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Lecture 45
Prof Duncan Shaw
Applications - finding genes
• Currently much interest in medical research, in finding the
genes causing disease
• Sometimes the gene can be guessed at (e.g. by already
knowing what goes wrong in disease) - this is called
“Candidate gene” analysis
• Occasionally, disease is caused by a chromosome
abnormality (e.g. Duchenne muscular dystrophy) - then you
just find the gene that is disrupted
• Usually, have to find the gene by genetic mapping - use
affected families and DNA polymorphisms all over genome
- analyse linkage
The Human Genome
• Is very large - 3,000,000,000 bp of DNA per copy
• Contains 35,000 genes
• About 1,000,000 sites where there is DNA
sequence variation (mostly with no effect on
phenotype)
• We can use these “polymorphisms” to find disease
genes by following their inheritance in families
• They can easily be visualised using DNA
technology
Cystic Fibrosis
• Autosomal recessive disease affecting about
1/2000
• Mucus builds up in lungs, leading to infection,
often death in 20s
• Gene was identified by genetic mapping (using CF
families)
• This approach (also applied to many other genetic
diseases) uses 100s of DNA polymorphisms all
over genome
A type of DNA polymorphism
chromosome
....CACACACACACA.... No. of CAs varies (alleles)
PCR
DNA fragments
Electrophoresis
1
2
3
4
5
large
small
Strategy for finding the gene
• Collect families with the disease (100 or
more)
• Clinical diagnosis. Affected/unaffected. All
same disease?
• Obtain blood samples for DNA extraction
• Analyse genotypes for all polymorphisms in
all families’ DNA samples - find out where
disease gene is located in the genome
Tracking the gene in families
1
1
3
2
3
2
4
5
6
linked
unlinked
4 5
6
Finding the right gene
chromosome
DNA fragments
gene
Clone in
bacteria or
yeast
no
Mutation in patients
but not controls?
yes
Correct gene!
The Cystic Fibrosis Gene
• Located on chromosome 7q
• Expressed in tissues affected by CF, i.e. Lung,
sweat gland, pancreas, nasal epithelium
• Codes for a 1480-amino acid protein involved in
transport of Cl- in and out of cells
• 70% of cases have 3 bases missing from gene,
causing one phenylalanine to be missing from
protein - DF508
• Rest of cases include 100s of different mutations
The CF Gene (continued)
• Because DF508 is so common, it might give an
advantage to carriers - increased cholera
resistance?
• People at risk can tested for carrier status,
pregnancies at risk can be diagnosed prenatally
• If you know specific mutation in family, can test
DNA directly - extract DNA from mouthwash or
amniocentesis, test by PCR reaction on CF gene
• If you don’t know mutation in family, test
indirectly using linkage analysis.......
Diagnosis by Linkage
1,2
2,2
1,2
1,2
?
1,2
1,2
1,1
2,2
1,2
2,2
A,B
C,D
A,C
A,D
haplotypes
informative
1,1
1,2
1,2
1,2
1,2
1,2
uninformative
A,C
A,B
D,E
C,D
A,C
?
Carrier status
CF diagnosis using 2 linked
markers (“haplotype”)
Gene Therapy for CF
• Gene therapy is the treatment of disease by
introducing an active copy of defective gene
– Ex vivo - remove some cells (e.g. bone marrow
or blood) from patient, replace gene, return
cells to patient
– In vivo - deliver gene direct to target, i.e. lungs
in CF
• Animal studies (rats) gave promising
results, now in clinical trials…...
Gene Therapy for CF (continued)
Nasal spray
CF patients
Functional CF gene,
wrapped up in lipid
droplets: “liposomes”
Outcome: 20% of
normal ion transport restored
to nasal epithelium