Dmd Flashcards
Incidence of dmd and BMD
1: 3500 DMD
1: 18000 BMD
Types of DMD mutations and BMD
60-65% out of frame deletions 1+exons
5% exon duplication
Rest nonsense or frameshift
BMD = in frame mutations (85%deletions)
Dmd onset and symptoms?
Before 5 years Progressive muscular weakness-proximal Muscular pseudohyoertrophy (chunky calves) Dilated cardiomyopathy Mean age of death 25
Symptoms BMD
Late learning to walk
Muscle weakness around 11
Loose ability t walk 40-50 yrs
No LD
Symptoms female carriers
5-10% cramps Usually in 30s 20% dilated cardiomyopathy -5yr echo/ecg Proximal weakness and asymmetric Carriers of BMD less affected
Management DMd?
Physio and splints to prevent contractures
Scoliosis - surgery
Cardiomyopathy- echo and ecg every 2 years until 10 - annually after
Management BMD
May require walking frames/ wheelchairs
Echo/ECG every 5 years
What percentage of mothers of affected are carriers?
60% carriers
But 10-20% gonadal mosaicism
Risk to future affected sons if mum NOT carrier?
10% affected son
10% risk carrier daughter
Based on results, pedigree and bayes
PND PGD
Mutation rate in dystrophin gene ?
~ 1/3 de novo
3 options for de novo mutations?
1) occur in egg at probands conception
2) after conception - mosaic
3) mutation present in mothers egg cells
Situation where female has classic dmd?
1) an x-autosome translocation alters normal x inactivation
2) Turner syndrome
3) uniparental disomy (2 from ma)
4) skewed x-inactivation
5) father has BMD and mother a carrier
Location of DYstophin ?
Xp21.2
What is the DAPC and what does it do?
Dystrophin associated protein complex
- Forms links between actin cytoskeleton and the extra cellular matrix
- stabilises sarcolema during repeated rounds of contraction and relaxation (nb. Muscle integrity)
Dystrophin domains?
FOUR
1) amino terminal (binds actin filaments)
2) rod like domain- 24 spectrin-like triple helical coiled coils (much dispensable)
3) cysteine rich domain
4) carboxy terminal - interacts with membrane proteins sarcoglycan and day
Dystrophin levels is DMD and BMD?
Dmd- virtually absent
BMD- 10-40%
Pathogenesis of lack of dystrophin?
- disruption of link between cytoskeleton actin and extracellular matrix
- cell membrane is fragile and can be mechanically damaged
- deficiency disrupts subsarcolenmal mitochondria localisation, promotes inefficiency, restricts atp generating capacity
What other DAPC members are implicated in muscle wasting disorders?
Laminin alpha2 causes MDC1A
Meridian deficient congenital muscular dystrophy
Size of dystrophin?
2.4 Mb - ink 0.3% of genomic sequence present in mature transcript
79 exons
14 kb mRNA
How many promoters and how many tissue specific?
7 different promoters
3 tissue specific - brain, muscle purkinji(large to small) dif exon 1
Hotspots for deletions?
Proximal - exons 2-20
Distal - exons 45-55
Frameshift hypothesis?
Deletions that disrupt the translational reading frame = more severe
Deletions leaving frame intact =milder BMD
Correct for 92% of cases
Exceptions to frameshift hypothesis?
- Deletions in protein binding domain are severe even if in frame
- deletion may affect splicing
- deletions affecting central rod domain are milder
What percentage of dmd/BMD are caused by duplications
5-10%
Difficult for frameshift mutation as orientation and position usually not known
Percentage point mutations
Dmd 25-35% (usually premature termination codons)
BMD 10-20% ( Missense, splice site, nonsense at 3’ end that avoids nonsense mediated decay).
What mutations exclusively cause DMD related cardiomyopathy? (DCM)
Mutations that disrupt a muscle specific isoform
Eg. Muscle specific promotor - compensated for in other
CK levels for DMD, BMD, DCM, female carriers DMD, female carriers BMD
DMD- 100% >10x normal Ck BMD- 100% 5x Dcm- most have increased CI Carrier dmd -50% 2-10x Carrier BMD- 30% 2-10x
3 non genetic methods to test for DMD
1) cK levels
2) muscle biopsy
3) immunohistochemisty
What to expect of dmd muscle biopsy
1) increase in variation of fibre size diameter
2) increase in fibrous connective tissue
3) large rounded hyaline fibres
4) foci of degeneration and regeneration
Immunohistochemisty results with anti-dystrophin antibody
Dmd patients show absence of staining
BMD show reduction in staining
Steps of MLpA
1) Denaturation
2) hybridisation of adjacent probes
3) ligation of adjacent probes
4) pcr of all lighted probes
What are the MLPA kits and what is sensitivity?
P034 exons 1-10 21-30 41-50 61-70
P035 exons 11-20+ promoter, 31-40, 51-60, 71-79
Sensitivity 72%
Benefit of array for dmd?
Loss or gain of sequence at intronic breakpoints
What percentage of point mutations not detected by Sanger?
2% due to complex rearrangements
Alternative to PB seq?
Sequence cDNA from muscle - detect rna mutations - try confirm with DNA but not always possible (i.e. Intronic)
Risk of recombination across gene if using micro satellite markers?
10%
Normal MLPA?
Only excludes 72%
If clinical diagnosis- can offer seq
Pos MLPA?
- If single exon- confirm by second method
- Check whether in frame or out using Leiden muscular dystrophy site
- if first or last exon deleted- could extend into neighbouring gene
If familial mutation is unknown?
MLPA of woman in fam with highest risk. If not identified- linkage
If mutation not found in mum?
If fam mutation known- mum gonadal?
If fam not known- carrier risk reduced- bayes
Risk of carrier if mum or grandmother of affected (bayes)
2/3 for mum
1/3 for gran
Recombination frequencies calculation?
If none seen- work out ave risk of double recombination between markers and multiplying by % of coding region between markers
Antisense oligonucleotides
Alter splicing of dmd precursor mRNA by targeting exons affected by deletion/mutation
- cause skipping of targeted exon from mature mRNA = convert dmd to BMD
What antisense rna molecule targeted in trial?
Exon 51 skipping in clinical trial (13% of dmd)
Prob with antisense rna?
Only transient
Muscle tissue must be present - can’t reverse muscle loss
How do read through drugs work?
Induce protein making machinery to read through premature stop codons
2 types of read through drugs?
Aminoglycoside antibiotics (gentamicin) - can’t be administered orally
Ataburen (ptc124) non amnioglycoside
- orally and less toxic (phase 2)
