Editing Cystic Fibrosis

==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 [http://www.labtestsonline.org/understanding/analytes/trypsinogen/test.html 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 
*&gt; 60 mmol/L = CF 
*40 mmol/L and 60 mmol/L are borderline 
*&lt; 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)

====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.

===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===