Cystic Fibrosis Flashcards
Inheritance pattern of CF
Autosomal recessive
CF: Affected gene and protein
Affected CFTR gene on chromosome 7 = affected CFTR protein
CF: Normal location and function of affected protein
Normally a glycosylated chloride channel on epithelial cell membranes
- sinuses
- liver
- pancreas
CF: How most common variant alters folding of protein
F508del mutation = Class II:trafficking defect; CFTR protein is misfolded and cannot reach cell surface
F = phenylalanine
3 different tiers of lab testing for CF
- Sweat Chloride Testing
- CFTR Genetic Analysis
- CFTR Physiologic Testing
Differentiate classes of CF
- based on quantity of protein
Class I: no functional CFTR protein
Class II: trafficking defect; misfolded CFTR protein cannot reach cell surface
Class III: defective channel regulation; CFTR protein reaches cell surface but channels cannot open
Class IV: decreased channel conductance; CFTR protein reaches cell but channel function is decreased
Class V: reduced CFTR synthesis; not enough functioning CFTR protein on cell surface
Class VI: decreased CFTR stability; CFTR protein created but does not work at the cell membrane
Describe CRMS/ CFSPID
CF transmembrane conductance regulator-related metabolic syndrome OR CF screen positive inconclusive diagnosis:
- Positive IRT screen and negative sweat Cl (<30 mmol/L), has 2 CFTR mutations, one of which is unknown significance
OR
- Positive IRT screen and intermediate sweat Cl (30-59 mmol/L), has one or no CFTR mutations
BOTH SHOULD GET CFTR GENE SEQUENCING; delayed onset or unaffected carriers
IRT = trypsinogen
CF: Treatment
-
Potentiators
- increases CFTR channel transport of Cl
- for Class III variant -
Correctors
- facilitates processing and trafficking of CFTR = increase CFTR on cell surface
- increases stability of misfolded protein
- for Class II variant (F508del) -
Potentiator/ Corrector Combo
- does both
Describe NBS of CF
- Immunoreactive trypsinogen (IRT); detects high levels of trypsinogen in CF
- sandwich immunoassay
- europium is released from labeled antibody when it binds trypsinogen = fluorescence
- directly proportional to [trypsinogen]
Why is trypsinogen detected in NBS for CF ?
Trypsinogen levels are increased in CF:
- trypsinogen produced by pancreas
- trypsinogen cleaved to trypsin in intestinal mucosa
- trypsin digests proteins > amino acids
Describe the Sweat Chloride Test
- CFTR normally moves water (and NaCl) from pores back to the cell (sweat pore becomes hypertonic)
- iontophoresis of pilocarpine to stimulate sweat production by sweat gland
- sweat is collected in a capillary tube with blue dye
- [Cl-] is measured by coulometry = increased in CF
Predictive testing
Suggests severity of disease
Prognostic testing
Determines which therapies are beneficial
Describe CFTR genotyping workflow (7)
Luminescent genetic assay:
- combines PCR and flow cytometry
- “Luminex xTag assay”
- DNA extraction from EDTA
- Multiplex PCR
- Exonuclease 1 and SAP
- Allele-specific PCR
- Hybridization to beads
- Strepatvidin binds to biotinylated nucleotide; Phycoerythrin is the fluorescent reporter
- Flow cytometry measurements
SAP = Shrimp alkaline phosphatase
Describe purpose of Exo1&SAP
After Multiplex PCR
Exonuclease 1: degrades unused primers
- cleaves ss primers in 3’ to 5’ direction
Shrimp Alkaline Phosphatase: destroys unused dNTPs
- dephosphorylation = unable to form phosphodiester bonds
