MLPH Genotype—Melanin Phenotype Correlation in Dilute Dogs

Coat color dilution in dogs is a specific pigmentation phenotype caused by a defective transport of melanosomes leading to large clumps of pigment. It is inherited as a Mendelian autosomal recessive trait and may be accompanied by hair loss, the so-called color dilution alopecia (CDA), or black hair follicular dysplasia (BHFD). We previously identified the noncoding c.-22G>A transition in the melanophilin gene (MLPH) as a candidate causative mutation for the dilute phenotype. We have now extended our study and genotyped 935 dogs from 20 breeds segregating for dilute coat color. The dilute-associated A allele segregates in many different breeds suggesting an old mutation event. We also investigated skin biopsies of dogs suspected of having either CDA or BHFD, and our data clearly indicate that the dilute mutation is required but not sufficient to develop clinical signs of the disease. The risk to develop CDA/BHFD seems to be breed specific. Interestingly, 22 out of 29 dogs with clinical signs of CDA/BHFD have clumped melanin in the epidermis, the follicular epithelium, and the hair shafts, whereas in dilute dogs without clinical disease, clumped melanin is only found in the follicular epithelium and the hair shafts but not in the epidermi

Four novel candidate causal variants for deficient homozygous haplotypes in Holstein cattle.

Mendelian variants can determine both insemination success and neonatal survival and thus influence fertility and rearing success of cattle. We present 24 deficient homozygous haplotype regions in the Holstein population of Switzerland and provide an overview of the previously identified haplotypes in the global Holstein breed. This study encompasses massive genotyping, whole-genome sequencing (WGS) and phenotype association analyses. We performed haplotype screenings on almost 53 thousand genotyped animals including 114 k SNP data with two different approaches. We revealed significant haplotype associations to several survival, birth and fertility traits. Within haplotype regions, we mined WGS data of hundreds of bovine genomes for candidate causal variants, which were subsequently evaluated by using a custom genotyping array in several thousand breeding animals. With this approach, we confirmed the known deleterious SMC2:p.Phe1135Ser missense variant associated with Holstein haplotype (HH) 3. For two previously reported deficient homozygous haplotypes that show negative associations to female fertility traits, we propose candidate causative loss-of-function variants: the HH13-related KIR2DS1:p.Gln159* nonsense variant and the HH21-related NOTCH3:p.Cys44del deletion. In addition, we propose the RIOX1:p.Ala133_Glu142del deletion as well as the PCDH15:p.Leu867Val missense variant to explain the unexpected low number of homozygous haplotype carriers for HH25 and HH35, respectively. In conclusion, we demonstrate that with mining massive SNP data in combination with WGS data, we can map several haplotype regions and unravel novel recessive protein-changing variants segregating at frequencies of 1 to 5%. Our findings both confirm previously identified loci and expand the spectrum of undesired alleles impairing reproduction success in Holstein cattle, the world's most important dairy breed

Mining massive genomic data of two Swiss Braunvieh cattle populations reveals six novel candidate variants that impair reproductive success

Background: This study was carried out on the two Braunvieh populations reared in Switzerland, the dairy Brown Swiss (BS) and the dual-purpose Original Braunvieh (OB). We performed a genome-wide analysis of array data of trios (sire, dam, and offspring) from the routine genomic selection to identify candidate regions showing missing homozygosity and phenotypic associations with five fertility, ten birth, and nine growth-related traits. In addition, genome-wide single SNP regression studies based on 114,890 single nucleotide polymorphisms (SNPs) for each of the two populations were performed. Furthermore, whole-genome sequencing data of 430 cattle including 70 putative haplotype carriers were mined to identify potential candidate variants that were validated by genotyping the current population using a custom array. Results: Using a trio-based approach, we identified 38 haplotype regions for BS and five for OB that segregated at low to moderate frequencies. For the BS population, we confirmed two known haplotypes, BH1 and BH2. Twenty-four variants that potentially explained the missing homozygosity and associated traits were detected, in addition to the previously reported TUBD1:p.His210Arg variant associated with BH2. For example, for BS we identified a stop-gain variant (p.Arg57*) in the MRPL55 gene in the haplotype region on chromosome 7. This region is associated with the ‘interval between first and last insemination’ trait in our data, and the MRPL55 gene is known to be associated with early pregnancy loss in mice. In addition, we discuss candidate missense variants in the CPT1C, MARS2, and ACSL5 genes for haplotypes mapped in BS. In OB, we highlight a haplotype region on chromosome 19, which is potentially caused by a frameshift variant (p.Lys828fs) in the LIG3 gene, which is reported to be associated with early embryonic lethality in mice. Furthermore, we propose another potential causal missense variant in the TUBGCP5 gene for a haplotype mapped in OB. Conclusions: We describe, for the first time, several haplotype regions that segregate at low to moderate frequencies and provide evidence of causality by trait associations in the two populations of Swiss Braunvieh. We propose a list of six protein-changing variants as potentially causing missing homozygosity. These variants need to be functionally validated and incorporated in the breeding program

A Heterozygous Missense Variant in MAP2K2 in a Stillborn Romagnola Calf with Skeletal-Cardio-Enteric Dysplasia

RASopathies are a group of developmental disorders caused by dominant mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) cell signaling pathway. The goal of this study was to characterize the pathological phenotype of a Romagnola stillborn calf with skeletal-cardio-enteric dysplasia and to identify a genetic cause by whole-genome sequencing (WGS). The calf showed reduced fetal growth, a short-spine, a long and narrow face, cardiac defects and heterotopy of the spiral colon. Genetic analysis revealed a private heterozygous missense variant in MAP2K2:p.Arg179Trp, located in the protein kinase domain in the calf, and not found in more than 4500 control genomes including its sire. The identified variant affecting a conserved residue was predicted to be deleterious and most likely occurred de novo. This represents the first example of a dominant acting, and most likely pathogenic, variant in MAP2K2 in domestic animals, thereby providing the first MAP2K2-related large animal model, especially in respect to the enteric malformation. In addition, this study demonstrates the utility of WGS-based precise diagnostics for understanding sporadic congenital syndromic anomalies in cattle and the general utility of continuous surveillance for rare hereditary defects in cattle.Skeletal dysplasias encompass a clinical-, pathological- and genetically heterogeneous group of disorders characterized by abnormal cartilage and/or bone formation, growth, and remodeling. They may belong to the so-called RASopathies, congenital conditions caused by heterozygous variants in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) cell signaling pathway. Herein, an affected calf of the Italian Romagnola breed was reported showing a skeletal-cardio-enteric dysplasia. We identified a most likely disease-causing mutation in the MAP2K2 gene by whole-genome sequencing (WGS). The MAP2K2 gene is known to be related with dominant inherited cardio-facio-cutaneous syndrome in humans, but it was so far unknown to cause a similar disease in domestic animals. We assume that the identified missense variant that was predicted to impair the function of the protein, occurred either within the germline of the dam or post-zygotically in the embryo. Rare lethal diseases such as the skeletal-cardio-enteric dysplasia in livestock are usually not characterized to the molecular level, mainly because of the lack of funds and diagnostic opportunities. Precise WGS-based diagnostics enables the understanding of rare diseases and supports the value of monitoring cattle breeding populations for fatal genetic defects

Exploring RNA polymerase regulation by NMR spectroscopy

RNA synthesis is a central process in all organisms, with RNA polymerase (RNAP) as the key enzyme. Multisubunit RNAPs are evolutionary related and are tightly regulated by a multitude of transcription factors. Although Escherichia coli RNAP has been studied extensively, only little information is available about its dynamics and transient interactions. This information, however, are crucial for the complete understanding of transcription regulation in atomic detail. To study RNAP by NMR spectroscopy we developed a highly efficient procedure for the assembly of active RNAP from separately expressed subunits that allows specific labeling of the individual constituents. We recorded [(1)H,(13)C] correlation spectra of isoleucine, leucine, and valine methyl groups of complete RNAP and the separately labeled β’ subunit within reconstituted RNAP. We further produced all RNAP subunits individually, established experiments to determine which RNAP subunit a certain regulator binds to, and identified the β subunit to bind NusE

Thermotoga maritima NusG : domain interaction mediates autoinhibition and thermostability

NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures

A de novo variant in the bovine ADAMTSL4 gene in an Original Braunvieh calf with congenital cataract

Inherited forms of cataract are a heterogeneous group of eye disorders known in livestock species. Clinicopathological analysis of a single case of impaired vision in a newborn Original Braunvieh calf revealed nuclear cataract. Whole-genome sequencing of the parent-offspring trio revealed a de novo mutation of ADAMTSL4 in this case. The heterozygous p.Arg776His missense variant affects a conserved residue of the ADAMTSL4 gene that encodes a secreted glycoprotein expressed in the lens throughout embryonic development. In humans, ADAMTSL4 genetic variants cause recessively inherited forms of subluxation of the lens. Given that ADAMTSL4 is a functional candidate gene for inherited disorders of the lens, we suggest that heterozygosity for the identified missense variant may have caused the congenital cataract in the affected calf. Cattle populations should be monitored for unexplained cataract cases, with subsequent DNA sequencing a hypothesized pathogenic effect of heterozygous ADAMTSL4 variants could be confirmed

Genome-Wide Analyses for Osteosarcoma in Leonberger Dogs Reveal the CDKN2A/B Gene Locus as a Major Risk Locus

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1–13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10−4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA