Lecture 06 - Cystic Fibrosis Flashcards
What is cystic fibrosis
an autosomal recessive genetic disorder
What is a rare way to get CF
get 2 mutations from one parent
What is the effected gene in CF
CFTR on chromosome 7
How many amino acids are in the CFTR protein
1480
What is the function of the CFTR protein
a chloride channel in the lungs, liver and GI systems
What is the most common mutation
the F508 mutation
What percent of people with CF have the Class I mutation
22%
What percent of people with CF have the Class II mutation
88%
What percent of people with CF have the Class III mutation
6%
What percent of people with CF have the Class IV mutation
6%
What percent of people with CF have the Class V mutation
5%
What percent of people with CF have the Class VI mutation
5%
What is the presentation of a normal CFTR
CFTR protein is created and moves to the cell surface allowing the transfer of chloride and water
What is the presentation of a Class I CFTR mutation
no functional CFTR protein is created
What is the presentation of a Class II CFTR mutation
CFTR protein is created but misfolds keeping it from moving to the cell surface (traffic defect)
What is the presentation of a Class III CFTR mutation
CFTR protein is created and moves to the cell surface but the channel gate does not open (defective regulation)
What is the presentation of a Class IV CFTR mutation
CFTR protein is created and moves to the cell surface but the channel is faulty (decreased channel conductance)
What is the presentation of a Class V CFTR mutation
Normal CFTR protein is created and moves correctly to the cell surface but not enough protein is made (reduce synthesis)
What is the presentation of a Class IV CFTR mutation
CFTR protein is created but it does not work properly at the cell membrane (decreased stability)
What is the F508 mutation
amino acid 508 is deleted which effects protein folding
What parts of the body are most effected by CF
sinuses, lungs, liver, pancreas, vas deferens
What is the gold standard test for diagnosing CF
the sweat chloride test
What sweat chloride results are negative for CF
</=29 mmol/L
What sweat chloride results are inconclusive for CF
30-59 mmol/L
What sweat chloride results are diagnostic for CF
> /= 60 mmol/L
What else is used to diagnose CF
CFTR genetic analysis and CFTR physiologic testing
How are newborns in alberta screened for CF
immunoassay for immunoreactive trypsinogen
Why is the newborn screen just a screen and not diagnostic
because people with only one mutation or other pancreatic issues can also have increased levels
What is the method of the CF screen
a sandwich assay using europium
How are cutoffs for the newborn screen determined
based on the population
What are the cutoffs at the UofA hospital
> /= 60 ug/L sent for secondary testing
<53 ug/L not sent for secondary testing
between 53 and 59 ug/L are sent for secondary testing to ensure that no CF is missed
What is the sweat chloride test
iontophoresis of pilocarpine to stimulate sweat productions which are collected into a coiled capillary tube that contains blue dye the concentration of Cl is then measured using coulometry
What is CFTR genotyping
determines the CFTR DNA sequences and can be used to suggest severity of disease as well as determine which therapies could be beneficial
What is the luminex CF X-tag genotype assay
a combination of allele specific PCR and flow cytometry
Polystyrene beads with different concentrations of dyes are used to identify
specific genotypes
Capture oligos specific to the 5’ end of one of the primers in the alleles
specific PCR are covalently bounded to the beads via a polyimide bonds to
carboxyl groups that coat the beads
How does the luminex CF X-tag genotype assay work
- DNA extraction from EDTA blood sample
- Multiplex PCR (16 amplicons)
- SAP/Exo treatment to degrade unincorporated primers and
nucleotide - Allele specific PCR
- Hybridization to beads
- Reporter addition Streptavidin – Phycoerythrin
- Flow cytometry measurements
What does exonuclease I do
degrade unincorporated primers
What does Shrimp Alkaline Phosphatase do
destroy unincorporated dNTPs
What is the reporter in CF genotype assay
phycoerythrin coupled with streptavidin
What is Ivacafter
a treatment that increases the probability that the CFTR channel is open to increase chloride transportation (used for Class III mutations)
What is Lumacaftor
a treatment that increased the stability of the p.Phe508del misfold protein allowing more proteins to be trafficked to the cell membrane (not effective alone)
What is Tezacaftor
a corrector that improves cellular processing and trafficking of F508del CFTR
What is Orkambi
a combination of lumacaftor and ivacaftor
What is Symdeko
a combination of Texacaftor and Ivacaftor
What is elexacaftor
a CFTR corrector and potentiator that stabilizes F508del CFTR
What is trikafta
a combination of elexacaftr, tezacaftor and ivacaftor
What is heterogeneity
CFTR protein activity = CFTR quantity X CFTR function
What is a concern to take into consideration regarding Trikafta
it can cross the placenta at infant therapeutic levels meaning if the mom is on the drug then the baby may test false negative at NBS
What can cause false positives in the chloride sweat test
improper testing technique
active smoking the day of the test
other medical conditions
specific medications
