Peter John M.Phil, PhD Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences & Technology (NUST)

Position Dependent Strategies

Functional complementation in transgenic mice A mouse gene has been identified by constructing transgenic mice Using non mutant BAC clones from a candidate region, crossing them to mice carrying the mutation

Human MYO15 gene DFNB3 had been mapped to a location that corresponded in the mouse to the location of the deafness gene shaker-2 Transgenic mice were constructed using BACs from the shaker-2 candidate region, and a BAC that corrected the shaker-2 phenotype was identified.

Human MYO15 gene This led to identifying the shaker-2 gene Human MYO15 gene was then isolated based on its close homology to the mouse gene, Its position within the DFNB3 candidate region confirmed, Mutations demonstrated in DFNB3 affected people.

Functional Complementation in Transgenic Mice

Functional Complementation in Transgenic Mice The shaker-2 mouse mutation was identified by finding a wild-type clone that corrected the defect.

Functional complementation in mammalian cell lines Mammalian cell lines have been generated that are deficient in DNA repair They show abnormal responses following exposure to UV irradiation or chemical mutagens. These mutant cells, can be transformed by fragments of normal human DNA or human chromosomes in order to produce a repair-competent phenotype.

Functional complementation in mammalian cell lines The ability of transferred chromosomes or clones to correct the uncontrolled growth of tumor cell lines has been used to help locate and then identify tumor suppressor genes

Positional cloning First step in positional cloning is to define the candidate region Initial localization from genetic mapping defines a candidate region of 10 Mb or more. The next step is to collect as many families as possible and establish a dense cover of polymorphic markers across the region.

Positional cloning Suitable markers may be found by database searching. Otherwise YACs, BACs and cosmids must be isolated from the candidate region and screened for polymorphisms. Pairs of closely spaced markers define the positions of the closest recombinations on either side of the disease locus.

Positional cloning This is decided by inspecting individual haplotypes. Linkage disequilibrium may allow very high resolution mapping

Candidate Region Search cDNA library screening, using as probes genomic clones from the candidate region CpG island identification, to seek the regions of under-methylated DNA which often lie close to gene

Chromosomal aberrations chromosomal break can cause a loss-of-function phenotype if it disrupts the coding sequence of a gene, or separates it from a nearby regulatory region. The breakpoint provides a valuable clue to the exact physical location of the disease gene.

Deletions and duplications Chromosomal deletions cause abnormalities due to loss of genes Microdeletions can be identified by several methods. FISH mapping. Hybridization-based restriction mapping.

Positional candidate strategies Identify candidate genes by a combination of their Map position Expression Pattern Function or homology

Positional candidate strategies Predictions of the biochemical function of an unknown disease gene are often proved wrong once the gene is isolated. candidate regions identified by positional cloning usually contain dozens of genes. It can be very time-consuming to identify every transcript from the region, and excessively laborious to screen them all for mutations.

Criteria for candidate gene Appropriate expression pattern Candidate gene should have an expression pattern consistent with the disease phenotype. Expression of candidate genes can be tested by RT-PCR or Northern blotting, but the best method for revealing the exact expression pattern is in situ hybridization against mRNA in tissue sections

Appropriate function Candidate genes may also be suggested on the basis of a close functional relationship to a gene known to be involved in a similar disease. The genes could be related by encoding a receptor and its ligand, or other interacting components in the same metabolic or developmental pathway.

Homology to a relevant human gene or EST Selecting candidate disease genes by homology is often more successful using model organisms Identification of transcripts often comes from matching genomic sequence generated from the candidate region against unmapped ESTs in the databases. Finding a match suggests the presence of an exon in the genomic DNA

Homology to a relevant gene in a Model Organism Powerful means of selecting good candidates from among a set of human genes is therefore to search the databases for evidence of homologous genes in these well-studied model organisms Such data might include the pattern of expression and the phenotype of mutants

Confirming a candidate gene Mutation screening Restoration of normal phenotype in vitro by using cell lines Production of a mouse model of the disease, a transgenic mouse model can be constructed.

Functional Analysis Once a candidate gene is confirmed, the next step is to understand its function Understanding the molecular pathology may also lead to insight into related diseases, and hopefully eventually to more effective treatment including perhaps gene therapy

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