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
When is linkage analysis performed?
Deletion can not be found in boy, but we want to find out which family members have the disease chromosome
Describe how linkage analysis is performed
Tracking of mutated dystrophin allele through the family, using a marker (i.e. not the exons, maybe repeats in an intron)
The marker must be polymorphic so that a person’s two X chromosomes can be differentiated
Uses CA repeats - STRs (polymorphic short tandem repeats)
Different numbers of repeats present in the population
Analysing the STR lengths, we can track which X chromosome is associated with the condition, and thus determine who is and isn’t a carrier
Describe when carrier testing would be performed, and how
Boy born with mutation in Dystrophin
→ we want to know which female relatives may be carriers
Perform MLPA
What are current treatments for DMD?
Steroids is the only therapy proven to work
There is currently no cure, but there are new avenues being explored
List some future therapies for DMD
There is hope on the horizon: Clinical trials currently happening
- Gene therapy
• Modified mini-genes (because the gene is so large)
• More like Becker phenotype - Upregulation of alternative proteins
• Utrophin (dystrophin homologue)
• This homologue is expressed in the foetus
• Possibility of up-regulating this - Anti-sense oligonucleotides
• Allow ‘skipping’ of exons
• Shorter, partially functioning protein
• Replicates the Becker phenotype - Restoration of dystrophin production
• If the mutation gives a premature STOP codon
• Allow read through
• Ataluren (in trials for CF as well, common mechanism)
• Gentamycin
• (amino glycosides..?) - Inhibition of protein degradation
• Increases muscle mass
• Flavocoxoid
• Pentoxifylline - Myoblast transfer therapy
• Myoblasts containing normal dystrophin gene implanted - Stem cell therapy
• Stem cells containing normal gene implanted
• iPS: normal gene put back into an iPS, then given to the person
What is the name of the protein complex in which dystrophin plays an important role?
Dystrophin associated protein complex (DAPC)
When are boys normally diagnosed with DMD?
Around 5 years of age
This is quite a late diagnosis
What is the life expectancy with boys with DMD?
Around 19 years
What are manifesting heterozygosity?
Carrier women may experience some symptoms:
• Cardiomyopathy
Due to skewed X inactivation
Affected X chromosome more expressed than the normal chromosome.
e.g. 90-10%
Due to mosaicism
When can de novo Dystrophin mutation occur?
Occurs in:
• The embryo
• In the developing eggs (gonadal mosaicism)
What is the screening test for DMD?
Blood test for elevated CK
20-50x the normal level
Normal: 1000 IU/L
DMD: 10 000 IU/L
NB Muscle degeneration with other aetiologies can lead to elevated serum CK
Describe the histology of BMD
- More variable than DMD
* Some muscle fibres have Dystrophin, some don’t
What are the aetiologies of other types of dystrophies?
Mutations in the many other proteins that are involved in DAPC
Describe the structure of the Dystrophin protein
- N terminal: binds actin
- Rod domain: spectrin like repeats
- Cysteine-rich domain
- C terminal: binds transmembrane proteins of DAPC
