1. Molecular Genetics of Muscle Disorders Chapter 13 (pp. 369-376) 650 different Muscles !

2. 650 different Muscles ! Skeletal Muscle For locomotion (voluntary) Cardiac Muscle Heart muscles (involuntary) Smooth Muscle Lining the wall of interior organs (involuntary)

3. More than 1000 genes required for proper muscle formation and functioning: Providing energy to the cell (respiration) Structure of muscles Involved in contraction process Muscular Dystrophy Mutations have been found in all of these categories

4. Figure 13.1 Muscle Cells: Can be very long (12 inches) Multinucleate

6. One nucleus per cell Many nuclei per cell because The cell is soooo long! One nucleus per cell

7. Calcium enters the cell after receiving a nerve impulse to do so, and leaves the cell during the ‘relaxation stage’.

8. Thin Myofilaments Thick Myofilaments

10. Mitochondria

11. Figure 13.3 Non-covalent interactions Signaling system

12. Extracellular Matrix (collagen, etc.)

14. All linked to a Nucleus

15. A lot of good, easy-to-understand information is at this < hyperlink

16. Four Domains

17. Binds to Actin (240 amino acids) Four Domains

18. Binds to Actin (240 amino acids) Central rod 24 repeats of ≈125 amino acids = 3000 a.a. Binds to Syntrophins, Dystrobrevin, etc. (420 amino acids) Four Domains

19. Binds to Actin (240 amino acids) Central rod 24 repeats of ≈125 amino acids = 3000 a.a. Binds to Dystroglycan (280 amino acids) Four Domains

20. Binds to Actin (240 amino acids) Central rod 24 repeats of ≈125 amino acids = 3000 a.a. Binds to Dystroglycan (280 amino acids) Binds to Syntrophins, Dystrobrevin, etc. (420 amino acids) Four Domains

21. X-Linked, Recessive 1 in 3300 male births Few symptoms until about 5 1/3-1/2 have learning disability First, not able to “keep up” with other children Then will start to “waddle” Then starts to walk on toes Curvature of spine Then uses hands to hold self up Wheelchair only by 10 years Chest muscles weaken, making breathing difficult Respiratory failure (death) at 17 Duchenne Muscular Dystrophy Heart & Lung muscles weaken

22. Muscles of shoulders & pelvis weaken first

23. Becker Muscular Dystrophy A milder form of the same disease (and mutation of the same gene, Dystrophin) 1 in 30,000 males Affected males live longer

24. Largest gene found in nature = 2.4 million bases Dystrophin 79 Exons

25. Largest gene found in nature = 2.4 million bases Dystrophin 79 Exons Duchenne Muscular Dystrophy is generally caused by frameshift (or missense mutations) at the 3 rd or 4 th domains Causing a truncated protein Becker Muscular Dystrophy is generally caused by internal parts of the proteing being missing cDNA cloned in 1987

26. How would you go about cloning this gene?

28. 2/3 of cases are inherited, 1/3 are sporadic (new mutations) Second human gene cloned by “Positional Cloning” (the third was Cystic Fibrosis gene) Dystrophin Lots of different labs were looking for this gene, and they took different approaches

29. Biopsy of normal and DMD muscle And did gel electrophoresis of the proteins 1987

30. Biopsy of normal and DMD muscle And did gel electrophoresis of the proteins 1987 Observed a lack of large MW proteins in DMD muscles

31. Immunoblots “Western Blots”

32. Normal DMD

33. Cytogenetic analysis of one DMD patient showed a thinner chromosome band here.

34. Made Hamster Somatic Cell Hybrids with that guy’s Chromosome X

35. Made Hamster Somatic Cell Hybrids with this guy’s Chromosome X And probed with 20 random pieces of DNA from a genomic library of Chromosome X (from a healthy person). ….19 probes hybridized, 1 didn’t !

36. Koenig, et al., 1987 Cell, Vol. 50: 509. Example of a DMD RFLP Digested with XmnI

37. As they got closer to figuring out that the DMD gene was in the middle of the short arm of the X chromosome they used various fragments of the Genomic Library to screen a cDNA Library. The cDNA Library was made from fetal muscle tissue (female).

38. A good candidate for a cDNA for DMD would be something large !

39. 1987 Reported that the most common difference between the healthy DMD gene and the mutated one was a 2000 bp deletion….it was present in about half of 104 boys with the disease.

40. 1988

41. Binds to Actin (240 amino acids) Central rod 24 repeats of ≈125 amino acids = 3000 a.a. Binds to Dystroglycan (280 amino acids) Binds to Syntrophins, Dystrobrevin, etc. (420 amino acids) Four Domains

42. Was initially mysterious because the smaller delection was associated With the more-severe form of MD. 76 exons

43. http://www.bms.ed.ac.uk/research/others/smaciver/MD%20cause.htm

44. Neuromuscular Disorders, 2004 Volume 14:650-658 The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre. Tuffery-Giraud S, Saquet C, Chambert S, Echenne B, Marie Cuisset J, Rivier F, Cossee M, Philippe C, Monnier N, Bieth E, Recan D, Antoinette Voelckel M, Perelman S, Lambert JC, Malcolm S, Claustres M. Laboratoire de Genetique Moleculaire et Chromosomique, Institut Universitaire de Recherche Clinique (IURC), CHU de Montpellier, 641 avenue du Doyen G. Giraud, 34093 Montpellier Cedex 5, France. tuffery@igh.cnrs.fr Tuffery-Giraud SSaquet CChambert SEchenne BMarie Cuisset JRivier FCossee MPhilippe C Monnier NBieth ERecan DAntoinette Voelckel MPerelman SLambert JCMalcolm SClaustres M At the DNA level: (89 cases) Substitution = 57% Deletion = 32% Duplication = 9% Insertion =2% At Protein Level: Frameshift = 55% Nonsense = 39% Deletion = 6% * Our textbook says 65% deletions

46. 2003 79 Exons in all

47. Healthy muscles DMD muscles http://www.neuro.wustl.edu/neuromuscular/pathol/dmdpath.htm * Remember this protein is fairly near the cell surface Cytochemical staining for Dystrophin makes diagnosis fast

48. What can be done with boys with DMD? At early stages Physical Therapy Splints, Crutches, Wheelchairs Surgery At later stages Cardiological medicines Assisted breathing devices (later stages) Prednisone, a corticosteroid, similar to a hormone produced by the adrenal glands. Lots of side effects: weight gain, bone brittleness, cataracts, mood changes… …more aggressive)

49. Used adenoviral vector

51. Hemophilia Color Blindness High Blood Pressure

52. Largest gene found in nature = 2.4 million bases Dystrophin 79 Exons Alternative splicing

54. Multiplex PCR PCR using multiple pairs of primers (representing exons) 9 primer pairs