Identification of c.483C>T polymorphism in the caprine tyrosinase-related protein 1 (TYRP1) gene

Tyrosinase-related protein 1 (TYRP1) has been shown to play a fundamental role in pigmentation both in human and mouse. In this work, we aimed to characterize the variability of the caprine TYRP1 gene and investigate its segregation in a wide array of goat breeds. By partially sequencing the coding region of the TYRP1 gene in 18 individuals from eight different breeds, we were able to identify a synonymous nucleotide substitution at exon 3 (c.483C>T). An extensive survey of Iberian and Balearic (N=175), Italian (N=99), Swiss (N=54), Asian (N=14), Canarian (N=92) and North African (N=117) goats with different coat colours was carried out. We found that the C-allele has a different distribution in European vs African breeds, being almost fixed in the latter. Moreover, the C-allele showed an increased frequency in white coated breeds (Girgentana, Grigia Molisana, Blanca de Rasquera and Saanen) when compared with those displaying a dark pigmentation (Cilentana Nera, Azpi Gorri and Murciano-Granadina). This could be due to genetic drift, migration and other factors associated with the demographic history of breeds under analysis or to a genetic hitchhiking event (c.483C>T frequencies would be shaped by a neighbouring causal mutation differentially selected in white and black goats). More refined studies will be needed to distinguish between these two alternative explanations.


Introduction
The molecular basis of coat colour inheritance in goats is very complex as demonstrated by recent studies targeting genes with known effects on pigmentation. Fontanesi et al. (2009) characterized the variability of the caprine melanocortin receptor 1 (MC1R) gene identifying one nonsense, three missense and one silent single nucleotide polymorphisms (SNP). Unfortunately, none of these mutations showed a clear association with coat colour when analysing their segregation in a pool of 317 goats belonging to six breeds with different pigmentation patterns (Fontanesi et al., 2009). Moreover, sequence analysis of the caprine agouti signaling protein (ASIP) gene has evidenced the existence of a duplicated copy, as previously demonstrated in sheep (Norris and Whan 2008), and three missense SNP. Whilst the ASIP copy number polymorphism might be associated with white colour in the Girgentana and Saaanen breeds, the three missense SNP did not shown any significant effect on pigmentation (Fontanesi et al., 2010).
Another molecule with known effects on the phenotypic variation of coat colour is tyrosinase-related protein 1 (TYRP1), a melanosomal enzyme deeply involved in eumelanin synthesis (Sarangarajan and Boissy 2001). Classical studies performed in mouse revealed that TYRP1 loss-of-function alleles are associated with a brown coat (Bennett and Lamoreux 2003). In cattle, a non-conservative H434Y substitution at the bovine TYRP1 enzyme has been related with the Dun coat of Dexter cows (Berryer et al., 2003), while in Soay sheep there is strong evidence that replacement of a highly conserved cysteine at position 290 by phenylalanine results in a lighter colour (Gratten et al., 2007).
Given its fundamental role in pigmentation, we have characterized the sequence variability of the coding region of the caprine TYRP1 gene and investigated its segregation in a wide array of caprine breeds from the Iberian Peninsula and Balearic Islands, Italy, Canary Islands, Asia and North Africa.

Materials and methods
Nucleic acids isolation and complementary DNA synthesis Genomic DNA extraction from hair follicles was performed with the DNeasy Blood and Tissue kit (Qiagen Iberia SL, Barcelona, Spain), while protocols reported by Zidi et al., (2008) were employed to purify DNA from blood samples. A Nanodrop spectrophotometer ND-1000 (SG Servicios Hospitalarios, Barcelona, Spain) was used to measure the concentration of genomic DNA preparations. We also isolated total RNA, with the RiboPure kit (Applied Biosystems, Sant Andreu de Llavaneres, Spain), from skin samples obtained from Palmera, Majorera and Tinerfeña goats at the slaughterhouse. Total RNA concentration was estimated with an Agilent 2100 Bioanalyzer equipment (Agilent Technologies, Barcelona, Spain) and reverse transcribed to complementary DNA with the Thermoscript RT-PCR System kit (Invitrogen, Barcelona, Spain). Both genomic DNA and cDNA were used as templates in sequencing experiments.
Sequencing reactions were carried out with the BigDye Terminator v1.3 Cycle Sequencing Kit (Applied Biosystems, Sant Andreu de Llavaneres, Spain) and primers listed in Table  1. Genotyping was performed in a Sequenom MassARRAY iPLEX platform at the Spanish National Genotyping Centre (CeGen, Santiago de Compostela, Spain). A total of 551 individuals were genotyped. These goats belonged to the following geographical areas: Italy ( Canary Islands (N=92): Tinerfeña (N=38), Palmera (N=38) and Majorera (N=16).
Switzerland (N=54): Saanen (N=54). Asia (N=14): Cashmere (N=14). TYRP1 frequencies were compared taking into consideration the geographical distribution of each breed (population analysis). A second comparison was made between white (Girgentana, Grigia Molisana, Blanca de Rasquera and Saanen) and black breeds (Cilentana Nera, Azpi Gorri and Murciano-Granadina). Instead of taking pictures from each one of the 551 goats under analysis and defining colour on an individual basis, we considered that each breed is represented by a single coat colour that corresponds to the one defined in the racial standard or, in the lack of Badaoui et al.  it, to the one that is more typically observed. Although this procedure simplifies the obtaining of phenotypes, it also diminishes the power of the statistical analysis because we did not take into account that a certain level of coat colour heterogeneity exists in each breed.

Results and discussion
In the current work we have characterized the near-complete sequence of the goat TYRP1 coding region (GenBank accession number: HQ391904). Analysis with the Prosite software (http://expasy.org/prosite, Figure 1) showed that the goat TYRP1 protein sequence has a conserved epidermal growth factor (EGF)-like domain represented by two hits (amino acid positions are numbered according to the TYRP1 coding sequence, with position 1 indicating the first nucleotide of the translation initiation codon): EGF-like domain signature 1 [99-110] and laminin-type EGF-like (LE) domain signature . Furthermore, two histidine rich copper-binding sites, i.e. tyrosinase CuA-binding region signature [215][216][217][218][219][220][221][222][223][224][225][226][227][228][229][230][231][232] and tyrosinase and hemocyanins CuB-binding region signature [397][398][399][400][401][402][403][404][405][406][407][408], were found to be evolutionary conserved. According to Jackson et al. (1992), the EGF-like domain might allow the interaction of TYRP1 with other melanoso-mal proteins to form a multienzymatic complex, while the copper-binding domains might have an important catalytic role (Furumura et al., 1998) Partial sequencing of the TYRP1 coding region in 18 individuals from diverse breeds allowed us to identify a synonymous c.483C>T SNP mapping to exon 3 (Figure 2). It is necessary to stress that our sequencing approach targeted a limited number of exons, so additional variability might be found by analysing other TYRP1 regions, such as introns and 3'UTR, as well as by sampling other goat populations. We investigated the segregation of the c.483C>T polymorphism in 551 goats belonging to 16 breeds by using a Sequenom MassARRAY iPLEX platform (Figure 3, Table  2). This analysis evidenced that the C-allele is almost fixed in Moroccan and Tunisian goats, and fixed in Canarian (Palmera, Tinerfeña and Majorera) breeds. This strong similarity in allelic frequencies between African and Polymorphism of the goat TYRP1 gene  Canarian goats is expectable given the close relationship between both geographical areas. Remarkable coincidences have been found between the different dialects spoken by the indigenous populations of the Canary Islands and the Amazigh language (Farrujia 2004). Moreover, analysis of mitochondrial and Ychromosome markers have revealed the presence of Berber haplotypes in the gene pool of Canary islanders at higher frequencies than in that of Iberian colonizers, a feature that suggests that these haplotypes entered the Canary islands as a consequence of a migratory movement originated at North Africa (Maca-Meyer et al., 2004;Fregel et al., 2009). In summary, our result is consistent with the hypothesis of a North African ancestry for Canarian goats but this interpretation still needs to be proved by surveying a representative number of genetic markers. We also observed that the C-allele has an increased frequency in white (Girgentana, Grigia Molisana, Blanca de Rasquera and Saanen) vs black (Cilentana Nera, Azpi Gorri and Murciano-Granadina, Tinerfeña was excluded because the C-allele is fixed) breeds. In this way, the frequency of the C-allele was around 0.57 and 0.86 in black and white goats, respectively (Table 2). This result can be explained in two ways. First, demographic and evolutionary forces might have differentially shaped TYRP1 allele frequencies in these two groups of goats. In this case, we would assume that the TYRP1 polymorphism is neutral and that differences (0.57 vs 0.86) observed in the frequencies of the C-allele are fortuitous. Alternatively, the increased frequency of the Callele in the white goat group might have been caused by genetic hitchhiking i.e. the synonymous polymorphism we have analysed is linked to an unknown causal mutation affecting melanogenesis. There are several factors that make difficult to discriminate between these two alternative hypotheses. In the first place, the goat groups we have analysed are composed by different breeds i.e. there is a substantial population substructure, a feature that might favour the emergence of spurious associations. Moreover, several of the analysed breeds do not display homogeneous coat colours limiting the power of our analysis. Finally, epistatic interactions between pigmentation genes occur very often, implying that the specific genetic background of each breed can differentially affect the penetrance of the TYRP1 genotype. By these reasons, trying to infer associations between pigmentation genes and coat colour based on comparing allelic frequencies between breeds is not expected to be a very fruitful approach. A more informative strategy would be to examine the co-segregation of the C-and T-alleles and pigmentation in within-breed crosses between individuals with divergent phenotypes for this trait. Once associations were confirmed, it would be necessary to sequence the 16.8 kb transcription unit of TYRP1 in related individuals with different coats to identify the causal mutation.

Conclusions
We have identified a synonymous c.483C>T SNP in the goat TYRP1 gene. Although this substitution is not expected to produce a functional change, we have observed an increased frequency of the C-allele in white vs black goats. Additional studies should be performed at the within breed level to evaluate the relative impact of demographic vs selection factors on the variability of the goat TYRP1 locus.