Cystic Fibrosis: Molecular Basis Flashcards
Be aware of discovery of the gene (CFTR) involved in causing cystic fibrosis Explain the molecular basis of cystic fibrosis - the CFTR protein and its structure Explain the role of CFTR in epithelial cells Distinguish the classes of CFTR gene mutations/variants and describe the most common variant Recognise the techniques for how variants are tested, especially panels
CF is characterised by:
- fat and protein malabsorption
- failure to thrive
- lung disease
- abnormal electrolyte composition in sweat
Median life expectancy
1950s vs currently
1950s: few months
Currently: >40 years
How was the gene responsible for CF identified?
1983 - fundamental defect identified as abnormal cAMP-mediated regulation of chloride transport (sweat ducts)
1985 - gene associated with CF mapped to chromosome 7q31.2 by linkage in families (still a technique used today in gene discovery for inherited conditions)
1989 - gene identified by positional cloning
CFTR gene codes for a:
Cl- ion channel protein
>Cystic fibrosis transmemebrane conductance regulator (CFTR protein)
*gene name is italicised, protein is normal font
CFTR protein is a member of the:
- ABC (ATP-binding cassette) superfamily of membrane transporters
- Not an active transporter unlike other ABC transporter proteins
- Gated chloride channel protein
- Passage of ions occurs by diffusion down concentration gradient (not active transport)
- Not an active transporter unlike other ABC transporter proteins
CFTR protein is regulated by:
- cAMP-dependent phosphorylation
- Regulates anion (mostly Cl- and HCO3-) transport, and mucociliary clearance
- Role in immunity/inflammation
- Expressed in epithelial cells (apical, i.e. lumen-facing, membranes) in wide variety of tissues
**Important to understand that the inflammation and infection is not solely related to impaired mucociliary function, but also affected by a basic role of this protein in immunity and infection
CFTR Protein Domains
3D structure
CFTR Protein domains (5 in total)
Describe the Membrane-spanning domains (MSD)
- Two membrane spanning domains (MSD1 and 2):
- form channel for passage of chloride ions (Transmembrane = forms the Cl- channel)
CFTR Protein domains (5 in total)
Describe the Nucleotide binding domains:
- Two nucleotide-binding domains (NBD 1 and 2):
- bind and hydrolyse ATP (Intracellular)
CFTR Protein domains (5 in total)
Describe the Regulatory domain:
- Regulatory (R) domain
- Several sites phosphorylated by cAMP-dependent protein kinase
- e.g. Protein Kinase A (Intracellular)
- Several sites phosphorylated by cAMP-dependent protein kinase
Activation of CFTR channel relies on ____
Phosphorylation
CFTR Closed State is the ______ state:
Dephosphorylated state
May have 1 ATP molecule permanently bound
>
Allows for rapid phosphorylation and dephosphorylation (dependent on the binding of the 2nd ATP molecule)
CFTR Open State is the _____ state:
Phosphorylated state
2 ATP molecules bound
Allows Cl- out of cells down concentration gradient (Channel closes when ATP hydrolyses)
Summary slide of the CFTR channel protein closed/states
*remember that activation of CFTR channel relies on phosphorylation
Describe the molecular mechanism of how CFTR changes from closed to open state:
- PKA phosphorylates R domain
- disrupts interaction between NBD1 and R domain, allowing ATPs to bind at NBD
- 2 ATP bind
- ATP-induced dimerisation of NBDs cause conformational change in the TMD region
- Channel opens
- Channel closes when ATP hydrolyses
Describe CFTR protein interactions with other proteins:
Carboxy-terminal anchored to cytoskeleton & kept close to other proteins which influence CFTR functions, which:
Carboxy-terminal anchored to cytoskeleton & kept close to other proteins which influence CFTR functions
- which influence CFTR functions, such as:
- conductance
- regulation of other channels (E.g. ENaC: Epithelial Sodium Channels - most important protein that CFTR interacts with in CF)
- signal transduction
- localisation at apical plasma membrane *F508del mutation affects NBD-1
CFTR function in the airways (lungs)
Normal
- Cl- ions move out of cell down concentration gradient
- Na+ ions and water move into the cell
- @Airway Surface Liquid (ASL)
CFTR function in the airways (lungs)
Cystic Fibrosis
- @ASL
- Cl- ions build up within the cell
- Even greater movement of Na+ ions and water into the cell to balance the internal Cl- ion concentration
- Results in dehydrated mucus in the airways
- *CFTR involved in regulating the function of ENaC (inhibitory role)
- Essentially because ENaC not inhibited, also contributes to greater Na and H2O into cell
CFTR function in airways (lungs)
Summary slide
CFTR function in lumen of sweat duct
Normal
- Extracellular high [Cl-]
- Intracellular Low [Cl-]
- Cl- ions move into cell down conc. Gradient (Together with Na+ ions and H2O)
- Sweat (salt and water) secreted into duct by gland
- Both Na+ and Cl- are normally reabsorbed by the duct cells before reaching the skin surface
CFTR function in lumen of sweat duct
Cystic Fibrosis
- Cl- ions cannot enter cell
- Na+ ions and water also remain in sweat duct
- Elevated Na+ and Cl- levels in the sweat (*Diagnostic test = sweat test)
CFTR function in lumen of sweat duct
Summary slide
Effects of CFTR dysfunction (mindmap)
Inflammasome = inflamatory proteosome (protein complexes which degrade unneeded or damaged proteins by proteolysis)
Impaired innate immunity = leaves airways vulnerable to inflammation and infections
Mucus abnormalities = much drier, stickier mucus
*Chronic infection and inflammation can lead to lung injury >> most common cause of death in CF
There are more than 2000 known CFTR variants.
CF-causing =
Non CF-causing =
Variant of varying clinical consequence =
Variant of unknown significance =
CF-causing = Pathogenic
Non CF-causing = Benign
Variant of varying clinical consequence = Variable penetrance
Variant of unknown significance = VUS
There are more than 2000 known variants
Variant of varying clinical consequence (Variable penetrance) =
Variants associated with a phenotype or disease in some PTs but not all
>>can be considered a “modifying” variant
>>In some PTs contributes to disease, in others, doesn’t
More than 2000 known variants:
Currently 322 most common variants =
Pathogenic (CF-causing) variants
>Useful to have other variants categorised as well to help you interpret findings from genomic testing where you may not be specifially looking for a CFTR variant
Frequency of CFTR variants/mutations
Majority of variants found in:
Exons 4, 8, 14, 20 (hotspots)
List the types of variants by frequency:
- Missense (40.23%)
- sub 1 a.a. for another
- still transcription or translation of full protein - Frameshift (15.88%)
- add/delete b.p.
- contributes particularly to Class 1 type variants - Splicing (11.66%)
- Nonsense (8.30%)
- no protein/truncated protein produced - In-frame in/del (1.96%)
- most common CF variant (F508del) is an in-frame deletion
Why is knowing the different classes of CFTR mutations/varaints useful?
Ans:
There is some overlap in classes that can be helpful in providing treatment for some PTs because…
There is some overlap in classes that can be helpful in providing treatment for some PTs because…
it could be that a combination of treatments works best to target multiple impacts from variants
CFTR Class I Variants:
Class I
Absent protein, normal mRNA abundance
>Usually make nonsense/FS/splice site variants
>lead to unstable RNA which is degraded, thus no protein produced
*MSD1
CFTR Class II variants:
Class II
Defective processing
(maturation, premature degradation)
>typically abnormal protein folding, thus defective processing in ER & Golgi appatarus
>CFTR protein typically degraded before it reaches plasma membrane
>Usually missense variant
*NBD1, e.g. F508del
CFTR Class III Variants:
Class III
Defective regulation
(e.g. decreased ATP binding and hydrolysis)
*NBD2 e.g. S1255P
CFTR Class IV Variants:
Class IV:
Defective/reduced ion conductance or channel gating due to alteration of Cl- channel
>associated with translocated protein that reaches membrane
>BUT missense variant causes defective/reduced ion chanel conductance or channel gating
*MSD1, e.g. R117H
CFTR Class V Variants:
Class V:
Reduced number of transcripts/protein production
(e.g. promoter or splicing abnormality)
\>specifically related to class V \>similar impact in overall reduced amt. of CFTR protein produced
*MSD1
CFTR Class VI Variants:
Class VI:
Instability at cell surface = accelerated turnover
>end result = less CFTR protein produced
CFTR Variants
Summary slide 1
CFTR Variants
Summary slide 2
Which CFTR class varaints affect which CFTR protein domains?
- MSD 1
- Splice variant intron 4 donor site (G > T)
- Class 1 (absent protein, normal mRNA abundance)
- Class V (reduced number of transcripts)
- R117H
- Class IV (defective conduction due to alteration of Cl- channel)
- Splice variant intron 4 donor site (G > T)
- NBD1
- F507del*
- Class II (defective processing)
- F507del*
- NBD2
- S1255P
- Class III (defective regulation)
- S1255P
- Instability at cel; surface
- Class VI
- Q1412X
F508del
Most well known/most common worldwide
Describe CFTR variant class, and what F508del does to cause CF?
Class II (defective processing/trafficking)
F508del causes protein to misfold
>
abnormal protein retained (majority) in endoplasmic reticulum (ER)
>
In ER, abnormal protein targeted for degradation (does not get transported to Golgi then membrane = absence of CFTR protein)
F508del
Most well known/most common worldwide
Is any CFTR trafficked to cell membrane at all?
*Understanding ‘defect’ can help target therapies
~3% of CFTR trafficked to cell membrane
*non-functional (Class III and IV properties)
*Example of how classes can overlap
>That residual protein acts as Class III (defective ATP binding/hydrolysis) or Class IV (defective/reduced ion channel conductance/gating)
>>May be useful to try multiple treatments targeted at other classes
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for CFTR Class I variants:
- Class I
- Absent functional CFTR
- Often nonsense/stop mutations
- Can be corrected by compounds that allow ‘read through’of mRNA (override the mRNA)
- e.g. aminoglycoside antibiotics, such as ataluren
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for CFTR Class II variants:
- Class II
- Absent functional CFTR
- Defective processing (maturation, premature degradation)
- ‘correctors’ to improve processing
- get CFTR protein out of ER into Golgi to cell surface membrane
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for CFTR Class III variants:
- Class III
- Defective channel regulation
- Decreased ATP binding/hydrolysis
- ‘potentiators’ to activate protein
- overcome the defective reguation of ATP binding/hydrolysis
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for CFTR Class IV variants:
- Class IV
- Defective CFTR channel
- Defective/reduced ion conductance/channel gating
- flavinoid compounds to augment channel function
- (increase open probability)
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for CFTR Class V varaints:
- Class V
- Reduced protein production
- Scarce functional CFTR
- Often splicing variants
- Drugs/treatments that increase levels of correctly spliced RNA
- Increase amt. of CFTR protein at cell membrane
Know variant = identify target for treatment
Molecular diagnosis useful in providing prognostic info to PT (how severe you expect disease to be)
High throughput screening assays have been used to screen drug libraries.
Q) Describe drug classes used for each CFTR variant class: i.e. recall everything
- Class I
- Absent functional CFTR
- Often nonsense/stop mutations
- Can be corrected by compounds that allow ‘read through’of mRNA (override the mRNA)
- e.g. aminoglycoside antibiotics, such as ataluren
- Class II
- Absent functional CFTR
- Defective processing (maturation, premature degradation)
- ‘correctors’ to improve processing
- get CFTR protein out of ER into Golgi to cell surface membrane
- Class III
- Defective channel regulation
- Decreased ATP binding/hydrolysis
- ‘potentiators’ to activate protein
- overcome the defective reguation of ATP binding/hydrolysis
- Class IV
- Defective CFTR channel
- Defective/reduced ion conductance/channel gating
- flavinoid compounds to augment channel function
- (increase open probability)
- Class V
- Reduced protein production
- Scarce functional CFTR
- Often splicing variants
- Drugs/treatments that increase levels of correctly spliced RNA
- Increase amt. of CFTR protein at cell membrane
Phe508del/F508del/ΔF508
Normal CFTR gene
vs
Cystic Fibrosis F508del variant in CFTR gene
Q) What happens to the genetic code to cause F508del?
- @ 5’ side
- Normal CFTR gene
- 507 = Isoleucine (ATC)
- 508 = Phenylalanine (TTT)
- 509 = Glycine (GGT)
- F508del variant (Class II, in-frame deletion of both 5’ and 3’ strands)
- *507 = Isoleucine (AT-, –T)
- *508 = Glycine (GGT)
- End up with a deletion of Phe but you do not disrupt the reading frame
1) F508del accounts for ~____% of variants in people from _____ ancestry
2) Homozygosity (2 F508del alleles) for F508del in ~___% of PTs with CF
1) ~70-75%, Northern European ancestry
2) Homozygosity for F508del in ~50% of PTs with CF
*Decreasing frequency moving down towards southern european countries
Q) What is the current test for F508del in CF?
*most of the testing in VIC is done in the Royal Children’s Hospital, next year will start using Next-Gen Sequencing
Perform PCR
>analyse products after acrylamide gel electrophoresis
Nucleotides 1653-1655 deleted in abnormal allele
N = normal allele
CF = allele with variant
F508del first variant that was tested for diagnostically
Now more varaints included
1) Describe how the testing panels work
2) Previously in Victoria, a total of ___ variants were tested in the ______, together accounting for ~___% of varaints
3) A further __ variants were tested in the _______
*SNP analysis (small nucleotide polymorphism)
1) Testing typically used PCR ± restriction enzymes
(RFLPs - restriction fragment length polymorphisms)
*Number of varaints in testing panels differs throughout the world - constantly evolving
*Some variants are specific to particular ethnic groups
2) 12 variants, initial diagnostic panel, ~80% of variants
3) Further 7 variants, extended panel
