L8 Muscle development and disease

Question 1

Myostatin

Answer 1

released by muscle cells to inhibit further muscle overgrowth - same gene leads to same phenotype

Question 2

Muscle anatomy

Answer 2

see onenote diagram

muscle is arranged hierarchically

in fish, each fibre is attached at either end of the somite

Question 3

Key proteins - laminin and dystrophin

Answer 3

see onenote

anchor extracellular matrix to the structural proteins inside the cell to allow contractile force to develop

Laminin - extracellular link to dystroglycan and integrin complexes

Dystrophin - intracellular protein linking dystroglycan to actin

Question 4

Key proteins - actin and myosin

Answer 4

actin and myosin are key intracellular filaments that slide across each other to produce force e.g. during muscle contraction (shortening)

Question 5

Birefringence for muscle integrity

Answer 5

see onenote

Birefringence - optical property of material that reflect polarised light differently

because of its regular structure, muscle is highly birefringent and will “glow” under polarised light - can visualise muscle defects

The more organised you are, the more light you reflect

Question 6

Muscular dystrophies

Answer 6

• more than 30 types
• different proteins in different muscle groups affected by different mutations
• characterised by progressive degeneration of muscle
• severe MDs have early childhood onset and die in early 30s (lung/heart failure)

Question 7

Epistasis

Answer 7

see onenote

when one gene masks the phenotypic effect of another

dominant epistasis 12:3:1
recessive epistasis 9:3:4

duplicate dominant epistasis 15:1

duplicate recessive epistasis 9:7 = complementation

Question 8

dominant epistasis

Answer 8

12:3:1

As soon as there is one dominant allele “A”, it doesn’t matter what the other allele is, “B”

Question 9

Recessive epistasis

Answer 9

9:3:4

As soon as you are homozygous for the recessive allele, it doesn’t matter what “B” is

Question 10

Duplicate dominant

Answer 10

15:1

As soon as you have a “A” or “B” you get a particular phenotype and the other allele doesn’t matter

Question 11

Duplicate recessive

Answer 11

9:7

Group all the ones with homozygous recessive “a”, recessive “b”, or recessive both

Complementation, if one pathway isn’t working due to the duplicate recessive, you won’t get a particular phenotype

Question 12

Epistatic relationship of MD proteins

Answer 12

see onenote slides

when the phenotype does not add up independently

If they’re not epistasis, the phenotypic effect would be additive

Negative epistasis
- Phenotype worse than you expect

Positive epistasis
- Phenotype not as bad as you expect, may be equal to a mutant

Reciprocal sign epistasis
- Having a double mutant somehow reverse the phenotype

NEED TO BE ABLE TO PICK WHICH EPISTASIS IT IS FROM LOOKING AT THE BARS IN THE GRAPH

Question 13

Epistasis: zebrafish muscle mutants

Answer 13

dystroglycan/dystrophin double mutant is NO worse than the individual mutants as they act in the same complex = positive epistasis

Question 14

Merosin - congenital muscular dystrophy (CMD)

Answer 14

see onenote slides

candyfloss zebrafish mutants lack functional laminin 2 protein

mutations cause premature stops in highly conserved aa regions in globular domain of laminin alpha2

CMD is not a developmental defect but a fibre attachment defect that only manifests when force is produced e.g. muscle contraction

Question 15

Duchenne/Becker’s muscular dystrophy

Answer 15

see onenote

DMD - out of frame

BMD - in frame

different MDs resulting from different mutations in the SAME protein

• both due to mutation in dystrophin gene
• main different NOT mutation type but whether the result is out/in frame

severity in DMD results from loss of functional rest of the protein

milder BMD results from affecting a portion but restoring the next exon

skip the affected exon and restore reading frame to turn DMD into BMD - exon skipping

Question 16

Animal models of DMD

Answer 16

see onenote

there are different animal models with different mutations, degree to which they match human cases is very variable

Question 17

zebrafish models of DMD

Answer 17

see onenote slides

3 different zebrafish mutant DMD modesl

3 Single bp mutation resulting in a stop codon => premature stop codon

in vivo - can follow disease progression and test therapeutic approaches

Question 18

zebrafish models of DMD: sapje

Answer 18

see onenote slides

histology phenocopies human DMD

birefringence allows in vivo assessment of phenotype

Has premature stop codon in the middle

Question 19

exon skipping using splice MO

Answer 19

see onenote

block normal splice site using splicing MO but as cryptic splice sites cannot always be predicted, the MO can have different results

using PCR at known locations, length of resulting mRNA or sequence can be used to distinguish between these possibilities

Question 20

zebrafish models of DMD - exon skipping in sapje

Answer 20

see onenote slides

Used 2 morpholino

• 1 targeted at exon 32
• 1 targeting mutation site

use of both MO successfully caused skipping of exon 32 with no other alterations to the protein coding DNA

Question 21

Exon skipping trials in human DMD

Answer 21

see onenote

No established way of injecting morpholino yet