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The Medical Genetics of Dystrophinopathies

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

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The Medical Genetics of Dystrophinopathies

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  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, 7th 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, 8th ed., 2016 p. 233-238, 270-271 Case 14. Duchenne Muscular Dystrophy

  4. Muscular dystrophies (MD) • are primary diseases ofmuscle characterized by muscle weakness and abnormalitiesof muscle fibers on histopathologicalexamination. • 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

  5. 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

  6. Clinical Phenotypes of DMD and BMD

  7. 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

  8. 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

  9. 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 Gowers Maneuver (sign)（Gowers征）

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Inheritance of DMD • Mutation rate • Calculated at 1 in 10,000 • 10-fold higher than for most other genes • Normal male produces 8 x 107 sperms/day, and therefore a sperm with a new DMD mutation every 10-11 seconds

  20. 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

  21. 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

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

  23. 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)

  24. 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

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

  26. 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)

  27. The Dystrophin-Glycoprotein Complex at the Cell Membrane

  28. 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

  29. Deletion Distribution in DMD and BMD

  30. multiplex ligation-dependent probeamplification (MLPA)

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

  32. 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?

  33. 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

  34. 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

  35. 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

  36. 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.

  37. DMD gene: effects of mutations and exon skipping

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