Lecture 32 - Muscular Dystrophies - Molecular Basis Flashcards Preview

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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
• 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


Third filaments


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:
• 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
• Brain

Smaller isoforms:
• 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:

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

1. Silent
• No change in protein product

2. Missense:
• Amino acid change in protein product

3. Nonsense:
• Causes premature stop codon, and stop in protein production

B. Deletions / insertions

1. Frameshift

2. 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?

• 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

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
• Chemoattractants

5. Degeneration during repeated cycles of muscle contraction:
• Apoptosis
• Necrosis
• Inflammation
• Fibrosis

6. 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)

1. Selected exons amplified with PCR
• Selected exons based on which exons are commonly mutated
• Maybe 5 exons

2. Gel electrophoresis

Deletion detection:
• 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
• 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

• 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

Actin skeleton

• C-terminus really important for assembly of DAPC

Transmembrane proteins in sarcolemma
• Sarcoglycans
• Dystroglycans




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


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

Exon-spanning codon:
• 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

Reduced levels

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?



In general, which sorts of mutations cause DMD more and less commonly?

More common:
• Large deletions

Less common:
• Duplications
• Point mutations