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Lectures3-4 - Discovery of Human Disease Genes Gene...

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Gene identification by genetic complementation Analysis of candidate gene/s Positional cloning following linkage analysis Bionformatics using genome information Discovery of Human Disease Genes Functional complementation of mammalian cell lines from patients or mice- e.g. DNA repair disorders and tumor suppressor genes. Functional complementation of yeast mutants - e. g. metabolic defects. Complementation of animal models of disease - using knock-outs or transgenic disease models. Isolation of activated oncogenes using NIH 3T3 cells. Gene identification by genetic complementation Requires prior knowledge of genes in the vicinity of the disease gene or of the underlying defect in the disease. Bioinformatic analysis of known genes, expression properties, model systems, localization studies. Genes cloned by this strategy include retinitis pigmentosa, familial hypertrophic cardiomyopathy and Li-Fraumeni syndrome. Analysis of candidate gene/s No prior knowledge of function of the gene needed. Location of gene is necessary by linkage to genetic markers (RFLPs, microsatellite repeats or other linked alleles). Step 1 - Gene localization - use linkage analysis to determine which chromosome is the gene located on? Map as finely as possible relative to genetic markers to narrow down to 10-100 putative genes. Step 2 - Gene identification - which gene is mutated in patients with disease? i.e. test candidate genes. Is the gene, mRNA, protein or protein function affected? Positional cloning of human disease genes
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Steps in Positional Cloning Several genes such as Huntington’s, Duchenne Muscular Dystrophy and cystic fibrosis were cloned this way. CFTR cloned in this way by Lap-Chee Tusi et al. in 1985. Linkage analysis was used to map gene to chromosome 7. Testing of linkage with more markers mapped disease gene to 1.5 Mb interval between MET and the marker D7S8. Chromosome jumping and cloning in YACs were used to define candidate genes. Patients were found with mutations in CFTR. By 1990, it was known that CF patients lacked a chloride transporter. The fully cloned CFTR gene complemented cells from CF patients. Today, bioinformatics tools of the 12 genes in 1.5 Mb interval would reveal only 3 putative membrane proteins and only one (CFTR) with the right tissue-specific expression pattern. Example of Positional Cloning Linkage Analysis Slides from web European School of Genetic Medicine Paolo Forabasco What is Linkage Analysis? Linkage analysis is a statistical tool used to define the location of a given disease gene on a chromosome by asking whether the disease gene is linked closely to a known, polymorphic genetic marker. The tighter the linkage, the closer the disease gene is to the marker .
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A first step in positional cloning and identification of candidate disease genes is to identify where the disease gene is located. This is done by linkage mapping.
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