Flashcards in Lecture 32 - Muscular Dystrophies - Molecular Basis Deck (34):
List important proteins in the following components:
• Thick filaments (A band)
• Thin filaments (I-band)
• M line
• Third filaments
• Intermediate filaments
Don't need to know all the details
Just appreciate that each component has a complex protein structure
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
• 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
• Alternative promoter usage and splicing of pre-mRNA
4 long isoforms:
• l, m, c, p
• Skeletal muscle
• Cardiac muscle
• Smooth muscle
How large is the skeletal and cardiac muscle Dystrophin isoform?
How many bp is the:
• Dystrophin gene
• Dystrophin mRNA transcript?
Gene: 2.4 Mbp
mRNA: 14 kb
Describe the structure of the Dystrophin protein
Which are the most important bits?
1. N-terminal domain
• Actin binding domain
2. Rod domain
• Spectrin like repeats
• Shorter forms have fewer of these repeats
3. Cysteine rich domain
4. 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
• No change in protein product
• Amino acid change in protein product
• Causes premature stop codon, and stop in protein production
B. Deletions / insertions
2. Code out of frame (shifted) downstream
• 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?
• 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)
• 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
1. Loss of DAPC
2. Sarcolemma rendered fragile, muscle fibres become susceptible to injury
• Tearing of sarcolemma during muscle contraction, because force can't be properly transmitted
3. Enhanced Ca2+ influx through Ca2+/stretch-activated channels
4. Activation of the inflammatory response, expression of:
• Inflammatory mediators
5. Degeneration during repeated cycles of muscle contraction:
6. Disrupted muscle architecture, weakness
What is the animal model for DMD?
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 --
1. Selected exons amplified with PCR
• Selected exons based on which exons are commonly mutated
• Maybe 5 exons
2. Gel electrophoresis
• Missing exons on the gel
• Only a certain number of exons investigated (e.g. 8 out of 79)
• Only deletions detected
• Does not detect point mutations (nonsense mutations)
-- MLPA --
1. PCR amplification of all 79 exons
2. Analysis with capillary electrophoresis
3. Detection of amount of gene
• 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
• Evenly spaced muscle fibres
• Regular staining
• Uniform size
• Little connective tissue
• Disordered spacing of muscle fibres
• Non-uniform staining
• Variable fibre size
• Necrotic muscle fibres
• Much connective tissue
• Inflammation: invasion by macrophages
• 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
• Travels less because it is larger
• Smaller proteins: travel further on the gel
Which is the most common human muscular dystrophy in childhood?
Describe the structure of DAPC
• C-terminus really important for assembly of DAPC
Transmembrane proteins in sarcolemma
How much do exons contribute to the size of the Dystrophin gene?
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
What varies in the various Dystrophin isoforms?
• C-terminus domain
• Cysteine-rich domain
• Actin-binding domain (only found in skeletal muscle isoform)
• Spectrin-like domain (variable n° of repeats)
Compare deletion of in-frame and exon spanning codons
In frame exons:
• Individual codons contained within an exon
• Deletion of the exon does not result in out-of-frame shift
• Codon spans two exons
• Deletion of exon results in out-of-frame shift
Which sorts of exons are deleted in DMD and BMD?
DMD: deletion of out-of-frame exons (codons span two exons)
BMD: deletion of in-frame exons (codon contained within an exon)
If a boy was suspected to have DMD but the DNA test came back negative, what is the next port of call?
1. DNA sequencing (Sanger)
2. Muscle biopsy
• Really don't want to do this because it is quite invasive
What is observed in the other proteins associated with DAPC in the muscle biopsy?
eg. Sarcoglycans, Aquaporins
This is not due to mutations in the genes, but rather secondary effects of the mutation in Dystrophin
Dystrophin is required for their stabilisation in the membrane
When is inflammatory cell infiltrate generally observed in DMD muscle biopsies?
Early-stage muscle pathology
What is the hallmark of late-stage muscle pathology in DMD?
Fibrous connective tissue
Why are de novo mutations in Dystrophin reasonably common?
How common are they?
Due to the sheer size of the gene (2.4 Mb)
Do novo mutations are responsible for 1/3 of DMD cases
To which chromosome does the Dystrophin gene map?