Lecture 11: Duchenne Muscular Dystrophy (an X-linked disease) Flashcards Preview

BIO227- Human Genetic Disorders > Lecture 11: Duchenne Muscular Dystrophy (an X-linked disease) > Flashcards

Flashcards in Lecture 11: Duchenne Muscular Dystrophy (an X-linked disease) Deck (29)
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1

Summarise what is muscular dystrophy

- Genetic disease affecting skeletal muscles
- Characterized by progressive muscle weakness and wasting, and loss of motor skills
- X-linked recessive most common: Duchenne and Becker (Becker is less severe)
- More than 1 in 3500 males born
- Onset age from infancy to adulthood
- Most end up wheelchair-bound

2

Define DMD

- The most common of several childhood muscular dystrophies
- An inherited disorder X-linked recessive with progressive degeneration of muscle
- Onset is generally before age 6 years
- Muscle loss usually not noticed until observation of unusual walking and/or talking around the age of 3
->parents spot issue first by comparing to siblings= allows early detection

3

State the incidence rates of DMD for men and women

- About 1 in every 3,500 to 5,000 males is born with DMD
- rarer for women

4

Describe the phenotype of female carriers of DMD

- Much rarer in females
->experience some symptoms: weaker muscles in the back, legs and arms that fatigue easily
-> Carriers may have heart problems, shortness of breath or failure to do moderate exercise
-heart problem picked later on in life or undetected
- they try to avoid exercise-> because symptoms
->Untreated heart problems, can be serious, even life-threatening

5

State the cause of DMD

- DMD caused by a mutation in the gene that produces an important muscle protein called dystrophin

6

Describe what dystrophin is

- Muscles are made up bundles of muscle fibres
- Interdependent proteins along the membrane surrounding each fiber- keep muscle cells working properly
->dystrophin is an interdependent protein
- Dystrophin acts as a spring
- When dystrophin missing = DMD
 Constant muscle contraction/ relaxation → weaken & destroys muscles

7

Explain the different types of muscular dystrophy

- Becker muscular dystrophy - inability to produce functional dystrophin
- Dystrophin: not 100% non-functional
- Limited function = less severe

- Duchenne muscular dystrophy – inability to produce dystrophin
- No dystrophin production or production of only non-functional dystrophin
- No function = more severe disease

8

When do you suspect DMD and what are the ways of diagnosis

When:
- if no family history of MD -> when not walking by >16-18 months
-> Gowers' signs
- if pos. family history of MD-> any abnormal muscle function
- patient has unexplained increase in transaminases

Screening:
- creatine kinase
-> conc. high= more testing
-> conc. low= another diagnosis

Confim diagnosis:
- testing for mutation found
- muscle biopsy to see if dystrophin protein absent
- gene sequencing if both test before doesn't give results

Pos. result- confirmed
dystrophinopathy diagnosis

9

Explain what happens after post-diagnosis

- Muscle biopsy is optional -may distinguish DMD from milder phenotypes
- Referral to specialist multidisplinary follow-up is required

- Genetic counselling needs to be offered to any ‘at risk’ female family members
- Patient and family support and contact with patient/family support organisations should be offered

10

Give the clinical features of DMD

- Early onset: signs appear before 6 years of age
- Delayed development of motor skills
- Difficulty in keeping balance
- Progressive muscle weakness/fatigue
- Respiratory muscles eventually involved in muscle weakness and fatigue
- Pseudohypertrophy- enlargement of muscle tissue because it’s abnormal
- Contractures- when elastic tissue of muscle is replaced by non-elastic tissue, making them remain too tight for too long and become shorter
- Wheelchair dependent by early teens
- Death usually in late teens, early twenties

11

Give the symptoms of DMD

Early
- Delayed Onset Walking
- Difficulty in performing a standing jump
- Waddling when walking (like penguin)
- Difficulty standing up
- Enlarged Calves-> pseudohypertrophy ( swollen due to muscle damage)
Later
- Difficulty getting up from a chair
- Loss of ability to climb stairs
- Wide gaited walk with balance problems

More symptoms
- Fatigue
- Mental retardation
- Muscle weakness: begins in legs and pelvis, less severe in arms, neck
- Poor motor skills (running, hopping, jumping)
- Frequent falls
- Rapidly worsening weakness
- Progressive difficulty walking by age 10 may need braces soon after most confined to wheelchair

12

Describe and explain what the Gower Manoeuvre is

- Used to detect DMD
- Uses more of upper body muscle than lower to stand-> sign of DMD

- Immediate response-> roll over to use hands
- Wide gait present throughout manoeuvre
- Affected boys stand up by bracing their arms against their legs because of the weakness in the proximal muscles

13

Give the potential Complications of DMD

- Scoliosis – Curvature due to contractures and muscle weakness
- Cardiac
– Dilated cardiomyopathy
– Arrhythmia, shortness of breath and fatigue
- Respiratory
– Progressive weakening of the diaphragm
– Pneumonia or other respiratory infections leads to:
– Respiratory failure
- Cognitive impairment (non-progressive)
- Permanent, progressive disability
- Decreased mobility
- Decreased ability to care for self

- Individuals affected by DMD killed by failure of the heart muscles usually by age 25

14

Explain how muscle histology is used to diagnose DMD

- Histology of normal (A) and DMD (B) muscle biopsies
- Stained by H&E
A:
- Myofibers uniform size
- evenly space
- polygonal (uniform shape)
B:
- increased size variation - due to atropy and hypertropy
- necrosis
- fibrosis
- fatty replacements

- Muscle biopsies (brown stain)
- Stained with antibody against dystrophin
Normal:
- Regular cellular architecture
- dystrophin on all outer membranes
DMD:
- Irregular architecture
- dystrophin absent from surface of muscle fibers
Carrier:
- Biopsy from a female carrier of DMD
- patchy staining around outer cell membranes

15

How can DMD be inherited

- female carriers
-> because they have offspring who are affected or carriers
-> obligate carriers of the disease gene
x-linked recessive

16

How can DMD be inherited when both parents shown on pedigree as healthy

- Could be de Novo or undiagnosed DMD female carrier
- Female carrier phenotypic consequence misdiagnosed

17

Summarise the genetic consequences of MD

- Main cause:
– X-linked disease
–recessive mutation in X-chromosome ->Inherited from parents

- Females affected only if both X-chromosomes mutated
– Single mutation: normal as enough functional dystrophin produced
-> Usually unaffected, but a carrier of disease = have phenotypic consequence
– Double mutation-> affected
->very unlikely

- Males – single X-chromosome
– Mutation in X-chromosome = no dystrophin production

18

Summarise the inheritance of MD

- Unaffected carrier mother + unaffected father
– Mother: 1 mutated X-chr + 1 normal X-chr
– Father: 1 normal X-chr + 1 normal Y-chr

- Unaffected female
– Normal X-chr from mother + normal X-chr from father

- Carrier female
– Normal X-chr from father + mutated X-chr from mother
– Normal X-chr = enough functional dystrophin produced

- Unaffected male
– Normal X-chr from mother + normal Y-chr from father

- Affected male
– Mutated X-chr from mother + normal Y-chr from father
= No normal X-chr = no dystrophin production

19

Explain how DMD gene discovered and identified

- Chromosomal abnormality= boy- visible deletion at Xp21.3
= woman- balanced Xp21;21p12 translocation
- Identified by positional cloning= chrom. Abnormality show to have giant implications on dystrophin gene
- Has large no. exons + average small size for each of them
- Usually size larger

- Balanced translocations and inversions-> very useful = no net loss of DNA
- The underlying gene is expected to be close to or located at breakpoint

20

Describe the structure of dystrophin

- Functionally important domains are in the N- and C-terminal regions
- Dystrophin protein- forms a structural link between actin filaments of the cytoskeleton and the extracellular matrix
-> via a membrane bound dystrophin-associated protein (DAP) complex

21

Explain the functional role of dystrophin

- Dystrophin anchors the cytoskeleton of muscle cells to the extracellular matrix
->via the dystrophin glycoprotein complex
- Complex includes sarcoglycans (mutations cause limb-girdle muscular dystrophies) + dystroglycans
- Muscle cells that lack dystrophin:
->mechanically fragile
->fail after a few years= progressive weakness

22

Explain how the deletions in dystrophin gene lead to DMD

- Large In-frame deletions of central exons in the dystrophin gene:
- Can remove many of the central exons
- Reading frame is not disrupted =associated with Becker muscular dystrophy
- There is no premature termination codon (TER)
- Exons at front + back of deletion join

- Large out of frame deletions of central exons in the dystrophin gene:
- Can remove many of the central exons
- If a single central exon deleted where the nucleotides lost is not divisable by 3= frame shift
- Premature termination codon (pTER) follows-often next exon triggering nonsense-mediated decay of dystrophin mRNA
- Ensures failure to make any protein = causing DMD

23

Explain how DMD can be diagnosed

- Blood Creatine phosphokinase (CPK) test
- Damaged muscles can release creatine kinase into blood
- Elevated levels ->muscle injury: trauma or muscular dystrophy

- Electromyography (EMG)
- Measuring electric signaling to and from the muscle
- rule out neurodegenerative diseases +confirm a muscle disease

- Muscle biopsy
- Microscopic analysis of a sample of muscle tissue
- identify absence of dystrophin + characteristics associated with muscular dystrophies

- Genetic testing
- Testing for mutations in muscular dystrophy related genes
- Determine the exact form of muscular dystrophy

24

Explain how exon deletions in DMD are screened

PCR
- Exon-specific primers used
- Based on:
->9 selected exons of the dystrophin gene were amplified from the DNA of affected

- PCR product run on an electrophoretic gel
->Each exon gives a band of a characteristic size

- ALWAYS NEED MARKER LANE-> many fragments of known size

- Boys have 1 X chrom. ->any deletion shows as missing bands
- Exon deletions arrowed
->Lane 3: large deletion or a technical failure?
- Boys with no deletions:
-> may have deletions in other exons
->OR point mutations
-> OR duplications
= causing loss of function

25

Explain the principles of MLPA

- Detects copy number changes over a broad range of DNA lengths
- Can scan for intragenic deletions and duplications by assessing copy number of exons
- Useful for disorders with high frequency intragenic deletions ->DMD
- Use alongside DNA sequencing to scan for point mutations

- Sample DNA placed in thermocycler+ heated= DNA strands separate= denaturation
- Probes added to sample DNA and target multiple exons
->probes are paired
->probe has stuffer fragment- each probe has unique length= amplification product of probe can be identified
- Hybridise overnight
->hybridise to adjacent sequences within target
- DNA ligase added to mixture-> ligates pair of probes together
- Probe amplified by PCR
->DNAP + DNA nucleotides + forward and reverse primers added-> forward primer fluorescently labelled
- Capillary electrophoresis separates PCR products
->uses standard sized fragments that have different label

26

Explain how results from MLPA can be used to diagnose DMD

- Capillary electrophoresis
- Paired red (normal) and blue peaks (sample) (125–475 bp) = individual exons
- Large exons can require partly overlapping probes
- Deletion of seven exons (arrowed) is shown: blue peak reduced by ~50% -heterozygous deletion
- Peak order is sometimes not the same as for gene exons

27

How can you distinguish between homozygous and heterozygous deletions

- Homozygous= complete deletion of peak
- Heterozygous= peak reduced by 50%

28

Give the different DNA analysis that is used for MD

- MPLA->for deletions of exons and duplications
-> for DMD and BMD
- direct sequencing-> small variations
- Immunochemistry- H&E and monoclonal antibodies

29

Outline the mechanism for DMD

- Mechanical stress caused by dystrophin absence
- leads to sarcolemmal disruption + calcium channel activation
- Increased intracellular calcium
->activates:
- calcium-dependent proteases
- chemokines
- cytokines
- Inflammation and necrosis retards muscle regeneration
- Necrosis accompanied by fibrosis and fatty tissue infiltration in muscle
- Fibrosis: formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process