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L3 Clinical Genomics > DMD related disorders > Flashcards

DMD related disorders Flashcards

1
Q

What does MLPA stand for?

A

Multiplex Ligation Dependent Probe Amplification

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2
Q

What is MLPA? (4)

A
  • 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
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3
Q

Why is MLPA used for DMD diagnosis?

A

80% of DMD variants are exon deletions and duplications causing Duchenne or Beckers muscular dystrophy

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4
Q

What are the steps in MLPA? (6)

A
  • Denaturation of patient DNA
  • Hybridisation of MLPA probes
  • Ligation
  • Amplification
  • Fragment separation
  • Analysis
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5
Q

What is the structure of MLPA probes? (5)

A
  • 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
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6
Q

What indicates a heterozygous deletion?

A

When the fluorescence peak for the patient is half of the control

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7
Q

What indicates a heterozygous duplication?

A

When the fluorescence peak for the patient is double of the control

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8
Q

What are muscular dystrophinopathies? (5)

A
  • 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)
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9
Q

What are DMD related muscular dystrophies? (3)

A
  • Duchenne and Becker muscular dystrophy
  • X-linked inheritance
  • DMD gene
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10
Q

Why do dystrophinopathy have such severe symptoms? (3)

A
  • 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
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11
Q

What are the main types of muscular dystrophinopathies? (7)

A
  • 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)
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12
Q

What is the incidence of DMD gene related dystrophinopathy?

A

1 in 3500 male infants

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13
Q

What does the DMD gene code for?

A

Dystrophin

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14
Q

Where is the DMD gene located?

A

X chromosome at p21.2

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15
Q

What is the inheritance of DMD related dystrophinopathy?

A

X-linked recessive manner

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16
Q

What are the characteristics of DMD related dystrophinopathy? (2)

A
  • Muscle weakness
  • Ranging from mild to severe
17
Q

What are the features of the DMD gene? (6)

A
  • 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
18
Q

What is the function of dystrophin? (4)

A
  • 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
19
Q

What is caused by a defect in dystrophin? (2)

A
  • 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
20
Q

What are the features of Duchenne muscular dystrophy (DMD) in males? (9)

A
  • 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
21
Q

What are the features of Becker muscular dystrophy (BMD) in males? (5)

A
  • Milder
  • Later onset skeletal muscle weakness
  • Don’t need a wheelchair until much later
  • Cardiomyopathy in teens
  • Longer lifespan than Duchenne
22
Q

What is the chance that a heterozygous female carrier will pass on the pathogenic DMD gene in each pregnancy?

A

50%

23
Q

What symptoms do female carriers of pathogenic DMD variant have? (3)

A
  • 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
24
Q

What would happen to offspring if a male with DMD did reproduce (doesn’t usually happen)? (2)

A
  • All daughters would be carriers (one X from mother, one X from father)
  • All sons would be unaffected (X from mother, Y from father)
25
Q

What are the variants associated with the DMD gene? (6)

A
  • 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
26
Q

What is the difference between DMD variants in Beckers and Duchennes? (6)

A
  • 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
27
Q

How do you go about genetic testing for DMD disorders? (5)

A
  • 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
28
Q

What does it mean if an MLPA screening shows 0 for a region of the DMD gene? (2)

A
  • Homozygous deletion if female
  • Hemizygous deletion if male
29
Q

How are deletion variants classified in DMD? (4)

A
  • 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
30
Q

How are duplication variants classified in DMD? (3)

A
  • Likely to be pathogenic
  • Most technologies like MLPA don’t indicate where a duplication is located
  • May not disrupt the gene
31
Q

What is the treatment for DMD? (6)

A
  • 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
32
Q

What new therapies are being developed for DMD? (2)

A
  • Exon skipping technologies
  • Induced pluripotent stem cells
33
Q

What is antisense mediated exon skipping? (4)

A
  • 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
34
Q

What is an example of exon skipping therapy? (5)

A
  • 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
35
Q

How are induced pluripotent stem cells used for DMD? (3)

A
  • 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

L3 Clinical Genomics (14 decks)

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