Cystic Fibrosis - Scott Flashcards
What is the inheritance pattern of cystic fibrosis?
autosomal recessive
What kind of mutation causes cystic fibrosis? Gene?
inactivating mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene
What is the CFTR? (i.e. what does it translate into?
chloride ion channel
(found on apical surface of epithelial cells lining airways, pancreatic ducts, intestine and other tissues)
What are 90% of deaths in cystic fibrosis due to?
respiratory failure
Besides lung pathology, what additional problems become more prevalent as people live longer?
- Liver- CF liver disease
- Pancreas- pancreatic insufficiency, CF diabetes
- Intestine- obstruction, malabsorption, cancer
- Male fertility
What was the median survival age for cystic fibrosis in 1970? 2013?
1970 → 16 yoa
2013 → 37.4 yoa
What kind of treatment strategy is helping people with cystic fibrosis live longer better quality lives?
- Treatment at specialized centers
- close monitoring
- rapid intervention especially pulmonary and GI manifestations
How does the normal CFTR ion channel work?
- CFTR is a chloride and bicarbonate ion channel
- When channel opens → Cl- and HCO3- ions flow through it (down electrochemical gradient)
- The electrochemical gradient across the airway epithelial apical cell membrane favors the flow of ions out of the cell, into the extracellular space
- Increased concentration of Cl- and HCO3- ions outside cell → creates osmotic conditions for flow of H2O out of the cell.
What is the structure of the normal CFTR?
- Membrane spanning domains (MSD) form a pore for chloride ion channel.
- Two nucleotide binding domains (NBD) and a Regulatory domain provide regulatory sites that promote opening of channel
- NBD binds ATP
- R has phosphorylation sites for PKA
What is the normal activity of the CFTR ion channel?
- Exist in two states- open and closed
- Open conformation forms aqueous pore
- Ions flow in direction of electrochemical gradient
- Constantly moving between open and closed conformations
- Activity = Amount of time spent in open conformation is regulated or gated
- Pore has size and electrochemical properties that allow only specific ions to flow through
What three things does the CFTR channel control?
- CFTR controls luminal ion concentrations and pH
- CFTR channel controls movement of H2O to apical surface of airway epithelial cell.
Apical export of Cl- by CFTR maintains luminal concentration of what?
H2O
Opening of the CFTR channel requires what two signals?
- protein kinase A,(PKA) → phosphorylates R domain
- 2 ATPs bind NBD domains
What are the five steps in the regulation of the CFTR channel?
- External stresses → activate cAMP
- Cyclic AMP intercellular signaling pathway
- Activation of PKA
- Phosphorylation of R domain
- ATP binding
Why is it important that > 1900 mutations have been identified in cystic fibrosis, only 5 mutations occur in > 1% of cases, and about 160 mutations account for > 95% of cases?
- Mutation based diagnostic tests are feasible
- Possible to design diagnostic tests to identify mutations
- Molecular based therapies are feasible → possible to design molecular based therapies
What are the major classifications of CFTR mutations?
- More Severe:
- Class 1 = no protein produced
- Nonsense mutation creates stop codon
- Class 2 = defective protein folding
- degradation of protein
- Class 3 = defective gating or regulation of
- channel opening
- Class 1 = no protein produced
- Less Severe:
- Class 4 = defective in ion transport
- Class 5 = normal CFTR produced but decreased amounts
What specific CF allele is associated with Class 1 CFTR mutations and is due to a nonsense mutation that creates stop codon resulting in no protein produced?
G542X
(5% of CF alleles)
What specific CF allele is associated with Class 2 CFTR mutations and is due to defective protein folding which results in degradation of protein?
F508del
(70% of CF alleles)
What specific CF allele is associated with Class 3 CFTR mutations and is due to defective gating or regulation of channel opening?
G551D
(4% of CF alleles)
For the more severe CFTR mutations like Class I, II, III; what percent of CFTR activity remains?
≤ 1% CFTR activity remaining
For the more severe CFTR mutations like Class I, II, III; how early is typical diagnosis?
usually in first year
For the more severe CFTR mutations like Class I, II, III; what is the median survival?
37.4 years
For the more severe CFTR mutations like Class I, II, III; what are the associated health problems?
- Pancreatic insufficient
- At risk for CF-related diabetes, liver disease
For the less severe CFTR mutations like Class IV and V; what is the percentage of remaining CFTR activity?
about 5% CFTR activity remaining
For the less severe CFTR mutations like Class IV and V; when is the typical diagnosis?
May have late presentation
For the less severe CFTR mutations like Class IV and V; what is the median survival?
Survival to 50 yoa not uncommon
What is the result of the CFTR mutation G542X?
- Mutation creates stop codon at amino acid 542
- Results in:
- Decreased mRNA and/or non-functional protein
- Results in:
What is the result of the CFTR mutation G551D?
- Ion channels constantly move between open and closed conformations
- Mutation makes it unable to respond to ATP (opening signal)
- Results in = greatly increased time in closed conformation
What is the result of the CFTR mutation F508del?
- Almost complete loss of functional protein
- Folding defect leads to degradation of most
- Small fraction of protein at cell surface is defective in gating, stability
What is the most common CFTR mutation that is responsible for 70% of alleles, 50% homozygous, and 40% one allele?
F508del
What is the principle defect in the F508del mutation?
folding
Why is the synthesis of CFTR protein considered a complex process?
- Takes place at the endoplasmic reticulum (ER) membrane
- Requires intra-domain folding and inter-domain interactions
- Requires interaction with series of chaperones
What does incorrect folding ultimately result in?
(Hint: F508del)
- altered interaction with chaperones
- retention in the ER and activation of quality control pathways
- degradation by the proteasome
Can you predict the severity of disease based on which mutation class the cystic fibrosis is classified?
NO
- Mutation class does not predict severity of disease in individuals
- All mutations in “Severe” category cause defect in pancreatic ducts → pancreatic enzyme insufficiency
- Those with “Severe” mutations tend to have worse pulmonary disease
- But factors that affect immune system and development of fibrosis also affect outcome
- “Severe” mutations are necessary but not sufficient for development of CF-related diabetes and liver disease
How does loss of chloride ion channel function cause manifestations of cystic fibrosis?
- CFTR is essential to maintain protection:
- Expansion of mucin
- Luminal epithelial surface is protected from pathogens by mucin layer and by movement of cilia
- Maintains airway surface liquid volume (ASL) which helps in clearance of mucus
- Expansion of mucin
- In absence of CFTR: mucin is descicated, ineffective barrier
Why does inactivation of CFTR affect respiratory function?
Loss of luminal H20 and ion balance
Why does inactivation of CFTR affect respiratory function?
CF is characterized by unresolved, excessive and ineffective inflammation.
- Decreased ASL (water layer) → ineffective mucin barrier failure to clear bacteria
- Constant activation of inflammatory pathways in airway epithelial cell
- LPS > TLR4 > MyD88 > NFκB
- Increased mucin secretion (which is ineffective)
- Inflammatory mediators (IL-1β, TNF-α, IL-8) → Neutrophil recruitment
- Neutrophils release elastase → Damage CFTR itself and epithelial cell
- DAMPs stimulate further inflammatory signaling
What is the pathology behind cystic fibrosis?
- Decreased ASL(water) layer → build up of viscous mucous, failure to clear bacteria
- Characteristic CF bacterial infections- Stapholococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia
- Persistent colonization and desiccated mucus → inflammation
- Damaging agents- free radicals, ROS, proteases, Increased mucus production
- Infection, inflammation and mucus plugging → long term structural damage
- bronchiolectasis, bronchiectesis
- Ultimate outcome is respiratory failure
What is the key clinical issue in cystic fibrosis?
- Airway infection:
- Infection appears early → 40-60% pediatric patients have Pseudomonas aeruginosa
- Tend to be pathogens not found in non-CF population
- Haemophilus influenzae
- Staphylococccus aureus MRSA
- Pseudomonas aeruginosa
- Burkholderia cepacia
- Difficult to eradicate
What is the goal in treatment of CF? How is it accomplished?
- Goal = prevent pulmonary damage
- Chest physiotherapy to improve drainage
- Aggressive treatment of infection (antibiotics)
- Hypertonic saline and Dnase aerosols to make mucus less viscous
- Anti-inflammatories
What is the pathology behind pancreatic duct blockage in CF patients?
- Epithelial cells lining pancreatic duct express CFTR
- Secrete HCO3-
- Luminal HCO3- recruits H2O and neutralizes secretions
- Prevents formation of protein plugs in pancreatic duct
- Loss of CFTR function → blockage of duct by secreted protein plugs
- Leakage of proteolytic enzymes, inflammation → destruction of tissue
What percent of people with CF have pancreatic duct damage?
Pancreatic duct is damaged in ~100% of all CF individuals!
~85% require replacement therapy to prevent malabsorbtion of lipids, fat-based vitamins
(Pancreatic phenotype is directly dependent on severity of genetic mutation)
How is CF related to diabetes?
- Pancreatic duct related inflammation leads to destruction of pancreatic islet cells that make insulin
- (not autoimmune process, INFLAMMATORY process!)
- CF related diabetes is primarily due to loss of ability to make insulin
- Insulin resistance may also contribute as a result of chronic systemic inflammation
How common is CF-related diabetes?
CF related diabetes affects ~20% of CF individuals below 20 yoa
What are the intestinal manifestations of CF?
- CFTR is expressed in crypts of intestinal epithelium
- Loss of function → failure to secrete H2O
- leads to dehydration of lumen, viscous mucus as in airway epithelium
- Loss of function → failure to secrete H2O
What are the classical intestinal conditions that CF patients may present with?
- Obstruction: meconium ileus
- occurs in 15% of newborn infants with CF
- only occurs if two “Severe” alleles
- Distal intestinal obstruction syndrome (DIOS) in older individuals
- Inability to absorb protein & fats
- most require nutritional supplementation
- Long term → individuals with CF have increased susceptibility to GI cancers
What is the pathology behind CF-associated liver disease?
- CFTR expressed in cholangiocytes that line the bile duct not hepatocytes or other liver cells
- CFTR is required to maintain luminal environment
- Absence leads to retention of toxic bile acids (taurocholic acid)
- Toxic bile acids build up → promote synthesis & secretion of MCP1 from both hepatocytes and cholangiocytes
- Recruitment and activation of hepatic stellate cells leads to fibrogenic myofibroblasts
- Excess collagen → fibrogenesis
How often does CF associated liver disease occur?
CF associated liver disease occurs 5-10% of CF individuals
(median age of diagnosis is 10 yoa)
Why does male infertility occur as a result of CF?
- Vas deferens is absent in virtually all male CF individuals
- becomes obstructed in the fetus or during infancy and is then reabsorbed
What is the basis of the “Sweat Test” for diagnosing CF?
- In sweat glands the CFTR promotes uptake of Cl- ions from the extracellular space with Na+ following
- decreased ability to absorb Cl- and Na+ results in increased NaCl in sweat
How is molecular genetic knowledge used for better testing and treatment?
- Widespread screening
- New treatments to correct specific mutation defects
How does molecular genetics make widespread screening of CF feasible?
- The gene that is mutated is known
- The DNA sequences of most mutations that cause the disease are known
- A panel of 23 mutations that account for 90% of mutations in northern European Caucasians and 70% of mutations in African Americans has been developed into a clinical test
Who should be screened for CF?
- Carrier screening
- ACOG recommendation is to offer genetic carrier screening to ALL individuals planning a pregnancy or seeking prenatal care
- Carrier screening- testing of family and more distant relatives of those with CF
- Prenatal testing
- Offered when 25% estimated chance of CF (two carriers)
- Screening of ALL newborns
- Diagnostic testing
What is the CF risk in ANY pregnancy?
1/2500 (in any pregnancy)
What is the detection rate of CF in genetic testing?
With genetic testing → 90% detection rate
What are the forms of prenatal testing used if both parents are carriers or parents have a previous child with CF (25% chance of CF)?
- Chorionic villus sampling 10-12 weeks for DNA mmutation testing
- Amniotic fluid 16-17 weeks for DNA mutation testing
- Also ultrasound for echogenic bowel
Why does early detection of CF in newborns make a difference?
- Nutrition:
- pancreatic enzyme replacement
- Pulmonary function:
- aggressive treatment to prevent obstruction & infection
What is the typical screening protocol in Newborn Screeing of CF?
- IRT immunoreactive trypsin = test of pancreatic function
- Part of panel done on heel stick blood sample. (screening step 1)
- DNA mutation analysis = based on panel representing vast majority of disease causing mutations (screening step 2)
- Sweat test = test of CFTR function (diagnostic)
What is the definitive diagnostic test for CF?
Sweat Test
- Sweating is induced by chemical treatment on skin
- Sweat released over 30 min is absorbed onto detective pad
- Cl- content is measured
(Testing of all family members is also done)
What are the current treatment strategies for CF?
- Aggressive, meticulous treatment of all possible manifestations
- Major objectives:
- Lungs- clear secretions, control infections
- Maintain nutrition
- Prevent intestinal obstruction
- Carried out by multidisciplinary care team in CF Centers accredited and funded by the Cystic Fibrosis Foundation
How successful have multidisciplinary care teams in CF Centers accredited and funded by the Cystic Fibrosis Foundation been at treating CF?
- 1955 → Most children with CF did not enter elementary school
- 2013 → Median predicted age of survival ~40
What are two new treatment strategies in CF?
- Lung transplantation- 60% survival after five years
- Described as way to extend life, not as cure
- Drugs to target specific deficiencies of CFTR mutations
What drugs were recently developed to correct specific CFTR mutations?
- Ivacaftor (Kalydeco)
- corrector for G551D
- Ataluren
- corrector for G542X
- Ivacaftor + Lumacaftor
- Lumacaftor is a corrector for F508Δ
How does Ivacaftor (Kalydeco) treat CF?
- Corrector for G551D
- Keeps channel in open conformation for higher percent of time, even without ATP gating
- FEV1 increase ~10%
- FDA approved Jan. 2012
- $300,000/y
How does Ataluren treat CF?
- Corrector for premature stop codon mutations (G542X)
- Causes ribosome to be less sensitive to stop codons
- Phase 3 clinical trials show some improvement, but not statistically significant
How does Ivacaftor + Lumacaftor treat CF?
- Lumacaftor is a corrector for F508Δ, promotes traffic of protein to the cell surface instead of degradation @ proteasome
- Combination → 4-7% improvement in FEV1
- Decrease in severe exacerbations
- FDA approval July, 2015
- ~$275,000/y
