations are generally in-frame deletions, i.e., maintain dystrophin expression Altered molecular weight Reduced levels

24. Western Blot in DMD and BMD Normal dystrophin MW = 427 kDa DMD –No dystrophin seen BMD –Truncated dystrophin observed –MWs differ in patients from different families

25. H&E ( 左 ) and Dystrophin Immunofluorescence ( 右 ) in DMD and BMD Muscle H&E –DMD: increased connective tissue and leukocytes Dystrophin IMF –Normal: localized to myocyte membrane –BMD: present but reduced –DMD: completely absent

26. Inheritance of DMD Mutation rate –Calculated at 1 in 10,000 10-fold higher than for most other genes –Normal male produces 8 x 10 7 sperms/day, and therefore a sperm with a new DMD mutation every 10-11 seconds

27. Inheritance DMD: XR, genetic lethal in males and overall frequency not changing –1/3 of cases are due to new mutations –2/3 of cases will have carrier mothers BMD: XR, with genetic fitness up to 70% of normal –High proportion of BMD cases are inherited –Only  10% of BMD due to new mutations

28. Female Carriers for DMD Majority have no clinical manifestations  70% have slightly increased serum CK Normal X chromosome inactivated in a critical proportion of cells in some –8% have significant muscle weakness, some with severe proximal muscle impairment

30. Generation of an X- autosome translocation with breakpoint in a female and how this results in the development of DMD

31. DMD Gene and Its Product Very large! –DMD 2.6 megabases (mb) 1.5% of X chromosome –Muscle DMD transcript ~14 kb –Muscle dystrophin protein 427 kD Large target => High mutation rate (at least in part)

32. DMD Gene ( www.dmd.nl ； www.umd.be/DMD/ ) Structurally complex –79 Exons –7 Tissue-specific promoters –Differential splicing => Numerous isoforms Tissue-specific isoforms Developmentally regulated isoforms

33. DMD Transcription/Translation Levels Match Clinical Phenotype Dystrophin protein most abundant –Skeletal muscle –Cardiac muscle –Brain But most tissues express at least one isoform

34. Dystrophin Structural protein Maintains muscle membrane integrity ( 维持肌细胞 膜的完整性 ) –Links actin cytoskeleton to laminin in extracellular matrix through dystroglycans ( dystrophin associated proteins, DAPs ) –Permits structural support in a moving membrane Mutations in other complex members are associated with muscular dystrophies –Sarcoglycans: limb girdle muscular dystrophy (LGMD) –Laminin: congenital muscular dystrophy (CMD)

35. The Dystrophin-Glycoprotein Complex at the Cell Membrane

36. Molecular Analysis of DMD and BMD 60% of DMD mutant alleles are deletions –Clustered in two regions 5’ half Central region: presumed to be due to slipped mispairing in spectrin-like repeats –Would expect to be in frame and 46% of deletions in the spectrin-like repeat region result in mild BMD  34% of DMD mutant alleles are point mutations –Randomly distributed

37. Deletion Distribution in DMD and BMD

38. multiplex ligation-dependent probe amplification (MLPA)

39. Carrier/Noncarrier Identification Possible in virtually all females when the lesion is known. Cannot pick up gonadal mosaicism

41. Counseling a Mother Without Prior Family and No Mutation in Her Scenario –Boy with DMD and identified mutation is first affected individual in family AND –Mother is not found to have a mutation in an accessible cell line, e.g., lymphocytes, buccal swab What is the conclusion?

42. Counseling a Mother Without Prior Family and No Mutation in Her Conclusion: –New mutation No likelihood of another affected boy Would be wrong in 5-15% of women with these findings! –Maternal mosaicism Mutation occurred during development and present in ova Probability of recurrence is significant

45. Therapy Symptomatic only at present-corticosteroids are beneficial and delay onset of wheel chair need for two years Isolation and characterization of DMD gene gave promise for effective therapeutics –Gene therapy Large size of cDNA Could use construct shortened by in frame deletion of spectrin-like repeats But problems of efficiency of delivery

46. Therapy Antisense therapy and exon skipping Creative approaches still needed –Immunosuppression –Up-regulation of dystrophin-related protein, e.g., utrophin, in muscles and brain –Stem cells from Muscle Bone marrow Fat

47. Antisense oligonucleotides （ AOs ） A short single-stranded nucleic acid, typically 15- 25 nucleotides in length, that has the ability to mediate therapeutic effects by directly interacting with pre-mRNA or mRNA in a sequence-specific manner. Therapeutic AOs are normally designed to bind to relevant exon-intron junctions in the pre- mRNA; blocking of splicing at that junction may induce skipping of an adjacent exon containing the harmful mutation.

49. DMD gene: effects of mutations and exon skipping

51. Exon skipping The use of molecular interventions to exclude an exon from a pre-mRNA that encodes a reading frame–disrupting mutation, thereby rescuing expression of the mutant gene.