Cystic Fibrosis Flashcards
What is the leading impact to CF?
Abnormally thick mucus
* produced by malfunction in transporting salt ions (Cl-) across epithelial cells lining duct
What are the clinical features affecting the RESPIRATORY system?
Frequent Coughing, Chronic Infections & Lung Damage
DUE TO:
* obstruction of bronchioles by mucus
* colonisation of bacteria (antibiotic-resistant strains)
* damage by inflammatory responses
What are the clinical features affecting the REPRODUCTIVE system?
Infertility in males & SUB-infertility in females
DUE TO:
* blockage of vas deferens = leads to fibrosis or atrophy
* cervical mucus in females = barrier of passage of sperm
* females may be anovulatory
What are the clinical features affecting the GASTROINTESTINAL system?
Poor Growth & Chronic Malabsorption
DUE TO:
- blockage of pancreatic ducts by mucus
- poor digestion of fats & proteins
What chromosome is the CFTR gene on?
7
What does the CFTR gene code for?
Ion channel protein & CFTR protein
ALSO:
* full transporter of four canonical domains === linked to single polypeptide chain
What processes is the CFTR anion pore gating regulated by:
- cAMP-dependent (Protein Kinase A) phosphorylation
- ATP binding
What are the domains of the CFTR protein?
4 Canonical Domains
* Membrane Spanning Domains (MSD - 1&2)
* Nucleotide Binding Domains (NBD - 1&2)
Regulatory Domain (R)
What is the role of the NBDs?
- bind & hydrolyse ATP
- ATP binding → channel OPENS
- ATP hydrolysed → channel CLOSES
What is the role of the MSDs?
- forms channel for passage of Cl-
What is the role of the R domain?
- Phosphorylated by cAMP
- Phosphorylation leads to fine tuning of channel function
How are the 4 Canonical Domains and R Domain linked?
They are linked into a single polypeptide chain
How do interactions between the MSDs & NBDs occur?
Via coupling helices
What do the channel opening & closing require in the CFTR molecule mechanism?
- Opening: Phosphorylation & Nucleotide (ATP) Binding
- Closing: Hydrolysis
What is the 2-step activation process in CFTR molecular mechanism?
- PKA phosphorylation
- ATP binding
DETAILED:
1. R domain blocks channel but can disengage and allow channel to be turned on by PKA adding P group which acts as a temporary lock and allows channel to flow freely.
2. ATP binding occurs
3. Eventually, dephosphorylation of P occurs and blocks channel again & resets to allow ^^ again
What is the gating cycle of the fully
phosphorylated CFTR channel?
-
Flow of Cl- ions = ATP-bound closed channels open to pre-hydrolytic open state
* ATP-induced dimerisation of NBD 1&2 = conformational changes in MSD 1&2 - Channel closure == ATP → ADP = NBD dimer disruption
What happens to the NBD domains (NBD 1 & 2) in the presence of ATP?
Relates to the dimers
- NBD 1 & 2 combine head-to-tail forming dimers.
- Two ATP molecules are trapped between these dimers.
What are the differences between NBD1 and NBD2?
- NBD1: head region has a “degenerate site” for ATP (weak binding, upper site 1)
- NBD2: tail region has a “catalytic site” for ATP (strong binding, lower site 2)
Explain what the 2 ATP is used for in NBD1 & NBD2
- both ATP need to bind
THEN:
- ATP1 undergoes hydrolysis = proper gating
- ATP2 either remain bound (for rapid opening/closing) OR dissociate (so CFTR is more stably closed)
How does the R domain affect the channel?
Interaction depends on phosphorylation state.
- dephosphorylated = wedges itself between the two halves of the channel = prevents NBD dimerisation & channel opening.
- phosphorylated = adopts a permissive state = allows the channel to function.
What part of the CFTR protein interacts with other cellular proteins?
C-terminus
* acts like an anchor
Why is the CFTR protein anchored to the cytoskeleton?
To be positioned near other proteins that influence its functions.
List some of the functions influenced by proteins interacting with CFTR.
- Conductance (regulating flow of ions)
- Regulation of other channels (e.g., ENaC channel)
- Signal transduction (cellular communication)
- Localisation at apical plasma membrane (positioning)
How does the CFTR function in NORMAL vs CF airways?
Lungs
Normal
* CFTR regulates movement of Cl- and Na+ ions.
* Balanced movement keeps airway surface liquid (ASL) hydrated
* Active transport: ENaC channels move Na+ into cells.
* Passive movement: Cl- follows Na+ out, maintaining hydration.
CF
* Defective CFTR disrupts ion regulation.
* Uninhibited ENaC causes excessive Na+ absorption.
* No functional CFTR for Cl- movement out of cells.
* ASL becomes depleted, hindering mucus clearance.
How does the CFTR function in NORMAL vs CF sweat ducts?
Normal
- Cl- ions, Na+ ions & water move into cell
- Sweat secreted in duct by gland
- Na+ & Cl- reabsorbed by duct cells before reaching skin surface
CF
- Cl- ions unable to enter cell == Na+ ions and water remain in sweat ducts
- resulting in elevated Na+ & Cl- levels in sweat
How does CFTR function in NORMAL vs CF pancreas?
Normal
- secretes bicarbonate
- bicarbonates moves into pancreatic duct
- mechanism: directly via CFTR & via bicarbonate chloride exchangers
CF
- absence of CFTR-dependent bicarbonate & Cl- secretion = abnormal acidic & low volume secretions = activation of proteolytic enzyme within gland
- == pancreatic inflammation & destruction
What are the CFTR gene variant classes?
6 classes
- Class I: No CFTR produced
- Class II: Defective processing - misfolds = trafficking defect
- Class III: Defective regulation - channel gate doesnt open properly
- Class IV: Reduced ion conductance-function of channel is faulty
- Class V: reduced CFTR production
- Class VI: accelerated turnover from the cell surface = decreased CFTR stability
What are the variant class-specific therapies?
- Class I: Aminoglycoside antibiotics = allows ‘read through’ of mRNA
- Class II: ‘Correctors’ to improve processing
- Class III: ‘Potentiators’ to activate protein
- Class IV: Flavonoid compounts = augment channel function == increase open probability
- Class V: splicing variants = increase levels of correctly spliced RNA
What is the most common CFTR gene variant?
F508del
What class of mutation is F508del?
Class II - possible drug target
- severe folding defect == premature degradation of most of translated protein in ER
- Located in NBD 1
- Defective processing
What is happening in F508del?
- 508 Phenylalanine deletion
- 1 base from 507 Isoleucine and 2 bases from 508 Phenylalanine DELETED
What is the inheritance pattern of CF?
Autosomal recessive
What is the carrier frequency and the most affected ethnicity?
1 in 25 & northern Europe
What is the incidence of CF?
1 in 2500-3000 live births
What are the Heterozygote (carrier) selective advantage?
Protection against diseases - Cholera & Typhoid fever
Describe the CFTR genotype-phenotype correlation
Limited correlation - class of CFTR variant & phenotype
- Class I,II,III mutation = pancreatic insufficiency (strong correlation) & more severe lung disease
- Class IV,V mutation = pancretic sufficiency & milder lung disease
What is the penetrance of CF?
Complete penetrance for severe CF-causing variant = individuals carry these disease-causing variants on BOTH CFTR alleles == develop CF
Incomplete penetrance = certain combinations of underlying causativ CFTR variants
What is the expressivity of CF?
Variable expressivity
- phenotype associated with disease-causing variant = differs between individuals
- genetic & environmental modifiers contribute
What are the genetic testing for CF?
Diagnostic testing
- Confirmation of diagnosis in symptomatic individuals
Cascade testing
Newborn screening
Population carrier screening
What are the special groups of CFTR gene variants?
Incompletely penetrant variants
1. PolyT tract c.1210-12T[5_9] (intron8)
2. c.350G>A p.Arg117His (‘R117H’ in exon4)
- penetrance depends on polyT tract variant on same allele
State the disease-causing CFTR gene variant
On allelle 1 & 2 respectively.
LP/P variant (F508del) & R117H & 5T
For an individual with F508del variant on one allele and an R117H variant on the other, what ‘_‘T polymorphism is more likely to symptoms of CF
5T polymorphism
- maximises effect of R117H = act as disease-causing variant
NOT 9T polymorphism since:
- minimises effect
Describe the sweat test
- measures concentration of Cl excreted
- pilocarpine iontophoresis induces sweating
- pilocarpine = parasympathomimetic == acts on cholinergic receptors
- Normal value: Cl<40mmol/L (<30 in infant)
- CF diagnosis: Cl>60mmol/L
Describe Newborn screening
- Identifies babies at risk of developing CF
- Allows early and pre-symptomatic therapy
- RT test = heel prick onto blood spot cards (2-3 days old)
- Elevated IRT == further testing
- Genetic testing using known common LP/P gene variants == only 12 common
- both = CF
- heterozygous = further testing = sweat test
- none = no CF
Describe Cascade testing
- Identification of carriers who are relatives of a diagnosed individual
- Information can be provided about reproductive risk
- only 12% opt-in
Describe Population carrier screening
- Identification of carriers in the general population
- Pre-conception carrier screening for reproductive purposes
Define genetic modifiers in term of CF?
- influences phenotypeof CF BUT cannot cause CF
- e.g. can influence how severe CF symptoms are