DMD

Question 1

What are dystrinopathies

Answer 1

a group of genetic hereditary disorders.

Question 2

How many types of dystrionpathies

Answer 2

Over 40 types

Question 3

How do they have progressive course?

Answer 3

They all have a progressive course, by which I mean the severity gets worse as an affected individual ages.

Question 4

What are dystrionpathies based on?

Answer 4

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.

Question 5

Other than muscle, what else are affected? what type of disorder does it mean that it is?

Answer 5

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/

Question 6

Why do dystrinopathies have such severe symptoms?

Answer 6

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.

Question 7

Name the types of MD

Answer 7

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

Question 8

DMD gene related dytropinopathies
How often does it occur?
What does it encode for adn where is the gene located?
How is it inherited?

Answer 8

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.

Question 9

WHat does mutation of DMD cause?

Answer 9

Mutation of DMD causes group of conditions that cause muscle weakness

Question 10

Muscle disease ranging from mild to severe, includes:

Answer 10

Duchenne muscular dystrophy (DMD)
Becker muscular dystrophy (BMD)

Question 11

How was DMD discovered to cause Duchenne muscular dystrophy?

Answer 11

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.

Question 12

Describe the DMD gene

Answer 12

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

Question 13

Describe dystrophin

Answer 13

In skeletal and cardiac muscle fibre cells

Rod shaped cytoplasmic protein

Part of Dystroglycan complex

Strengthen muscle fibres and protect them from injury

Question 14

What happens if there’s a defect in dystrophin

Answer 14

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

Question 15

Duchenne Muscular Dystrophy (DMD) - Males

Answer 15

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

Question 16

Becker Muscular Dystrophy (BMD) - Males

Answer 16

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

Question 17

X-linked recessive inheritance

Answer 17

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.

Question 18

Describe female carriers

Answer 18

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

Question 19

Three types of DMD gene variants

Answer 19

2/3 pathogenic variants are de novo
Two hotspots for variants
- Exons 3-8
- Exons 44-50
~70% have deletion of >1 exon

Question 20

Distribution of variants in DMD protein

Answer 20

~ 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

Question 21

DMD gene variants Becker-MD

Answer 21

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

Question 22

DMD gene variants Duchenne-MD

Answer 22

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

Question 23

Genetic testing for gene variants

Answer 23

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

Question 24

If looking for a known familial pathogenic variant, what genetic techqniue is used for one variant?

Answer 24

IF we are looking for known familial pathogenic variants, then sanger sequencing is an option for that one variant.

Question 25

What must be considered for diagnostic testing? Therefore what must we use?

Answer 25

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.

Question 26

Whats the quickest strategy to identify pathogenic variant in DMD?

Answer 26

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.

Question 27

Multiplex Ligation-dependent Probe Amplification MLPA

Answer 27

Gene dosage analysis
Or copy number variation (CNV)
Measures quantity of a single gene
Deletions
Duplications
Exon by exon

Question 28

MLPA Probes

Answer 28

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

Question 29

4 steps of MLPA
What does MLPA probe hybrisise to

Answer 29

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

Question 30

What is MLPA?

Answer 30

Multiplex ligation dependent probe amplification, MLPA, is a PCR based method for detecting exonic deletions and duplications in a single gene

Question 31

What can microarray analysis detect?
After microarray analysis what would it be necessary to do?

Answer 31

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.

Question 32

Whats the quickest strategy to identify a pathogenic variant in DMD?

Answer 32

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.

Question 33

How is MLPA analysied?

Answer 33

Analysed via measuring relative quantity of the DNA fragments versus control

Each probe related to an exon (look at slide)

Question 34

DMD treatment

Answer 34

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

Question 35

New therapies

Answer 35

Exon skipping
Induced pluripotent stem cells

Question 36

Antisense mediated exon skipping

Answer 36

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

Question 37

Exon Skipping e.g. Eteplirsen

Answer 37

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

Question 38

Exon Skipping e.g. Eteplirsen, Stop variant in exon 51

Answer 38

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

Question 39

Induced pluripotent stem cells & gene editing

Answer 39

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

Question 40

IPSC and gene editing in DMD patient

Answer 40

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