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

Question 1

Summarise what is muscular dystrophy

Answer 1

• 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

Question 2

Define DMD

Answer 2

• 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

Question 3

State the incidence rates of DMD for men and women

Answer 3

• About 1 in every 3,500 to 5,000 males is born with DMD

- rarer for women

Question 4

Describe the phenotype of female carriers of DMD

Answer 4

• 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

Question 5

State the cause of DMD

Answer 5

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

Question 6

Describe what dystrophin is

Answer 6

• 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

Question 7

Explain the different types of muscular dystrophy

Answer 7

• 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

Question 8

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

Answer 8

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

Question 9

Explain what happens after post-diagnosis

Answer 9

• 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

Question 10

Give the clinical features of DMD

Answer 10

• 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

Question 11

Give the symptoms of DMD

Answer 11

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

Question 12

Describe and explain what the Gower Manoeuvre is

Answer 12

• 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

Question 13

Give the potential Complications of DMD

Answer 13

• 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

Question 14

Explain how muscle histology is used to diagnose DMD

Answer 14

• 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

Question 15

How can DMD be inherited

Answer 15

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

Question 16

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

Answer 16

• Could be de Novo or undiagnosed DMD female carrier

- Female carrier phenotypic consequence misdiagnosed

Question 17

Summarise the genetic consequences of MD

Answer 17

• 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

Question 18

Summarise the inheritance of MD

Answer 18

• 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

Question 19

Explain how DMD gene discovered and identified

Answer 19

• 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

Question 20

Describe the structure of dystrophin

Answer 20

• 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

Question 21

Explain the functional role of dystrophin

Answer 21

• 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

Question 22

Explain how the deletions in dystrophin gene lead to DMD

Answer 22

• 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

Question 23

Explain how DMD can be diagnosed

Answer 23

• 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

Question 24

Explain how exon deletions in DMD are screened

Answer 24

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

Question 25

Explain the principles of MLPA

Answer 25

• 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

Question 26

Explain how results from MLPA can be used to diagnose DMD

Answer 26

• 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

Question 27

How can you distinguish between homozygous and heterozygous deletions

Answer 27

• Homozygous= complete deletion of peak

- Heterozygous= peak reduced by 50%

Question 28

Give the different DNA analysis that is used for MD

Answer 28

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

Question 29

Outline the mechanism for DMD

Answer 29

• 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