What is DAPC?
- dystrophin associated protein complex
- also known as DAGC (glycoprotein)
- anchored to ECM, w/i sarcolemma and dystrophin links to actin
What is a gene?
- 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
What is gene expression?
- genomic DNA
- pre mRNA
- mature mRNA
- different splicing
- protein
What is the average gene?
- consists of 8-9 exons
- is spread across 3,000 bases
- produces a processed gene message ~1000 letters (amino acids) long
What is the dystrophin gene?
- 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
What is duchenne muscular dystrophy?
- 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
What are the isoforms of dystrophin?
Dp260, Dp140, Dp116, Dp71
What is utropin?
- embryonic homologue
- looks similar to dystrophin
What are the domains of dystrophin?
- 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
What is the reading frame of dystrophin?
- certain exons are in-frame and others have codons spanning the exon:exon junctions
- shifting the reading frame → DMD
What is the genetics of DMD?
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
Wat are nonsense mutations?
- 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
What are frameshift mutations?
- 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
What are in-frame deletions?
- deletion doesn’t change overall gist of message
- dystrophin still produced but shorter than usual (= truncated protein)
- typical of BMD mutations
What are duplications?
- 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
How does dystrophin act as a cellular anchor?
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
What happens when you lose dystrophin?
- 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
What is the role of dystrophin in calcium homeostasis?
- 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
What is the consequence of dystrophin mutations?
- 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
How do dystrophin mutations cause DMD and BMD?
- 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
What is DNA diagnosis?
- 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
What is MLPA?
- multiplex ligation-dependent probe amplification in DMD
- determines the relative copy number of all exons within gene simultaneously
- deletions or duplications
What is a normal biopsy?
- regularly sized fibres
- pink
- uniform
- nuclei on the periphery
What is a biopsy in DMD?
- 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
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