Cystic Fibrosis: Molecular Basis and Genetics Flashcards
Recall a brief overview on CF
Mention the organ systems affected by CF and how is affected.
Recall the respiratory clinical features of CF.
Recall the gastrointestinal clinical features of CF.
Recall the reproductive clinical features of CF.
Describe the CFTR gene.
Describe the characteristic and function of CFTR protein.
Recall the five domains of CFTR protein.
Recall the following schematic diagram of CFTR showing its domain organisation.
Activation of CFTR channel relies on ___________________.
Recall the CFTR molecular mechanism.
MSD1 and MSD2 are represented by the dark and light blue rectangle respectively. NBD1 and NBD2 are represented by the dark and light green shape respectively, while the purple oval represents the R region.
Phosphorylation of the R region by PKA disrupts the interaction between NBD1 and R region, allowing ATP to bind at the NBDs. ATP binding induces the dimerisation of the NBDs, which causes a conformational change in the MSD region into an open conformation. The channel opens and Cl- flux occurs across the membrane.
Hydrolisation of ATP at one of the NBDs causes destabilization of the NBD dimer. Release of Pi and ADP rests the protein into a closed conformation and closes the channel gate.
Note: ATP may remain bound to one of the NBD, allowing for rapid transition back to open-ready state (after addition of another ATP). Alternatively, ATP is released and CFTR is restored to closed state.
Recall how CFTR protein interacts with other protein.
Recall CFTR function in airways and the effects of CF.
Recall the following diagram showing effects of CFTR dysfunction.
Majority of CFTR gene variants/mutations are found in _______________________.
Recall the classes of CFTR mutations/variants.
Recall the following table further elaborating the classes of CFTR mutations.
Recall examples of CFTR variants/mutations.
Describe the variant/mutation F508del in CF.
F508del accounts for ~ 70-75% of variants in people from northern European ancestry
Homozygosity for F508del in ~50% of patients with CF
Recall mutation/variant class-specific therapies for CF.
Recall a test for F508del in CF.
Testing typically used PCR ± restriction enzymes (RFLPs – restriction fragment length polymorphisms)
Recall testing of variants by SNP analysis
- Multiplex PCR
- single reaction, multiple primers, fluorescent tags
- Assay involves primer extension (mini-sequencing) of each hybridised primer so that each product incorporates a coloured fluorescent tag (dNTP) and the colour of the peak depends on which dNTP has been added (ie determined by the SNP):
- A= green, C = blue, T = red, G= black
- Analysis of each variant (ie single nucleotide polymorphism, SNP) performed in 2 batches (multiplex A and B) on capillary gel electrophoresis
Recall the process of SNP genotyping by MALDI-TOF MS
Matrix-Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry
Can genotype large number of samples (several hundreds to thousands) and a medium number of SNPs (tens to hundreds)
Recall the genetics of CF.
Recall the worldwide prevalence of CF.
Recall the typical pedigree of CF.
An obligate carrier is an individual who may be clinically unaffected but who must carry a gene mutation based on analysis of the family history; usually applies to disorders inherited in an autosomal recessive and X-linked recessive manner.
Recall the correlation between genotype and phenotype of CF.
Recall the clinical penetrance of R117H variant of CF.
_______________, ______________, _____________________ not well correlated with CFTR variants although under strong genetic influence
Recall the ratio between genetic and environmental modifiers on various aspects of CF.
Recall studies with twins and siblings in regards to CF.
How to identify genetic modifiers of CF?
Recall the use and limitations of GWAS in finding DNA sequence variants (SNPs) that could act as genetic modifiers for CF.
Recall some genetic modifiers of different features of CF.
Recall genetic modifiers of lung disease in CF.
Recall genetic modifiers of other features in CF.
Poorer health outcomes of CF associated with:
Recall the different purposes of CF testing.
Recall newborn screening for CF.
Recall the CF newborn screening protocol.
Recall reproductive options for CF carrier couples.
Recall CFTR function in the lumen of duct sweat.
