Multiple independent de novo mutations are associated with the development of schistosoma reflexum, a lethal syndrome in cattle.

Schistosoma reflexum (SR) is a lethal congenital syndrome characterized by U-shaped dorsal retroflexion of the spine and exposure of abdominal viscera. SR is usually associated with severe dystocia. The syndrome is thought to be inherited as a Mendelian trait. We collected a series of 23 SR-affected calves from four breeds (20 Holstein, one Red Danish, one Limousin, one Romagnola) and performed whole-genome sequencing (WGS). WGS was performed on 51 cattle, including 14 cases with parents (trio-based; Group 1) and nine single cases (solo-based; Group 2). Sequencing-based genome-wide association studies with 20 Holstein cases and 154 controls showed no association (above Bonferroni threshold; P-value<3 ×10-09). Assuming a monogenic recessive inheritance, no region of shared homozygosity was observed, suggesting heterogeneity. Alternatively, the presence of possible dominant acting de novo mutations were assessed. In Group 1, heterozygous private variants, absent in both parents, were found in seven cases. These involved the ACTL6A, FLNA, GLG1, IQSEC2, MAST3, MBTPS2, and MLLT1 genes. In addition, heterozygous private variants affecting the genes DYNC1LI1, PPP2R2B, SCAF8, SUGP1, and UBP1 were identified in five cases from Group 2. The detected frameshift and missense variants are predicted to cause haploinsufficiency. Each of these 12 affected genes belong to the class of haploinsufficient loss-of-function genes or are involved in embryonic and pre-weaning lethality or are known to be associated with severe malformation syndromes in humans and/or mice. This study presents for the first time a detailed genomic evaluation of bovine SR, suggesting that independent de novo mutations may explain the sporadic occurrence of SR in cattle.

Identification of genomic regions associated with differences in fleece type in Huacaya and Suri alpacas (Vicugna pacos).

The difference in fleece type is the distinguishing trait between the two types of alpacas (Vicugna pacos), Huacaya and Suri. The Suri fleece type has been found to be inherited dominantly over the Huacaya type, resulting in offspring with the Suri phenotype. The aim of our study was to map genomic regions associated with the two different fleece types. In this study, 91 alpacas (54 Huacayas and 37 Suris) from Germany and Switzerland were genotyped using the 76k alpaca SNP array. Only 59k chromosome-localised markers map to the alpaca reference assembly VicPac3.1, and after quality control 49 866 SNPs, were retained for population structure assessment and to conduct a genome-wide association study. Both principal component and neighbour-joining tree analysis showed that the two fleece-type cohorts overlapped rather than forming two distinct clusters. Genome-wide significantly associated markers were observed in the scaffold region of chromosome 16 (NW_021964192.1), which contains a cluster of keratin genes. A haplotype predominantly found in Suri alpacas has been identified which supports dominant inheritance. Variant filtering of nine whole-genome sequenced alpacas from both fleece types in the critical interval of 0.4 Mb did not reveal perfect segregation of either fleece type for specific variants. To our knowledge, this is the first study to use the recently developed species-specific SNP array to identify genomic regions associated with differences in fleece type in alpacas. There are still some limitations, such as the preliminary status of the reference assembly and the incomplete annotation of the alpaca genome.

Ear type in sheep is associated with the MSRB3 locus.

Ear morphology is an important determinant of sheep breeds. It includes different variable traits such as ear size and erectness, suggesting a polygenic architecture. Here, we performed a comprehensive genome-wide analysis to identify regions under selection for ear morphology in 515 sheep from 17 breeds fixed for diverse ear phenotypes using 34k SNP genotyping data. GWASs for two ear type traits, size and erectness, revealed a single genome-wide significant association on ovine chromosome 3. The derived marker alleles were enriched in sheep with large and/or floppy ears. The GWAS signal harboured the MSRB3 gene encoding methionine sulphoxide reductase B3, which has already been found to be associated with different ear types in other species. We attempted whole-genome resequencing to identify causal variant(s) within a 1 Mb interval around MSRB3. This experiment excluded major copy number variants in the interval, but failed to identify a compelling candidate causal variant. Fine-mapping suggested that the causal variant for large floppy ears most likely resides in a 175 kb interval downstream of the MSRB3 coding region.

De novo stop-lost germline mutation in FGFR3 causes severe chondrodysplasia in the progeny of a Holstein bull.

Fifteen cases of chondrodysplasia characterized by disproportionate dwarfism occurred in the progeny of a single Holstein bull. A de novo mutation event in the germline of the sire was suspected as cause. Whole-genome sequencing revealed a single protein-changing variant in the stop codon of FGFR3 gene on chromosome 6. Sanger sequencing of EDTA blood proved that this variant occurred de novo and segregates perfectly with the observed phenotype in the affected cattle family. FGFR3 is an important regulator gene in bone formation owing to its key role in the bone elongation induced by FGFR3-dimers. The detected paternally inherited stop-lost variant in FGFR3 is predicted to add 93 additional amino acids to the protein's C-terminus. This study provides a second example of a dominant FGFR3 stop-lost variant as a pathogenic mutation of a severe form of chondrodysplasia. Even though FGFR3 is known to be associated with dwarfism and growth disorders in human and sheep, this study is the first to describe FGFR3-associated chondrodysplasia in cattle.

New genomic features of the polled intersex syndrome variant in goats unraveled by long-read whole-genome sequencing.

In domestic goats, the polled intersex syndrome (PIS) refers to XX female-to-male sex reversal associated with the absence of horn growth (polled). The causal variant was previously reported as a 11.7 kb deletion at approximately 129 Mb on chromosome 1 that affects the transcription of both FOXL2 and several long non-coding RNAs. In the meantime the presence of different versions of the PIS deletion was postulated and trials to establish genetic testing with the existing molecular genetic information failed. Therefore, we revisited this variant by long-read whole-genome sequencing of two genetically female (XX) goats, a PIS-affected and a horned control. This revealed the presence of a more complex structural variant consisting of a deletion with a total length of 10 159 bp and an inversely inserted approximately 480 kb-sized duplicated segment of a region located approximately 21 Mb further downstream on chromosome 1 containing two genes, KCNJ15 and ERG. Publicly available short-read whole-genome sequencing data, Sanger sequencing of the breakpoints and FISH using BAC clones corresponding to both involved genome regions confirmed this structural variant. A diagnostic PCR was developed for simultaneous genotyping of carriers for this variant and determination of their genetic sex. We showed that the variant allele was present in all 334 genotyped polled goats of diverse breeds and that all analyzed 15 PIS-affected XX goats were homozygous. Our findings enable for the first time a precise genetic diagnosis for polledness and PIS in goats and add a further genomic feature to the complexity of the PIS phenomenon.

A genome-wide significant association on chromosome 15 for congenital entropion in Swiss White Alpine sheep.

Entropion is a known congenital disorder in sheep presumed to be heritable but no causative genetic variant has been reported. Affected lambs show a variable inward rolling of the lower eyelids leading to blindness in severe cases. In Switzerland, the Swiss White Alpine (SWA) breed showed a significantly higher prevalence for entropion than other breeds. A GWAS using 150 SWA sheep (90 affected lambs and 60 controls), based on 600k SNP data, revealed a genome-wide significant signal on chromosome 15. The 0.2 Mb associated region contains functional candidate genes, SMTNL1 and CTNND1. Pathogenic variants in human CTNND1 cause blepharocheilodontic syndrome 2, a rare disorder including eyelid anomalies, and SMTNL1 regulates contraction and relaxation of skeletal and smooth muscle. WGS of a single entropion-affected lamb revealed two private missense variants in SMTNL1 and CTNND1. Subsequent genotyping of both variants in 231 phenotyped SWA sheep was performed. The SMTNL1 variant p.(Asp452Asn) affects an evolutionary conserved residue within an important domain and represents a rare allele, which occurred also in controls. The p.(Glu943Lys) variant in CTNND1 represents a common variant unlikely to cause entropion as the mutant allele occurred more frequently in non-affected sheep. Therefore, we propose that these protein-changing variants are unlikely to explain the phenotype. Additionally, WGS of three further disconcordant pairs of full siblings was carried out but revealed no obvious causative variant. Finally, we conclude that entropion represents a more complex disease caused by different non-coding regulatory variants.

A deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits.

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the at mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an at allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit at allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 at -associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the at allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.

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 bS oay and two novel loss-of-function variants. We propose to designate the frame-shift variant TYRP1:c.86_87delGA as bVS 1 and the nonsense variant TYRP1:c.1066C>T as bVS 2 . Interestingly, the bVS 1 allele occurs only in local breeds of Switzerland whereas the bVS 2 allele seems to be more widespread across Europe.

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 (KITPINZ ) 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 KITPINZ variant occurs in some white spotted animals of the Swiss breeds Evolèner and Eringer. The introgression of the KITPINZ 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.

Two MC1R loss-of-function alleles in cream-coloured Australian Cattle Dogs and white Huskies.

Loss-of-function variants in the MC1R gene cause recessive red or yellow coat-colour phenotypes in many species. The canine MC1R:c.916C>T (p.Arg306Ter) variant is widespread and found in a homozygous state in many uniformly yellow- or red-coloured dogs. We investigated cream-coloured Australian Cattle Dogs whose coat colour could not be explained by this variant. A genome-wide association study with 10 cream and 123 red Australian Cattle Dogs confirmed that the cream locus indeed maps to MC1R. Whole-genome sequencing of cream dogs revealed a single nucleotide variant within the MITF binding site of the canine MC1R promoter. We propose to designate the mutant alleles at MC1R:c.916C>T as e1 and at the new promoter variant as e2 . Both alleles segregate in the Australian Cattle Dog breed. When we considered both alleles in combination, we observed perfect association between the MC1R genotypes and the cream coat colour phenotype in a cohort of 10 cases and 324 control dogs. Analysis of the MC1R transcript levels in an e1 /e2 compound heterozygous dog confirmed that the transcript levels of the e2 allele were markedly reduced with respect to the e1 allele. We further report another MC1R loss-of-function allele in Alaskan and Siberian Huskies caused by a 2-bp deletion in the coding sequence, MC1R:c.816_817delCT. We propose to term this allele e3 . Huskies that carry two copies of MC1R loss-of-function alleles have a white coat colour.

Canine NAPEPLD-associated models of human myelin disorders.

Canine leukoencephalomyelopathy (LEMP) is a juvenile-onset neurodegenerative disorder of the CNS white matter currently described in Rottweiler and Leonberger dogs. Genome-wide association study (GWAS) allowed us to map LEMP in a Leonberger cohort to dog chromosome 18. Subsequent whole genome re-sequencing of a Leonberger case enabled the identification of a single private homozygous non-synonymous missense variant located in the highly conserved metallo-beta-lactamase domain of the N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD) gene, encoding an enzyme of the endocannabinoid system. We then sequenced this gene in LEMP-affected Rottweilers and identified a different frameshift variant, which is predicted to replace the C-terminal metallo-beta-lactamase domain of the wild type protein. Haplotype analysis of SNP array genotypes revealed that the frameshift variant was present in diverse haplotypes in Rottweilers, and also in Great Danes, indicating an old origin of this second NAPEPLD variant. The identification of different NAPEPLD variants in dog breeds affected by leukoencephalopathies with heterogeneous pathological features, implicates the NAPEPLD enzyme as important in myelin homeostasis, and suggests a novel candidate gene for myelination disorders in people.