Download Muscle Diseases-06

Molecular Genetics of Muscle Disorders
Chapter 13 (pp. 369-376)
650 different
Muscles !
Skeletal Muscle
• For locomotion (voluntary)
Cardiac Muscle
• Heart muscles (involuntary)
Smooth Muscle
• Lining the wall of interior organs
(involuntary)
650 different
Muscles !
More than 1000 genes required for
proper muscle formation and functioning:
• Providing energy to the cell (respiration)
• Structure of muscles
• Involved in contraction process
Mutations have been found
in all of Dystrophy
these categories
Muscular
Muscle Cells:
• Can be very long (12 inches)
• Multinucleate
Figure 13.1
Many nuclei per cell because
The cell is soooo long!
One nucleus per cell
One nucleus per cell
Calcium enters the cell after receiving
a nerve impulse to do so, and leaves
the cell during the ‘relaxation stage’.
Thick Myofilaments
Thin Myofilaments
Mitochondria
Non-covalent
interactions
Signaling
system
Figure 13.3
Extracellular Matrix (collagen, etc.)
All linked
to a Nucleus
A lot of good,
easy-to-understand
information is at this
< hyperlink
Four Domains
Four Domains
Binds to Actin (240 amino acids)
Four Domains
Central rod
24 repeats of
≈125 amino acids
= 3000 a.a.
Binds to Syntrophins,
Dystrobrevin, etc.
(420 amino acids)
Binds to Actin (240 amino acids)
Four Domains
Binds to
Dystroglycan
(280 amino acids)
Central rod
24 repeats of
≈125 amino acids
= 3000 a.a.
Binds to Actin (240 amino acids)
Four Domains
Binds to
Dystroglycan
(280 amino acids)
Central rod
24 repeats of
≈125 amino acids
= 3000 a.a.
Binds to Syntrophins,
Dystrobrevin, etc.
(420 amino acids)
Binds to Actin (240 amino acids)
• X-Linked, Recessive
Duchenne
Muscular Dystrophy • 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
Heart &failure
Lung muscles
weakenat 17
• Respiratory
(death)
Muscles of shoulders &
pelvis weaken first
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
Dystrophin
Largest gene found in nature = 2.4 million bases
79 Exons
Dystrophin
Largest gene found in nature = 2.4 million bases
79 Exons
Duchenne Muscular Dystrophy is generally caused by
frameshift (or missense mutations) at the 3rd or 4th domains
Causing a truncated protein
Becker Muscular Dystrophy is generally caused by internal
parts of the proteing being missing
cDNA cloned in 1987
How would you go about cloning this gene?
Dystrophin
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)
Lots of different labs were looking for this
gene, and they took different approaches
1987
Biopsy of normal and DMD muscle
And did gel electrophoresis of the proteins
1987
Biopsy of normal and DMD muscle
And did gel electrophoresis of the proteins
Observed a lack
of large MW
proteins in DMD
muscles
Immunoblots
“Western Blots”
Normal
DMD
Cytogenetic analysis of one DMD patient
showed a thinner chromosome band here.
Made Hamster Somatic Cell Hybrids with that guy’s Chromosome X
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 !
Example of a DMD RFLP
Digested with XmnI
Koenig, et al., 1987 Cell, Vol. 50: 509.
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).
A good candidate for
a cDNA for DMD
would be something
large !
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.
1988
Four Domains
Binds to
Dystroglycan
(280 amino acids)
Central rod
24 repeats of
≈125 amino acids
= 3000 a.a.
Binds to Syntrophins,
Dystrobrevin, etc.
(420 amino acids)
Binds to Actin (240 amino acids)
76 exons
Was initially mysterious because the smaller delection was associated
With the more-severe form of MD.
http://www.bms.ed.ac.uk/research/others/smaciver/MD%20cause.htm
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)
At Protein Level:
Substitution = 57%
Deletion = 32%
Duplication = 9%
Insertion =2%
Frameshift = 55%
Nonsense = 39%
Deletion = 6%
* Our textbook says 65% deletions
79 Exons in all
2003
Healthy muscles
DMD muscles
Cytochemical staining for Dystrophin
makes diagnosis fast
* Remember this protein is fairly near the cell surface
http://www.neuro.wustl.edu/neuromuscular/pathol/dmdpath.htm
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)
Used adenoviral vector
Hemophilia
Color Blindness
High Blood Pressure
Dystrophin
Largest gene found in nature = 2.4 million bases
79 Exons
Alternative splicing
Multiplex PCR
PCR using multiple pairs of primers (representing exons)
9 primer pairs