Lecture 31 - Molecular Basis of Muscular Dystrophies Flashcards Preview

Molecule to Malady > Lecture 31 - Molecular Basis of Muscular Dystrophies > Flashcards

Flashcards in Lecture 31 - Molecular Basis of Muscular Dystrophies Deck (45):
1

Function of dystrophin

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

2

Which chromosome is dystrophin on?

Xp21

3

Number of sarcoglycans

Four

4

Exons in an average gene

8-9

5

Size of average gene

~3000 bases

6

Size of average mRNA

~1000 bases

7

Proportion of X chromosome taken up by dystrophin

1%

8

Size of dystrophin gene

79 exons
2.4 megabases

9

Proportion of dystrophin gene that is exons

0.6%

10

Proportion of dystrophin mutations that are de novo

~1/3

11

How is dystrophin differently expressed in different tissues?

Different gene transcripts lead to different protein isoforms

12

What is BMD?

Becker muscular dystrophy
A milder phenocopy of DMD

13

What do mutations in dystrophin lead to?

Duchenne muscular dystrophy
Becker muscular dystrophy

14

How are different dystrophin isoforms formed?

Alternative promotor usage and splicing of mRNA

15

Isoforms of dystrophin
1)
2)

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

16

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

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

17

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

1) CNS
2) Retina
3) Kidney

18

Most common isoform of dystrophin found in skeletal and cardiac muscle

Long isoform
427kDa protein, 3685 amino acids

19

Feature of skeletal isoform dystrophin that is absent from other isoforms

Actin-binding domain

20

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

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

21

Dystrophin mutation that results in DMD

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

22

Dystrophin mutation that results in BMD

Shortening of spectrin-like rod domain.
Protein is still partially functional

23

Missense mutation

Changes amino acid encoded

24

Nonsense mutation

Stop codon

25

Proportion of DMD mutations that are nonsense

15%

26

Can nonsense mutations be picked up on MLPA?

Not normally

27

Proportion of DMD mutations that are frameshift

60%

28

Typical feature of BDM mutations

In-frame mutations

29

Proportion of DMD mutations that are duplications

5%

30

Can duplications be identified with MLPA?

Yes

31

What determines whether a duplication will cause DMD or BMD?

If it is an in-frame mutation or not

32

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

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

33

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

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

34

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

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

35

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

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

36

How is DMD modelled?

In mdx mice

37

When are elevated inflammatory mediators observed in muscle?

Prior to onset of DMD

38

Proportion of DMD caused by large partial deletions

65%

39

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

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

40

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

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

41

How can dystrophin be stained for?

Immunohistochemistry
Fluorescent antibody probe

42

Early pathological findings of DMD muscle
1)
2)

1) Invasion of muscle by phagocytes
2) Necrotic fibres are pale with an NADH stain

43

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

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

44

When staining for dystrophin, how do histological slides appear?

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

45

What does Western blotting tell us?
1)
2)

1) Quantifies amount of protein in a specific tissue
2) Determines the size of a protein