Cystic Fibrosis

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Contents

Cystic Fibrosis

General background information

  • 1 / 2500 newborns (Caucasian)
  • Life expectancy of only 30 years

Mode of inheritance

  • Autosomal recessive
    • Most common autosomal recessive disease of Caucasian children.

Single important gene

  • CFTR (Cystic Fibrosis transmembrane conductance regulator)
  • 27 exons, 1480 amino acids
  • A chloride channel that is activated by cAMP
  • Over 1400 know mutations
    • 70% of cases are the deltaF508 mutant


  • Two transmembrane domains (MSD1 / 2) form a pathway for the Cl- ion.
  • Two intracellular domains (NBD1 / 2) flank the transmembrane domains and are important for processing and regulation of CFTR, respectively.
    • NBD1 and NBD2 are ATP-binding domains
  • R domain's position determines if channel is open or closed.
    • R's position is determined by phosphorylation via PKA.

Etiology

  • There is ethnic variation in the frequency of alleles and in the frequency of carriers and in the frequency of disease. 
Some polymorphisms?
  • deltaF508:
    • 70% of Caucasian disease-cuasing cases
    • A 3-base deletion: AT(CTT)T or A(TCT)TT; (Ile, Phe)
      • Last codon of 507 (aa) and first two of 508 (aa) or
      • Last two codons of 507 (aa) and first one of 508 (aa)
    • Either way, it removes the phenylalanine at position 508


  • Mutations are classified:
    • Class 1: protein is completely absent
      • Mutations in the MSD1 exon cause a splicing defect and complete absence of the protein
      • Class 1 mutations are commonly nonsense mutations
      • Includes G542X and R553X; drastically decreases mRNA levels for CFTR
    • Class 2: processing of the protein is defective
      • Mutations of the NBD1 domain cause defective processing
      • Contains deltaF508 mutation (70%)
      • Can result in abnormal protein folding and lack of CFTR at cell membrane
      • Can result in poor regulation of ORCC by CFTR
    • Class 3: regulation of the protein is defective
      • Mutations of the NBD2 domain cause defective regulation
    • Class 4: conduction of the Cl- ion is defective
      • Mutations of the MSD1 domain cause defective Cl- transport


  • CFTR is broken in cystic fibrosis.
  • CFTR's borked phenotype also affects ORCC and ENaC, too.
    • ORCC is the outwardly rectifying chloride channel
    • ENaC is the amilioride-sensitive sodium channel (epithelial Na channel)

CFTR and Intron 8 / Exon 9

  • Intron 8 of the CFTR gene contains a stretch of Thiamines (Ts)
  • When the stretch of Ts is 7, or 5 nucleotides long, exon 9 is likely to get spliced out.
    • When 9Ts are present, 100% of mRNA transcripts include exon 9.
    • When 5Ts are present, 10% of mRNA transcripts include exon 9.

Pathogenesis

  • Onset can be neonatal to adult.
  • Life expectancy is 30 years.
  • Boys live longer than girls


  • Other factors that can affect the phenotype include:
    • Severity of the CFTR polymorphisms (could be one or many, could be missense or exon loss)
    • Modifying loci exist
    • Environmental factors


  • Phenotypic variability is seen in CF.
    • There are "classic" and "nonclassic" presentations of cystic fibrosis.
    • Pancreatic sufficiency (PS) and pancreatic insufficiency (PI) can each occur in CF pts.


  • Modifier genes and environmental factors have role in severity of disease and involvement of the lungs
    • Think about genes associated with inflammation and infection
    • TGF-beta, MBL2 (mannose-binding lectin 2), others
    • Exposure to second hand smoke and how TGFB1 and CFTR respond has been shown to be important

Genotype-Phenotype Correlation

  • The genotype strongly predicts the pancreatic phenotype in cystic fibrosis
    • DeltaF508 and null alleles generate PI (pancreatic insufficiency)
    • DeltaF508 generates elevated sweat chloride
    • Partially active alleles generate PS (pancreatic sufficient) phenotypes


  • The genotype poorly predicts pulmonary phenotype in cystic fibrosis
    • DetlaF508 may cause mild to severe pulmonary disease
    • Reasons are not completely clear
    • Perhaps environmental?


  • The genotype does not correlate with meconium ileus, DIOS, liver disease, or diabetes at all in cystic fibrosis
    • DIOS is distal intestinal obstruction syndrome
    • NB: these almost never occur in the presence of a PS mutation

Phenotypic information

  • Pleomorphic in nature, affecting respiratory, gastrointestinal, and many other systems
  • Decreased life expectancy (30 years)


  • Respiratory:
    • Primary cause of death
      • Airways get congested and cause death
    • Chronic cough
    • Recurrent infections
    • Bronchiectasis (chronic dilation of bronchi)


  • Gastrointestinal
    • Meconium ileus (thick meconium congestion)
    • Steatorrhea (fatty stools)
    • Failure to thrive
    • Recurrent pancreatitis
    • Neonatal jaundice
    • Liver cirrhosis
    • hepatic failure is second most common cause of death


  • Other:
    • Endocrine: diabetes mellitus
    • Musculoskeletal: clubbing
    • Reproductive: absence / aplasia of vas deferens, infertility
      • NB: 95% of males are infertile
    • Integumentary: elevated sweat chloride (salty baby syndrome); >60 mEq / L (60 mmol / l)


CF in the airway epithelium

  • Healthy state:
    • Beta agonist elevates cAMP levels.
    • cAMP activates PKA.
    • PKA phosphorylates R subunit of CFTR.
    • Chloride is actively moved into the lumen of the airway.
    • Na follows passively between cell tight junctions.
    • H20 follows passively through cell membranes.
  • Disease state:
    • Beta agonist elevates cAMP levels.
    • cAMP activates PKA.
    • No CFTR is present for PKA to phosphorylate (activate).
    • Chloride cannot be moved into the lumen because CFTR is borked.
    • Na doesn't follow.
    • Very little water ends up in the lumen.


  • Reduced chloride secretion lead to a depletion of airway surface liquid (dehydration of lung surface)
  • Mucus layer of the lung may become adherent to cell surfaces and disrupt the cough and cilia dependent clearance of mucus
  • Mucus obstructs airflow, provides a niche favorable to pathogenic organisms, inhibits function of antimicrobial peptides
  • Inflammatory response releases cytokines and enzymes that damage bronchials
  • Recurrent cycles of infection, inflammation, and tissue destruction decrease amount of functional lung tissue and lead to pulmonary disease and respiratory failure
    • Sometimes the body just builds a wall around the infection and thus tissue dies.
  • Bronchiectasis
    • injury to lungs in which airways are stretched out, scarred, and can no longer move air in or out
    • Ecta means "stretched out" in Greek.
  • Lung disease is more severe when exposed to environmental factors such as cigarette smoke

CF in the pancreas

  • Deficient secretion of pancreatic enzymes
    • lipase, trypsin, chymotripsin
    • The trypsinogen test looks for elevated trypsinogen which indicates abnormal pancreatic activity ref. This test is followed up with genetic testing.
  • Loss of CFTR chloride transport into pancreatic ducts leads to the retention of exocrine enzymes in the pancreas.
  • Retention of enzymes causes fibrosis of the pancreas
  • Normal digestion can be restored through pancreatic enzyme supplements
  • 5-15% of patients are pancreatic sufficient
    • have enough pancreatic exocrine function for normal digestion have enough pancreatic exocrine function for normal digestion
    • have better growth and overall prognosis
  • Some individuals with idiopathic chronic pancreatitis carry mutations in CFTR and lack other clinical signs of CF
    • Idiopathic means the etiology is different from person to person; "we don't know why that is happening."


CF sweat phenotype

  • Sweat sodium and chloride concentrations are increased
  • Loss of CFTR leads to no reabsorbing of chloride in sweat gland duct
    • Causes reduction in electrochemical gradient that drives sodium entry across apical membrane and increased chloride concentration in sweat
  • Sweat test used to diagnose CF
    • small electrode is placed on the skin to stimulate the sweat gglands
    • amount of chloride is measured
  • > 60 mmol/L = CF
  • 40 mmol/L and 60 mmol/L are borderline
  • < 40 mmol/L are considered negative for CF
  • Sweat is normal in 1-2% of patients with CF
  • So this is clinical / phenotypic heterogenity.

CF vas deferens phenotype

  • 95% of males with CF lack vas deferens
    • congenital bilateral absence of the vas deferens (CBAVD)
    • Caused by problem in development of Wolffian duct
  • Females have some reduction in fertility
    • Some otherwise well males have CBAVD Some otherwise well males have CBAVD associated with mutations in CFTR
    • May be associated with heterozygous or homozygous CFTR condition


Diagnosis

  • 23 mutations are recommended for testing / screening
    • 9 are intronic mutations, (the rest are exonic mutations) 8 are missense, 4 are nonsense, and 2 are in-frame deletions (like deltaF508)

Treatment

  • Therapy depends on the nature of the defect.
  • We have gotten better at treating CF over the last four decades.
  • A better understanding of the disease pathogenesis enables better therapy
  • Remember that CF is a multi-system disease (pleomorphic)
  • It has been shown that pts "do better" when treated by a multi-discipline team
  • Treat the symptoms, whether related to CFTR issues or not


  • Pulmonary therapies (problem:correction)
    • absent CFTR : use CFTR modulators and correctors
    • decreased airway surface liquid : restore ion transport
    • disrupted mucocilliary clearance : mechanically clear airway of mucus
    • colonization and chronic infection : apply antibmicrobials
    • neutrophil-dominated inflammation : apply anti-inflammatory therapy


  • Aminoglycoside therapy
    • First, aminoglycosides act as antibiotics
      • Effective agains pseudomonas aerruginosa (a particularly important pathogen in CF pulmonary issues).
    • Second, for nonsense mutations, high doses of aminoglycosides can increase read-through.
    • Some studies show an increase in CFTR function under high doses of aminoglycosides in nonsense mutation CF pts.
    • Gentamicin is an example of an aminoglycoside being used.
    • Note that there is some toxicity to these high doses.
    • We are looking for more effective drugs like ataluren and PTC124


  • Other treatments:
    • Improve trafficking of CFTR with chaperones, correctors, and PBA (sodium-r-phenylbutyrate
      • Chaperones: help with folding 
***Correctors:
***PBA:
    • Improve channel function with potentiators
      • Help the channel move Cl- appropriately
      • VX-770 as an example
      • Given orally
      • Shown to help for at least one known mutation: G551D

Recent research

5 important facts

Not to be confused with

  • Several associated "monosymptomatic disorders": CBAVD (congenital bilateral absence of vas deferens), recurrent idiopathic pancreatitis


  • Congential bilateral absence of vas deferens (CBAVD)
    • Causes 2-5% of male infertility
    • 70% have >1 CF mutation
    • Can be a deltaF508 carrier
    • 33% have one CF mutation and a 5T variant
    • 20% have one CFTR mutation
    • 20% have two CFTR mutations
    • 1% have two 5T variants
    • May not develop pulmonary disease at all
    • As with CF, modifier genes and environmental factors have role in severity of disease and involvement of the lungs


Questions and answers

Cystic fibrosis transmembrane conductance regulator (CFTR)

  • Found at 7q31
    • Spans 190kb of DNA
    • 27 exons in codingg reggion
    • Encodes large integral membrane protein of about 170 kD
  • Chloride channel located in the apical membrane of the epithelial cells
  • Five domains
    • Two membrane spanning domains (with 6 transmembrane sequences)
    • Two nucleotide (ATP) binding domains
    • A regulatory domain with multiple phosphorylation sites
  • Pore of chloride channel formed by the 12 transmembrane segments
  • ATP is bound and hydrolyzed by nucleotide binding domains, energy released is used to open and close channel

CFTR functions as apical epithelial chloride channel

  • Dysregulated sodium transport is also important in pathophysiology of CF
  • cAMP-regulated chloride channel that regulates other ion channels
  • Maintains hydration of secretions within airways secretions within airways and ducts through the transport of chloride and inhibition of sodium uptake

Mechanisms of CFTR dysfunction

  • Class 1: defect in protein production, generates unstable RNAs or has premature stop codon premature stop codon
  • Class 2: defective protein processing due to misfolding of protein
    • Delta-F508 is part of this class
      • delta F508 was identified by Riordan et al., and it is a 3-bp deletion of the phenylalanine codon at position 508. ref
      • Delta-F508 does not fold normally enough to allow exit from the ER 
What is the mechanism by which correct folding is "checked"?
*No one in our class, nor the professor knew.
*It may have to do with histone acetylation, which is surprising ref.
*After reading some articles, it seems that the mechanism isn't known but it is suspected that chaperone proteins in the ER which can signal for destruction bind to the hydrophobic regions that (when properly folded) should not be accessible because they are found in the core of the protein.
      • Delta-F508 also exhibits defects in stability and activation
  • Class 3: disrupt regulation and nucleotide binding domains of proteins
  • Class 4: deffective chloride conduction due to mutations in membrane spanning domains
  • Class 5: reduced number of CFTR transcripts
  • Class 6: proteins synthesized normally but are unstable at the cell surface

Delta-F508 mutation and allelic heterogeneity

  • A change in one of the exons is class 4, problems in the channel.
  • 508 and some others affect processing and are in the nucleotide binding domain.

Allelic heterogeneity in CFTR

  • Deletion of phenylalanine at position 508 (Delta-F508) is most common defect accounting for about 70% of all CF alleles in whites
    • Delta-F508 allele probably comes from a single origin
    • Only 7 other mutations are more frequent than 0.5% in these individuals
  • 1600 CF gene sequence variants are associated with disease
    • Nearly half are missense mutations
    • Remainder are point mutations 
Does "point mutation" mean single-nucleotide changes that do not change the amino acid?
    • Less than 1% are genomic rearrangements
    • Few missense mutations have been subjected to functional analysis

CFTR and locus heterogeneity

  • Mutations in epithelial sodium channel (ENaC)
  • Families have non-classical presentations including CF-like pulmonary infections, less severe intestinal disease, elevated sweat chloride levels
  • Functional interaction between CFTR protein and ENaC 
What is the interaction?
  • CF demonstrates locus heterogeneity—if mutations are not found in CFTR then abnormalities in ENaC must be considered

Clinical heterogeneity in CF

  • Results from allelic heterogeneity, modifying loci, or nongenetic factors
  • CFTR genotype is a good predictor of exocrine pancreas function
    • Homozygous Delta-F508 mutation or predicted null alleles usually have pancreatic insufficiency
    • Alleles that allow for synthesis of partially functional CFTR are associated with pancreatic sufficiency
  • CFTR genotype is poor predictor of severity of pulmonary didisease
    • In Delta-F508 mutation patients the severity of lung disease is variable
    • Modifier for CFTR (TGFß1) seems to be associated with more severe lung disease

Population screening for CF

  • Not clear if early identification significantly improves long term prognosis
  • Improved nutrition for pancreatic enzymes is important
  • Screening can only account for 85% of the mutations
  • Usually screen for Delta-F508 and 22 less common but not rare mutations
  • Prenatal diagnosis by DNA analysis can be done at 10-12 weeks
    • Not considered accurate enough.
    • Measuring intestinal enzymes in amniotic fluid is no longer used due to high false positive rate
  • Up to 25% of pregnant women undergo CF carrier testing (2004)

Treatment of individuals with CF

  • Control pulmonary infection
    • Clearance of pulmonary secretions
    • control pulmonary infection (antibiotics)
  • Improve nutrition
    • pancreatic enzyme replacement
    • adequate nutrition
    • prevention of intestinal obstruction
  • Only effective treatment of resppiratory failure is lung transplantation
  • Extensive counseling to deal with psychological effects of having a chronic fatal disease
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