Lecture 31 - Molecular Basis of Muscular Dystrophies

Question 1

Function of dystrophin

Answer 1

Links dystroglycan complex (which links to ECM) to muscle fibre

Question 2

Which chromosome is dystrophin on?

Answer 2

Xp21

Question 3

Number of sarcoglycans

Answer 3

Four

Question 4

Exons in an average gene

Answer 4

8-9

Question 5

Size of average gene

Answer 5

~3000 bases

Question 6

Size of average mRNA

Answer 6

~1000 bases

Question 7

Proportion of X chromosome taken up by dystrophin

Answer 7

1%

Question 8

Size of dystrophin gene

Answer 8

79 exons

2.4 megabases

Question 9

Proportion of dystrophin gene that is exons

Answer 9

0.6%

Question 10

Proportion of dystrophin mutations that are de novo

Answer 10

~1/3

Question 11

How is dystrophin differently expressed in different tissues?

Answer 11

Different gene transcripts lead to different protein isoforms

Question 12

What is BMD?

Answer 12

Becker muscular dystrophy

A milder phenocopy of DMD

Question 13

What do mutations in dystrophin lead to?

Answer 13

Duchenne muscular dystrophy

Becker muscular dystrophy

Question 14

How are different dystrophin isoforms formed?

Answer 14

Alternative promotor usage and splicing of mRNA

Question 15

Isoforms of dystrophin
1)
2)

Answer 15

1) 4 long isoforms (l, m, c, p)

2) Smaller isoforms

Question 16

Where are the long isoforms of dystrophin expressed?
1)
2)
3)
4)

Answer 16

1) Skeletal muscle
2) Cardiac muscle
3) Smooth muscle
4) Brain

Question 17

Where are the short isoforms of dystrophin expressed?
1)
2)
3)

Answer 17

1) CNS
2) Retina
3) Kidney

Question 18

Most common isoform of dystrophin found in skeletal and cardiac muscle

Answer 18

Long isoform

427kDa protein, 3685 amino acids

Question 19

Feature of skeletal isoform dystrophin that is absent from other isoforms

Answer 19

Actin-binding domain

Question 20

Domains in full-length dystrophin
1)
2)
3)
4)

Answer 20

1) N-terminal actin-binding domain
2) Rod domain of spectrin-like repeats (variable length)
3) Cysteine-rich domain
4) C-terminal domain, that allows assembly of the dystrophin-associated protein complex

Question 21

Dystrophin mutation that results in DMD

Answer 21

Loss of C-terminal domain, some of spectrin-like rods

Question 22

Dystrophin mutation that results in BMD

Answer 22

Shortening of spectrin-like rod domain.

Protein is still partially functional

Question 23

Missense mutation

Answer 23

Changes amino acid encoded

Question 24

Nonsense mutation

Answer 24

Stop codon

Question 25

Proportion of DMD mutations that are nonsense

Answer 25

15%

Question 26

Can nonsense mutations be picked up on MLPA?

Answer 26

Not normally

Question 27

Proportion of DMD mutations that are frameshift

Answer 27

60%

Question 28

Typical feature of BDM mutations

Answer 28

In-frame mutations

Question 29

Proportion of DMD mutations that are duplications

Answer 29

5%

Question 30

Can duplications be identified with MLPA?

Answer 30

Yes

Question 31

What determines whether a duplication will cause DMD or BMD?

Answer 31

If it is an in-frame mutation or not

Question 32

How does dystrophin link muscle fibres with the ECM?
1)
2)
3)

Answer 32

1) N-terminal links to F-actin
2) C-terminal links to dystrophin-associated protein complex in the sarcolemma
3) DAPC has an extracellular anchor, which links to the ECM

Question 33

What effect does the linkage of the cytoskeleton with the ECM have in muscles?
1)
2)
3)

Answer 33

1) Stabilises sarcolemma during muscle contraction and relaxation
2) Transmits force generated by contraction to the ECM
3) Linkage allows DAPC to be involved in cell signalling

Question 34

Effect of losing dystrophin
1)
2) a, b, c, d
3)
4)
5)

Answer 34

1) Loss of DAPC at the sarcolemma
2) Makes the sarcolemma very fragile, which leads to:
a) Ca2+ influx (disregulation)
b) Apoptosis, necrosis
c) Inflammation
d) Fibrosis

3) Disrupted muscle architecture
4) Signalling defects
5) Secondary loss of other proteins

Question 35

Effect of dystrophin mutations on cellular Ca2+
1)
2)
3)
4)

Answer 35

1) Increases amount of intracellular Ca2+
2) Increased Ca2+ influx through ca2+ stretch channels
3) This might lead to activation of the inflammatory response
4) This is observed in mdx mice

Question 36

How is DMD modelled?

Answer 36

In mdx mice

Question 37

When are elevated inflammatory mediators observed in muscle?

Answer 37

Prior to onset of DMD

Question 38

Proportion of DMD caused by large partial deletions

Answer 38

65%

Question 39

MLPA
1)
2)
3)
4)
5)
6)

Answer 39

1) Multiplex ligation-dependent probe amplification
2) A way to detect gene duplications or deletions
3) A PCR process
4) Use PCR primers for ‘hotspots’ where duplications or deletions often occur
5) Run results on a gel, can see here if a region is absent or overexpressed
6) Determines relative number of of all exons within a gene simultaneously

Question 40

Characteristics of DMD muscle biopsy
1)
2)
3)
4)
5)
6)
7)

Answer 40

1) Variable fibre size
2) Hypercontracted (opaque) muscle fibres)
3) Muscle fibre degeneration and regeneration
4) Normal or immature muscle fibre internal architecture
5) Absent dystrophin
6) Reduced sarcoglycans, aquaporins
7) Increased fibrosis within a muscle

Question 41

How can dystrophin be stained for?

Answer 41

Immunohistochemistry

Fluorescent antibody probe

Question 42

Early pathological findings of DMD muscle
1)
2)

Answer 42

1) Invasion of muscle by phagocytes

2) Necrotic fibres are pale with an NADH stain

Question 43

Late-stage pathological findings of DMD muscle
1)
2)
3)

Answer 43

1) Increased endomysial connective tissue
2) Variable fibre size
3) Hypercontracted muscle fibres

Question 44

When staining for dystrophin, how do histological slides appear?

Answer 44

Normal - dystrophin around edges of fibres
DMD - Dystrophin absent
BMD - Dystrophin reduced

Question 45

What does Western blotting tell us?
1)
2)

Answer 45

1) Quantifies amount of protein in a specific tissue

2) Determines the size of a protein