Cystic Fibrosis: Molecular Basis

Question 1

CF is characterised by:

Answer 1

• fat and protein malabsorption
• failure to thrive
• lung disease
• abnormal electrolyte composition in sweat

Question 2

Median life expectancy

1950s vs currently

Answer 2

1950s: few months

Currently: >40 years

Question 3

How was the gene responsible for CF identified?

Answer 3

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

Question 4

CFTR gene codes for a:

Answer 4

Cl- ion channel protein

>Cystic fibrosis transmemebrane conductance regulator (CFTR protein)

*gene name is italicised, protein is normal font

Question 5

CFTR protein is a member of the:

Answer 5

• 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)

Question 6

CFTR protein is regulated by:

Answer 6

• 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

Question 7

CFTR Protein Domains

3D structure

Answer 7

Question 8

CFTR Protein domains (5 in total)

Describe the Membrane-spanning domains (MSD)

Answer 8

• Two membrane spanning domains (MSD1 and 2):
  
  • form channel for passage of chloride ions (Transmembrane = forms the Cl- channel)

Question 9

CFTR Protein domains (5 in total)

Describe the Nucleotide binding domains:

Answer 9

• Two nucleotide-binding domains (NBD 1 and 2):
  
  • bind and hydrolyse ATP (Intracellular)

Question 10

CFTR Protein domains (5 in total)

Describe the Regulatory domain:

Answer 10

• Regulatory (R) domain
  
  • Several sites phosphorylated by cAMP-dependent protein kinase
    
    • e.g. Protein Kinase A (Intracellular)

Question 11

Activation of CFTR channel relies on ____

Answer 11

Phosphorylation

Question 12

CFTR Closed State is the ______ state:

Answer 12

Dephosphorylated state

May have 1 ATP molecule permanently bound
>
Allows for rapid phosphorylation and dephosphorylation (dependent on the binding of the 2nd ATP molecule)

Question 13

CFTR Open State is the _____ state:

Answer 13

Phosphorylated state

2 ATP molecules bound
Allows Cl- out of cells down concentration gradient (Channel closes when ATP hydrolyses)

Question 14

Summary slide of the CFTR channel protein closed/states

*remember that activation of CFTR channel relies on phosphorylation

Answer 14

Question 15

Describe the molecular mechanism of how CFTR changes from closed to open state:

Answer 15

1. PKA phosphorylates R domain
2. disrupts interaction between NBD1 and R domain, allowing ATPs to bind at NBD
3. 2 ATP bind
4. ATP-induced dimerisation of NBDs cause conformational change in the TMD region
5. Channel opens
6. Channel closes when ATP hydrolyses

Question 16

Describe CFTR protein interactions with other proteins:

Carboxy-terminal anchored to cytoskeleton & kept close to other proteins which influence CFTR functions, which:

Answer 16

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

Question 17

CFTR function in the airways (lungs)

Normal

Answer 17

• Cl- ions move out of cell down concentration gradient
• Na+ ions and water move into the cell
• @Airway Surface Liquid (ASL)

Question 18

CFTR function in the airways (lungs)

Cystic Fibrosis

Answer 18

• @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

Question 19

CFTR function in airways (lungs)

Summary slide

Answer 19

Question 20

CFTR function in lumen of sweat duct

Normal

Answer 20

• 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

Question 21

CFTR function in lumen of sweat duct

Cystic Fibrosis

Answer 21

• 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)

Question 22

CFTR function in lumen of sweat duct

Summary slide

Answer 22

Question 23

Effects of CFTR dysfunction (mindmap)

Answer 23

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

Question 24

There are more than 2000 known CFTR variants.

CF-causing =

Non CF-causing =

Variant of varying clinical consequence =

Variant of unknown significance =

Answer 24

CF-causing = Pathogenic

Non CF-causing = Benign

Variant of varying clinical consequence = Variable penetrance

Variant of unknown significance = VUS

Question 25

There are more than 2000 known variants

Variant of varying clinical consequence (Variable penetrance) =

Answer 25

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

Question 26

More than 2000 known variants:

Currently 322 most common variants =

Answer 26

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

Question 27

Frequency of CFTR variants/mutations

Majority of variants found in:
Exons 4, 8, 14, 20 (hotspots)

List the types of variants by frequency:

Answer 27

1. Missense (40.23%)
   - sub 1 a.a. for another
   - still transcription or translation of full protein
2. Frameshift (15.88%)
   - add/delete b.p.
   - contributes particularly to Class 1 type variants
3. Splicing (11.66%)
4. Nonsense (8.30%)
   - no protein/truncated protein produced
5. In-frame in/del (1.96%)
   - most common CF variant (F508del) is an in-frame deletion

Question 28

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…

Answer 28

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

Question 29

CFTR Class I Variants:

Answer 29

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

Question 30

CFTR Class II variants:

Answer 30

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

Question 31

CFTR Class III Variants:

Answer 31

Class III

Defective regulation
(e.g. decreased ATP binding and hydrolysis)

*NBD2 e.g. S1255P

Question 32

CFTR Class IV Variants:

Answer 32

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

Question 33

CFTR Class V Variants:

Answer 33

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

Question 34

CFTR Class VI Variants:

Answer 34

Class VI:

Instability at cell surface = accelerated turnover

>end result = less CFTR protein produced

Question 35

CFTR Variants

Summary slide 1

Answer 35

Question 36

CFTR Variants

Summary slide 2

Answer 36

Question 37

Which CFTR class varaints affect which CFTR protein domains?

Answer 37

• 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)
• NBD1
  
  • F507del*
    
    • Class II (defective processing)
• NBD2
  
  • S1255P
    
    • Class III (defective regulation)
• Instability at cel; surface
  
  • Class VI
  • Q1412X

Question 38

F508del

Most well known/most common worldwide

Describe CFTR variant class, and what F508del does to cause CF?

Answer 38

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)

Question 39

F508del

Most well known/most common worldwide

Is any CFTR trafficked to cell membrane at all?
*Understanding ‘defect’ can help target therapies

Answer 39

~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

Question 40

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:

Answer 40

• 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

Question 41

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:

Answer 41

• 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

Question 42

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:

Answer 42

• Class III
  
  • Defective channel regulation
  • Decreased ATP binding/hydrolysis
    
    • ‘potentiators’ to activate protein
    • overcome the defective reguation of ATP binding/hydrolysis

Question 43

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:

Answer 43

• Class IV
  
  • Defective CFTR channel
  • Defective/reduced ion conductance/channel gating
    
    • flavinoid compounds to augment channel function
    • (increase open probability)

Question 44

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:

Answer 44

• 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

Question 45

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

Answer 45

• 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

Question 46

Phe508del/F508del/ΔF508

Normal CFTR gene
vs
Cystic Fibrosis F508del variant in CFTR gene

Q) What happens to the genetic code to cause F508del?

Answer 46

• @ 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

Question 47

1) F508del accounts for ~____% of variants in people from _____ ancestry
2) Homozygosity (2 F508del alleles) for F508del in ~___% of PTs with CF

Answer 47

1) ~70-75%, Northern European ancestry
2) Homozygosity for F508del in ~50% of PTs with CF

*Decreasing frequency moving down towards southern european countries

Question 48

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

Answer 48

Perform PCR
>analyse products after acrylamide gel electrophoresis

Nucleotides 1653-1655 deleted in abnormal allele

N = normal allele

CF = allele with variant

Question 49

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 _______

Answer 49

*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