Cystic Fibrosis

cystic fibrosis

most common fatal genetic disease in caucasian population

caused by loss of function mutations in gene encoding CFTR protein

autosomal recessive disease

deficient epithelial anion (Cl-) permeability

multiple organs affected

lung disease currently accounts for most of the morbidity and mortality

estimated that one in every 3600 children born in Canada has it, one in 25 Canadians carry one defective copy of the gene (carriers not affected)

CF airways

mucopurulent material, mucus plugging of smaller airways

CF and sweat glands

decreased reabsorption of NaCl by water-impermeable ductal epithelial cells leads to elevated sweat Cl-

pancreas and CF

approx 95% patients have some degree of dysfunction

enzyme insufficiency= malabsorption of fat and protein

small intestine and CF

meconium becomes thickened and congested in ileus—results in blockage

CFTR gene

encodes a cyclic AMP-regulated, ATP-gated Cl- channel

located on chromosome 7

CF symptoms

persistent cough with productive thick mucus

wheezing and shortness of breath

frequent chest infections, which may include pneumonia

bowel disturbances, such as intestinal obstruction or frequent oily stools

weight loss or failure to gain weight despite increased appetite

salty tasting sweat

infertility (men) and decreased fertility (women)

cystic fibrosis transmembrane conductance regulator (CFTR)

member of a large family of membrane transport proteins: ATP-binding cassette (ABC) transporters

ATP binding and hydrolysis by cytoplasmic NBD dimer is coupled to opening and closing of a single anion channel pore formed by the MSDs

ATP-binding cassette (ABC) transporters

their proteins have two nucleotide binding domains (NBDs) that dimerize in order to bind and hydrolyze ATP, plus two membrane spanning domains (MSDs) that come together to form a pathway for transmembrane substrate transport

membrane spanning domains (MSDs)

unique ABC protein, doesn’t use ATP to power active transport

what regulates CFTR channel activity?

phosphorylation of a cytoplasmic regulatory domain (R domain), a region that is unique structurally to this channel

CFTR function

cAMP-mediated anion transport

in addition to Cl-, also transports HCO3-, glutathione, I-, and SCN- (thiocyanate)

regulates ion and water balance across epithelia, interacts with other ions channels (e.g. ENaC)

where are CFTRs present?

at apical membrane of epithelial cells

absence of functional CFTR results in

increased viscosity of secretions

ciliary dysfunction

mucus dehydration

mucus impaction

failure of MCC

repeated infections by opportunistic pathogens

neutrophilic inflammatory response

tissue damage

respiratory insufficiency

linked to over-activity of ENaC

most common CFTR mutation

F508del-CFTR, a deletion of phenylalanine at position 508

accounts for over 70% of all mutant alleles for class II

CFTR mutation class I

completely absent functional CFTR protein

• no functional CFTR protein made usually due to stop codon resulting in premature protein truncation

mutations: nonsense, frameshift, canonical splice

examples: Gly542X, Trp1282X, Arg553X, 621+1G→T

CFTR mutation class II

absent functional CFTR due to trafficking defect (protease destruction of misfolded CFTR)

• missense mutations and in-frame deletions disrupt CFTR folding and trafficking to surface, degraded in golgi

mutations: missense, amino acid deletion

examples: Phe508del, Asn1303Lys, Ile506del, Arg560Thr

CFTR mutation class III

defective channel regulation, perhaps by nascent CFTR post-ER

• defective channel regulation or gating

mutations: missence, amino acid change

examples: Gly551Asp, Gly178Arg, Gly551Ser, Ser549Asn

CFTR mutation class IV

decreased channel conductance, perhaps by nascent CFTR which leads to defective CFTR channel

• defective Cl- conductance; misshapen CFTR restricts movement of Cl-

mutations: missense, amino acid change

examples: Arg117His, Arg347Pro, Arg117Cys, Arg334Trp

CFTR mutation class V

reduced synthesis of CFTR, scarce functional CFTR (usually via production of incorrect RNA)

• reduced amount of CFTR usually due to alternative splicing that disrupts mRNA processing

mutations: splicing defect, missense

examples: 3849+10kbC→T, 2789+5G→A, 3120+1G→A, 5T

CFTR mutation class VI

decreased CFTR stability in membrane, life cycle much shorter

• increased turnover of CFTR at cell surface

mutations: missense, amino acid change

examples: 4326delTC, Gln1412X, 4279insA

newborn screening for CF

test most common 20-30 mutations

these programs demonstrated to reduce disease severity and cost of care

prevent delayed/missed diagnoses

MCC in CF airway

in absence of CFTR, unrestrained Na+ hyperabsorption occurs with loss of Cl- secretion, result is decreased PCL and failure of this

PCL regulation by active ion transport- in excess

Na+ absorption via ENaC is dominant

Cl- is absorbed passively via the paracellular pathway

PCL regulation by active ion transport- PCL volume low

ENaC is inhibited which makes the apical membrane potential more negative, generating a driving force for Cl- secretion

abnormal ion transport in CF results in

mucostasis

mucostasis

loss of functional CFTR leads to increased Na+, Cl-, and water absorption across large airways

absence of CFTR releases ENaC from tonic inhibition

lack of PCL results in this and formation of thickened mucus

plaques and plugs which adhere to CF airway surfaces

experimentally, CF airway epithelia excessively absorb

ASL, deplete PCL, los ciliary-dependent mucus transport

CFTR and HCO3-

transport this, leading to changes in pH of ASL

bacterial killing highly dependent on pH so a change may result in reduced functioning innate antimicrobial peptides

airway mucus also dependent on pH for normal functioning, so alterations with reduced anion concentrations might cause defects in mucus tethering and detachment, increased viscosity

CFTR and glutathione

transports this, less of this important antioxidant in ASL to deal with oxidative stress

CF and excessive inflammatory/neutrophil response

get this in absence of infection

S aureus and P aeruginosa common pathogens

recurrent and persistent bacterial infections

cytokine response (IL-8) leads to (this immune cell) recruitment to airways

although recruited to combat pathogens, activation can damage surrounding lung tissue via release of oxidants and proteases→loss of lung function

CFTR-deficient cells exhibit

innate pro-inflammatory state

higher concentrations of pro-inflammatory mediators (IL-6, -8, -1 beta) with decreased levels anti-inflammatory IL-10

signaling abnormalities and aberrant intracellular processes, with increased transcription of pro-inflammatory mediators

excessive activation of transcription factors such as NFkB and AP-1

CF neutrophils have reduced

phagocytic capacity

on death, CF neutrophils release

DNA, which increases mucus viscosity and makes everything worse

oxidases with increase oxidative stress

result is bronchiectasis

net result of exaggerated pro-inflammatory signaling/failure to clear bacteria is

accumulation of PMNs and their products

constitutive activation of NFkB signaling results in

increased amount of ROS generated by neutrophils

increased levels of ROS associated with

increased IL-8 production, defective autophagy, and reduced CFTR expression

antioxidant expression in CF

impaired

GSH secretion reduced, as CFTR dysfunction impairs trafficking of GSH into the ASL via CFTR

thiocyanate (SCN-, potent) reduced trafficking via defective CFTR

result is decreased ability to kill opportunistic pathogens such as P aeruginosa and S aureus

increased oxidative stress in CF airway

constitutive activation of NFkB, mediates production of proinflammatory chemokines (IL-8)

PMNs recruited and persist in airway, release ROS in effort to kill bacteria, ROS burden increased

all activates MAPK signaling, which increases IL-8 production→more PMNs

defective CFTR unable to transport GSH and SCN- into ACL, limiting cell’s ability to neutralize stress

therapeutic strategies in CF

1. broad-based inflammatory modulators

2. antibacterials with anti-inflammatory properties

3. modulators of intracellular signaling

4. inhibitors of neutrophil influx

5. inhibitors of neutrophil products

6. anti-oxdants

7. anti-proteases

8. hyperosmolar agents

9. CFTR potentiators

10. CFTR correctors

11. gene therapy to replace CFTR

therapeutic strategies in CF- broad-based inflammatory modulators

corticosteroids, ibuprofen

therapeutic strategies in CF- antibacterials with anti-inflammatory properties

azithromycin

therapeutic strategies in CF- modulators of intracellular signaling

interferon gamma

therapeutic strategies in CF- inhibitors of neutrophil influx

anti-IL-8, CXCR2 antagonists

therapeutic strategies in CF- inhibitors of neutrophil products

dornase-alfa (recombinant human deoxyribonuclease I, which cleaves DNA)

therapeutic strategies in CF- antioxidants

N-acetyl cysteine

therapeutic strategies in CF- anti-proteases

alpha1-protease inhibitor

therapeutic strategies in CF- hyperosmolar agents

hypertonic saline, mannitol

therapeutic strategies in CF- CFTR potentiators

increase activity of defective CFTR at cell surface (apical) either by acting on gating defects or conductance defects

improve Cl- transport through CFTR: Ivacaftor (Kalydeco)

useful for gating mutations, such as G551D (4-5%)

clinical benefits: reduced sweat Cl-, increased FEV1, weight gain, improved quality of life

therapeutic strategies in CF- CFTR correctors

overcome defective protein processing that normally results in production of misfolded CFTR, allows increased trafficking of CFTR to plasma membrane

rescue the trafficking defect, e.g delta F508

Lumacaftor

therapeutic strategies in CF- gene therapy to replace CFTR

ineffective to date

viral vector receptors located in basolateral cell membrane; repeated administration of viral vectors hindered by development of antibodies

adenovirus-associated vectors cannot hold genes as large as CFTR

G551D mutation

most common in class III, channel gets to cell surface and fails to open correctly

potentiators increase open probability of channel

Lumacaftor

corrector, target class II defects, the most common

deltaF508 carried by approx 90% of CF population, 50% homozygous, 90% heterozygous

acts as a molecular chaperone during protein folding, only modest effects on sweat Cl-

combination therapies

new wave, combination of lumacaftor/ivacaftor (Orkambi): for patients with 2 copies of F508-del-CFTR mutation

trikafta (elexacaftor/ivacaftor/tezacaftor): for patients 12+ with CF who have at least one F508del mutation

elexacaftor is a second generation corrector, tezacaftor is a potentiator

production correctors

theoretical, promote read-through of premature termination codons in mRNA which results in truncated and often non-functional protein

5-10% of CF-causing mutations

agent that suppress normal proofreading function of ribosome result in full length protein despite PTC, se generating more CFTR protein production

e.g. atalurin for class I mutation