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CF Genes & Genetics
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All references used and a copy of this file are online
http://users.ox.ac.uk/~trin0791/Lecture/References.htm
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can Gene therapy vector design
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can Gene therapy vector design Molecular assay development
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can Gene therapy vector design Molecular assay development Laboratory automation
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can Gene therapy vector design Molecular assay development Laboratory automation Animal studies – mouse
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What do I do for the Consortium?
Job title?
Job description?
Try to advance or support our research wherever I think I can Gene therapy vector design Molecular assay development Laboratory automation Animal studies – mouse Software tools, databases, websites
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Working for the Consortium
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Thanks to the rest of the Consortium
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CF Genes & Genetics
Background
Genetics of CF
How it affects us in the lab?
The future
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James Watson & Francis Crick 1953
"We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). This structure has novel features which are of considerable
biological interest."
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What is the structure of DNA?
Four different nucleoside bases
Adenine A
Cytosine C
Thymine T Guanine G
Very strong sugar-phosphate backbone ACGTCTGAGTATGGGAGT
Base-pairing between the two strands
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What is the structure of DNA?
Four different nucleoside bases
Adenine A - T
Cytosine C - G
Thymine T - A Guanine G - C
Very strong sugar-phosphate backbone ACGTCTGAGTATGGGAGT
Base-pairing between the two strands
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How does DNA code for proteins?
Was not immediately obvious
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Amino acid triplet code
Crick, Brenner et al., 1961
DNA is copied (transcribed) into RNA
RNA is read (translated) and a protein produced
3 DNA bases (codon) = 1 Amino acid (the building blocks of proteins)
4 bases maximum 3 in a codon 64 possible triplet codes
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Each triplet codes for only 1 amino acid
Some amino acids are coded for by multiple codons
This code is almost completely universal across all forms of life
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1 gene codes for 1* protein
*There may be multiple versions of the protein made by the gene
A gene may code for 100s or 1000s of amino acids
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Every human cell has exactly the same genes
Prior to the human genome project, estimate was 100,000+ genes
Nerve cell Photoreceptors Macrophage
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Every human cell has exactly the same genes
It turns out, we have just over 30,000
Mutations in just 1 gene – CFTR causes CF
DNA from blood – screen for CFTR mutations
Nerve cell Photoreceptors Macrophage
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Arranged on 23 chromosomes
3000,000,000 base pairs of DNA
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Only 2% of it actually codes for proteins
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98% is ‘junk DNA’
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98% regulates the 2% that does the work
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Human genome headlines
On average any two humans differ only once in 1500 bases
Still means 1.4 million common mutations Men have slightly more
Everyone has the same genes
In the same order
No genetic basis for race
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Variations stand out
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Variations stand out
When they occur, mutations can be easily identified
Direct sequencing of DNA
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Variations stand out
Design assays to specifically amplify the mutation/wt sequence
There are systems that can screen for 100s of mutations at a time
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CFTR is located on chromosome 7
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Total sequence around 250,000 bp
Only the red sections here code for the protein 4400 bp (1.8%) 1480 amino acid protein
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CFTR is completely unremarkable
Known as a ‘housekeeping’ gene
Typically expressed at very low levels in airway cells
1-2 copies of mRNA per cell (my own estimate from nasal epithelia)
There are over 1900 mutations that can cause CF
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Why are there so many mutations in CFTR?
1/25 Caucasians are carriers – don’t get CF
Must be some evolutionary advantage to being a carrier
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Carriers were more resistant to diarrhea
Carrier mice have lower CFTR activity in the intestine
Secrete less water in response to intestinal infections
Medieval European carriers less likely to die from dehydration?
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Nice theory but - less evidence of in humans
Carriers seem to have normal CFTR chloride activity in intestine
Frequency of CF too high for late emergence of cholera epidemics
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Theory makes a lot of sense, but not clear
Still most likely explanation but not 100%
‘Founder effects’ can be very subtle over 1000s of years
Definitive experiment? 1000 carriers and 1000 non carriers Give them cholera Any volunteers?
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Why are mutations of this gene such an issue?
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CFTR is a real ‘keystone’ protein
Very complex trans-membrane protein
Folding crucial
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Regulates chloride transport across epithelia
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Is CFTR just a chloride channel?
No, other ions as well – bicarbonate
Evidence that its involved in Pseudomonas killing as well
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Interacts with many other proteins
Loss of CFTR function will influence the function of these proteins too
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No-one fully understands the role(s) of CFTR
If you think you do – you haven’t realised how complicated it is yet.
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Mutations fall into 6 different classes
Non-CF 1 2 3 4 5&6 Production Processing Regulation Activity Abundance
G542X ΔF508 G551D Q1412X
Stops translation early
Miss folding of the protein
Gets to surface No activity
Some functional protein
Mild/absent lung disease
Spectrum of severity
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G551D almost works!
1 2 3 G542X ΔF508 G551D
Translated – Folds– Processed – Gets to membrane – No ATP binding
Very specific issue for G551D CFTR – ‘just’ needs activation
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Kalydeco very specific for G551D CFTR
1 2 3 G542X ΔF508 G551D
Random drug screening is expensive
High chance of a specific activity on just G551D
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ΔF508 is much harder to fix
1 2 3 G542X ΔF508 G551D
ΔF508 CFTR fails much earlier – exquisite removal of unfolded proteins
You need a drug which can refold it and get it all the way to membrane
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ΔF508 – off target effects?
1 2 3 G542X ΔF508 G551D
Far more likely to have non-specific effects
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How much random screening can you afford?
Far more likely to have non-specific effects
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Genetics is usually very subtle
CFTR is one of the few genes where it’s not
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This shouldn’t affect us?
All gene therapists need to do is get a copy of the coding sequence
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You can buy virtually any gene online
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The CFTR sequence has been known since 1989
Hasn’t it…?
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Spring 2006
2-3 years work
We had our clinical trial plasmid design
Plasmid vector tested and ready – the CFTR sequence was functional
Manufacturing slot booked
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Collaborators in Japan noticed something
The CFTR sequence had subtly changed in March
Two changes Position 620 now Histidine not Asparagine (N620H) Position 833 now Phenylalanine not Leucine (L833F)
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Sequencing genes is a numbers game
N620H was simply an error in 1989 that took a long time to be noticed
L833 was in the original 1989 sequence
F833 is by far the more common variation (90%)
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The original sequence was functional
Had normal CFTR electrics
CFTR protein on a gel
Used in every other CF gene therapy trial
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Lets not bother changing it!
90% certain it would make no difference
90% certain isn’t enough
Risky – cheap – quick
Guaranteed – expensive - slower
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Lets not bother changing it!
90% certain it would make no difference
90% certain isn’t enough
Risky – cheap – quick
Guaranteed – expensive – slower
BOTH!!!
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The result was our clinical trial plasmid pGM169
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We had to do more work – boo hoo!!
Not all CFTR variations change its function
The notion of a single fixed gene sequence is misleading
Prone to human error
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Cost a lot of money
Why is research so expensive sometimes We noticed this issue really early but were on a tight timeline For 6 weeks everything else got shelved Had to spend money on a backup strategy
What if the new ‘correct’ sequence was actually worse? We had to test it in multiple models A slight hint that it may be more active in cell culture models
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Was it worth it?
25 previous gene therapy trial for CF
None of them used the most common functional CFTR sequence
Ours does
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The future?
Mid 1990s – several hundred machines
12 months - $ millions = The human genome project
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The 1000 Genome Project
Sequence 1000 human genomes
Map every common instanceof variation
$1000-$10,000 each in 3-4 days
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Genome sequencing as a diagnostic tool
Oxford Nanopore MinION
Sequence a whole genome <$100
Still a few years away
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Genome sequencing as a diagnostic tool
Oxford Nanopore MinION
Sequence a whole genome <$100
Still a few years away
Massive implications Earlier diagnosis Modifier genes Better outcome predictions ‘Smarter’ trials
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Are the NHS ready for this?
Prof. Crook, NDM, Oxford
Replace microbiological screening with genome sequencing
Infrastructure in place, embedded within the hospital and GP surgeries Tight geographical mapping of infections
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Are the NHS ready for this?
Prof. Crook, NDM, Oxford
Replace microbiological screening with genome sequencing
Infrastructure in place, embedded within the hospital and GP surgeries Tight geographical mapping of infections
![Page 68: CF Genes & GeneticsCF Genes & Genetics Background Genetics of CF How it affects us in the lab? The future James Watson & Francis Crick 1953 ... Frequency of CF too high for late emergence](https://reader033.vdocuments.us/reader033/viewer/2022060416/5f143a2783efdf604243158d/html5/thumbnails/68.jpg)
How will this affect CF?
Routine genome based screening of sputum
Better infection control
Less infections
Reduce the spread/development of antibiotic resistant strains
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Conclusions
CF Mutations Database http://www.genet.sickkids.on.ca
Scientists always say something revolutionary is around the corner
In terms of the technology driving genetics it really is true
Potentially huge benefits