A chromosome-scale fishing cat reference genome for the evaluation of potential germline risk variants.

The fishing cat, Prionailurus viverrinus, faces a population decline, increasing the importance of maintaining healthy zoo populations. Unfortunately, zoo-managed individuals currently face a high prevalence of transitional cell carcinoma (TCC), a form of bladder cancer. To investigate the genetics of inherited diseases among captive fishing cats, we present a chromosome-scale assembly, generate the pedigree of the zoo-managed population, reaffirm the close genetic relationship with the Asian leopard cat (Prionailurus bengalensis), and identify 7.4 million single nucleotide variants (SNVs) and 23,432 structural variants (SVs) from whole genome sequencing (WGS) data of healthy and TCC cats. Only BRCA2 was found to have a high recurrent number of missense mutations in fishing cats diagnosed with TCC when compared to inherited human cancer risk variants. These new fishing cat genomic resources will aid conservation efforts to improve their genetic fitness and enhance the comparative study of feline genomes.

Suspected malnutrition-induced reversible feline skin fragility syndrome in a cat with congenital axial deformities.

A stray cat, an intact female Japanese domestic shorthair cat of unknown age (suspected to be a young adult), was rescued. The cat was lethargic and thin and had marked skin fragility, delayed wound healing without skin hyperextensibility, and hind limb proprioceptive ataxia and paresis. Survey radiography, computed tomography, and magnetic resonance imaging revealed congenital vertebral anomalies, including thoracolumbar transitional vertebrae, scoliosis resulting from a thoracic lateral wedge-shaped vertebra, and a kinked tail, and a dilated spinal cord central canal. Through nutritional support, the cat's general condition normalized, followed by a gradual and complete improvement of skin features. Whole-genome sequencing was completed; however, no pathogenic genetic variant was identified that could have caused this phenotype, including congenital scoliosis. A skin biopsy obtained 7 y after the rescue revealed no remarkable findings on histopathology or transmission electron microscopy. Based on clinical course and microscopic findings, malnutrition-induced reversible feline skin fragility syndrome (FSFS) was suspected, and nutritional support was considered to have improved the skin condition. Key clinical message: This is the second reported case of presumed malnutrition-induced reversible FSFS and was accompanied by long-term follow-up.

Syndrome de fragilité cutanée réversible induit par la malnutrition soupçonné chez un chat avec des difformités axiales congénitales. Un chat errant, une femelle intacte de race japonaise à poil court et d'âge inconnu (suspecté être une jeune adulte), a été secourue. La chatte était léthargique et maigre, et avait une fragilité marquée de la peau, un retard dans la guérison de plaies sans hyperextensibilité de la peau, et une ataxie proprioceptive et parésie des membres postérieurs. Des radiographies, un examen par tomodensitométrie, et de l'imagerie par résonnance magnétique ont révélé des anomalies congénitales des vertèbres, incluant des vertèbres transitionnelles thoraco-lombaires, une scoliose résultant d'une vertèbre thoracique en forme de coin, une queue pliée, et un canal central de la moelle épinière dilaté. Grâce à un soutien nutritionnel, la condition générale du chat s'est stabilisée, suivi d'une amélioration graduelle et complète des caractéristiques de la peau. Le séquençage du génome complet a été effectué; toutefois, aucune variation génétique pathogénique n'a été identifiée qui aurait pu causer ce phénotype, incluant la scoliose congénitale. Une biopsie cutanée obtenue 7 j après le sauvetage n'a révélé aucune trouvaille spéciale à l'histopathologie ou par microscopie électronique à transmission. Basé sur le déroulement clinique et l'examen microscopique, le syndrome de fragilité cutanée réversible félin induit par la malnutrition (FSFS) était suspecté, et le soutien nutritionnel a été considéré comme ayant amélioré la condition cutanée.Message clinique clé :Ce cas est le deuxième cas rapporté de FSFS induit par la malnutrition soupçonné et a fait l'objet d'un suivi à long terme.(Traduit par Dr Serge Messier).

Association of a novel dystrophin (DMD) genetic nonsense variant in a cat with X-linked muscular dystrophy with a mild clinical course.

X-linked muscular dystrophy in cats (FXMD) is an uncommon disease, with few reports describing its pathogenic genetic variants. A 9-year-old castrated male domestic shorthair cat was presented with persistent muscle swelling and breathing difficulty from 3 years of age. Serum activity of alanine aminotransferase, aspartate transaminase, and creatine kinase were abnormally high. Physical and neurological examinations showed muscle swelling in the neck and proximal limb, slow gait, and occasional breathing difficulties. Electromyography showed pseudomyotonic discharges and complex repetitive discharges with a "dive-bomber" sound. Histopathology revealed muscle necrosis and regeneration. Whole-genome sequencing identified a novel and unique hemizygous nonsense genetic variant, c.8333G > A in dystrophin (DMD), potentially causing a premature termination codon (p.Trp2778Ter). Based on a combination of clinical and histological findings and the presence of the DMD nonsense genetic variant, this case was considered FXMD, which showed mild clinical signs and long-term survival, even though immunohistochemical characterization was lacking.

Classification of feline hypertrophic cardiomyopathy-associated gene variants according to the American College of Medical Genetics and Genomics guidelines.

Introduction: The correct labeling of a genetic variant as pathogenic is important as breeding decisions based on incorrect DNA tests can lead to the unwarranted exclusion of animals, potentially compromising the long-term health of a population. In human medicine, the American college of Medical Genetics (ACMG) guidelines provide a framework for variant classification. This study aims to apply these guidelines to six genetic variants associated with hypertrophic cardiomyopathy (HCM) in certain cat breeds and to propose a modified criterion for variant classification. Methods: Genetic samples were sourced from five cat breeds: Maine Coon, Sphynx, Ragdoll, Devon Rex, and British Short- and Longhair. Allele frequencies were determined, and in the subset with phenotypes available, odds ratios to determine the association with HCM were calculated. In silico evaluation followed with joint evidence and data from other publications assisting in the classification of each variant. Results: Two variants, MYBPC3:c.91G > C [A31P] and MYBPC3:c.2453C > T [R818W], were designated as pathogenic. One variant, MYH7:c.5647G > A [E1883K], was found likely pathogenic, while the remaining three were labeled as variants of unknown significance. Discussion: Routine genetic testing is advised solely for the MYBPC3:c.91G > C [A31P] in the Maine Coon and MYBPC3:c.2453C > T [R818W] in the Ragdoll breed. The human ACMG guidelines serve as a suitable foundational tool to ascertain which variants to include; however, refining them for application in veterinary medicine might be beneficial.

Precision medicine using whole genome sequencing identifies a novel dystrophin (DMD) variant for X-linked muscular dystrophy in a cat.

BACKGROUND: Muscular dystrophies (MDs) are a large, heterogeneous group of degenerative muscle diseases. X-linked dystrophin-deficient MD in cats is the first genetically characterized cat model for a human disease and a few novel forms have been identified. HYPOTHESIS/OBJECTIVES: Muscular dystrophy was suspected in a young male domestic shorthair cat. Clinical, molecular, and genetic techniques could provide a definitive diagnosis. ANIMALS: A 1-year-old male domestic shorthair cat presented for progressive difficulty walking, macroglossia and dysphagia beginning at 6 months of age. The tongue was thickened, protruded with constant ptyalism, and thickening and rigidity of the neck and shoulders were observed. METHODS: A complete neurological examination, baseline laboratory evaluation and biopsies of the trapezius muscle were performed with owner consent. Indirect immunofluorescence staining of muscle cryosections was performed using several monoclonal and polyclonal antibodies against dystrophy-associated proteins. DNA was isolated for genomic analyses by whole genome sequencing and comparison to DNA variants in the 99 Lives Cat Genome Sequencing dataset. RESULTS AND CLINICAL IMPORTANCE: Aspartate aminotransferase (687 IU/L) and creatine kinase (24 830 IU/L) activities were increased and mild hypokalemia (3.7 mmol/L) was present. Biopsy samples from the trapezius muscle confirmed a degenerative and regenerative myopathy and protein alterations identified by immunohistochemistry resulted in a diagnosis of a in dystrophin-deficient form of X-linked MD. A stop gain variant (c.4849C>T; p.Gln1617Ter) dystrophin was identified by genome sequencing. Precision/genomic medicine efforts for the domestic cat and in veterinary medicine support disease variant and animal model discovery and provide opportunities for targeted treatments for companion animals.

Beta-mannosidosis in a domestic cat associated with a missense variant in MANBA.

A 6-month-old cat of unknown ancestry presented for a neurologic evaluation due to progressive motor impairment. Complete physical and neurologic examinations suggested the disorder was likely to be hereditary, although the signs were not consistent with any previously described inherited disorders in cats. Due to the progression of disease signs including severely impaired motor function and cognitive decline, the cat was euthanized at approximately 10.5 months of age. Whole genome sequence analysis identified a homozygous missense variant c.2506G > A in MANBA that predicts a p.Gly836Arg alteration in the encoded lysosomal enzyme ß -mannosidase. This variant was not present in the whole genome or whole exome sequences of any of the 424 cats represented in the 99 Lives Cat Genome dataset. ß -Mannosidase enzyme activity was undetectable in brain tissue homogenates from the affected cat, whereas α-mannosidase enzyme activities were elevated compared to an unaffected cat. Postmortem examination of brain and retinal tissues revealed massive accumulations of vacuolar inclusions in most cells, similar to those reported in animals of other species with hereditary ß -mannosidosis. Based on these findings, the cat likely suffered from ß -mannosidosis due to the abolition of ß -mannosidase activity associated with the p.Gly836Arg amino acid substitution. p.Gly836 is located in the C-terminal region of the protein and was not previously known to be involved in modulating enzyme activity. In addition to the vacuolar inclusions, some cells in the brain of the affected cat contained inclusions that exhibited lipofuscin-like autofluorescence. Electron microscopic examinations suggested these inclusions formed via an autophagy-like process.

Single-haplotype comparative genomics provides insights into lineage-specific structural variation during cat evolution.

The role of structurally dynamic genomic regions in speciation is poorly understood due to challenges inherent in diploid genome assembly. Here we reconstructed the evolutionary dynamics of structural variation in five cat species by phasing the genomes of three interspecies F1 hybrids to generate near-gapless single-haplotype assemblies. We discerned that cat genomes have a paucity of segmental duplications relative to great apes, explaining their remarkable karyotypic stability. X chromosomes were hotspots of structural variation, including enrichment with inversions in a large recombination desert with characteristics of a supergene. The X-linked macrosatellite DXZ4 evolves more rapidly than 99.5% of the genome clarifying its role in felid hybrid incompatibility. Resolved sensory gene repertoires revealed functional copy number changes associated with ecomorphological adaptations, sociality and domestication. This study highlights the value of gapless genomes to reveal structural mechanisms underpinning karyotypic evolution, reproductive isolation and ecological niche adaptation.

Limited historical admixture between European wildcats and domestic cats.

Domestic cats were derived from the Near Eastern wildcat (Felis lybica), after which they dispersed with people into Europe. As they did so, it is possible that they interbred with the indigenous population of European wildcats (Felis silvestris). Gene flow between incoming domestic animals and closely related indigenous wild species has been previously demonstrated in other taxa, including pigs, sheep, goats, bees, chickens, and cattle. In the case of cats, a lack of nuclear, genome-wide data, particularly from Near Eastern wildcats, has made it difficult to either detect or quantify this possibility. To address these issues, we generated 75 ancient mitochondrial genomes, 14 ancient nuclear genomes, and 31 modern nuclear genomes from European and Near Eastern wildcats. Our results demonstrate that despite cohabitating for at least 2,000 years on the European mainland and in Britain, most modern domestic cats possessed less than 10% of their ancestry from European wildcats, and ancient European wildcats possessed little to no ancestry from domestic cats. The antiquity and strength of this reproductive isolation between introduced domestic cats and local wildcats was likely the result of behavioral and ecological differences. Intriguingly, this long-lasting reproductive isolation is currently being eroded in parts of the species' distribution as a result of anthropogenic activities.

Genetic swamping of the critically endangered Scottish wildcat was recent and accelerated by disease.

The European wildcat population in Scotland is considered critically endangered as a result of hybridization with introduced domestic cats,1,2 though the time frame over which this gene flow has taken place is unknown. Here, using genome data from modern, museum, and ancient samples, we reconstructed the trajectory and dated the decline of the local wildcat population from viable to severely hybridized. We demonstrate that although domestic cats have been present in Britain for over 2,000 years,3 the onset of hybridization was only within the last 70 years. Our analyses reveal that the domestic ancestry present in modern wildcats is markedly over-represented in many parts of the genome, including the major histocompatibility complex (MHC). We hypothesize that introgression provides wildcats with protection against diseases harbored and introduced by domestic cats, and that this selection contributes to maladaptive genetic swamping through linkage drag. Using the case of the Scottish wildcat, we demonstrate the importance of local ancestry estimates to both understand the impacts of hybridization in wild populations and support conservation efforts to mitigate the consequences of anthropogenic and environmental change.

Cardiomyopathy associated 5 (CMYA5) implicated as a genetic risk factor for radial hemimelia in Siamese cats.

OBJECTIVES: The present study aimed to determine the inheritance pattern and genetic cause of congenital radial hemimelia (RH) in cats. METHODS: Clinical and genetic analyses were conducted on a Siamese cat family (n = 18), including two siblings with RH. Radiographs were obtained for the affected kittens and echocardiograms of an affected kitten and sire. Whole genome sequencing was completed on the two cases and the parents. Genomic data were compared with the 99 Lives Cat Genome data set of 420 additional domestic cats with whole genome and whole exome sequencing data. Variants were considered as homozygous in the two cases of the siblings with RH and heterozygous in the parents. Candidate variants were genotyped by Sanger sequencing in the extended pedigree. RESULTS: Radiographs of the female kitten revealed bilateral absence of the radii and bowing of the humeri, while the male kitten showed a dysplastic right radius. Echocardiography suggested the female kitten had restrictive cardiomyopathy with a positive left atrial-to-aortic root ratio (LA:Ao = 1.83 cm), whereas hypertrophic cardiomyopathy was more likely in the sire, showing diastolic dysfunction using tissue Doppler imaging (59.06 cm/s). Twenty-two DNA variants were unique and homozygous in the affected kittens and heterozygous in the parents. Seven variants clustered in one chromosomal region, including two frameshift variants in cardiomyopathy associated 5 (CMYA5) and five variants in junction mediating and regulatory protein, P53 cofactor (JMY ), including a missense and an in-frame deletion. CONCLUSIONS AND RELEVANCE: The present study suggested an autosomal recessive mode of inheritance with variable expression for RH in the Siamese cat family. Candidate variants for the phenotype were identified, implicating their roles in bone development. These genes should be considered as potentially causal for other cats with RH. Siamese cat breeders should consider genetically testing their cats for these variants to prevent further dissemination of the suspected variants within the breed.

Precision medicine using whole genome sequencing in a cat identifies a novel COL5A1 variant for classical Ehlers-Danlos syndrome.

BACKGROUND: Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue disorders occurring in both human and veterinary patients. The genetics of these disorders are poorly described in small animal patients. HYPOTHESIS/OBJECTIVES: Define the clinical manifestations and genetic cause of a suspected form of EDS in a cat. ANIMALS: A 14-week-old male domestic medium hair cat was presented with skin hyperextensibility and fragility. The classic tragic facial expression was observed as well as chronic pruritus and mild hyperesthesia. METHODS: Blood samples and a skin biopsy sample were collected from the affected cat. Clinical examinations, histology, electron microscopy and whole genome sequencing were conducted to characterize the clinical presentation and identify possible pathogenic DNA variants to support a diagnosis. Criteria defining variant pathogenicity were examined including human disease variant databases. RESULTS: Histology showed sparse, disorganized collagen and an increase in cutaneous mast cells. Electron microscopy identified ultrastructural defects commonly seen in collagen type V alpha 1 chain (COL5A1) variants including flower-like collagen fibrils in cross-section. Whole genome sequencing and comparison with 413 cats in the 99 Lives Cat Genome Sequencing Consortium database identified a novel splice acceptor site variant at exon 4 in COL5A1 (c.501-2A>C). CONCLUSIONS AND CLINICAL IMPORTANCE: Our report broadens the current understanding of EDS in veterinary patients and supports the use of precision medicine techniques in clinical veterinary practice. The classification of variants for pathogenicity should be considered in companion animals.

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.

CTSK variant implicated in suspected pyknodysostosis in a domestic cat.

Case summary: A 9-month-old entire male domestic longhair cat presented with a history of pathological fractures, chronic musculoskeletal pain and poor growth. Multiple facial and skeletal abnormalities were identified on physical examination and advanced imaging (CT and radiographs). A variant in CTSK was identified in the affected cat following whole-exome sequencing (WES). The cat was managed symptomatically with diet, environmental modifications and analgesia. Relevance and novel information: This is the first report of a cat with a similar clinical presentation and genetic variant to the hereditary human genetic disorder pyknodysostosis. In this case, WES was performed, which often facilitates the diagnosis of various hereditary disorders (ie, a conceptual framework for practicing feline genomic medicine). Despite the severe skeletal and appendicular abnormalities described, the cat was alive more than 2 years after its initial presentation.

Genetics of randomly bred cats support the cradle of cat domestication being in the Near East.

Cat domestication likely initiated as a symbiotic relationship between wildcats (Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density (i.e., rodents). Humans benefited from the cats' predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor's (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species.

X-linked myotubular myopathy associated with an MTM1 variant in a Maine coon cat.

OBJECTIVE: Describe the clinical course and diagnostic and genetic findings in a cat with X-linked myotubular myopathy. CASE SUMMARY: A 7-month-old male Maine coon was evaluated for progressively worsening gait abnormalities and generalized weakness. Neurolocalization was to the neuromuscular system. Genetic testing for spinal muscular atrophy (LIX1) was negative. Given the progressive nature and suspected poor long-term prognosis, the owners elected euthanasia. Histopathology of skeletal muscle obtained post-mortem disclosed numerous rounded atrophic or hypotrophic fibers with internal nuclei or central basophilic staining. Using oxidative reactions mediated by cytochrome C oxidase and succinic dehydrogenase, scattered myofibers were observed to have central dark staining structures and a "ring-like" appearance. Given the cat's age and clinical history, a congenital myopathy was considered most likely, with the central nuclei and "ring-like" changes consistent with either centronuclear or myotubular myopathy. Whole genome sequencing identified an underlying missense variant in myotubularin 1 (MTM1), a known candidate gene for X-linked myotubular myopathy. NEW OR UNIQUE INFORMATION PROVIDED: This case is the first report of X-linked myotubular myopathy in a cat with an MTM1 missense mutation. Maine coon cat breeders may consider screening for this variant to prevent production of affected cats and to eradicate the variant from the breeding population.

Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

De novo Mutations in Domestic Cat are Consistent with an Effect of Reproductive Longevity on Both the Rate and Spectrum of Mutations.

The mutation rate is a fundamental evolutionary parameter with direct and appreciable effects on the health and function of individuals. Here, we examine this important parameter in the domestic cat, a beloved companion animal as well as a valuable biomedical model. We estimate a mutation rate of 0.86 × 10-8 per bp per generation for the domestic cat (at an average parental age of 3.8 years). We find evidence for a significant paternal age effect, with more mutations transmitted by older sires. Our analyses suggest that the cat and the human have accrued similar numbers of mutations in the germline before reaching sexual maturity. The per-generation mutation rate in the cat is 28% lower than what has been observed in humans, but is consistent with the shorter generation time in the cat. Using a model of reproductive longevity, which takes into account differences in the reproductive age and time to sexual maturity, we are able to explain much of the difference in per-generation rates between species. We further apply our reproductive longevity model in a novel analysis of mutation spectra and find that the spectrum for the cat resembles the human mutation spectrum at a younger age of reproduction. Together, these results implicate changes in life-history as a driver of mutation rate evolution between species. As the first direct observation of the paternal age effect outside of rodents and primates, our results also suggest a phenomenon that may be universal among mammals.

The TNNT2:c.95-108G>A variant is common in Maine Coons and shows no association with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common and potentially fatal heart disease in many cat breeds. An intronic variant in TNNT2, c.95-108G>A, was recently reported as the cause of HCM in the Maine Coon. The aim of this study was to determine this variant's allele frequency in different populations and its possible association with HCM. Based on 160 Maine Coon samples collected in Belgium, Italy, Sweden and the USA, the variant's allele frequency was estimated to be 0.32. Analysis of the 99 Lives feline whole genome sequencing database showed that the TNNT2 variant also occurs in other breeds, as well as mixed-breed cats. Comparison of 31 affected and 58 healthy cats did not reveal significantly increased odds for HCM in homozygotes. Based on the combined evidence and in agreement with the standards and guidelines for the interpretation of sequence variants, this variant is currently classified as a variant of unknown significance and should not be used for breeding decisions regarding HCM.