The Story so far

As mentioned earlier, a lot of emphasis has been placed in developing expression maps of the human and mouse genomes which will assist the process of disease-gene identification (Durkin at a/; 1992; Polymeropoulos at a!., 1992 and 1993; Okubo at a!., 1992; Murakawa at a!., 1994; Takahashi and Ko, 1993; Southern, 1992). This is probably a reflection of the fact that both in mice and humans, the identification of mutations associated with developmental anomalies was greatly enhanced by the availability of candidate genes and comparative mapping information (reviewed by Copeland at a!., 1993). The molecular basis for the majority of mouse mutants was established through the candidate gene approach.

The integration of mouse and human genetic maps provide an important tool in the search for the genetic cause of developmental anomalies in both species. Several examples illustrate the value of comparative mapping. In 1990 the gene for Waardenburg Syndrome Type I (WSl) was localised to region 2q37 (Foy at a!., 1990). This region is homologous to a part of mouse chromosome 1 which harbours the gene responsible for the developmental mutant splotch (Sp), WSI belongs to a group of four auditory-pigmentary syndromes characterised by abnormal pigmentation of the skin, hair and eyes, loss of hearing and mild facial dysmorphism (Waardenburg, 1951; Arias, 1971). The cause is thought to be the absence of melanocytes, of neural crest origin, from the affected tissues (Hughes at a i, 1994). splotch was classified as a semidominant mutation and exists in several spontaneous (Dickie, 1964; Silvers, 1979) and X-irradiation induced alleles (Beechey and Searle, 1986). Heterozygotes for the first allele (Sp), which arose spontaneously (Russell, 1947), have white spotting of the belly and occasionally on the back, feet, and tail (Silvers, 1979). On certain genetic backgrounds, heterogygotes for the Sp^ (delayed; Dickie, 1964) alelle develop craniofacial abnormalities (cited by Steel and Smith,

1992). The disruption of pigmentation of the iris and hypopigmentation of the hairs and the craniofacial anomalies seen in Sp^/+ heterozygotes, coupled with comparative mapping information, led to the suggestion that splotch and WSI could be caused by defects in homologous loci (Foy et al., 1990). The murine paired box- containing gene 3, Pax3, was mapped to mouse chromosome 1 and found to be associated with the splotch mutation (Epstein at al., 1991; Goulding at a i, 1993). This led to the discovery of mutations in the human homologue of Pax3, PAX-3, in WSI patients (Tassabehji at al., 1992 and 1993). Unlike the hearing impairement of WSI patients, some splotch heterozygotes show no impairement of the auditory system (Steel and Smith, 1992). Mutations in the PAX-3 gene also cause the Klein- Waardenburg syndrome, WS-III (Hoth at ai, 1993).

The Waardenburg syndrome type II gene (WSII) has recently been mapped to human chromosome 3 (Hughes at al., 1994), close to the human homologue of the mouse microphthalmia (mi) gene (Tachibana at ai, 1994). The WSII gene is also inherited in a dominant mode and, unlike Waardenburg syndrome type I, is not associated with facial dysmorphism (Arias, 1971). The microphthalmia mouse mutant was suggested as a candidate model for some forms of the Waardenburg syndrome (Asher and Friedman, 1990) and was found to be caused by mutations in a basic-helix-loop-helix-zipper (bHLH-ZIP) protein (Hodgkinson at a i, 1993). Sequence analysis of the human homologue of the mi gene, MITF, revealed point mutations in two familial cases of WSII (Tassabehji at ai, 1994). Other examples of homologous-gene mutations causing similar phenotypes include the human condition aniridia (AN) and the mouse mutation small eye (Say) and the shakar-1 {sh1)rx)ouse mutant and Usher type IB (Ton at ai, 1991; Hill at ai, 1991; Jordan at a i, 1992; reviewed by Darling and Abbott, 1992; Gibson at ai, 1995; Well at a i, 1995).

Mouse mutations which are associated with deletions or other chromosomal rearrangements are naturally more amenable to gene identification, since the chromosomal rearrangements themselves are relatively easy to characterise (Bultman at ai, 1991; Bultman at ai, 1992). The agouti (a) locus, for example, has at least 19 different alleles (Silvers, 1979; Green, 1989). This locus controls the production of eumelanin (black or brown), and pheomelanin (yellow) pigment granules by the melanocytes in the hair follicle. The eumelanin production is also influenced by alleles present in other loci (Silvers, 1979). The starting strain which led to the identification of this gene carried an inversion which disrupted both the agouti and the limb deformity loci on mouse 2 (Woychik at ai, 1985, Woychik at a i, 1990). The molecular characterisation of the breakpoints led to the identification of the agouti gene which was shown to be associated with several rearrangements in different agouf/alleles (Bultman at ai, 1992). Several other alleles of this locus have since been characterised (Bultman at ai, 1994; Michaud at ai, 1993; Miller at a i,

From a total of about 50 mouse mutations cloned, only in three cases was there no chromosomal rearrangements. These loci are the mouse nude (nd), obese (ob) and shaker-1 (sh1), which were discovered through the isolation and analysis of transcribed sequences in the candidate regions (Nehls et al., 1994; Zhang et a!.,

1994; Gibson et a/., 1995). Crucial to these discoveries was the technique of exon trapping’ (Buckler et a!., 1991; Hamaguchi et a!., 1992). Briefly, DNA fragments from the candidate region are subcloned into an intron present in a mammalian expression vector. After transfection of a suitable eukaryotic cell line, the cytoplasmic messenger RNA (mRNA) is screened by PCR amplification using oligonucleotide primers that flank the engineered intron. If this has acquired an exon-containing DNA fragment, the PCR product will be larger than expected. These clones are then used to screen complementary DNA (cDNA) libraries, derived from tissues thought to expressed the defective gene, for obtaining full-length transcripts. In the case of the nude gene, some 50 putative exons were used to screen the expression pattern of their parent genes and one was selected for further analysis; this eventually led to the identification of the gene involved and the causative mutation (Nehls et a!.,

1994).