1.

TOXIN

2. ANIMAL PHENOTYPE

3. PHYSIOLOGY

DISCOVERY OF ION CHANNELS

4. REQUIRES AN ION TO LIVE

5. HUMAN DISEASE (ABNORMAL EKG)

IDENTIFICATION OF ION CHANNELS BY INHERITED DISEASE

positional cloning and d candidate gene approach Cardiac Arrythmias (Long QT syndrome)

Identification of many disease related genes

Search for a disease gene begins with linkage analysis. In this approach, the aim is to find out the rough location of the gene relative to another DNA sequence called a genetic marker marker, which has its position already known:

Principle P i i l of f li linkage k analysis: l i P Paternal t l (bl (blue) ) and d maternal (red) chromosomes aligned in a germ cell, a cell that gives rise to eggs or sperm. The capital letters represent the paternal alleles and th lower the l case l letters tt represent t the th maternal t l alleles for three genes: ABC. The middle panel shows recombination, which involves crossing over of DNA strands between the paired chromosomes. h Th The maternal t l and d paternal t l alleles ll l are mixed (recombined) and these mixed chromosomes are passed to the sperms or eggs. If A is the disease gene and B and C are genetic markers recombination is likely to occur much markers, more frequently between A and C than it is between A and B. This allows the disease gene to be mapped relative to the markers B and C.

IDENTIFICATION OF HUMAN ION CHANNEL RELATED DISEASES

LONG-QT syndrome KvLQT1 K channel HERG K channel SCN5A Na channel

1.4 Determining Distance Between Loci Linkage between two genes results in fewer recombination events than non non-recombination recombination events. events In this example example, we know the loci are linked because there are no recombinants. The proportion of gametes of each type (recombinant and non-recombinant) can be used to predict the distance between the two loci; in general general, the more recombinants there are, the farther apart the loci are.

In linkage analysis, genes are mapped relative to one another by the likelihood of recombination between the mutant gene (HERG) to known regions of the chromosome with highly polymorphic alleles (microsatellites). If there is linkage between the microsatellites and the loci where HERG is located located, the recombination rate will be very low.

marker marker K channels K channels marker

Above is the pedigree structure and genotypic analysis of one family with inherited LQT syndrome. Disease carrying individuals are closed squares and circles. Males are squares, females circles. Haplotypes (linked gene loci) for polymorphic markers (D7S505 (D7S505, D7S636 D7S636, D7S483) linked to LQT are shown under each individuals. Haplotypes cosegregating with the disease phenotype are indicated by a box. Haplotype analyses indicate that the LQT phenotype in these kindreds is linked to markers on chromosome 7q35-36.

Below is a pedigree structure of one family and the results of PCR amplification using primer pair 3, 9 and the effect of the mutation on the predicted structure of the HERG protein. There two PCR products associated with the diseased individual indicating that one allele carries the deletion of HERG leading to the Long QT syndrome