Cystic Fibrosis

From Iusmgenetics

Current revision as of 14:36, 13 December 2011

[edit] Cystic Fibrosis

[edit] General background information

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

[edit] Mode of inheritance

• Autosomal recessive
  • Most common autosomal recessive disease of Caucasian children.
• Pleomorphic: lungs, pancreas, endocrine, reproductive, etc.

[edit] 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 (know this: inframe, drop of one aa: phe)
  • Some pts have more than one disease causing mutation

• 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.

[edit] Etiology

• There is ethnic variation in the frequency of alleles and in the frequency of carriers and in the frequency of disease.
• Some polymorphisms: some benign mutations are around in the population
• 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
  • Either way, it leaves the Ile (because ATT and ATC both code for Ile)

• Mutations are classified (don't need to know what each does):
  • 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)

[edit] CFTR and Intron 8 / Exon 9

• Intron 8 of the CFTR gene contains a stretch of Thiamines (Ts)
• As the stretch of Ts is shortened, and particularly at the length of 5, 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.
• When CF is found in the presence of R117H, there is inefficient splicing of the 5T variant, resulting a reduced full-length transcription.
  • R117H results in functional protein and so is considered a milder form of CF.
  • High levels of R117H yield CBAVD
  • Low levels of R117H yield Chronic lung diease

[edit] 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

[edit] 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

[edit] 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
    • Great growing substance for bacteria
  • 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)

[edit] 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

[edit] 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."

[edit] 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.

[edit] CF vas deferens phenotype

• 95% of males with CF lack vas deferens
  • Congenital Bilateral absence of the vas deferens (CBAVD)
    • Causes 2-5% of male infertility
  • 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

[edit] 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)

[edit] CF and Newborn Screening

• The newborn screen for CF in Indiana measures immunoreactive trypsin (IRT).
• If immunoreactive trypsin is high, a DNA test is done.
• The DNA test detects 46 of the most common CFTR changes / mutations
  • The test may find 1 or more mutation in the pt.
  • Note that the implication of a positive mutation identification in CFTR is not always clear.
• A sweat test is done to confirm a cystic fibrosis disease state in the newborn.
• Counseling and treatment follow the screening / genetic confirmation / sweat confirmation.

[edit] 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
  • Note that not all of these are realized.

• 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 so we must consider the balance.
  • We are looking for more effective drugs like ataluren and PTC124

• Other treatments:
  • You don't need to know the chemicals)
  • Improve trafficking of CFTR with chaperones, correctors, and PBA (sodium-r-phenylbutyrate)
    • Chaperones: help with folding and getting to the surface of the cell
    • "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

[edit] Recent research

[edit] 5 important facts

[edit] 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 the 5T variant
    • Note that 5T variant is on an otherwise normal locus
  • 20% have one CFTR mutation
  • 20% have two CFTR mutations
  • 1% have two 5T variants
    • Results in normal protein but a deficient amount
  • 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
  • Recall R117H is associated with CBAVD and inefficient splicing of the 9th exon.

[edit] Questions and answers