1. Lecture 45 Prof Duncan Shaw

2. 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

3. 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

4. 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

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

6. 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

7. Tracking the gene in families 12 3456 1 2 3 4 5 6 linked unlinked

8. Finding the right gene chromosome DNA fragments Clone in bacteria or yeast gene Mutation in patients but not controls? no yes Correct gene!

9. 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

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

11. Diagnosis by Linkage 1,2 2,2 1,1 1,2 2,2 1,2 1,1 1,2 A,B C,D A,CA,D ? uninformative informative haplotypes A,CD,E A,BC,D A,C ? Carrier status

12. CF diagnosis using 2 linked markers (haplotype)

13. 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…...

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