Week 2 Cystic Fibrosis Cumulative Lecture, Module, Article Flashcards
what are some issues/symptoms that arise due to CF? (4 broad ones)
salty sweat, damage to respiratory system (lung disease due to mucus), chronic digestive issues (pancreatic insufficiency), infertility in males
what type of inheritance is CF?
Autosomal Recessive
what is the cystic fibrosis gene?
Cystic Firbrosis Transmembrane Coductance Regulator Gene (CFTR gene)
CFTR is a member of what super family? what is unique about CFTR within this superfamily?
the ATP-Binding Cassette (ABC) transporters superfamily. CFTR contains an R domain that is not seen in other ABC transporters. Also CFTR does not move ions against their gradients like many ABC transporters do
where is the CFTR gene located? How big is it? how big is the protein in produces?
7q31.2. 180,000 bp. 1480 AA
what is the structure of CFTR? (domains)
Five domains: (2) Membrane spanning domains (MSD1 and MSD2) each with 6 transmembrane alpha helices. (2) Nucleotide Binding Domains (NBD1 and NBD2) and an R domain
What is the function of the MSD?
forms chloride ion chanel
what is the function of the NBD?
bind and hydrolyze ATP to open/close ion gate
what is the function of the R domain?
responsible for CFTR activation. Ion channels only open when R is phosphorylated by PKA and ATP is bound at NBDs
CFTR is described as being a ______ activated and ______ gated ion channel.
cAMP activated and ATP gated
how is cAMP involved in CFTR activation?
cAMP–>activates PKA–>PKA phosphorylates and activates the R domain of CFTR
give the sequence of events in CFTR opening
- R domain is phosphorylated by PKA
- Phosp. of R allows ATP to bind NBD1
- Hydrolysis of ATP by NBD1 transiently opens ion channel
- PKA phosphorylates more sites on R
- NBD2 can bing ATP which stabilizes the open ion channel
- Hydrolysis and release of ADP closes the ion channel
- R domain dephosphorylation will also close the channel
what is the function of CFTR? (2)
conducts Chloride ions across the PM. CFTR also plays a role in regulating activity/expression of other ion channels, cytoskeletal elements and signal transduction. This occurs through the C-terminal
in what type of cells is CFTR found? where within these cells is it located?
Epithelial cells. Apical (lumenal) PM
in general, what direction does CFTR move chloride ions?
when active CFTR moves chloride ions down their concentration gradient. This is generally our of epithelial cells (secreted). The exception is seen in sweat glands wher Cl is reabsorbed.
what occurs in a Class I CFTR mutation?
CFTR is not produced at all (premature stop codon)
what occurs in class II CFTR mutations?
defective protein processing causes CFTR to be degraded before it reaches the PM. misfolded CFTR
what occurs in class III CFTR mutations?
defective channel regulation or gating. gating defect
what occurs in a class IV CFTR mutations?
defective chloride conductance (restricted Cl movement thru channel.)
what occurs in a class V CFTR mutation?
reduced amount of CFTR protein (alternative splicing)
what occurs in a class VI CFTR mutation?
accelerated turnover of surface CFTR
what is the most common class of CFTR mutation? what is the most common mutation that leads to this? what percent of CF cases does this correlate to?
Class II. caused by DeltaF508 (deletion of Phe at position 508). about 70% of CF cases have this mutation
deltaF508 causes which structural change in CFTR?
a change in NBD1
outline normal sweat duct fucnciton
- isotonic secretion enters the secretory coil
- due to Na’s high electrochemical gradient it is reabsorbed through epithelial Na carriers (ENaC)
- Cl is electricaly attracted to Na and follows Na into the cell through apical CFTR
- the duct is impermeable to water so water cannot follow the ions
- the sweat duct excretes a hypotonic sweat
what are the two portions of a sweat duct?
secretory coil, and reabsorptive coil
outline the sweat duct seen in CF
Cl movement is essentially gone. Na movement is also reduced suggesting CFTR activates ENaC in SWEAT DUCTS. Very little reabsorption of Na and Cl results in a salty sweat
describe ion and water movement in normal epithelial cells of the lungs
- CFTR allows Cl to move down its gradient and be secreted from epithelial cells into the respiratory mucosa (lumen)
- CFTR also suppresses Na ion channels (ENaC) limiting the amount of Na that enters the epithelial cell from the lumen
- high lumenal NaCl draws water out of the cells and contributes to the airway surface liquid (ASL)
What is the role/fxn of Airway surface liquid?
provides a microenvironment for beating cilia to clear mucus
describe ion and water movement in epithelial cells of the lungs in individuals with CF (low-volume model)
- lack of CFTR prevents Cl from being secreted to lumen
- ENaC are not inhibited and Na and H20 move into the cell (reabsorbed)
- decreased water in lumen decreases ALS which decreases the ability of cilia to move mucus and makes the mucus more viscous
How does cilia’s inability to move mucus in the airways lead to scarring of the lungs/airways?
stagnant mucous leads to mucus obstructions, infections, and inflammation. these all contribute to scarring of the lungs.
generally, what kills most people with CF?
end-stage lung disease from excessive scarring of the lungs.
in patients with CF the ability to _____ Cl is impaired
secrete
describe the function of normal CFTR in pancreatic function
CFTR secretes Cl to the lumen. H20 follow along with HCO3 to modulate pH
what effects are seen in the pancreas of people with CF?
CFTR does not secrete Cl into lumen. Less water moves to lumen. Viscous pancreatic juice is produced and enzymes are not delivered to duodenum (blocked pancreatic ducts).
How can CF lead to diabetes?
Blocked pancreatic ducts trap pancreatic enzymes. Trapped enzymes damage pancreatic cells affecting the production of insulin and causing diabetes
a patient comes in with less than 1% CFTR function, what are the likely clinical features?
pancreatic insufficiency, pulmonary infection, positive sweat test, congenital absence of vas deferens
a patient comes in with less than 4.5% CFTR function, what are the likely clinical features?
pulmonary infection, positive sweat test, absence of vas deferens
a patient comes in with less than 5% but more than 4.5% CFTR function, what are the likely clinical features?
positive sweat test, absence of vas deferens
a patient comes in with less than 10% but more than 5% CFTR function, what are the likely clinical features?
absence of vas deferens
a patient comes in with 10% CFTR function, what are the likely clinical features?
none this person wont have CF
in individuals with CF, what classes of mutations will likely lead to pancreatic insufficiency?
Class I, Class II, Class III
we have seen that CF can impair lung function, pancreatic function and produce salty sweat? Describe the correlation between genotype and phenotype seen in these symptoms.
pancreatic: strong
sweat chloride: moderate
lung fxn: weak
To treat CF, do we need to drastically improve CFTR function?
No, we have seen that CFTR function over 10% is sufficient to eliminate symptoms
what potentially explains why 4-5% of caucasians are heterozygous for CF yet the rate of random mutations causing CF never exceeds 1%?
when cholera was a big issue being recessive for CF (having half the amount of CFTR) meant people would not lose as much water due to cholera toxin
how does cholera toxin work?
- A1 subunit of cholera toxin ADP-ribosylates alpha subunit of Gs-protien
- Gs-protein can no longer hydrolyze GTP to GDP and is always active
- Gs continually activates adenylyl cylcase which increases the amount of cAMP
- cAMP activates PKA which phosphorylates and activates CFTR
- Chloride is continuously secreted and water follows salt
- excess water loss and fatal dehydration
what is the average life expectancy in the US for individuals with CF?
40s
what does NBS stand for (not a protein)
Newborn Screening
what is the carrier frequency (CF) for A. Jewish, White-non jews, and african americans
AJ: 1 in 24
CnJ: 1 in 25
AA: 1 in 61
What is quantitiative pilocarpine iontophoresis? When should it be used
this is the CF sweat test. This should be the first diagnostic test for CF when there is clinical suspicion of CF
what is Nasal potential difference test? when is it used
Measures salt transport in and out of nasal cells in response to different salt solution. A test for CF. difficult to perform and not done at all CF centers. mostly done for research
A sweat [Cl] less than 40 mmol/L should e interpreted as…
normal
a sweat [Cl} greater than 60 mmol/L should be interpreted as…
positive diagnosis of CF
non-sense, frame shift, and splice site mutaions typically lead to which type of CF?
Type I. Reduced or absent CFTR transcription
Even if deltaF580 can be made to make it to the PM (type II), what is its likely fate?
it will become a class VI mutation and be taken into the cell too early
what would a therapy for class I mutations in CF have to accomplish?
the compound would have to allow for read-through of premature stop codons
what would a therapy for class II mutations in CF have to accomplish?
the compound would have to help properly fold and locate CFTR to the cell surface
what would a therapy for class III mutations in CF have to accomplish?
Potentiator. Improve CFTR function to transport Cl
p. Gly551Asp (G551D) is what class of mutation? how common is this mutation? can it be fixed?
Class III. second most common mutation at 4% of CF popn. can be treated with Kalydeco (ivacaftor)
what is the difference between a cis acting variant and a trans acting variant?
cis-acting are on the SAME chromosome (two different mutations on the same chromosome, affecting the same gene)
trans-acting are on homologous chromosomes. Each gene on each chromosome is affected
what is the significance of the poly-T tract?
CFTR has a poly-T tract located in the non-coding region (intron 8). These Ts can influence splicing of exon 9 in pre-mRNA
what types of poly-T are seen? which are “normal” which are detrimental?
9T (normal), 7T (normal), 5T (detrimental)
if a person is homozygous for 5T (5T/5T), will they have CF?
no. there will still be enough normal CFTR made (10%)
what does Kayldeco (ivacaftor) treat?
Type III mutation of CF p.G551D
what are TG tracts in CFTR gene?
located adjacent to the poly-T tracts of CFTR gene. Also alter splicing.
in general increasing/decreasing the number of poly-T and TG tracts makes its effects on CFTR more detrimental
decreasing the length of T-tracts (5T)
Increasing the number of TGs (TG12 and TG13 are the most detrimental)
The effects of TG tracts and poly-T tracts show how….
cis-acting variants can cause more significant symptoms than each variant alone
if a person is homozygous for 5T 13TG alleles what is the likely outcome?
may act as CF-causing mutation, but likely mild. risk for male infertility
if a person is homozygous for 5T 11TG allels what is the likely outcome/
unlikely to act as a CF-causing mutation. still a risk for male infertility
how do you tell if a mutation is cis- (on the same chromosome) or trans- (on different chromosome) for a mutation. Ex; a person is p.Arg117His and 5T/7T are they p. Arg117His 5T and 7T or p.Arg117His 7T and 5T
mutation analysis will not give info on cis or trans. parental testing may be the best bet.
historicaly p. Phe508del is generally with what poly-T tract?
9T
Congenital bilateral absence of vas deferens: genotype/phenotype correlation
CBAVD nearly always occurs. CBAVD may be present even in the absence of pancreatic/lung findings
what is a compound heterozygote?
The presence of two different mutant alleles at a particular gene locus, one on each chromosome of a pair.
what is the difference between a classical and non-classical mutation in CFRT
classical: severe
non-classical: mild
if a compound heterozygote has one classical mutation (PI) and one noncalssical muation (PS), will they likely be PI or PS?
PS. the mild mutation typically trumps the severe mutation
what are 5 CF genetic tests
targeted mutation testing, mutation panels, sequence analysis, del/dup testing, sequence and del/dup analysis
when would you use a targeted mutation test?
when you are looking for one or two specific mutations (you know what you are looking for!). most often done for familial mutations
when would CF mutation panels suggested to be used?
CF carrier screening is recommended to every preconceptional or pregnant woman
how many specific mutations does a minimal panel for CARRIER screening look for?
23 specific mutations
how does sequence analysis work?
reads through and looks for mutations in all important areas of the gene (exons, introns, splice sites)
what percent of mutations will sequence analysis detect?
98%
what are some downfalls of sequence analysis in CF genetic testing?
detects unclassified CFTR variants (difficult to interpret)
does not detect large deletions or duplications
why wont sequence analysis detect large deletions or duplications in DNA?
only reads what info is present. does not measure dosage of each chromosome. two copies of CFTR or 1 copy of CFTR would give same result.
what is del/dup analysis
measures the relative abundance of DNA present in a cell. gives you info about dosage and therefore dup/del
what % of CFTR mutations are due to del/dup?
about 2%
what is the most thorough genetic testing clinically available for CF?
sequence+del/dup analysis
what is the issue with sequence + del/dup analyis?
slow turn around time, more expensive than other tests
what is the difference between the sweat test and genetic tests?
sweat test tells you if you have CF, genetic test tells you why you have CF
Linda just had a sequence + del/dup analysis done for CF and came back negative. Does this mean she does not have CF/is not a carrier?
NO!
How are newborns screened for CF?
IRT from a heelstick Immunoreactive trypsinogen (which is a pancreatic enzyme precursor elevated in babies with CF) is acquired from blood taken from the heel
a sweat test cant be performed immediately on a newborn, why?
sweat chloride is normally elevated in a newborn, want to ensure you can get sweat. Baby comes back just before five weeks
If a baby has an IRT
negative screen, low risk no follow up needed
if a newborn has an IRT within the top 4th%ile for that day what is the course of action?
mutation panel to test for 42 common mutations
- no mutations found: low risk, negative screen
- 1 mutation found: moderate risk, arrange sweat chloride
- 2 mutations found: high risk, arrange for DNA blood testing
what is Orkambi used for?
treatment of class two CF specifically people who have F508del CFTR mutation. (improves CFTR by 6% does not improve sweat test)
what is meconium ileus?
bowel obstruciton that occurs when meconium of child is thicker and stickier than normal (meconium: childs first poo)
what are some clinical clues to diagnosing a CF? (5)
meconium ileus, failure to thrive, chronic respiratory disease with sinus disease, rectal prolapse, newborn screening
what are some complications that arise from a failure to diagnose CF during infancy? (7)
hypo- chloremia, natremia, proteinemia. and vitamin A, D, E, K defficiency
why are CF patients vitamin A,D,E,K deficient?
pancreas cant absorb fat soluble vitamins
what is one body system that CF doesnt impact?
CNS
what is the most common bacteria in CF patients 17 and up?
Pseudomonas Aeruginosa
what are the usual treatments used on someone with CF to improve quality and length of life? (6)
nasojejunal feeds, enzyme replacement, Vitamins added (ADEK), antibiotics, mucolytics, chest physiotherapy
the C-terminal of CFTR has what three proteins? these interact with what receptor known to play a role in intracellular signaling
Thr, Arg, Leu which anchor to PDZ-type receptors
the high-salt model of CF effects on the lungs suggests what?
suggests airways behave in a similar fashion to sweat ducts. elevated sodium chloride levels in airways inactivate endoegenous antimicrobial peptides that predispose patients to bacterial infections
What drug has been approved (FDA) to treat Type III p. G551D? How does this drug fxn
Kalydeco AKA Ivacaftor. This drug is a potentiator meaning it increases the effectiveness of CFTR regulatory domains (R and NBD1/2
What drug has been approved to treat Type II mutation delF508?
Orkambi. This is a combination of Kalydeco AKA ivacaftor(potentiator) and lumacaftor
what is the chief contributor of morbidity and mortality in CF patients
mucosal obstruction of exocrine glands
