Prof Duncan Shaw

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Lecture 45

• Prof Duncan Shaw

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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

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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

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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

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A type of DNA polymorphism
chromosome
....CACACACACACA....
No. of CAs varies (alleles)
PCR
DNA fragments
1 2 3 4 5
Electrophoresis
large
small
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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

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Tracking the gene in families
1 2 3 4 5 6
1
2
3
4
5
6
linked
unlinked
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Finding the right gene
chromosome
DNA fragments
gene
Clone in bacteria or yeast
Mutation in patients but not controls?
no
yes
Correct gene!
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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

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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 dont know mutation in family, test
  indirectly using linkage analysis.......

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Diagnosis by Linkage
1,2
1,2
C,D
A,B
1,2
1,2
1,1
2,2
A,C
A,D
2,2
2,2
?
haplotypes
informative
A,C
D,E
1,2
1,2
1,2
1,1
A,B
C,D
1,2
1,2
1,2
1,2
?
uninformative
A,C
Carrier status
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CF diagnosis using 2 linked markers (haplotype)
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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...

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Gene Therapy for CF (continued)
Nasal spray
CF patients
Outcome 20 of normal ion transport restored to
nasal epithelium
Functional CF gene, wrapped up in lipid
droplets liposomes