A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism.

The domestic cat (Felis catus) numbers over 94 million in the USA alone, occupies households as a companion animal, and, like humans, suffers from cancer and common and rare diseases. However, genome-wide sequence variant information is limited for this species. To empower trait analyses, a new cat genome reference assembly was developed from PacBio long sequence reads that significantly improve sequence representation and assembly contiguity. The whole genome sequences of 54 domestic cats were aligned to the reference to identify single nucleotide variants (SNVs) and structural variants (SVs). Across all cats, 16 SNVs predicted to have deleterious impacts and in a singleton state were identified as high priority candidates for causative mutations. One candidate was a stop gain in the tumor suppressor FBXW7. The SNV is found in cats segregating for feline mediastinal lymphoma and is a candidate for inherited cancer susceptibility. SV analysis revealed a complex deletion coupled with a nearby potential duplication event that was shared privately across three unrelated cats with dwarfism and is found within a known dwarfism associated region on cat chromosome B1. This SV interrupted UDP-glucose 6-dehydrogenase (UGDH), a gene involved in the biosynthesis of glycosaminoglycans. Importantly, UGDH has not yet been associated with human dwarfism and should be screened in undiagnosed patients. The new high-quality cat genome reference and the compilation of sequence variation demonstrate the importance of these resources when searching for disease causative alleles in the domestic cat and for identification of feline biomedical models.

The practical use of genome sequencing data in the management of a feline colony pedigree.

BACKGROUND: A higher prevalence of inherited disorders among companion animals are often rooted in their historical restricted artificial selection for a variety of observed phenotypes that eventually decreased genetic diversity. Cats have been afflicted with many inherited diseases due to domestication and intense breed selection. Advances in sequencing technology have generated a more comprehensive way to access genetic information from an individual, allowing identification of putative disease-causing variants and in practice a means to avoid their spread and thus better pedigree management. We examine variants in three domestic shorthair cats and then calculated overall genetic diversity to extrapolate the benefits of this data for breeding programs within a feline colony. RESULTS: We generated whole genome sequence (WGS) data for three related cats that belong to a large feline pedigree colony. Genome-wide coverage ranged from 27-32X, from which we identified 18 million variants in total. Previously known disease-causing variants were screened in our cats, but none carry any of these known disease alleles. Loss of function (LoF) variants, that are in genes associated with a detrimental phenotype in human or mice were chosen for further evaluation on the comparative impact inferred. A set of LoF variants were observed in four genes, each with predicted detrimental phenotypes as a result. However, none of our cats displayed the expected disease phenotypes. Inbreeding coefficients and runs of homozygosity were also evaluated as a measure of genetic diversity. We find low inbreeding coefficients and total runs of homozygosity, thus suggesting pedigree management of genetic relatedness is acceptable. CONCLUSIONS: The use of WGS of a small sampling among a large feline colony has enabled us to identify possible disease-causing variants, their genotype state and measure pedigree management of genetic diversity. We contend a limited but strategic sampling of feline colony individuals using WGS can inform veterinarians of future health anomalies and guide breeding practices to ensure healthy genetic diversity.

Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

A domestic cat whole exome sequencing resource for trait discovery.

Over 94 million domestic cats are susceptible to cancers and other common and rare diseases. Whole exome sequencing (WES) is a proven strategy to study these disease-causing variants. Presented is a 35.7 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. Whole exome sequencing was conducted on 41 cats with known and unknown genetic diseases and traits, of which ten cats had matching whole genome sequence (WGS) data available, used to validate WES performance. At 80 × mean exome depth of coverage, 96.4% of on-target base coverage had a sequencing depth > 20-fold, while over 98% of single nucleotide variants (SNVs) identified by WGS were also identified by WES. Platform-specific SNVs were restricted to sex chromosomes and a small number of olfactory receptor genes. Within the 41 cats, we identified 31 previously known causal variants and discovered new gene candidate variants, including novel missense variance for polycystic kidney disease and atrichia in the Peterbald cat. These results show the utility of WES to identify novel gene candidate alleles for diseases and traits for the first time in a feline model.

Diagnostic Ultrasound Detection of Changes in Femoral Muscle Mass Recovery after Tibial Plateau Levelling Osteotomy in Dogs.

OBJECTIVE: The goal of this study was to develop a clinically feasible ultrasound (US) protocol that can detect changes in thigh muscle mass in dogs after stifle surgery. The primary aim of this study was to compare previously described US measurement locations of the canine thigh for detecting changes in muscle mass in dogs recovering from tibial plateau levelling osteotomy (TPLO). STUDY DESIGN: This was a prospective, exploratory pilot study. Adult dogs (n = 7) undergoing pet-owner elected TPLO were enrolled. Twelve different US measurements were performed in triplicate by a single experienced observer. Measurements were performed at 0, 2, 4 and 8 weeks after surgery at a proximal and distal location along the femur. Data from all available time points and locations were analysed for the main effect of time within modalities. RESULTS: A total of 1,008 US measurements were performed. Measurements of the transverse sectional area of the rectus femoris muscle detected significant (p ≤ 0.05) muscle loss between weeks 0 and 2 at the lateral and medial aspects of the distal location (19% and 15% respectively). Measurements of the thigh muscle thickness were significantly (p < 0.01) increased between 2nd- and 8th- week time points at the lateral aspect of the proximal location (26%). CONCLUSION: The proximal femoral location, measured from the lateral aspect, appears to be the most suitable US measurement for detecting increases in femoral muscle mass in dogs recovering from TPLO. The provided pilot data suggest that further research evaluating this outcome measure is indicated.

Metabolic Differences between Dogs of Different Body Sizes.

INTRODUCTION: The domesticated dog, Canis lupus familiaris, has been selectively bred to produce extreme diversity in phenotype and genotype. Dogs have an immense diversity in weight and height. Specific differences in metabolism have not been characterized in small dogs as compared to larger dogs. OBJECTIVES: This study aims to identify metabolic, clinical, and microbiota differences between small and larger dogs. METHODS: Gas chromatography/mass spectrometry, liquid chromatography/tandem mass spectrometry, clinical chemistry analysis, dual-energy X-ray absorptiometry, and 16S pyrosequencing were used to characterize blood metabolic, clinical, and fecal microbiome systems, respectively. Eighty-three canines from seven different breeds, fed the same kibble diet for 5 weeks, were used in the study. RESULTS: 449 metabolites, 16 clinical parameters, and 6 bacteria (at the genus level) were significantly different between small and larger dogs. Hierarchical clustering of the metabolites yielded 8 modules associated with small dog size. CONCLUSION: Small dogs had a lower antioxidant status and differences in circulating amino acids. Some of the amino acid differences could be attributed to differences in microflora. Additionally, analysis of small dog metabolites and clinical parameters reflected a network which strongly associates with kidney function.

A High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination.

High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location.

Effects of postexercise feeding of a supplemental carbohydrate and protein bar with or without astaxanthin from Haematococcus pluvialis to exercise-conditioned dogs.

OBJECTIVE: To characterize the postprandial nutrient profiles of exercise-conditioned dogs fed a supplemental carbohydrate and protein bar with or without astaxanthin from Haematococcus pluvialis immediately after exercise. ANIMALS: 34 exercise-conditioned adult Husky-Pointer dogs. PROCEDURES: The study had 2 phases. During phase 1, postprandial plasma glucose concentration was determined for dogs fed a bar containing 25% protein and 18.5% or 37.4% maltodextrin plus dextrin (rapidly digestible carbohydrate; RDC), or dry kibble (30% protein and 0% RDC) immediately after exercise. During phase 2, dogs were exercised for 3 days and fed a bar (25% protein and 37.4% RDC) with (CPA; n = 8) or without (CP; 8) astaxanthin or no bar (control; 8) immediately after exercise. Pre- and postexercise concentrations of plasma biochemical analytes and serum amino acids were determined on days 1 and 3. RESULTS: Phase 1 postexercise glucose concentration was increased when dogs were provided the 37.4% RDC bar, but not 0% or 18.5% RDC. On day 3 of phase 2, the CPA group had the highest pre-exercise triglyceride concentration and significantly less decline in postexercise glucose concentration than did the CP and control groups. Mean glucose concentration for the CP and CPA groups was significantly higher than that for the control group between 15 and 60 minutes after bar consumption. Compared to immediately after exercise, branched-chain amino acid, tryptophan, leucine, and threonine concentrations 15 minutes after exercise were significantly higher for the CP and CPA groups, but were lower for the control group. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs fed a bar with 37.4% RDCs and 25% protein immediately after exercise had increased blood nutrient concentrations for glycogen and protein synthesis, compared with control dogs.