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.

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 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.

Identification of small and large genomic candidate variants in bovine pulmonary hypoplasia and anasarca syndrome.

The pulmonary hypoplasia and anasarca syndrome (PHA) is a congenital lethal disorder, which until now has been reported in cattle and sheep. PHA is characterized by extensive subcutaneous fetal edema combined with hypoplasia or aplasia of the lungs and dysplasia of the lymphatic system. PHA is assumed to be of genetic etiology. This study presents the occurrence of PHA in two different cattle breeds and their genetic causation. Two PHA cases from one sire were observed in Slovenian Cika cattle. Under the assumption of monogenic inheritance, genome-wide homozygosity mapping scaled down the critical regions to 3% of the bovine genome including a 43.6 Mb-sized segment on chromosome 6. Whole-genome sequencing of one case, variant filtering against controls and genotyping of a larger cohort of Cika cattle led to the detection of a likely pathogenic protein-changing variant perfectly associated with the disease: a missense variant on chromosome 6 in ADAMTS3 (NM_001192797.1: c.1222C>T), which affects an evolutionary conserved residue (NP_001179726.1: p.(His408Tyr)). A single PHA case was found in Danish Holstein cattle and was whole-genome sequenced along with its parents. However, as there was no plausible private protein-changing variant, mining for structural variation revealed a likely pathogenic trisomy of the entire chromosome 20. The identified ADAMTS3 associated missense variant and the trisomy 20 are two different genetic causes, which shows a compelling genetic heterogeneity for bovine PHA.

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.

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.

An ABCA12 missense variant in a Shorthorn calf with ichthyosis fetalis.

Two clinical forms of ichthyosis in cattle have been reported, ichthyosis fetalis and congenital ichthyosis. Ichthyosis poses animal welfare and economic issues and the more severe form, ichthyosis fetalis, is lethal. A Shorthorn calf with ichthyosis fetalis was investigated and a likely causal missense variant on chromosome 2 in the ABCA12 gene (NM_001191294.2:c.6776T>C) was identified by whole genome sequencing. Mutations in the ABCA12 gene are known to cause ichthyosis fetalis in cattle and Harlequin ichthyosis in humans. Sanger sequencing of the affected calf and the dam confirmed the variant was homozygous in the affected calf and heterozygous in the dam. Further genotyping of 130 Shorthorn animals from the same property revealed an estimated allele frequency of 3.8%. The presented findings enable genetic testing for breeding and diagnostics.

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.

A non-coding regulatory variant in the 5'-region of the MITF gene is associated with white-spotted coat in Brown Swiss cattle.

Recently, the Swiss breeding association reported an increasing number of white-spotted cattle in the Brown Swiss breed, which is normally solid brown coloured. A total of 60 Brown Swiss cattle with variably sized white abdominal spots, facial markings and depigmented claws were collected for this study. A genome-wide association study using 40k SNP genotypes of 20 cases and 1619 controls enabled us to identify an associated genome region on chromosome 22 containing the MITF gene, encoding the melanogenesis associated transcription factor. Variants at the MITF locus have been reported before to be associated with white or white-spotted phenotypes in other species such as horses, dogs and mice. Whole-genome sequencing of a single white-spotted cow and subsequent genotyping of 172 Brown Swiss cattle revealed two significantly associated completely linked single nucleotide variants (rs722765315 and rs719139527). Both variants are located in the 5'-regulatory region of the bovine MITF gene, and comparative sequence analysis showed that the variant rs722765315, located 139 kb upstream of the transcription start site of the bovine melanocyte-specific MITF transcript, is situated in a multi-species conserved sequence element which is supposed to be regulatory important. Therefore, we hypothesize that rs722765315 represents the most likely causative variant for the white-spotting phenotype observed in Brown Swiss cattle. Presence of the mutant allele in a heterozygous or homozygous state supports a dominant mode of inheritance with incomplete penetrance and results in a variable extent of coat colour depigmentation.