Detecting genes in new and old mouse models for epilepsy: a prospectus through the magnifying glass
Introduction
A colleague, Dr Gregory Cox, is known for reminding his seminar audience that positional cloning is rather like a genetic ‘whodunnit’:
“When you have eliminated all which is impossible, then whatever remains, however improbable, must be the truth.” Sir Arthur Conan Doyle—The Adventure of the Blanched Soldier.
Indeed, the dicta of criminal detection provide remarkably appropriate parallels to the process of positional cloning, where the ultimate goal is to narrow the field so that all but one gene is physically separable from the mutation—the gene that remains must be the correct gene. To date, even with rapidly advancing mouse genomics technologies and resources, it is no small task to achieve the ultimate state of ‘eliminating the impossible.’ Assuming that the average resolution initially attained in a genetic mapping cross is about 2.5 cM (40 backcross, or N2 mice), that the average size of a gene expressed in the brain is 2 kb of coding sequence spanning 25 kb of genomic DNA, that there are about 50 000 genes expressed in the mouse brain and that 1 cM corresponds to about 2000 kb in mice, a 200-fold ‘enrichment’ is required before any one of the approximately 75 genes in that initial 2.5 cM region can be indicted (Fig. 1). Moreover, as of this writing less than half of those 75 genes are known and only a fraction have been placed on chromosomal maps. Therefore, until these feats are achieved, which genomics gurus predict will happen by the year 2005, to positionally clone a gene by eliminating all but the right gene relies on relentless perseverance.
However, this is not to say that whenever possible, researchers avail themselves of shortcuts that can be taken along the way. For example, even without a high resolution genetic map, if a known gene has been mapped previously to a chromosome and if one has a strong hypothesis about the basis of the defect and thinks the known gene is a viable candidate, then one can test the gene for defects in mutant mice. This reliance upon one’s belief in the hope of gaining strong circumstantial evidence can often produce a conviction, especially when there is more than one independent occurrence of a mutation in a gene (multiple alleles) and/or when a mutation occurred on a known genetic background.
“Circumstantial evidence is occasionally very convincing, as when you find a trout in the milk, to quote Thoreau’s example.” Sir Arthur Conan Doyle—The Adventure of the Noble Bachelor.
But those who have done a lot of positional cloning know all too well that with anything less than a strong prior hypothesis about a candidate gene, the pursuit can be a great waste of effort. Sir Arthur seems to have foretold the fate of many molecular geneticists:
“It is of the highest importance in the art of detection to be able to recognize out of a number of facts which are incidental and which vital. Otherwise your energy and attention must be dissipated instead of being concentrated.” Sir Arthur Conan Doyle—The Reigate Puzzle.
Despite such heinous caution, gene discovery by positional cloning has proven to be extremely successful in the past 3 years—predominantly for single gene disorders—epilepsy models included. The cloning stories behind the slow-wave epilepsy, tottering and stargazer mouse models for spike-wave epilepsy (Fletcher et al., 1996, Cox et al., 1997, Letts et al., 1998) are representative examples of how each gene-improbable or not-was identified at different stages of the process, of what the risks were and of the evidence required to stake a claim. These examples draw a stark contrast to the situation for genetically complex models (Seyfried, 1983, Frankel et al., 1994, Frankel et al., 1995b, Ferraro et al., 1997), i.e. efforts to identify alleles that vary commonly in populations, which either serve as modifier genes to ‘major’ alleles, or together combine to effect seizure disorders. In light of the large number of epilepsy-related genes that are likely to exist and of the potential value of identifying them, phenotype-driven mutagenesis screens (Rinchik, 1991, King et al., 1997, Brown, 1998, Schimenti and Bucan, 1998) may well represent a viable means to such an end.
Section snippets
Background and phenotypic characteristics
The autosomal recessive slow-wave epilepsy (swe) mutation arose in the SJL/J strain at JAX in 1993 (Cox et al., 1997). Affected mice are easily recognized by 11–14 days-of-age based on their ataxic gait and slightly smaller size at weaning compared to unaffected littermates. Mutants display a moderate to severe degree of locomotor ataxia that is most prominent in the hindlimbs, with a slow, wide-based gait and coarse truncal instability during movement. In addition, depending upon genetic
Background and phenotypic characteristics
The original tottering mutation arose spontaneously in the DBA/2 strain (Green and Sidman, 1962), and was the first of several mouse models with 5–7 Hz spike-wave seizures accompanied by behavioral arrest (Noebels, 1986). This series of CNS-restricted mutants, which includes tottering, lethargic, ducky and stargazer, were initially detected because of their moderate-to-severe ataxia. As in human absence patients and most of these mutants, tottering seizures respond to anti-absence drugs such as
Background and phenotypic characteristics
First described in 1990 (Noebels et al., 1990), the stargazer mutation arose spontaneously at The Jackson Laboratory on the A/J inbred mouse line. Although mutant mice were initially noticed for their distinctive head-tossing and ataxic gait, subsequent electrocorticography revealed recurrent spike-wave seizures when the animal was still, characteristic of absence epilepsy. The seizures were notably more prolonged and frequent than in the other 5–7 Hz spike-wave seizure models, lasting on
Acknowledgements
I thank Verity Letts and Gregory Cox (The Jackson Laboratory) for reviewing a preliminary version of this prospectus, the other members of our laboratory, past and present, for providing the inspiration behind this article and our extramural collaborators cited in the text. I would also like to credit a Chris Redmond (whom I do not know), who conveniently listed a number of quotes from stories by Sir Arthur Conan Doyle on the ‘Sherlockian Holmepage’
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