Novel variants in the KIT and PAX3 genes in horses with white‐spotted coat colour phenotypes

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part‐bred Arabians with sabino‐like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white‐increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18–W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.     

A complex structural variant at the KIT locus in cattle with the Pinzgauer spotting pattern

A specific white spotting phenotype, termed finching or line‐backed spotting, is known for all Pinzgauer cattle and occurs occasionally in Tux‐Zillertaler cattle, two Austrian breeds. The so‐called Pinzgauer spotting is inherited as an autosomal incompletely dominant trait. A genome‐wide association study using 27 white spotted and 16 solid‐coloured Tux‐Zillertaler cattle, based on 777k SNP data, revealed a strong signal on chromosome 6 at the KIT locus. Haplotype analyses defined a critical interval of 122 kb downstream of the KIT coding region. Whole‐genome sequencing of a Pinzgauer cattle and comparison to 338 control genomes revealed a complex structural variant consisting of a 9.4‐kb deletion and an inversely inserted duplication of 1.5 kb fused to a 310‐kb duplicated segment from chromosome 4. A diagnostic PCR was developed for straightforward genotyping of carriers for this structural variant (KIT^PINZ) and confirmed that the variant allele was present in all Pinzgauer and most of the white spotted Tux‐Zillertaler cattle. In addition, we detected the variant in all Slovenian Cika, British Gloucester and Spanish Berrenda en negro cattle with similar spotting patterns. Interestingly, the KIT^PINZ variant occurs in some white spotted animals of the Swiss breeds Evolèner and Eringer. The introgression of the KIT^PINZ variant confirms admixture and the reported historical relationship of these short‐headed breeds with Austrian Tux‐Zillertaler and suggests a mutation event, occurring before breed formation.     

Whole‐genome sequencing reveals a large deletion in the MITF gene in horses with white spotted coat colour and increased risk of deafness

White spotting phenotypes in horses are highly valued in some breeds. They are quite variable and may range from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for white spotting phenotypes in horses. For the present study, we investigated an American Paint Horse family segregating a phenotype involving white spotting and blue eyes. Six of eight horses with the white‐spotting phenotype were deaf. We obtained whole‐genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~63‐kb deletion spanning exons 6–9 of the MITF gene (chr16:21 503 211–21 566 617). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. PCR‐based genotyping revealed that all eight available affected horses from the family carried the deletion. The finding of an MITF variant fits well with the syndromic phenotype involving both depigmentation and an increased risk for deafness and corresponds to human Waardenburg syndrome type 2A. Our findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

A novel KIT deletion variant in a German Riding Pony with white‐spotting coat colour phenotype

White spotting phenotypes in horses may be caused by developmental alterations impairing melanoblast differentiation, survival, migration and/or proliferation. Candidate genes for white‐spotting phenotypes in horses include EDNRB, KIT, MITF, PAX3 and TRPM1. We investigated a German Riding Pony with a sabino‐like phenotype involving extensive white spots on the body together with large white markings on the head and almost completely white legs. We obtained whole genome sequence data from this horse. The analysis revealed a heterozygous 1273‐bp deletion spanning parts of intron 2 and exon 3 of the equine KIT gene (Chr3: 79 579 925–79 581 197). We confirmed the breakpoints of the deletion by PCR and Sanger sequencing. Knowledge of the functional impact of similar KIT variants in horses and other species suggests that this deletion represents a plausible candidate causative variant for the white‐spotting phenotype. We propose the designation W28 for the mutant allele.     

A de novo germline mutation of KIT in a white-spotted Brown Swiss cow

White-spotting coat colour phenotypes in cattle are either fixed characteristics of specific cattle breeds or occur sporadically owing to germline genetic variation of solid-coloured parents. A Brown Swiss cow showing a piebald pattern resembling colour-sidedness was referred for genetic evaluation. Both parents were normal solid-brown-coloured cattle. The cow was tested negative for the three known DNA variants in KIT, MITF and TWIST2 associated with different depigmentation phenotypes in Brown Swiss cattle. Whole-genome sequencing of the cow was performed and a heterozygous variant affecting the coding sequence of the bovine KIT gene was identified on chromosome 6. The variant is a 40 bp deletion in exon 9, NM_001166484.1:c.1390_1429del, and leads to a frameshift that is predicted to produce a novel 50 amino acid-long C-terminus replacing almost 50% of the wt KIT protein, including the functionally important intracellular tyrosine kinase domain (NP_001159956.1:p.(Asn464AlafsTer50)). Interestingly, among three available offspring, two solid-coloured daughters were genotyped as homozygous wt whereas a single son showing a slightly milder but still obvious depigmentation phenotype inherited a copy of the novel variant allele. The genetic findings provide strong evidence that the identified loss-of-function KIT variant most likely represents a de novo germline mutation that is causative owing to haploinsufficiency.     

Whole genome sequencing reveals a novel deletion variant in the KIT gene in horses with white spotted coat colour phenotypes

White spotting phenotypes in horses can range in severity from the common white markings up to completely white horses. EDNRB, KIT, MITF, PAX3 and TRPM1 represent known candidate genes for such phenotypes in horses. For the present study, we re‐investigated a large horse family segregating a variable white spotting phenotype, for which conventional Sanger sequencing of the candidate genes’ individual exons had failed to reveal the causative variant. We obtained whole genome sequence data from an affected horse and specifically searched for structural variants in the known candidate genes. This analysis revealed a heterozygous ~1.9‐kb deletion spanning exons 10–13 of the KIT gene (chr3:77,740,239_77,742,136del1898insTATAT). In continuity with previously named equine KIT variants we propose to designate the newly identified deletion variant W22. We had access to 21 horses carrying the W22 allele. Four of them were compound heterozygous W20/W22 and had a completely white phenotype. Our data suggest that W22 represents a true null allele of the KIT gene, whereas the previously identified W20 leads to a partial loss of function. These findings will enable more precise genetic testing for depigmentation phenotypes in horses.     

Increasing the accuracy of genomic prediction in pure‐bred Limousin beef cattle by including cross‐bred Limousin data and accounting for an F94L variant in MSTN

Explicitly fitting effects for major genes or QTL that account for a large percentage of variation in a whole genomic prediction model may increase prediction accuracy. This study compared approaches to account for a major effect of an F94L variant in the MSTN gene within the genomic prediction using bovine whole‐genomic SNP markers. Among the beef cattle breeds, Limousin have been known to have an F94L variant that is not present in Angus. The reference population in this study consisted of 3060 beef cattle including pure‐bred Limousin (PL), cross‐bred Limousin with Angus (LF) and pure‐bred Angus, genotyped using a BovineSNP50 BeadChip and directly for the MSTN‐F94L variant. We compared prediction accuracies in PL animals using the three datasets from only the PL population, admixed PL and LF (AL) or multibreed analysis using all of the PL, LF and Angus (MB) population according to four‐fold cross‐validation after K‐means clustering. The MSTN‐F94L variant was the most strongly associated with five traits (birth weight, calving ease direct, milk, weaning weight and yield grade) among the 13 measured traits in PL and AL populations. Fitting the MSTN‐F94L variant as a random effect, the genomic prediction accuracies for birth weight increased by 2.7% in PL, by 2.2% in AL and by 3.2% in MB. Prediction accuracies for five traits increased in the MB analysis. Fitting MSTN‐F94L as a fixed effect in PL, AL and MB analyses resulted in increased prediction accuracy in PL for eight traits. Prediction accuracies can be improved by including a causal variant in genomic evaluation compared with simply using whole‐genome SNP markers. Fitting the causal variant as a fixed effect along with markers fitted as random effects resulted in greater prediction accuracies for most traits. Causal variants should be genotyped along with SNP markers.     

A genome‐wide significant association on chromosome 2 for footrot resistance/susceptibility in Swiss White Alpine sheep

Footrot is one of the most important causes of lameness in global sheep populations and is characterized by a bacterial infection of the interdigital skin. As a multifactorial disease, its clinical representation depends not only on pathogen factors and environmental components but also on the individual resistance/susceptibility of the host. A genetic component has been shown in previous studies; however, so far no causative genetic variant influencing the risk of developing footrot has been identified. In this study, we genotyped 373 Swiss White Alpine sheep, using the ovine high‐density 600k SNP chip, in order to run a DNA‐based comparison of individuals with known clinical footrot status. We performed a case–control genome‐wide association study, which revealed a genome‐wide significant association for SNP rs418747104 on ovine chromosome 2 at 81.2 Mb. The three best associated SNP markers were located at the MPDZ gene, which codes for the multiple PDZ domain crumbs cell polarity complex component protein, also known as multi‐PDZ domain protein 1 (MUPP1). This protein is possibly involved in maintaining the barrier function and integrity of tight junctions. Therefore, we speculate that individuals carrying MPDZ variants may differ in their footrot resistance/susceptibility due to modified horn and interdigital skin integrity. In conclusion, our study reveals that MPDZ might represent a functional candidate gene, and further research is needed to explore its role in footrot affected sheep.     

Identification of two TYRP1 loss‐of‐function alleles in Valais Red sheep

The Valais Red sheep breed is a local breed of the Swiss canton Valais. Although the breed is characterised by its brown colour, black animals occasionally occur and the objective of this study was to identify the causative genetic variants responsible for the obvious difference. A GWAS using high‐density SNP data to compare 51 brown and 38 black sheep showed a strong signal on chromosome 2 at the TYRP1 locus. Haplotype analyses revealed three different brown‐associated alleles. The WGS of three sheep revealed four protein‐changing variants within the TYRP1 gene. Three of these variants were associated with the recessively inherited brown coat colour. This includes the known missense variant TYRP1:c.869G>T designated as b^Soay and two novel loss‐of‐function variants. We propose to designate the frame‐shift variant TYRP1:c.86_87delGA as b^VS1 and the nonsense variant TYRP1:c.1066C>T as b^VS2. Interestingly, the b^VS1 allele occurs only in local breeds of Switzerland whereas the b^VS2 allele seems to be more widespread across Europe.     

Genome‐wide analysis reveals artificial selection on coat colour and reproductive traits in Chinese domestic pigs

Pigs from Asia and Europe were independently domesticated from c. 9000 years ago. During this period, strong artificial selection has led to dramatic phenotypic changes in domestic pigs. However, the genetic basis underlying these morphological and behavioural adaptations is relatively unknown, particularly for indigenous Chinese pigs. Here, we performed a genome‐wide analysis to screen 196 regions with selective sweep signals in Tongcheng pigs, which are a typical indigenous Chinese breed. Genes located in these regions have been found to be involved in lipid metabolism, melanocyte differentiation, neural development and other biological processes, which coincide with the evolutionary phenotypic changes in this breed. A synonymous substitution, c.669T>C, in ESR1, which colocalizes with a major quantitative trait locus for litter size, shows extreme differences in allele frequency between Tongcheng pigs and wild boars. Notably, the variant C allele in this locus exhibits high allele frequency in most Chinese populations, suggesting a consequence of positive selection. Five genes (PRM1, PRM2, TNP2, GPR149 and JMJD1C) related to reproductive traits were found to have high haplotype similarity in Chinese breeds. Two selected genes, MITF and EDNRB, are implied to shape the two‐end black colour trait in Tongcheng pig. Subsequent SNP microarray studies of five Chinese white‐spotted breeds displayed a concordant signature at both loci, suggesting that these two genes are responsible for colour variations in Chinese breeds. Utilizing massively parallel sequencing, we characterized the candidate sites that adapt to artificial and environmental selections during the Chinese pig domestication. This study provides fundamental proof for further research on the evolutionary adaptation of Chinese pigs.     

Genetic variation in IRF4 expression modulates growth characteristics,tyrosinase expression and interferon‐gamma response in melanocytic cells

A SNP within intron4 of the interferon regulatory factor4 (IRF4) gene, rs12203592*C/T, has been independently associated with pigmentation and age‐specific effects on naevus count in European‐derived populations. We have characterized the cis‐regulatory activity of this intronic region and using human foreskin‐derived melanoblast strains, we have explored the correlation between IRF4 rs12203592 homozygous C/C and T/T genotypes with TYR enzyme activity, supporting its association with pigmentation traits. Further, higher IRF4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following UVR, an etiological factor in melanoma development. Since UVR, and accompanying IFNγ‐mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of IRF4 status. Manipulation of IRF4 levels followed by IFNγ treatment revealed a subset of chemokines and immuno‐evasive molecules that are sensitive to IRF4 expression level and genotype including CTLA4 and PD‐L1.     

Genome‐wide variability and selection signatures in Italian island cattle breeds

In the present study, a sample of 88 animals belonging to four local (Modicana, Sarda, Sardo‐Bruna and Sardo‐Modicana) and one cosmopolitan (Italian Brown Swiss) cattle breeds were genotyped with a medium density SNP beadchip and compared to investigate their genetic diversity and the existence of selection signatures. A total of 43 012 SNPs distributed across all 29 autosomal chromosomes were retained after data quality control. Basic population statistics, Wright fixation index and runs of homozygosity (ROH) analyses confirmed that the Italian Brown Swiss genome was shaped mainly by selection, as underlined by the low values of heterozygosity and minor allele frequency. As expected, local cattle exhibited a large within‐breed genetic heterogeneity. The F_ST comparison revealing the largest number of significant SNPs was Sardo‐Bruna vs. Sardo‐Modicana, whereas the smallest was observed for Italian Brown Swiss vs. Sardo‐Modicana. Modicana exhibited the largest number of detected ROHs, whereas the smallest was observed for Sardo‐Modicana. Signatures of selection were detected in genomic regions that harbor genes involved in milk production traits for Italian Brown Swiss and fitness traits for local breeds. According to the results of multi‐dimensional scaling and the admixture analysis the Sardo‐Bruna is more similar to the Sarda than to the Italian Brown Swiss breed. Moreover, the Sardo‐Modicana is genetically closer to the Modicana than to the Sarda breed. Results of the present work confirm the usefulness of single nucleotide polymorphisms in deciphering the genetic architecture of livestock breeds.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

Bos indicus introgression into (peri‐)alpine cattle breeds – evidence from the analysis of bovine whey protein variants

The major bovine whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG), exhibit breed‐specific genetic variation. The aim of this study was to identify possible new protein variants and determine the distribution of variants across a variety of 18 taurine and indicine cattle breeds applying a DNA‐based sequencing approach. To this end, the open reading frames of the respective genes (LALBA and LGB) were sequenced in 476 animals. Within the LALBA gene, a previously unknown synonymous and a previously undesignated non‐synonymous nucleotide exchange were identified. Furthermore, two known α‐LA variants (A and B) and four known β‐LG variants (A, B, C and W) were determined. The occurrence of typical indicine variants in some taurine cattle breeds, such as Suisse Eringer, German Hinterwälder and Hungarian Grey Steppe, further supports the hypothesis of ancient Bos indicus introgression into (peri‐)alpine cattle breeds.