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

....CACACACACACA....

chromosome

PCR

No. of CAs varies (alleles)

DNA fragments

Electrophoresis1 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 2

3 4 5 6

1 2 3 4 5 6

linked

unlinked

Finding the right gene

chromosome

DNA fragments

Clone inbacteria oryeast

gene

Mutation in patientsbut not controls?

no

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 - F508

• Rest of cases include 100s of different mutations

The CF Gene (continued)• Because F508 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 1,2

2,2 1,1

1,2 1,2

2,2 2,2

1,2 1,1

1,2 1,2

1,2 1,2

1,2 1,2

A,B C,D

A,C A,D?

uninformative

informative haplotypes

A,C D,E

A,B C,D

A,C ?

Carrier status

CF diagnosis using 2 linkedmarkers (“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)

Functional CF gene,wrapped up in lipid droplets: “liposomes”

Nasal spray

CF patients

Outcome: 20% of normal ion transport restoredto nasal epithelium