Lecture 32 - Muscular Dystrophies - Molecular Basis Flashcards
List important proteins in the following components: • Thick filaments (A band) • Thin filaments (I-band) • M line • Third filaments • Z-disc • Intermediate filaments
Don’t need to know all the details
Just appreciate that each component has a complex protein structure
Thick filaments:
• Myosin
Thin filaments:
• Actin
• Tropomyosin
• Troponin
M-line
Third filaments
Z-disc
Intermediate filaments
• Desmin
• Plectin
List the features of the Dystrophin gene
What diseases can mutations in it cause?
- Chromosome Xp21
- Second largest gene known
- 79 exons
- Large size makes it susceptible to mutations
- Various promoters → different transcripts in different tissues → tissue isoforms
Mutations cause: • DMD • BMD • X-linked cardiomyopathy • X-linked cramps-myalgia syndrome • Isolated quadriceps myopathy
List the features of DMD
• Common causes
• Characteristic features
- Most common human MD
- Most commonly caused by large deletions (out of frame)
- Less commonly caused by duplications or point mutations
• Characterised by necrosis, phagocytosis and regeneration of muscle fibres
What is an isoform?
Discuss the various isoforms of Dystrophin:
• Where they are found
• How they arise
Variant forms of the same protein
Formation:
• Alternative promoter usage and splicing of pre-mRNA
4 long isoforms: • l, m, c, p • Skeletal muscle • Cardiac muscle • Smooth muscle • Brain
Smaller isoforms:
• CNS
• Retina
• Kidney
How large is the skeletal and cardiac muscle Dystrophin isoform?
3685 aa
427 kDa
How many bp is the:
• Dystrophin gene
• Dystrophin mRNA transcript?
Gene: 2.4 Mbp
mRNA: 14 kb
→ Huge
Describe the structure of the Dystrophin protein
Which are the most important bits?
Multiple domains:
- N-terminal domain
• Actin binding domain - Rod domain
• Spectrin like repeats
• Shorter forms have fewer of these repeats - Cysteine rich domain
- C-terminal domain
• Allows assembly of DAPC
Most important bits:
• Actin binding domain
• Cysteine rich domain
• C-terminal domain
Rod domain is not so vital
Compare the Dystrophin protein in DMD and BMD
DMD: absent protein due to out-of-frame deletions
BMD: shorter, but still functional protein due to in-frame deletions
What are the various outcomes of base substitutions in coding regions?
What are some other types of mutations that can occur to cause DMD?
A. Base substitutions / point mutations
- Silent
• No change in protein product - Missense:
• Amino acid change in protein product - Nonsense:
• Causes premature stop codon, and stop in protein production
B. Deletions / insertions
- Frameshift
- Code out of frame (shifted) downstream
C. Duplications
• Disruption of reading frame → DMD
• Preservation of reading frame → BMD
Describe the importance of the various types of mutations in causing DMD
Large deletion causing frameshift: 60%
Point mutation → Nonsense mutations: 15%
Duplications causing frameshift: 5% (of one or more exons)
Which mutation types will be picked up with MLPA?
Which won’t?
Detected:
• Duplications
• Deletions
Not detect:
• Nonsense mutations (point mutations)
• Small duplications / deletions
Describe the role of Dystrophin in the DAPC
What is the function of the DAPC?
Links the internal cytoskeleton (myofibrils) to the ECM
N-terminus: binds F-actin
C-terminus: binds DAPC at the sarcolemma (through another protein, i.e. not directly)
DAPC function:
• Stabilisation of sarcolemma during cycles of contraction and relaxation through transmission of force generated in sarcomeres to ECM
• Cell signalling
Describe the effect of loss of Dystrophin
- Loss of DAPC
- Sarcolemma rendered fragile, muscle fibres become susceptible to injury
•Tearing of sarcolemma during muscle contraction, because force can’t be properly transmitted - Enhanced Ca2+ influx through Ca2+/stretch-activated channels
- Activation of the inflammatory response, expression of:
• Inflammatory mediators
• Chemoattractants
5. Degeneration during repeated cycles of muscle contraction: • Apoptosis • Necrosis • Inflammation • Fibrosis
- Disrupted muscle architecture, weakness
What is the animal model for DMD?
mdx mice
What is observed in mdx mice and DMD with regard to intracellular ion concentration?
Elevated intracellular calcium levels