Muscle diseases brief explanation-06.ppt

Muscles and its diseases

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. [email protected]
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