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L28 - Disease Mapping Be able to describe the steps in a positional…

- - - - Examination of Clonal Sequences
        Examing the clonal sequences revealed four candidate genes in the region
        Use existing knowledge to determine which candidates most plausibly contribute to diseased phenotype
        
        Additional Studies Eliminated three genes
        
        Further DNA sequencing in unaffected family memnbers and patients with the suspect genes revealed a 3-bp deletion in the gene of the CF patient
        
        CF Mechanism of Disease
        Gene for CF encodes a membrane protein that controls chloride movement into & out of cells
        Mutations cause channel to stay closed = chloride builds up in
        cells, thick mucous build up
- - - - Expression Array(No details given.
      - SNP Genotyping - Microarray SNP-Chip
        
        Probeswhich differe from one another at one base psosition will bind to different complimentary sequences. They fluoresce at unique base and allow the sequence to be read
    - - Targeting a Diseased gene - (Disease Target Panel)
        
        Genomic DNA is fragmented
        
        Fragment DNA is hybridised with RNA library ‘baits’
        
        These are designed to anneal to
        the genes of interest*
        
        The RNA baits contain a biotin label which makes them discernible
        
        The target DNA is purified from the total DNA by addition of streptavidin beads which coat the hybrids and which are magnetic
        
        Relies upon a Biotin-streptavidin-bead-magnet
        
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      - Whole Exome Sequencing
        Requires a comparison genotype, (e.g.family member).
        Largely uninformative if used on a single individual.
        
        Probes rely upon hybridisation seletcion to capture the coding portion of the genome for high throughput sequencing
        
        Reduced cost compared
        to whole genome
        
        Can pick up mutations in
        genes not already known
        to cause disease
      - Whole genome
        
        Not commonly used for diagnosis
        
        Costly
        
        Sensitive to splice site variants (intronic)
        
        Not useful for determining effect of non-coding sequence
        
        Limitations
        
        Repeats, >50% of human genome is repeats, these do not sequence/align well
        
        Difficult to identify structural variation - aligns to duplicates more than one
        
        Data storage and analysis huge computational power and cost
  - - - Variants and deleterious mutations in unknown gene(s)
      - Synonymous variants
      - Missense variants of uncertain significance in
        known genes
      - Variants in non coding regions
    - - Linkage
        
        Linkage is measured by the recombination fraction, θ= probability that, in any meiosis, there will be a recombination between adjacent loci.
        
        θ = 0.0: complete linkage
        
        θ = 0.5: no linkage
        
        The Closer LOCI are together, the less likely it is that they will be separated through recombination
        
        Thereby LOCI which are close together, are inherited together
        
        LOCI which are linked are in Linkage Disequilibrium
- - - - Strategies for Enriching information obtained through Linkage Analysis can be applied in any of these two scenarios
        
        Genetic Heterogeneity
        Reliance on low incidence families, as high incidence implies that there is more than one gene variant affecting disorder
        
        Incomplete Penetrance
        If low penetrance, than high incidence families are more informative = more individuals to detect the disease in.
- - - - Calculating LOD
        Calculate two different probabilities for obtaining a specific set of recombinants observed in a family
        
        1. First probability is calculated assuming independent assortment (No linkage)
        
        2. Second probability is calculated assuming a specific degree of linkage (RF = 0.0 - 0.5)
        
        Lod Score (Z)
        A test to estimate whether the likelihood of two loci being linked is greater than likelihood that the same two loci are unlinked
        
        Equation (Slide 23-25)