## 3 Molecular Characterization of Coat Color Affecting Genes in Rabbits
### A Mutation in the Tyrosine-Related Protein 1 (Tyrp1) Gene and the Brown Locus  
The _Brown_ locus in rabbits has been first described by family based studies in the last century that suggested the presence of a wild type \(B\) allele that can produce dense eumelanin throughout the coat and a recessive \(b\) allele that is unable to produce black pigmentation, with the result that only brown pigments are obtained [30; 62; 31]. Using a candidate gene approach derived by the fact that in mice mutations in the tyrosinase-related protein 1 (_Tyrp1_) gene are responsible for the same locus [79], the rabbit _Tyrp1_ gene was sequenced in brown rabbits as well as in rabbits with several other coat colors not affected by the _Brown_ locus [80]. A non-sense mutation in exon 2 (g.41360196G > A), leading to a premature stop codon at position 190 of the deduced protein sequence (p.W190 ter), was identified only in Havana rabbits that have a characteristic brown coat color. Therefore, this disrupting mutation was considered the only candidate mutation determining the recessive \(b\) allele at this locus in rabbit [80]. The KIT Gene is Responsible for the English Spotting Locus and the Megacolon Defect in the Checkered Giant Bred  
A dominant allele (_En_) is considered the determining genetic factor for the spotted phenotype, very variable in extent, attributed to the _English spotting_ locus. The recessive wild-type allele (_en_) determines a self-colored phenotype [81]. Rabbits with the heterozygous genotype _En/en_ have far larger patches of colored fur than rabbits with the homozygous _En/En_ genotype [30]. Heterozygous _En/en_ rabbits have the characteristic spotted patterns described in the English spot (or spotted), Butterfly, and Checkered Giant breeds [67]. This is a unique allelic combination that is needed to produce breed standards desired by fancy breeders. The patches of the spotted phenotypes can be of different colors according to the allelic combination at other loci. Other genes are probably involved in determining the degree of extension of the colored patterns versus the white areas [82]. Dominant homozygous _En/En_ rabbits are affected by an underlying megacolon defect and are usually subvital compared to the animals with the other two genotypes, i.e., \(En/en\) and \(en/en\)[30; 82; 83; 84; 85; 86; 87]. The defect has incomplete penetrance, it is influenced by environmental factors (age, diet, stressors) and is recessive, as \(En/en\) rabbits are not affected [87]. Etiopathogenic analyses of rabbits having this megacolon pointed out that abnormalities of the enteric nervous system throughout the colon might be involved in this defect [85; 86; 88]. Several genes that are implicated both in coat color spotted patterns and in similar disfunctions of the digestive tract have been described in rodents and other mammals. Two of these genes were also investigated in association studies with the coat color and megacolon phenotypes in rabbits. The endothelin receptor B (_EDNRB_) gene was first excluded to be involved in these traits in Checkered Giant rabbits [89]. Subsequently, the analysis of _KIT_ gene markers in Checkered Giant families indicated that a synonymous polymorphism in exon 5 of this gene is associated with both traits [87]. The _KIT_ gene encodes the mast/stem cell growth factor receptor that is mainly involved in the differentiation of melanoblasts and in regulating their migration from the neural crest along the dorsolateral pathway to reach the final destinations in the skin [90; 91]. _KIT_ gene expression in the gut musculature is prominent only in interstitial cells of Cajal (ICC) that are essential in gut motility [92]. _KIT_ gene expression in eccum and colon specimens of \(En/En\) rabbits resulted very low compared to the expression in the same tissues observed in \(en/en\) rabbits. Reduced and altered c-kit immunolabelled ICC, together with neuronal and ICC abnormalities in eccum and colon tissues, were identified in \(En/En\) rabbits [87].