Duchene muscular dystrophy and gene therapy Flashcards
What is a muscular dystrophy?
Genetic disorders that primarily, but not exclusively affect the skeletal muscles, they cause progressive weakness and loss of muscle mass
- The genes that are affected are the ones responsible for making proteins responsible for protecting the muscle fibers from damage, each type of muscular dystrophy (Duchenne’s Muscular Dystrophy, Becker’s Muscular Dystrophy, Emery- Dreifuss Dystrophy, Facioscapulohumeral Dystrophy, etc) is caused by a genetic mutation that is specific to it, many of these mutations are inherited but some of them occurs spontaneously in the mother’s egg or the developing embryo and it is passed to the next generations
What is Duchenne muscular dystrophy?
An X-linked recessive, genetic disorder characterized by progressive muscle degeneration and weakness, it usually occurs in young boys, and people with a family history of muscular dystrophy are at higher risk and passing it on to their children
Describe DMD
- Affects 1 in 3,500 male children
- It is a muscle-wasting disease
- It has a 100% mortality rate, it has reduced in recent years due to the availability of gene therapy
- Its effects are usually seen in mild 20’s
- 1/3 of the affected individuals have a family history
- 2/3 is sporadic (non-inherited, spontaneous mutations)
Describe the DMD gene mutations
- It affects the short arm of X chromosome 21
- Introns make up 99.4% of the entire gene (non-coding sequence)
- The protein associated with this gene was identified and named dystrophin, where its lack can cause the muscles to be fragile and easily damaged
- The main type of DMD-Caausing mutation is intragenic deletions (60%), comprising 1 or more exons, 2nd most common is point mutation (30%) and the third is duplication (5-10%)
Describe the pathology of DMD
- As we said the gene encoding for dystrophin is mutated and thus:
- Dystrophin plays an important role in muscle contractions and osmolarity of the cell membrane
- The muscle cell won’t have strength, as dystrophin provides strength to the muscle cells by linking the internal cytoskeleton to the surface of the membrane (linker b/w proteins of cell membrane and F-actin of intracellular cytoskeleton)
- Without this structural support, the cell membrane becomes permeable as components from the outside of the cell enter building internal pressure until the cell bursts
- Under normal conditions, muscle stem cells repair any damage, but in DMD the damage is so extreme that stem cells are exhausted and repair can no longer occur
Describe the molecular makeup of dystrophin
- Intracellular rod-shaped protein with four major functional domains, localized in the inner surface of the sarcolemma (plasma membrane of skeletal muscle fibers)
1) N-terminal/actin binding site: Binds dystrophin to the membranes surrounding striated muscle fibers, cytoskeleton F-actin
2) C-terminal: Interacts with multiple proteins to assemble the dystrophin-associated protein complex (DAPC)
3) Rod-domain: contains 24 repeated proteins that maintain the molecular structure and give it its flexibility, spans the sarcolemma of skeletal and cardiac muscles
4) Cysteine-rich region: required to activate the protein binding
- Head (N-terminal) -> binds to F-actin
- Neck (Rod domain) -> flexibility
- Body (C-terminal) -> DAPC
- Dystrophin is like the neck tube of the Hoover, it connects the dusting brush to the body (connects the extracellular to the intracellular actin and myosin), Without the tube, the vacuum will never work -> without the dystrophin, the muscle will never work
What is the main pathology associated with DMD?
- Muscular weakness, can begin as early as 3 years
- Progressive weakness affects of Hip, pelvic area, thighs, and shoulder girdle musculature first, by the early teens, the heart and respiratory muscles also are affected.
- Weakness of the paraspinal makes walking difficult leads to:
1) Waddling gate
2) Lumbar lordosis
3) Forward thrusting of the abdomen
4) Scapular winging
5) Anteroposterior scoliotic curve
6) Joint contractures
7) Respiratory impairment (main cause of mortality)
8) Weight gain
Describe the inheritance pattern of DMD
- X-linked recessive inheritance pattern, where females have a 50% of being a carrier and males have a 50% of being affected, the pedigree will only show affected males
- We can’t have male carriers
Can girls be affected by DMD?
1) Turner syndrome
2) Skewed x Inactivation (Skewed x inactivation is defined as a pattern where 80% or more of the cells show a preferential inactivation of one X chromosome)
3) Independent mutation event (Independent mutation event is when severe damage occurs to one of the X chromosomes -> if we have left off with the mutated X (X) chromosome-> the girl will get the disease)
Yes, girls can be affected but it is rare:
- Rare conditions manifest in females and they are milder in the form such as Turner syndrome (XO)
- DMD is X-linked recessive if the X gene is affected with Xp21 mutations
- In females with XX - there might be no symptoms unless the X is severely damaged
- In female with XO - mild symptoms
In short what is the difference between DMD and BMD?
Normal -> long and functional protein
In DMD -> no functional protein at all (C-TERMINAL)
In BMD> short & partially functioning protein (C-TERMINAL)
What is Becker muscular dystrophy?
- Duchenne muscular dystrophy (DMD) is caused by nonsense (point mutation in a sequence of DNA that results in a premature stop codon) or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation (add single nucleotide that can transcript the codon to a.a lead to truncated partially function protein)
- A milder version of DMD
- It is when there is an Altered size and decreased amount of dystrophin
- In DMD (outframe mutation), exon 55 is deleted 44-56, while in BMD (Inframe mutation) exon 55 stays and 44-54 is deleted, and thus it is a milder version of DMD
What are the diagnostic tests for DMD?
1) CK (creatine kinase) test
2) Genetic testing (positive we stop, negative we continue to the other tests)
3) Muscle biopsy
4) Western blot
Describe the creatine kinase test
- Creatine kinase has 3 isoenzymes:
1) CK-MM (Found in skeletal muscles)
2) CK-MB (found in cardiac muscles)
3) CK-BB (Found in the brain)
- Normal CK is 40-300 Units/Liter, in affected individuals it is >1000U/L, which is an indicator for muscle cell destructions, that lead to its release into the blood stream
Describe the muscle biopsy test
- Muscle biopsy is painful and it is done under general anesthesia
- The microscopic cross-sectional image will show an extensive replacement of muscle fibers with fat cells, this will cause pseudohypertrophy as the muscle cells are phagocytosed and replaced by fat cells
- Usually done as the final step to confirm DMD
Describe the western blot test
- Done using blood or saliva samples
- Western blots work by showing the size of the protein, where large proteins show on the top and smaller ones are closer to the bottom
So to perform it:
1) Control: Normal dystrophin showing normal size
2) In BMD: the band will be shorter than the control
3) In DMD: there will be an absence of the dystrophin protein
- Western blot analysis based on
The separation of proteins by electrophoresis, then The binding of specific antibodies to the protein of interest
