DMD Flashcards
What are dystrinopathies
a group of genetic hereditary disorders.
How many types of dystrionpathies
Over 40 types
How do they have progressive course?
They all have a progressive course, by which I mean the severity gets worse as an affected individual ages.
What are dystrionpathies based on?
They are based on the degeneration and death of muscle fibres, which are not renewed, hence why this is a progressive disorder, with age more and more muscle is lost.
Other than muscle, what else are affected? what type of disorder does it mean that it is?
They are multi system disorders, on that its not just muscle that is affects, they also suffer from intellectual disability and heart problems, or cardiomyopathy/
Why do dystrinopathies have such severe symptoms?
The reason is simply that muscle is the most abundant body tissue.
It accounts on average for 23% of female weight and 40% of male weight.
Name the types of MD
Duchenne MD
Becker MD
Emery-Dreifuss MD – joints and heart
Myotonic dystrophy – adult onset muscle wasting
Limb Girdle MD – legs and arms
Distal MD - lower arms, hands, lower legs and feet.
Oculopharyngeal MD - upper eyelids and throat
DMD gene related dytropinopathies
How often does it occur?
What does it encode for adn where is the gene located?
How is it inherited?
DMD gene related dystrinopathy occurs in 1 in 3500 male infants.
The DMD gene encodes the dystrophin gene, located on chromosome X at p21.2
The DMD gene is inherited in an X-linked recessive manner.
WHat does mutation of DMD cause?
Mutation of DMD causes group of conditions that cause muscle weakness
Muscle disease ranging from mild to severe, includes:
Duchenne muscular dystrophy (DMD)
Becker muscular dystrophy (BMD)
How was DMD discovered to cause Duchenne muscular dystrophy?
In the early 80s, cytogeneticists were reporting numerous patients with muscular dystrophy having apparently balanced translocations involving an autosome and the X chromosome,
There were various autosome chromosomes involved, but the breakpoint on the X chromosomes was always Xp21.
This led to the hypothesis that there must be a gene that is disrupted by the translocations which then causes the muscular dystrophy phenotype.
The DMD gene was then cloned and the dystrophin protein identified.
Describe the DMD gene
Largest known human gene
Covering 2.3megabases(0.08% of the human genome)
Chromosome Xp21
Takes 16 hours to transcribe
Mature mRNAmeasures 14 kilobases
79-exons
encodes 3685 amino acid residues
The 79 exons only account for 0.6% of the gene, rest is large non-coding introns
Describe dystrophin
In skeletal and cardiac muscle fibre cells
Rod shaped cytoplasmic protein
Part of Dystroglycan complex
Strengthen muscle fibres and protect them from injury
What happens if there’s a defect in dystrophin
Skeletal & cardiac muscle cells with absence of or reduced expression of functionaldystrophin
Become damaged as the muscles repeatedly contract & relax with use
The damaged cells weaken & die over time
Causing characteristic muscle weakness & heart problems seen in DMD and BMD
Duchenne Muscular Dystrophy (DMD) - Males
Most common dystrinopathy
Age of onset 3 to 5 years old
Delayed walking
Muscle weakness, lower limbs
Psuedohypertophy- enlarged calves
Cardiomyopathy – heart disease (by 14 years)
Breathing problems - caused by deformed bones and muscle weakness
Wheel chair bound by 12
Becker Muscular Dystrophy (BMD) - Males
Milder
Later-onset skeletal muscle weakness
Wheelchair dependency (after age 16 years); although some remain ambulatory (capable of walking) into their 30s – key difference from DMD
Cardiomyopathy (diagnosed at ~14 years)
Life span to mid 40s
X-linked recessive inheritance
The DMD gene is transmitted in an X-linked recessive manner.
So a carrier Heterozygous female has a 50% chance of transmitting the DMD gene with a pathogenic variant in each pregnancy.
Sons who inherit the pathogenic variant will be affected wit Duchenne or Beckers;
Daughters who inherit the pathogenic variant are heterozygous and may have a range of clinical manifestations, though mild/
Its worth considering that although Males with DMD usually do not reproduce, if they did all their daughters would be obligate carriers and the sons of course would be unaffected as they would not inherit the X chromosome with the mutated DMD gene.
Describe female carriers
i.e one mutated DMD allele, one wild type DMD allele
~76% of DMD and 81% of BMD have NO symptoms
Mild to moderate muscle weakness (20% of DMD, 15% of BMD)
Cardiomyopathy (8% in DMD, none in BMD)
Features vary dependent on X-inactivation
X-inactivation may be skewed favouring either wild type or variant allele
Three types of DMD gene variants
2/3 pathogenic variants are de novo
Two hotspots for variants
- Exons 3-8
- Exons 44-50
~70% have deletion of >1 exon
Distribution of variants in DMD protein
~ 20% have small variants within exons.
Half of these (~10% of all patients) have nonsense mutations
~35% are small deletions or insertions that disrupt the reading frame (7% of all patients)
Remaining 15% (3% of all patients) have splice variants
DMD gene variants Becker-MD
Variants that do not alter reading frame = BMD
E.g. Inframe deletion or duplication, may include a number of exons
BMD phenotype occurs when some dystrophin is produced
But shorter-than normal dystrophin protein, which retains partial function
DMD gene variants Duchenne-MD
Those that alter reading frame / premature truncation = DMD
Variants that lead to absence of dystrophin expression
E.g. Splice variants, out of frame deletions, frameshifts, nonsense, large deletions
They produce a severely truncated dystrophin protein molecule that is degraded
Leading to the more severe DMD phenotype
Genetic testing for gene variants
Blood sample, DNA
Sanger sequence – familial point mutations
Multiplex ligation dependent probe amplification – exon deletions / duplications
Large variant’s can be detected via microarray
Confirm these via MLPA
Next Generation Sequencing ( very expensive) can detect deletions, duplications & point mutations - also copy number changes
If looking for a known familial pathogenic variant, what genetic techqniue is used for one variant?
IF we are looking for known familial pathogenic variants, then sanger sequencing is an option for that one variant.
What must be considered for diagnostic testing? Therefore what must we use?
For diagnostic testing we must consider that the majority of pathogenic variants in the DMD gene are large deletions.
Therefore we must use a mechanism that will detect these exonic copy number changes.
Whats the quickest strategy to identify pathogenic variant in DMD?
First use MLPA to exclude the exonic deletions and duplications, which will detect over 70% of positive cases, followed by NGS analysis of any of the negative cases to identify any point mutations.
Multiplex Ligation-dependent Probe Amplification MLPA
Gene dosage analysis
Or copy number variation (CNV)
Measures quantity of a single gene
Deletions
Duplications
Exon by exon
MLPA Probes
Consists of 2 halves
Each half has a gene specific sequence
Specific for an exon of a gene
unique size stuffer sequence – each probe has different sized stuffer – allows resulting fragments to be separated by size.
Universal primer for PCR
4 steps of MLPA
What does MLPA probe hybrisise to
Hybridsation: Probe hybridises to homologous exon sequences in gene
Note: Probe halves are not attached – PCR would not complete
Ligation: DNA probes ligated together with DNA ligase. PCR can proceed.
PCR with universal primers X and Y: exponential amplification of ligated probes only - PCR products
Fragment analysis: fragments separated by size using capillary electrophoresis
What is MLPA?
Multiplex ligation dependent probe amplification, MLPA, is a PCR based method for detecting exonic deletions and duplications in a single gene
What can microarray analysis detect?
After microarray analysis what would it be necessary to do?
Microarray analysis can also detect large copy number changes, particularly for the DMD gene as this is of course the largest human gene
it would be necessary to confirm it via another method such as MLPA, to be able to definitively decide which DMD exons were involved.
Whats the quickest strategy to identify a pathogenic variant in DMD?
To conclude, the quickest strategy to identify pathogenic variant in the DMD is to first use MLPA to exclude the exonic deletions and duplications, which will detect over 70% of positive cases, followed by NGS analysis of any of the negative cases to identify any point mutations.
How is MLPA analysied?
Analysed via measuring relative quantity of the DNA fragments versus control
Each probe related to an exon (look at slide)
DMD treatment
No cure
Steroids (Corticosteroids) to maintain muscle function & slow muscle weakening
- But has side effects: weight gain, decreased bone mineralization, & behavioural disturbances
Physical therapy - maintain muscle strength
Assisted ventilation
Surgeries
Cardiac transplantation
New therapies
Exon skipping
Induced pluripotent stem cells
Antisense mediated exon skipping
Antisense oligonucleotides (AONs)
Small modified pieces of DNA or RNA that specifically hybridize to a target exon
Hybridize to a target exon during the pre-mRNA splicing process
Hide the target exon from the splicing machinery,
Will be spliced out with its flanking introns
May make deletion larger
But, restores reading frame
Allows production of a partially functional dystrophin like those found in BMD
Exon Skipping e.g. Eteplirsen
Drug, Eteplirsen – contains AON
Promotes dystrophin production by restoring the translational reading frame of DMD through specific skipping of exon 51
14% of DMD patients have exon 51 premature truncating variants
Only good for 14% of patients
Exon Skipping e.g. Eteplirsen, Stop variant in exon 51
Eteplirsen has homologous sequence to exon 51
Hybridizes to exon 51
Causes it to be skipped during splicing
Corrects the translational reading frame
Leads to production of shortened functional dystrophin proteins
Relieves DMD symptoms
Induced pluripotent stem cells & gene editing
Induced pluripotent stem cells (iPSC) – derived from a mature cell
Reprogrammed to embryonic stem cell like state
Can differentiate to any human cell type
Disease modelling
Therapy – use patients own cells, gene edit pathogenic variant, transplant back
Huge potential e.g. Parkinsons
IPSC and gene editing in DMD patient
Generate iPSC from e.g. fibroblast
Gene edit DMD pathogenic variant - CRISPR/Cas9 or zinc finger nucleases
Differentiate iPSCs to myogenic progenitors
Transplant back into patient
Now has self derived cells producing normal DMD
