Lecture 32 - Muscular Dystrophies - Molecular Basis Flashcards Preview

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Flashcards in Lecture 32 - Muscular Dystrophies - Molecular Basis Deck (34):
1

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

2

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

3

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

4

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

5

How large is the skeletal and cardiac muscle Dystrophin isoform?

3685 aa
427 kDa

6

How many bp is the:
• Dystrophin gene
• Dystrophin mRNA transcript?

Gene: 2.4 Mbp

mRNA: 14 kb

→ Huge

7

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

8

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

9

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

10

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)

11

Which mutation types will be picked up with MLPA?
Which won't?

Detected:
• Duplications
• Deletions

Not detect:
• Nonsense mutations (point mutations)
• Small duplications / deletions

12

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

13

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

14

What is the animal model for DMD?

mdx mice

15

What is observed in mdx mice and DMD with regard to intracellular ion concentration?

Elevated intracellular calcium levels

16

What is MLPA?

Multiplex ligation-dependent probe amplification

17

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

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 --

1. PCR amplification of all 79 exons

2. Analysis with capillary electrophoresis

3. 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

18

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

19

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)

20

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

21

Which is the most common human muscular dystrophy in childhood?

DMD

22

Describe the structure of DAPC

Actin skeleton

Dystrophin
• C-terminus really important for assembly of DAPC

Transmembrane proteins in sarcolemma
• Sarcoglycans
• Dystroglycans

Laminin

ECM

23

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

24

How big is the Dystrophin protein compared to normal proteins

Dystrophin more than 3 times the normal length

Dystorphin: ~3600 aa
Normal: ~1000 aa

25

What varies in the various Dystrophin isoforms?

Invariant:
• C-terminus domain
• Cysteine-rich domain

Variant:
• Actin-binding domain (only found in skeletal muscle isoform)
• Spectrin-like domain (variable n° of repeats)

26

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

27

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)

28

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

29

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

30

When is inflammatory cell infiltrate generally observed in DMD muscle biopsies?

Early-stage muscle pathology

31

What is the hallmark of late-stage muscle pathology in DMD?

Fibrous connective tissue

32

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

33

To which chromosome does the Dystrophin gene map?

Xp21

34

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

More common:
• Large deletions

Less common:
• Duplications
• Point mutations