The spontaneous coat color mutant white nose (wn) maps to murine Chromosome 15

Fancy mice, having variation in coat color, were the progenitors of the earliest laboratory mice. These animals were readily amenable to genetic study because of their observable phenotypes. About 80 classical mutations affecting mouse coat color have been described to date. A number of the genes in which these mutations occur have now been cloned, leading to the finding that these mutations often correspond to human genetic disease loci. For example, dominant white spotting in mice is caused by mutations in the KIT receptor tyrosine kinase gene, and mutations at this locus are responsible for piebaldism in humans (Jackson 1994, 1997). In 1996 we noted the spontaneous appearance of a mouse with a white streak on its nose and belly in our C57BL/6-129/Sv transgenic colony. The founder animal was heterozygous for interruption of the gelsolin gene (Witke et al. 1995). However, after initial breeding experiments were performed, it became clear that the trait was segregating independent of the gelsolin locus, and was heritable as an autosomal recessive trait. Because the most striking initial feature of the phenotype was the linear white streak down the face of the C57BL/6-129/Sv mice, this trait was given the name white nose (wn). Analysis of several incross litters of animals possessing the trait revealed some variation in the phenotype (Fig. 1). There is a lack of pigmentation in a linear streak down the snout and on the ventral surface of all of the animals. However, the streak may run down the face of the mice from near the ears (Fig. 1a, middle animal); or it may start lower down, and a small separated patch lacking pigmentation may remain on the forehead (Fig. 1a, left animal). In similar fashion, variation may be seen in the ventral pigmentation defect, which may be of different width and have an irregular border (Fig. 1c, middle animal). In less than 5% of animals expressing the trait, an additional small patch lacking pigmentation is found on the dorsolateral body of the animals. The animals appeared to behave and to reproduce normally. Pathologic examination of four adult wn mice (2 males, age 3 months; 1 male and 1 female, age 1 year) revealed them to be of normal size and weight. There were no gross organ abnormalities, nor were tumors present. Histopathologic examination of the brain, heart, lungs, liver, spleen, kidney, and gut showed no significant findings. Hematology and chemistry tests, performed in two mice each, were within normal limits. These tests included a complete blood count, electrolytes, glucose, and liver function tests. Examination of the skin across the region of hypopigmentation by immunohistochemistry demonstrated no detectable S-100 staining in the hair follicles, indicating the absence of melanocytes (data not shown). No differences between male and female animals were noted. In order to map the wn locus in a backcross panel (Silver 1995), we utilized the C3H/HeJ strain (obtained from The Jackson Laboratory, Bar Harbor, Me.) after initial attempts using M. m. castaneus were unsuccessful. The C3H/HeJ strain was chosen, among others, because it is divergent enough to be distinguished from the C57BL/6 and 129/Sv strains with microsatellite markers, and, in addition, its dark coat color facilitates the clear distinction of the mutant phenotype. After an outcross mating between a male exhibiting the phenotype and C3H/HeJ females, backcross matings were initiated between the initial male and the female offspring. A panel of 48 backcross animals was thereby derived for linkage analysis. Twenty-two of these animals exhibited the characteristic markings shown in Fig. 1, consistent with Mendelian inheritance of an autosomal recessive trait. The murine chromosome containing the mutant gene underlying the trait was located with the strategy of interval haplotype analysis (Neuhaus and Beier 1998). Briefly, this method uses the determination of genotypes at proximal and distal markers on each murine chromosome to perform linkage analysis. Thirteen wn animals were genotyped, with 40 Massachusetts Institute of Technology (Mit) microsatellite primer pairs obtained from Research Genetics (Huntsville, Ala.). Although the markers were uninformative for 7 chromosomes, 11 chromosomes were eliminated as candidates (Chrs 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 19), and linkage to Chr 15 was identified (LOD score 3.9). To confirm this finding and to more precisely map the wn locus, we empirically tested 44 D15Mit microsatellite markers to determine their utility in the background of the C57BL/6-129/Sv and C3H/HeJ backcross. Of the markers, 23 were informative in the backcross animals, allowing the paternal and heterozygous phenotypes to be distinguished. As shown in Fig. 2a, three markers, D15Mit68, D15Mit31, and D15Mit1, had no recombination events within the wn locus (LOD score 14.4), confirming that wn maps to Chr 15. A single recombination event defined the proximal end of the critical interval (D15Mit187), and two recombination events defined the distal end (D15Mit241). These flanking microsatellite markers have been mapped to positions 43.3 and 46.4 cM, respectively, by the Mouse Genome Database (1998), indicating that we have mapped the wn locus to a 3.1-cM candidate interval. Analysis of recombination events in our mapping panel gives a somewhat larger estimate for the size of this region, 6.3 cM. No other known coat color mutation maps within this interval. The previously described coat color mutation belted (bt), consisting of the absence of pigmentation in a transverse band on the body (Murray and Snell 1945), has been mapped telomeric to D15Mit241 in one of our laboratories (D.Foernzler and D.R.Beier, unpublished observation). Also, examination of over 20 progeny Correspondence to: P.W. Marks Mammalian Genome 10, 750–752 (1999).