Cystic Fibrosis

From Iusmgenetics

Contents 1 Cystic Fibrosis 1.1 General background information 1.2 Mode of inheritance 1.3 Single important gene 1.4 Etiology 1.4.1 CFTR and Intron 8 / Exon 9 1.5 Pathogenesis 1.5.1 Genotype-Phenotype Correlation 1.6 Phenotypic information 1.6.1 CF in the airway epithelium 1.6.2 CF in the pancreas 1.6.3 CF sweat phenotype 1.6.4 CF vas deferens phenotype 1.7 Diagnosis 1.8 Treatment 1.9 Recent research 1.10 5 important facts 1.11 Not to be confused with 1.12 Questions and answers

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.
• Pleomorphic: lungs, pancreas, endocrine, reproductive, etc.

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.

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)

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.
  • High levels of R117H yield CBAVD
  • Low levels of R117H yield Chronic lung diease

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
    • 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)

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)
    • 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

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
  • 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:
  • 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 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.

Questions and answers