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
What is MLPA?
Multiplex ligation-dependent probe amplification
Describe the process of DNA diagnosis of mutations in the Dystrophin gene
Which sorts of mutations can be detected?
– Multiplex PCR of gene –
(Now outdated)
- Selected exons amplified with PCR
• Selected exons based on which exons are commonly mutated
• Maybe 5 exons - Gel electrophoresis
Deletion detection:
• Missing exons on the gel
Cons:
• Only a certain number of exons investigated (e.g. 8 out of 79)
• Only deletions detected
• Does not detect point mutations (nonsense mutations)
– MLPA –
- PCR amplification of all 79 exons
- Analysis with capillary electrophoresis
- Detection of amount of gene
Pros:
• Every single exon investigated
• Determines relative copy number of all exons within gene simultaneously
• Detect carriers
• Detects deletions and duplications
• Still does not detect small duplications / deletions or nonsense (point) mutations
Compare normal and Dystrophic muscle biopsies
Normal: • Evenly spaced muscle fibres • Regular staining • Uniform size • Little connective tissue
Dystrophic: • Disordered spacing of muscle fibres • Non-uniform staining • Variable fibre size • Necrotic muscle fibres • Much connective tissue • Inflammation: invasion by macrophages • Fibrosis • Muscle fibre degeneration and regeneration • Hypercontracted (opaque) muscle fibres
Early dystrophic changes:
• Macrophage infiltration
• Necrotic muscle fibres (pale NADH stain)
• Regenerating fibres
Late dystrophic changes: • Basophilic fibres (H&E stain) • Alkaline phosphatase positive fibres • 2C fibres • Increased endomysial connective tissue • Variable fibres size • Hypercontracted muscle fibres
Describe what immunohistochemistry can tell us about DMD
Abs fluorescently tagged, specific for dystrophin
Normal: fluorescence observed around the rim of muscle fibres
DMD: Absent protein
NB BDM: decreased staining (not completely absent)
What can Western blotting tell us about MD?
Compare normal and MD results
Proteins run on a gel
Western blotting can:
• Quantify the amount of a protein in a specific tissue
• Determine the size of a protein
Normal:
• Travels less because it is larger
BMD:
• Smaller proteins: travel further on the gel
Which is the most common human muscular dystrophy in childhood?
DMD
Describe the structure of DAPC
Actin skeleton
Dystrophin
• C-terminus really important for assembly of DAPC
Transmembrane proteins in sarcolemma
• Sarcoglycans
• Dystroglycans
Laminin
ECM
How much do exons contribute to the size of the Dystrophin gene?
Only 0.6%
Thus, the vast majority of the gene are intronic regions
How big is the Dystrophin protein compared to normal proteins
Dystrophin more than 3 times the normal length
Dystorphin: ~3600 aa
Normal: ~1000 aa
