Successful trio-based reverse genetic screen in an endangered local cattle breed

We scanned the genomic data of almost one thousand Evolèner cattle, representing the by far smallest local cattle population of Switzerland, whereof only pedigree records are available. A reverse genetic screen using 94k SNP markers in 585 trios mapped recessive deleterious loci using homozygous haplotype deficiency. We found two haplotypes, EH1 and EH2, with moderate allele frequencies of >0.10. Mining for candidate variants was carried out by linkage analysis of the predicted haplotype status and whole-genome sequencing variant catalogue of seven Evolèner bulls. This led to the detection of two perfectly linked missense variants affecting conserved residues: GBE1:p.Arg437Gln for EH1, and LRRC8A:p.Ala73Val for EH2. No homozygous animals were observed in >5,100 cattle of various breeds including Evolèner. The presented study showed that very limited data can lead to the identification of candidate variants and thereby help to improve reproduction success in an endangered indigenous breed of cattle

Uniformisation of Lead Tungstate Crystals for the CMS Electromagnetic Calorimeter Endcaps

Electromagnetic calorimeters based on dense, scintillating crystals have the potential to achieve extremely good energy resolution. However, care must be taken to minimise systematic effects such as variation of light collection efficiency with depth in the crystal. A HPMT has been used to accurately measure the non-uniformity of light collection in lead tungstate crystals for the CMS detector. It is found that the non-uniformity of crystals measured in the detector configuration is not much larger than the maximum tolerable limit. Shading the chamfered edges of the crystals with a pencil can reduce the non-uniformity by approximately 0.1%/X0. Roughening the chamfers achieves a similar reduction

Ear type in sheep is associated with the MSRB3

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

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

Identification of two TYRP 1 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 bSoay and two novel loss-of-function variants. We propose to designate the frame-shift variant TYRP1:c.86_87delGA as bVS1 and the nonsense variant TYRP1:c.1066C>T as bVS2. Interestingly, the bVS1 allele occurs only in local breeds of Switzerland whereas the bVS2 allele seems to be more widespread across Europe

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

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

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