lecture 8

Question 1

What is some of the history of CF?

Answer 1

• Recognised by midwives for hundreds of years: “the infant that tastes of salt will surely die…”
• CF first described in 1938
• • identified mucous plugs in pancreas in babies dying of malnutrition
• • condition called ‘cystic fibrosis of the pancreas’

in 1948 there was a massive heatwave in NY and lots of young children and babies were presenting at the emergency department suffering from overhe ating/dehydration.
First time there was a link made between abnormally high salt levels in these children and cystic fibrosis.

• later, CF characterised by fat & protein malabsorption, failure to thrive, lung disease and abnormal electrolyte composition in sweat

Question 2

How was the gene responsible for CF identified?

Answer 2

• 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
• 1989: gene identified by positional cloning

Question 3

Describe the CFTR gene.

Answer 3

Codes for an ion channel protein.
Gene:
- 90kb DNA
- Coding region, 27 exons (NB old naming of exons in diagram below)
- Codes for large integral glycosylated membrane-spanning protein

Cystic fibrosis transmembrane conductance regulator:

• CFTR protein
• 170kD, 1480 amino acids

The gene was first described as having 24 exons and subsequently introns were detected in exons 6, 14, and 17 so that each of these exons were then numbered ‘a’ and ‘b’. In fact, now they are re-numbered again: 6b has become 7 etc
It’s clear that certain exons code for certain domains of the protein

Question 4

What is the CFTR protein?

Answer 4

• member of the ABC (ATP-binding cassette) superfamily of membrane transporters
• Chloride channel protein (different from other ABC transporters because passage of ions occurs by diffusion down concentration gradient)
• even though it is cAMP-dependent it is not active transport
• Regulated by cAMP-dependent phosphorylation
• Expressed in epithelial cells (apical, i.e. lumen-facing, membranes) in a wide variety of tissues

Five domains:

• two membrane-spanning domains (MSD1 and 2: form channel for passage of chloride ions)
• two nucleotide-binding domains (NBD1 and 2: bind and hydrolyse ATP)
• Regulatory (R) domain: several sites phosphorylated by cAMP-dependent protein kinase, e.g. protein kinase A

Activation of CFTR channel (from closed state to open state) relies on phosphorylation

Question 5

How does the CFTR protein interact with other proteins?

Answer 5

• TRL sequence at carboxy terminal
• carboxy-terminal anchored to cytoskeleton and kept close to other proteins which influence CFTR functions, such as:
• • conductance
• • regulation of other channels (e.g. ENaC: epithelial sodium channel)
• • signal transduction
• • localisation at apical plasma membrane

Question 6

What is the model of CF that relates to the movement of chloride ions in the lungs?

Answer 6

(simplified model)
Normal
- in the normal lung, the Cl- ions move out of the cell down a concentration gradient, while Na+ ions and water move into the cell
- depending on the tissue the direction of chloride ion movement might be different

CF
- in the lung from a person with CF, the CL- ions build up within the cell, resulting in an even greater movement of Na+ ions and water into the cell to balance the internal Cl- ion concentration. This results in dehydrated mucus in the airways.

Question 7

What is the model of CF that relates to the movement of chloride ions in the sweat ducts?

Answer 7

Normal

• in the normal sweat ducts, the Cl- ions move into the cell down a concentration gradient, together with Na+ ions and water.
• Sweat (salt and water) as secreted into the duct by the gland and both Na+ and Cl- are normally reabsorbed by the duct cells before reaching the skin surface

CF

• in the sweat ducts from a person with cystic fibrosis, the Cl- ions cannot enter the cell, so that Na+ ions and water also remain in the sweat duct.
• this results in elevated Na+ and Cl- levels in the sweat

Question 8

What are the CFTR mutations?

Answer 8

• cystic fibrosis mutation database
• more than 1900 known mutations:
• • CF-causing mutations
• • Non CF-causing mutations
• • Mutation of varying clinical consequence
• • Mutation of unknown significance

Question 9

What are the frequencies of the different kinds of mutations we can see in CFTR?

Answer 9

• Missense: 40.23%
• Frameshift: 15.88%
• Splicing: 11.66%
• Nonsense: 8.30
• In-frame in/del: 1.96
• Large in/del: 2.53
• Promoter: 0.77%
• Sequence variation: 13.87
• Unknown: 4.80%

The first three are the most serious, also nonsense.
Sequence variations tend to be the benign mutations. Unknown are unknown.

Majority of mutations are found in exons 4, 8, 14 and 20 (new numbering). 4, 8 and 20 contribute to MSDs, while 14 is part of the regulatory domain. This is in regards to the NUMBER of mutations but the most common single mutation is found in a NBD.

Question 10

What are the classes of CFTR mutations?

Answer 10

• Class I: No protein production. Typically nonsense, can be splice site or frameshift mutations. Generate a protein that is unstable and will be targeted for degradation. Don’t get any protein being made, don’t get any at the surface of the cell.
• Class II: Defective processing (maturation, premature degradation). Protein is transcribed and translated but there is something wrong with the trafficking - rarely makes it past the ER. But never makes it to the plasma membrane. Most common mutation is one of these.
• Class III: Defective regulation (e.g. decreased ATP binding and hydrolysis). Makes it to the plasma membrane. Protein channels don’t open properly (usually don’t open at all).
• Class IV: Defective/reduced ion conductance or channel gating. There is a little bit of opening of the channels. Probably not as severe as previous mutations.
• Class V: Reduced protein production (e.g. promoter or splicing abnormality).
• Class VI: accelerated turnover from cell surface. Pretty rare. Not able to function as efficiently.

Question 11

Give some examples of mutations of the different classes.

Answer 11

1. Splice mutation intron 4 leading to Class I mutation (sometimes class V??) MSD1.
2. R117H: class IV, defective conduction due to alterations of Cl channel. Missense mutation from Arginine to Histadine. Hard to predict phenotype with this mutation. In MSD1.
3. F508del: most common mutation. Class II defective processing. NBD1. Protein does not make it to cell surface.
4. S1255P. Class II Defective regulation.
5. Q1412X. Class VI Instability at the cell surface. Glutamine to a stop codon. Towards the carboxy terminus - not as severe as if the stop codon had appeared more at the N-terminus

Question 12

What is the most common mutation in cystic fibrosis?

Answer 12

F508del: (aka Phe508del/pF508del/deltaF508)

• class II (defective processing/trafficking)
• causes protein to misfold.
• mutant protein retained in endoplasmic reticulum (ER)
• In ER, mutant protein targeted for degradation
• Never makes it to cell surface.
• Understanding the defect can help target therapies

Question 13

How can we make mutation class-specific therapies?

Answer 13

High throughput screening assays have been used to screen drug libraries:

• Class I: often nonsense mutations; can be corrected by compounds (e.g. aminoglycoside antibiotics) that allow ‘read through’ of mRNA
• Class II: ‘correctors’ to improve processing
• Class III: ‘potentiators’ to activate protein
• Class IV: flavonoid compounds to augment channel function (increase open probability)
• Class V: often splicing mutations; increase levels of correctly spliced RNA

Question 14

Describe the F508del mutation.

Answer 14

• deletion of phenylalanine (aa508)
• actually not a deletion of the codon that specifies 508
• isoleucine at position 507 and glycine at position 509
• actually the third base of the preceding codon and the first two bases of 508 that are deleted
• because of redundancy (more than one codon for one aa) by chance the third base of 508 still produces isoleucine when it becomes the third base of 507 because of this redundancy (ATC –> ATT)

Question 15

What is the frequency of F508del in CF?

Answer 15

• these three deletions are the most common cause of CF worldwide: F508del accounts for ~70-75% of mutations in people from northern European descent
• frequency lessens as you go further into the mediterranean (still present) (migration?)
• in australia we have a high level of this mutation because many descendant from northern european countries
• there are some asian specific mutations
• Homozygosity for F508del in ~50% of patients with CF

Question 16

How do we test for F508del in CF?

Answer 16

• nucleotides 1653-1655 deleted in mutant allele
• perform PCR and analyse products after acrylamide gel electrophoresis (as opposed to standard gel)
• only looking at 3 base change in normal allele and mutant allele.
• N/N should have thick band at higher bp
• N/CF should have two bands
• CF/CF should have thick band at smaller bp
• this test doesn’t tell you if there is another mutation on another allele in heterozygote
• tests have been developed to try to look at multiple mutations simultaneously

Question 17

Why are mutation panels used in testing?

Answer 17

• F508del first mutation that was tested for diagnostically
• now more mutations included
• testing typically used PCR + restriction enzymes (RFLPs - restriction fragment length polymorphisms)
• Number of mutations in testing panels differs throughout the world – constantly evolving
• some mutations are specific to particular ethnic groups
• Previously, in Victoria a total of 12 mutations were tested in the initial diagnostic panel, together accounting for ~80% of mutations
• A further 7 mutations were tested in the extended panel
• now we test for more diagnostically

Question 18

How do we complete testing of mutations by SNP analysis?

Answer 18

• 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 (i.e. determined by the SNP):
  A=green, C= blue, T=red, G=black.
• Analysis of each mutation (i.e. SNP) performed in 2 batches (multiplex A and B on capillary gel electrophoresis

Question 19

What is the most recent CFTR mutation panel and testing in Vic?

Answer 19

• New diagnostic testing panel includes 38 (40?) mutations - accounting for ~91% of mutations in our Victorian population
• Victorian Clinical Genetics Services recently changed technology for detecting mutations: still SNP genotyping but measuring MASS of the allele-specific products following PCR
• MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry)
• can genotype large numbers of samples (several hundreds to thousands) and a medium number of SNPs (tens to hundreds)

Question 20

What is the process of MALDI-TOF MS?

Answer 20

1. sample prep
2. multiplex PCR
3. Purification and primer extension steps
4. Mix the PCR products with a special compound that they then spot onto chips. They heat the plate to v. high temperatures –> causes ionisation of the PCR products and then they will go into a gas phase and they will set up a potential difference and accelerate the particles through to the spectrometer. Measure the mass-charge ratio. Time of Flight
5. Run on MALDI-TOF machine
6. analysis

technology has gone from simple PCR to complex but high throughput, fast, accurate machines.