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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