DMD related disorders Flashcards
What does MLPA stand for?
Multiplex Ligation Dependent Probe Amplification
What is MLPA? (4)
- Method for detecting copy number variation (CNV) (deletions/duplications) within genes
- More cost and time effective than using NGS for CNV detection
- Microarrays can do CNVs but the location of array probes is unreliable, no specific probes for exons so might miss CNVs
- Best test for conditions where the causative pathogenic variants are exon deletion or duplication
Why is MLPA used for DMD diagnosis?
80% of DMD variants are exon deletions and duplications causing Duchenne or Beckers muscular dystrophy
What are the steps in MLPA? (6)
- Denaturation of patient DNA
- Hybridisation of MLPA probes
- Ligation
- Amplification
- Fragment separation
- Analysis
What is the structure of MLPA probes? (5)
- One probe is made of 2 oligonucleotide halves, each with a hybridisation sequence that is specific to a particular gene location e.g. exon
- The target sites of each half are immediately adjacent
- Right half has a unique size stuffer sequence so can be separated later by electrophoresis
- Each probe has different sized stuffer
- Probes contain universal PCR primer sites
What indicates a heterozygous deletion?
When the fluorescence peak for the patient is half of the control
What indicates a heterozygous duplication?
When the fluorescence peak for the patient is double of the control
What are muscular dystrophinopathies? (5)
- A group of X-linked muscle disorders
- Over 40 known disorders
- Progressive nature
- Based on degeneration and death of muscle fibres which aren’t renewed ^ progressive
- Multi-system disorders (cardiomyopathy and intellectual disabilties)
What are DMD related muscular dystrophies? (3)
- Duchenne and Becker muscular dystrophy
- X-linked inheritance
- DMD gene
Why do dystrophinopathy have such severe symptoms? (3)
- Muscle is the most abundant body tissue
- Accounts for 23% of female weight and 40% of male weight
- Muscle has functions in all areas of the body
What are the main types of muscular dystrophinopathies? (7)
- Duchenne MD (most common)
- 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, feet)
- Oculopharyngeal MD (upper eyelids and throat)
What is the incidence of DMD gene related dystrophinopathy?
1 in 3500 male infants
What does the DMD gene code for?
Dystrophin
Where is the DMD gene located?
X chromosome at p21.2
What is the inheritance of DMD related dystrophinopathy?
X-linked recessive manner
What are the characteristics of DMD related dystrophinopathy? (2)
- Muscle weakness
- Ranging from mild to severe
What are the features of the DMD gene? (6)
- Largest known human gene
- Located at Xp21
- Takes 16 hours to transcribe
- Makes up 0.08% of the genome
- 79 exons, rest is massive non coding introns
- Encodes 3685 amino acid dystrophin protein
What is the function of dystrophin? (4)
- In skeletal and cardiac muscle fibres
- Rod shaped cytoplasmic protein
- Vital part of the dystroglycan complex which connects the actin filaments in a muscle fibre to the extracellular matrix
- Functions to strengthen muscle fibres and protect them from injury when contracting and relaxing
What is caused by a defect in dystrophin? (2)
- Absence/reduced dystrophin, skeletal and cardiac muscle cells gradually become damaged with use so weaken and die over time
- Causes characteristic muscle weakness and heart problems seen in DMD and BMD
What are the features of Duchenne muscular dystrophy (DMD) in males? (9)
- Severe
- Most common dystrophinopathy
- Age of onset 3-5 years old, wheelchair bound by 12
- Delayed walking
- Muscle weakness particularly of lower limbs
- Pseudohypertrophy (enlarged calves)
- Cardiomyopathy by 14 years old
- Breathing problems
- Average lifespan less than 30 years
What are the features of Becker muscular dystrophy (BMD) in males? (5)
- Milder
- Later onset skeletal muscle weakness
- Don’t need a wheelchair until much later
- Cardiomyopathy in teens
- Longer lifespan than Duchenne
What is the chance that a heterozygous female carrier will pass on the pathogenic DMD gene in each pregnancy?
50%
What symptoms do female carriers of pathogenic DMD variant have? (3)
- 76% of Duchenne and 81% of Beckers female carriers have no symptoms
-May have some clinical manifestations but mild (mild muscle weakness or heart problems for Duchenne carriers) - Features may vary dependent on X-inactivation favouring WT or variant allele
What would happen to offspring if a male with DMD did reproduce (doesn’t usually happen)? (2)
- All daughters would be carriers (one X from mother, one X from father)
- All sons would be unaffected (X from mother, Y from father)
What are the variants associated with the DMD gene? (6)
- Most DMD mutations are large deletions of more than one exon in over 70% cases (DMD and BMD mostly caused by large exonic deletions)
- Two hotspots: exons 3-8 and exons 44-50
- Point mutations (20%, half of which are nonsense) and duplications are the minority
- The remaining mutations are a mix of deletions/insertions causing frameshifts and splice site changes
- 2/3 of mutations are de novo
- Missense mutations very rarely pathogenic
What is the difference between DMD variants in Beckers and Duchennes? (6)
- Beckers do not alter the reading frame
- Inframe deletions or duplications maybe including a number of exons
- Some dystrophin is produced which may be shorter than normal dystrophin but has partial function = milder than Duchennes
- Duchennes alter the reading frame, resulting in premature truncation of dystrophin
- The faulty dystrophins are subject to NMD resulting in absence of dystrophin expression = more severe
- Can be splice variants, out of frame deletions causing frameshifts, nonsense and large multi exon deletions
How do you go about genetic testing for DMD disorders? (5)
- Patient blood sample
- Sanger sequencing is an option if looking for a known familial pathogenic variant
- Majority of pathogenic variants in DMD gene are large deletions so use MLPA (detecting deletions/duplications in a single gene)
- Microarray finding would need to be confirmed via another method to decide which DMD exons are involved
- MLPA will identify exonic deletions and duplications for 70% of cases, if not NGS analysis to identify point mutations
What does it mean if an MLPA screening shows 0 for a region of the DMD gene? (2)
- Homozygous deletion if female
- Hemizygous deletion if male
How are deletion variants classified in DMD? (4)
- Exon/whole gene deletions generally considered pathogenic
- DMD likely diagnosis where reading frame is disrupted
- BMD likely diagnosis where reading frame is maintained
- Literature for similar deletion variants is helpful
How are duplication variants classified in DMD? (3)
- Likely to be pathogenic
- Most technologies like MLPA don’t indicate where a duplication is located
- May not disrupt the gene
What is the treatment for DMD? (6)
- No cure
- Corticosteroids to maintain muscle function and slow weakening (side effects: weight gain, decreased bone mineralisation, behavioural disturbances)
- Physical therapy
- Assisted ventilation
- Surgery
- Heart transplant
What new therapies are being developed for DMD? (2)
- Exon skipping technologies
- Induced pluripotent stem cells
What is antisense mediated exon skipping? (4)
- Antisense oligonucleotides (AONs) = small pieces of DNA or RNA that hybridise to target exon during pre-mRNA splicing
- Hides the target exon from splicing machinery so it is spliced out with its flanking introns
- May make the deletion larger but restores the reading frame
- Allows production of a partially functional dystrophin like in BMD
What is an example of exon skipping therapy? (5)
- Eteplirsen
- Has homologous sequence to exon 51, hybridises, causes 51 to be spliced out
- Restores the reading frame
- Causes production of shortened functional dystrophin and relieves symptoms
- 14% of DMD patients have exon 51 premature truncation variants
How are induced pluripotent stem cells used for DMD? (3)
- Derived from a mature cell, reprogrammed to embryonic stem cell like state
- Can differentiate into any human cell type
- Use patients own cells, gene edit the pathogenic variant and transplant back healthy DMD-containing cells
