Lecture 27 - Muscular Dystrophy Flashcards
What are muscular dystrophies?
What are some common pathological features?
What is the most common type?
Group of inherited disorders of muscle
Common features:
• Muscle fibre necrosis
• Phagocytosis of muscle fibres
Duchenne muscular dystrophy (DMD) is the most common type
List some clinical signs of DMD
- Gower sign
- Hypotonia
- Delay in walking
- Toe walking
- Clumsiness, falling
- Muscle pseudo hypertrophy
- Lumbar lordosis
- Protuberant abdomen
- IQ < 75
- Speech delay
What is hypotonia?
Floppy muscles
Why muscle ‘pseudo’-hypertrophy?
Muscles look hypertrophied
However, the muscle cells haven’t bigger, rather there is excess fat and connective tissue
Describe the molecular pathogenesis of DMD
- Mutation in Dystrophin
- Forces can not be transmitted during muscle contraction
- Shearing / tearing of sarcolemma
- Membrane instability and Ca2+ influx through stretch activated channels
- Activation of proteolytic and lipolytic enzymes
- Degradation of muscle
- Regeneration of muscle (up to a certain point)
In which cells is Dystrophin expressed?
All muscle cells (sub-sarcolemma)
Brain
What is the role of Dystrophin in muscle cells?
Forms link between actin (cytoskeleton) and extracellular matrix
Describe DAPC
‘Dystrophin-associated protein complex’
Intracellular-TM-extracellular complex
Components: • Actin cytoskeleton • Dystrophin • Transmembrane proteins • Laminin • ECM
What happens when muscle degeneration is in excess of muscle regeneration?
DMD
What is an important complication of DMD?
Describe this
Scoliosis
As a result of the muscle degeneration
Spinal abnormality, whereby it is to one side
Surgery to insert metal rod
Describe treatments and prognosis for DMD
Prognosis:
• Wheelchair usually by the age of 12
• Death late teens-20’s
• Due to respiratory / cardiac failure
Treatment:
• Steroids (? decreased inflammatory response)
• Occupational therapy
• Physiotherapy
Compare DMD and BMD: • Severity • Prognosis • Fertility • Aetiology • Prevalence • Dystrophin protein
(Becker muscular dystrophies)
Severity:
• DMD: more severe
• BMD: milder
Prognosis:
• DMD: wheelchair by 12
• BMD: may never need a wheelchair, survive into adulthood
Fertility:
• DMD: Zero, do not live long enough to reproduce
• BMD: reduced (0.7 fitness)
Aetiology:
Both due to mutations in Dystrophin gene, but they are allelic variants, as they are due to different sorts of mutations in this gene
Prevalence:
• DMD: 1/3500, more common
• BMD: 1/20000, rarer
Dystrophin protein:
• DMD: absent (non-functional)
• BMD: partially functional
What is the inheritance of DMD?
X-linked recessive
Why can’t women have DMD?
Since it is X-linked recessive, the father would have to have the disease. However, people with DMD are infertile
NB Women may show symptoms due to imbalanced X inactivation if they are carriers
Why are only 2/3 of isolated cases of DMD due to inheritance from mother?
1/3 are due to de novo mutations
Give some general features of the Dystrophin gene
- Xp21
- 79 exons
- 7 promoters
- Very large gene (2° largest)
What is the significance of the 7 different promoters in the Dystrophin gene?
Under different promoters, different isoforms are made.
These isoforms are tissue specific: • Cortex • Muscle • Purkinje • Retinal etc.
Regulation of gene expression in various tissues resulting in different isoforms of the protein being produced
Describe the importance of reading frame in DMD and BMD
DMD: out of frame deletion
• Non-functional protein
• mRNA degraded
BMD: in frame deletion
• Shorter protein, which is still functional
Describe the various methods of diagnosis of DMD and BMD
- Screening: blood test
• Creatine kinase - Muscle biopsy
• Gold standard
• Only done if DNA testing result isn’t clear
• Distinctive muscle histology observed - DNA tests
a. Direct testing
• 3 multiplex PCRs (outdated)
• MLPA (used now)
b. Indirect testing
• Linkage analysis
What is creatine kinase?
What is observed with it in DMD?
What is the normal range?
Enzyme found in muscles
Because muscle is degenerating, there is a leak of specific CK into the blood stream
In DMD:
• Elevated CK serum levels (20-50x)
• 12,000 U/L
Normal:
• 40-240 U/L
Describe pathology of muscle biopsy in DMD
- Abnormal variation in diameter of muscle (atrophy and hypertrophy
- Focal necrotic fibres
- Focal regenerative fibres
- Focal inflammatory cell infiltrate
- Replacement of muscle fibres with fat and fibrous connective tissue
- <5% dystrophin positive fibres
Describe direct DNA testing for DMD diagnosis in the past
3 multiplex PCRs, each with numerous primers
100% of deletions detected
All 79 exons are amplified by PCR
Exons then run on a gel
Some exons will be missing → disease
Drawbacks:
• Not quantitative; has been superseded by MLPA
What type of mutations predominantly give rise to DMD?
65% (Out-of-frame) deletions
5%: Duplications
15%: Point mutations
→ These are hard to catch with DNA testing
Describe current direct DNA testing Describe: • The probe • The procedure • Analysis
Why is this better than previous direct DNA testing technologies?
MLPA: multiplex ligation-dependent probe amplification
- PCR based + Capillary electrophoresis
- Detection of deletions or duplication in all 79 exons
- Does not detect cases due to point mutations
MLPA probe:
• Each is specific for the exon to be tested
• Two parts
– will only ligate if the specific exon is present –
• Target sequences A and B complementary to exon sequences
• Universal primers
Procedure:
- Denaturation of DNA
- Hybridisation of probes (only if specific exon is present)
- Ligation of probes
- PCR amplification
- Detection and analysis (length of axons)
Results positive for DMD:
• Certain exons will be shorter (half the length) of control exons
Can deliver quantative information:
• i.e. in carriers, half of the gene product is produced
• Thus can be used to detect carriers
