Lecture 31 - Molecular Basis of Muscular Dystrophies Flashcards
Function of dystrophin
Links dystroglycan complex (which links to ECM) to muscle fibre
Which chromosome is dystrophin on?
Xp21
Number of sarcoglycans
Four
Exons in an average gene
8-9
Size of average gene
~3000 bases
Size of average mRNA
~1000 bases
Proportion of X chromosome taken up by dystrophin
1%
Size of dystrophin gene
79 exons
2.4 megabases
Proportion of dystrophin gene that is exons
0.6%
Proportion of dystrophin mutations that are de novo
~1/3
How is dystrophin differently expressed in different tissues?
Different gene transcripts lead to different protein isoforms
What is BMD?
Becker muscular dystrophy
A milder phenocopy of DMD
What do mutations in dystrophin lead to?
Duchenne muscular dystrophy
Becker muscular dystrophy
How are different dystrophin isoforms formed?
Alternative promotor usage and splicing of mRNA
Isoforms of dystrophin
1)
2)
1) 4 long isoforms (l, m, c, p)
2) Smaller isoforms
Where are the long isoforms of dystrophin expressed? 1) 2) 3) 4)
1) Skeletal muscle
2) Cardiac muscle
3) Smooth muscle
4) Brain
Where are the short isoforms of dystrophin expressed?
1)
2)
3)
1) CNS
2) Retina
3) Kidney
Most common isoform of dystrophin found in skeletal and cardiac muscle
Long isoform
427kDa protein, 3685 amino acids
Feature of skeletal isoform dystrophin that is absent from other isoforms
Actin-binding domain
Domains in full-length dystrophin 1) 2) 3) 4)
1) N-terminal actin-binding domain
2) Rod domain of spectrin-like repeats (variable length)
3) Cysteine-rich domain
4) C-terminal domain, that allows assembly of the dystrophin-associated protein complex
Dystrophin mutation that results in DMD
Loss of C-terminal domain, some of spectrin-like rods
Dystrophin mutation that results in BMD
Shortening of spectrin-like rod domain.
Protein is still partially functional
Missense mutation
Changes amino acid encoded
Nonsense mutation
Stop codon
Proportion of DMD mutations that are nonsense
15%
Can nonsense mutations be picked up on MLPA?
Not normally
Proportion of DMD mutations that are frameshift
60%
Typical feature of BDM mutations
In-frame mutations
Proportion of DMD mutations that are duplications
5%
Can duplications be identified with MLPA?
Yes
What determines whether a duplication will cause DMD or BMD?
If it is an in-frame mutation or not
How does dystrophin link muscle fibres with the ECM?
1)
2)
3)
1) N-terminal links to F-actin
2) C-terminal links to dystrophin-associated protein complex in the sarcolemma
3) DAPC has an extracellular anchor, which links to the ECM
What effect does the linkage of the cytoskeleton with the ECM have in muscles?
1)
2)
3)
1) Stabilises sarcolemma during muscle contraction and relaxation
2) Transmits force generated by contraction to the ECM
3) Linkage allows DAPC to be involved in cell signalling
Effect of losing dystrophin 1) 2) a, b, c, d 3) 4) 5)
1) Loss of DAPC at the sarcolemma
2) Makes the sarcolemma very fragile, which leads to:
a) Ca2+ influx (disregulation)
b) Apoptosis, necrosis
c) Inflammation
d) Fibrosis
3) Disrupted muscle architecture
4) Signalling defects
5) Secondary loss of other proteins
Effect of dystrophin mutations on cellular Ca2+ 1) 2) 3) 4)
1) Increases amount of intracellular Ca2+
2) Increased Ca2+ influx through ca2+ stretch channels
3) This might lead to activation of the inflammatory response
4) This is observed in mdx mice
How is DMD modelled?
In mdx mice
When are elevated inflammatory mediators observed in muscle?
Prior to onset of DMD
Proportion of DMD caused by large partial deletions
65%
MLPA 1) 2) 3) 4) 5) 6)
1) Multiplex ligation-dependent probe amplification
2) A way to detect gene duplications or deletions
3) A PCR process
4) Use PCR primers for ‘hotspots’ where duplications or deletions often occur
5) Run results on a gel, can see here if a region is absent or overexpressed
6) Determines relative number of of all exons within a gene simultaneously
Characteristics of DMD muscle biopsy 1) 2) 3) 4) 5) 6) 7)
1) Variable fibre size
2) Hypercontracted (opaque) muscle fibres)
3) Muscle fibre degeneration and regeneration
4) Normal or immature muscle fibre internal architecture
5) Absent dystrophin
6) Reduced sarcoglycans, aquaporins
7) Increased fibrosis within a muscle
How can dystrophin be stained for?
Immunohistochemistry
Fluorescent antibody probe
Early pathological findings of DMD muscle
1)
2)
1) Invasion of muscle by phagocytes
2) Necrotic fibres are pale with an NADH stain
Late-stage pathological findings of DMD muscle
1)
2)
3)
1) Increased endomysial connective tissue
2) Variable fibre size
3) Hypercontracted muscle fibres
When staining for dystrophin, how do histological slides appear?
Normal - dystrophin around edges of fibres
DMD - Dystrophin absent
BMD - Dystrophin reduced
What does Western blotting tell us?
1)
2)
1) Quantifies amount of protein in a specific tissue
2) Determines the size of a protein
