Cytoskeleton Protein Defects Flashcards
fragile cytoskeleton of RBCs
hemolytic anemia
complete absence of cytoskeletal protein dystrophin
Duchenne’s muscular dystrophy
dystrophin protein is present but abnormal
becker’s muscular dystrophy
hemolytic anemia characterized by spherical and …….
fragile RBCs that lyse and release hemoglobin
hereditary spherocytosis
in types of anemia, bone marrow cannot work fast enough to produce RBCs, so …..
the spleen will begin producing RBCs as well
hereditary spherocytosis cause
caused by mutations in genes for the erythrocyte membrane skeleton of RBCs
spectrin
osmotic fragility test
test the membrane strength of RBCs in a hypotonic solution
HS RBCs will burst
osmotic fragility curve
doing the osmotic fragility test over several trials w/ increasing hypotonic concentrations (decreased [NaCl])
normal RBCs will withstand the tests longer (closer to water) than HS RBCs
HS is the most common
most common hemolytic anemia in people of northern European descent
HS genes
most are identified but 10% of diseased - we do not know why they have HS
treatments for HS
- blood transfusions
- splenectomy - which removes spherocytes from circulation
- increase RBC# and Hb
DMD is the most common …..
fatal neuromuscular disorder
no cure
early 20s
DMD treatments
- nothing can alter course of disease
2. treatments to maintain general health and quality of life
DMD genetics
- x linked recessive
- dystrophin gene mutations
- genetic defect present at birth but does not show symptoms to ~3 yrs of age
function of dystrophin
provide structural stability to muscle cell membranes during contractions
dystrophin protein
- has 4 functional domains
- N-terminus binding to actin
- long spectrin like repeats
- cysteine rich
- bind to transmembrane proteins at c-terminus
dystrophin C-terminus
cysteine rich
binds to dystroglycans and syntrophins - transmembrane proteins
dystrophin is localized to
inner surface of muscle membrane
loss of dystrophin
results in destabilization of entire actin to membrane complex
dystrophic myopathy
progressive muscle degeneration with loss of functional muscle tissue over time w/ resulting weakness
clinical presentation of DMD
- elevated creatine kinase in blood (50-100x normal)
- slow walking, general weakness
- age of diagnosis ~5 years
- wheelchair dependent by 13 yrs
physical presentation of DMD
–necrosis of muscle fibers — replaced by fat or CT
leads to pseudohypertrophy
gower maneuver
because of weakened leg muscles, boys w/ DMD have a distinct way of rising from the floor
using their hands to walk up their legs
EMG
electromyography
test that is used to record the electrical activity of muscles
CRDs
CRDs
complex repetitive discharges
abnormal/spontaneous firing APs associated w/ membrane instability
seen on EMGs
significance of CRD presence
indicative of muscle membrane instability and muscle pathology
hallmark of testing for dystrophies
becker muscular dystrophy
- -milder form than Duchenne
- -similar symptoms to dmd but milder
- -wheelchair at 16
MD etiology
- -x linked recessive
- -dmd–dystrophin gone
- -bmd–50-80% of protein present
associated w/ frameshift mutations
dmd
western plot
or protein blot
indicates presence or absence of proteins
DMD dystrophin presence
<0.30%
BMD phenotype
- -variable in symptoms
- -no frameshifts
- -dystrophin present and abnormal in quantity or size
- -walk till 16
- -lifespan 45 yrs
DMD phenotype
- -frameshifts
- -no dystrophin
- -wheelchair by 12
- -lifespan up to 25 yrs
inheritance risk for MD
50% risk for affected sons
50% risk for daughters to be carriers
when mother is carrier
MD manifestation in females
- mild
- slight elevation of CK
- weak back, arms, legs
- fatigue easily
- heart problems/shortness of breath
cellular pathophysiology of MD
- sarcolemmas sustain mechanical injury
- Ca influx/oxidative stress
- reduced resting potential (close to threshold)
- fiber degeneration
- irreversible necrosis – replaced by fat or CT
How does MD result in fatality?
progressive respiratory weakness
chronic hypoxia
result in cardiac and respiratory failure
diagnostic tools for MD
- EMG
- blood test
- biopsy
- DNA study
- quantitative dystrophin analysis
prenatal MD diagnosis
- use fetal DNA – molecular analysis
2. preimplantation genetics – checking blastomere in IVF – transfer only healthy embryos
treatments for DMD
- use growth factors
- use gene therapy
- gene therapy microdystrophins
- exon skipping
- nonsense stop codon read-thru
describe DMD option 1 gene therapy
- replace dystrophin gene
but is too large for adeno-associated viruses
also try utrophin
ultimately does not work — immune issues
DMD usage of growth factors
- myostatin inhibits muscle cell growth
- knockout myostatin
- muscles grow very large
- but does not change that we are just making more DMD compromised muscle
utrophin
homologous dystrophin like protein
DMD option 2 gene therapy
- use microdystrophins
generate a protein like dystrophin but w/ less spectrin like repeats
works little but still immune issues
problem of dystrophin immunity
immune system has never seen this protein before so generates a response
T-cell mediated immunity against dystrophin proteins
DMD exon skipping
–in dmd deletions in gene cause frameshift mutations
therapy to skip the exons to get back into frame
works little but still immune issues
DMD stop codon skipping
–premature stop codon mutations which result in single nuc substitution
use a drug to skip or suppress the stop codon
–read-thru stop codon
works little but still immune issues
overall treatment strategies for DMD conclusions
all approaches have problems, typically immune responses
KCU investigating the dystrophin gene itself
dystrophin gene
has several different products by different promoters
dif. dystrophin sizes depending on where in the body it is
ex. muscle vs. retinal dystrophin
Dp260
retinal dystrophin
similar but basically smaller
DMD treatment w/ dp260
works in mice
but do not know the promoter for dp260 yet
describe how the osmotic fragility test indicates a patient has hereditary spherocytosis
–using this test we subject RBCs to a hypotonic solution to test the strength of their membranes
–in hs RBC membranes are weakened and will show up much weaker in a of test
describe the function of the erythrocyte membrane cytoskeleton and how defects in the EMS cause hereditary spherocytosis
–the cytoskeleton is responsible for giving RBCs their biconcave shape which gives them structural integrity which is important for them to survive the mechanical stress of passing thru the circulatory system
defects in ems result in deformed shapes of RBCs, which weakens them and greatly shortens their lifespan
EMS
erythrocyte membrane cytoskeleton
differentiate Duchenne muscular dystrophy vs. becker muscular dystrophy
Duchenne — dystrophin protein is absent
becker — dystrophin protein is abnormal in quantity or size
describe 2 therapeutic strategies to attempt to treat Duchenne muscular dystrophy
- treat patient with growth factors to knockout myostatin (which prevents muscle cell growth) to allow muscles to grow large but the muscles are still weakened by dmd
- gene therapy by using microdystrophins, creating a dystrophin-like protein, treating patient with this. works a little bit but the patient will have an immune response since the immune system has never encountered this protein before
