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BIOM30002 > lecture 32 > Flashcards

lecture 32 Flashcards

1
Q

What is DAPC?

A
  • dystrophin associated protein complex
  • also known as DAGC (glycoprotein)
  • anchored to ECM, w/i sarcolemma and dystrophin links to actin
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2
Q

What is a gene?

A
  • a segment of DNA (A, C, G and T) which carries information to make a protein or control expression of other genes
  • genes consist of information domains (exons) interrupted by non-coding sequence (introns)
  • genes are transcribed into mRNA, which is then translated to make a protein
  • the order of amino acids in the protein is determined by the nucleotide sequence of the gene
  • the order of amino acids determines the shape of the protein
  • shape of the protein determines its function
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3
Q

What is gene expression?

A
  • genomic DNA
  • pre mRNA
  • mature mRNA
  • different splicing
  • protein
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4
Q

What is the average gene?

A
  • consists of 8-9 exons
  • is spread across 3,000 bases
  • produces a processed gene message ~1000 letters (amino acids) long
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5
Q

What is the dystrophin gene?

A
  • localised to chromosome Xp21
  • second largest gene known, occupying 1% of the X-chromosome and 0.1% of the entire genome
    → 79 exons spanning 2.4 megabases, mRNA is over 14kb
    → exons account for only 0.6% of the gene, rest large intronic regions
    → large size makes it susceptible to mutations: 1/3 of all mutations de novo
  • differing transcripts occur in different tissues
  • mutations in dystrophin cause DMD and BMD
    → DMD: duchenne muscular dystrophy
    → BMD: becker muscular dystrophy (milder phenocopy of DMD)
    → also: X-linked cardiomyopathy, X-linked cramps-myalgia ssyndrome, isolated quadriceps myopathy
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6
Q

What is duchenne muscular dystrophy?

A
  • the most common human MD (1/3500 boys)
  • the dysrophin gene product is also known as dystrophin
  • the large DMD gene causes production of several isoforms
    → isoform = variant forms of the same protein
    → formed by alternative promoter usage and splicing of pre-mRNA
    → 4 long isoforms (l, m, c, p): skeletal, cardiac, smooth muscle, brain
    → smaller isoforms: central nervous system, retina, kidney
  • the predominant isoform found in skeletal and cardiac muscle is a 427 kDa protein predicted to contain 3685 amino acids
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7
Q

What are the isoforms of dystrophin?

A

Dp260, Dp140, Dp116, Dp71

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8
Q

What is utropin?

A
  • embryonic homologue

- looks similar to dystrophin

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9
Q

What are the domains of dystrophin?

A
  • full length dystrophin has four structural domains
    1. A (vital) N-terminal “actin binding” domain
    2. middle “rod” domain of spectrin like repeats
  • shorter forms with fewer repeats remain variably functional
    3. a cysteine-rich domain
    4. a carboxyl-terminal domain allowing assembly of the DAPC

3 and 4 found in all isoforms
2 of variable length amonst isoforms

actin binding most important for skeletal muscle
c-terminal attaches to sarcolemma/DAPC
rod domain maybe not as important

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10
Q

What is the reading frame of dystrophin?

A
  • certain exons are in-frame and others have codons spanning the exon:exon junctions
  • shifting the reading frame → DMD
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11
Q

What is the genetics of DMD?

A

in coding regions of genes there are three types of base substitutions
→ silent: i.e. no change in protein product
→ missense: amino acid change in protein produce
→ nonsense: causes a premature stop in protein production

other sorts of mutations in DMD:
- deletions/insertions
→ frameshift: base added or lost from amino acid sequence
→ code out of frame (shifted) downstream
- duplications

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12
Q

Wat are nonsense mutations?

A
  • premature stop signals
  • normal: iiiTHEiiiBADANDiiiOLDDiiiOGATETiiiHEFATiiiiCATENDiii
  • mutation changing A to E
    iiiTHEiiiBADEND ….

the bad end
corruption of message, loss of gene product
- 15% of all gene mutations in DMD
- usually not picked up on MLPA

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13
Q

What are frameshift mutations?

A
  • example: deletion of exon 3 (old D)
  • THE BAD AND OGA TET HEF ATC ATE NDi iii
  • corruption of genetic message, loss of product
    • occurs in ~60% of DMD cases and is out of frame
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14
Q

What are in-frame deletions?

A
  • deletion doesn’t change overall gist of message
  • dystrophin still produced but shorter than usual (= truncated protein)
  • typical of BMD mutations
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15
Q

What are duplications?

A
  • responsible for about 5% of all DMD cases
  • duplications shifting the reading frame cause DMD
  • duplications preserving the reading frame cause BMD
  • 2nd most common DMD mutation identified
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16
Q

How does dystrophin act as a cellular anchor?

A

dystrophin links the internal cytoskeleton to the ECM
- the N (amino)-terminus of dystrophin binds to F-actin and the carboxyl (C) terminus to the DAPC at the sarcolemma
- the DAPC links the actin cytoskeleton to the ECM
→ this stabilises the sarcolemma during cycles of contraction and relaxation
→ this also transmits force generated in the muscle sarcomeres to the ECM
- the DAPC is also involved in cell signalling

17
Q

What happens when you lose dystrophin?

A
  • causes loss of the DAPC at the sarcolemma
  • loss of this physical link renders the sarcolemma fragile
  • muscle fibres become susceptible to injury and degeneration during repeated cycles of muscle contraction
18
Q

What is the role of dystrophin in calcium homeostasis?

A
  • dystrophin has also been proposed to play a role in calcium homeostasis
  • mdx mice are the animal model for DMD
  • in mdx mice and DMD patients resting intracellular calcium levels are elevated in muscle
  • mdx muscles show enhanced calcium influx through calcium/stretch-activated channels, causing activation of the inflammatory response
  • elevated expression of inflammatory mediators and chemoattractants is seen in dystrophin-deficient muscles prior to the onset of weakness
  • dystrophic changes in muscle may related to inflammation due to aberrant Ca++ homeostasis
19
Q

What is the consequence of dystrophin mutations?

A 
- absence of structural proteins in muscle → 
membrane instability → 
-- calcium influx 
-- apoptosis, necrosis 
-- inflammation 
-- fibrosis 
- disrupted muscle architecture 
- signalling defects secondary loss of other proteins 
- weakness
20
Q

How do dystrophin mutations cause DMD and BMD?

A
  • mutations disrupting the reading frame (=frame-shift) of dystrophin cause loss of dystrophin and cause DMD
  • in BMD mutations maintain the reading frame → abnormal but partly functional dystrophin
  • 65% of DMD is caused by large partial deletions
  • 5% is caused by duplications of one or more exons
  • deletions and duplications : detected by multiplex ligation-dependent probe amplification (MLPA) analysis (PCR)
  • other patients have small duplications/deletions or point mutations
  • not detected by MLPA, may be found by other methods
21
Q

What is DNA diagnosis?

A
  • polymerase chain reaction
  • produces enormous numbers of copies of a specified DNA sequence
  • multiplex PCR of the dystrophin gene:
    • primer sets of selected exons
    • gel electrophoresis
22
Q

What is MLPA?

A
  • multiplex ligation-dependent probe amplification in DMD
  • determines the relative copy number of all exons within gene simultaneously
  • deletions or duplications
23
Q

What is a normal biopsy?

A
  • regularly sized fibres
  • pink
  • uniform
  • nuclei on the periphery
24
Q

What is a biopsy in DMD?

A
  • lose uniform architecture
  • fibrosis
  • inflammatory cells
  • different colours
  • variable fibre size
  • hypercontracted (opaque) muscle fibres
  • muscle fibre degeneration and regeneration
  • muscle fibre internal architecture: normal or immature
  • absent dystrophin in DMD (immunohistochemistry)
  • other membrane proteins:
    • sarcoglycans: reduced
    • aquaporing 4: reduced
  • increased fibrosis within muscle
  • invasion of fibres by macrophages
  • necrotic fibres are pale on NADH stain
25
Q

What is western blotting?

A
  • looking at protein on a gel
  • quantifies the amount of protein in specific tissue
  • detemrines size of protein

BIOM30002 (36 decks)

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