Molecular mechanisms in inherited disorders Flashcards
Mutation in what proteins results in Cystic Fibrosis?
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
What is CFTR?
membrane protein that acts as a chloride channel
CFTR belongs to what group of transporters?
group of ATP Binding Cassette (ABC) transporter
Describe the structure (domains) of CFTR?
It has 2 membrane spanning domains, two ATP binding domains and a regulatory domain that can be phosphorylated.
What causes activation of CFTR?
phosphorylation of regulatory domain by PKA
Why is there low Na and H20 in the lumen (mucous)?
the mutant CPTR can’t transport Cl from the cell into the lumen therefore Na will stay in cell to balance Cl’s negative charge.
Why is the mucous thick and viscid in pts w/ Cystic Fibrosis?
due to the low water and electrolyte concentration in the mucous.
Why do CF pts have a higher risk of developing respiratory infections?
The thick viscid mucus are prone to developing bacterial infections.
Respiratory infections are common causes of what in CF pts?
mortality and morbidity
How does the mutant CFTR effect the pancreas?
Leads to thick pancreatic secretions which obstruct pancreatic duct and destroy tissue. Results in deficiency of pancreatic enzymes.
What type of tissues replace pancreatic tissue in pts with cystic fibrosis?
Fibrotic tissue and fat
What symptoms are found in pts with CF?
Malabsorption, sterility, salty sweat, chronic pancreatitis, secondary biliary cirrhosis and recurring resp infections
Why is there defective protein digestion in pts with CF?
Blockage of pancreatic duct results in deficient secretion of pancreatic enzymes like lipase, trypsin, and chymotrypsin
Why is there steatorrhea in pts w/ CF?
B/C of the maldigestion of nutrients and the excretion of fat in stool due to deficiency of fat soluble vitamins esp Vit K (not stored in liver)
Why do many children w/ CF have protein malnutrition and delay in growth?
B/C of the deficient secretion of pancreatic enzymes which aid in the digestion of proteins
How is it possible to restore normal digestion and nutrition in pts w/ CF?
by providing pancreatic enzyme supplements
The viscid secretions in the intestine of CF pts causes what in kids?
meconium ileus and intestinal obstruction
Why are more than 95% of males with CF infertile?
B/C they don’t have a vas deferens; called Congenital bilateral absence of the vas deferens (CBAVD)
What is the normal role of CFTR in sweat glands?
to reabsorb NaCl from the sweat.
How is the role of CFTR different in sweat glands compared to that in lungs/ pancreas?
CFTR reabsorbs NaCl in sweat glands while in other tissues, it secretes Cl (opposite fxns)
The presence of excessive salt in sweat is diagnostic of what condition?
Cystic fibrosis
What chromosome contains the mutation for CFTR?
chromosome 7
What is the most common position for the mutation that results in CFTR?
position 508 (F508)
Is Cystic Fibrosis a x-linked disorder?
No, autosomal recessive disorder
What is the most common CFTR mutation and on what domain is it on?
3 bp deletion that eliminates phenylalanine residue from the ATP binding domain 1
What is the effect of phenylalanine deletion at position 508 on CFTR structure?
doesn’t cause change in reading frame but prevents protein from maturing and from reaching the plasma membrane
What drug is used to stimulate sweat glands in the sweat chloride test?
pilocarpine - used to collect sweat to measure chloride concentration in diagnosis of CF
What molecular diagnostic tests an be used to determine CF if the mutation is known?
ASO and Allele specific PCR test
Why is it possible to use PCR and Southern blotting to detect F508 mutations in pts w/ CF?
B/C the mutant gene (60 bp) is shorter than the normal gene (63pb) by 3 bp
Describe the G protein pathway involved in CFTR.
activated G-protein coupled receptor will activate adenylate cyclase which will increase levels of cAMP. this activates protein kinase A (PKA) to phosphorylate CFTR. This opens channel so that Cl can leave.
What type of mutation causes sickle cell anemia?
point mutation in 6th position of B-globin chain of hemoglobin where glutamic acid is replaced by valine
At what position does the point mutation occur?
postion 6 of B-globin chain of Hb
What effect does the substitution of valine for glutamic acid have on the structure of Hb?
Will create a hydrophobic pocket on the exterior surface of Hb b/c valine is hydrophobic and glutamic acid is hydrophobic
Why does Hb tend to aggregate, forming long filament like structures in pts w/ sickle cell anemia?
the mutation will create a hydrophobic pocket “sticky patch” forming site of attachment for nearby Hb molecules.
What effect does aggregation of Hb molecules have on the structure of RBC?
Distorts structure of RBC- sickled shape
What is the effect of sickled RBC on Bl flow?
blocks bl flow b/c can’t flow freely
Why do pts w/ sickle cell disease have anemia?
b/c the spleen removes the sickled and distorted RBCs often
Why would splenomegaly be present in pts w/ sickle cell disease?
B/c spleen is responsible for removing the sickled RBCs
What type of hyperbilirubinemia in found in sickle cell disease?
Pre-hepatic (hemolytic type)
What type of bilirubin accumulates in pts w/ Sickle cell anemia?
unconjugated (direct) and total bilirubin
Why will jaundice be present in pts w/ sickle cell anemia?
b/c of the excessive removal of sickled RBC from the bloood
Why is the urine of pts w/ sickle cell anemia normal?
the high levels of unconjugated bilirubin can’t be excreted in urine b/c bound to albumin
Why will people w/ sickle cell anemia develop pigmented gall stones?
b/c large amounts of conjugated bilirubin are secreted into bile
In electrophoresis, why would HbS (sickle cell form) move slower than HbA toward the positive end?
HbS has valine which is less negative than the glutamate in HbA (normal)
Why is it useful to use ASO test to detect sickle cell disease?
Makes it possible to identify carriers
What does RFLP analysis reveal about sickle cell disease and carriers in relation to restriction sites?
Normal: 3 restriction sites; in sickle cell anemia: loss of central restriction site producing longer fragment generated which will travel more slowly than normal band
What gene is mutant in Duchenne muscular dystrophy and Becker muscular dystrophy?
Dystrophin gene
Almost complete absence of functional dystrophin results in what disease?
Duchenne Muscular Dystrophy (DMD)
What causes Becker Muscular Dystrophy?
production of abnormal dystrophin or decreased amts of dystrophin
Why is Duchenne Muscular Dystrophy more common in males?
X-linked recessive disease
What is the most common muscular dystrophy?
DMD
Which muscular dystrophy is more milder? which muscular dystrophy has a later onset?
Becker Muscular Dystrophy for both b/c DMD will occur between ages 2-6 and most pts die in early 20s.
What is the differences between DMD deletions and BMD deletions?
DMD: cause frameshift mutations resulting in large deletions of exons (almost no protein)
BMD: in-frame mutations therefore protein is shorter but translated
Why will pts w/ DMD and BMD have trouble rising, climbing stairs and maintaining balance?
b/c DMD and BMD affects pectoral muslces, trunk and upper/lower legs -muscle weakness
What is Gowers’ maneuver?
distinctive way of risking from floor: get on hands/knees first, raise posterior, then “walk” hands up legs to raise their upper body
Gowers’ manuever is characteristic of what disease and why?
Duchenne muscular dystrophy; b/c of weakened leg muscles
What would lead to pseudohypertrophy and in what disease is it found?
replacement of normal muscle w/ connective tissue / fat; in pts with DMD
The largest gene encodes for what protein and where is it found?
Encodes for dystrophin; expressed in smooth, cardiac and skeletal muscle w/ lower levels in the brain
Why would cardiac abnormalities be found in pts w/ DMD resulting in their early death?
b/c gene for dystrophin also expressed in cardiac muscle
Describe structure of Dystrophin
has two globular heads with flexible rod-shaped center. One head binds to actin; other head (C-terminal domain) binds to protein in plasma membrane
How does Dystrophin anchor cystoskeleton of muscle cells to extracellular matrix?
by linking actin filaments to transmembrane proteins of muscle cell plasma mem.
Why is Dystrophin important?
Its role in anchorage of cytoskeleton to EC matrix stabilizes plasma mem and helps cell to withstand stress of muscle contraction.
What does the loss of dystrophin result in?
oxidative cellular injury and myonecrosis
How can we differentiate between DMD and BMD in western blot?
DMD: no band b/c of frameshift deletion; BMD: smaller protein size and decreased quantity when compared to normal
Microscopy of BMD/ DMD will show what when compared to normal?
increase in adipocytes and fibrous tissue
What will immunofluorescense microscopy show in pts w/ DMD, BMD?
BMD: reduced quantity of dystrophin; DMD: complete absence
What are serum creatine kinase (MM) in pts w/ muscular dystrophy?
high b/c CK MM indicates muscle damage
What is the serum CK-MM levels in females who are carriers for DMD?
high levels
Gowers’ manuever is characteristic of what disease and why?
Duchenne muscular dystrophy; b/c of weakened leg muscles
What would lead to pseudohypertrophy and in what disease is it found?
replacement of normal muscle w/ connective tissue / fat; in pts with DMD
The largest gene encodes for what protein and where is it found?
Encodes for dystrophin; expressed in smooth, cardiac and skeletal muscle w/ lower levels in the brain
Why would cardiac abnormalities be found in pts w/ DMD resulting in their early death?
b/c gene for dystrophin also expressed in cardiac muscle
Describe structure of Dystrophin
has two globular heads with flexible rod-shaped center. One head binds to actin; other head (C-terminal domain) binds to protein in plasma membrane
How does Dystrophin anchor cystoskeleton of muscle cells to extracellular matrix?
by linking actin filaments to transmembrane proteins of muscle cell plasma mem.
Why is Dystrophin important?
Its role in anchorage of cytoskeleton to EC matrix stabilizes plasma mem and helps cell to withstand stress of muscle contraction.
What does the loss of dystrophin result in?
oxidative cellular injury and myonecrosis
How can we differentiate between DMD and BMD in western blot?
DMD: no band b/c of frameshift deletion; BMD: smaller protein size and decreased quantity when compared to normal
Microscopy of BMD/ DMD will show what when compared to normal?
increase in adipocytes and fibrous tissue
What will immunofluorescense microscopy show in pts w/ DMD, BMD?
BMD: reduced quantity of dystrophin; DMD: complete absence
What are serum creatine kinase (MM) in pts w/ muscular dystrophy?
high b/c CK MM indicates muscle damage
What is the serum CK-MM levels in females who are carriers for DMD?
higher levels
