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Summary The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university‐based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping‐by‐sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo‐autosomal sexing marker (zinc‐finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats.

A neutered male domestic medium-haired cat presented at a veterinary neurology clinic at 20 months of age due to progressive neurological signs that included visual impairment, focal myoclonus, and frequent severe generalized seizures that were refractory to treatment with phenobarbital. Magnetic resonance imaging revealed diffuse global brain atrophy. Due to the severity and frequency of its seizures, the cat was euthanized at 22 months of age. Microscopic examination of the cerebellum, cerebral cortex and brainstem revealed pronounced intracellular accumulations of autofluorescent storage material and inflammation in all 3 brain regions. Ultrastructural examination of the storage material indicated that it consisted almost completely of tightly-packed membrane-like material. The clinical signs and neuropathology strongly suggested that the cat suffered from a form of neuronal ceroid lipofuscinosis (NCL). Whole exome sequence analysis was performed on genomic DNA from the affected cat. Comparison of the sequence data to whole exome sequence data from 39 unaffected cats and whole genome sequence data from an additional 195 unaffected cats revealed a homozygous variant in CLN6 that was unique to the affected cat. This variant was predicted to cause a stop gain in the transcript due to a guanine to adenine transition (ENSFCAT00000025909:c.668G > A; XM_003987007.5:c.668G > A) and was the sole loss of function variant detected. CLN6 variants in other species, including humans, dogs, and sheep, are associated with the CLN6 form of NCL. Based on the affected cat’s clinical signs, neuropathology and molecular genetic analysis, we conclude that the cat’s disorder resulted from the loss of function of CLN6. This study is only the second to identify the molecular genetic basis of a feline NCL. Other cats exhibiting similar signs can now be screened for the CLN6 variant. This could lead to establishment of a feline model of CLN6 disease that could be used in therapeutic intervention studies.

The domestic dog has evolved to be an important biomedical model for studies regarding the genetic basis of disease, morphology and behavior. Genetic studies in the dog have relied on a draft reference genome of a purebred female boxer dog named “Tasha” initially published in 2005. Derived from a Sanger whole genome shotgun sequencing approach coupled with limited clone-based sequencing, the initial assembly and subsequent updates have served as the predominant resource for canine genetics for 15 years. While the initial assembly produced a good-quality draft, as with all assemblies produced at the time, it contained gaps, assembly errors and missing sequences, particularly in GC-rich regions, which are found at many promoters and in the first exons of protein-coding genes. Here, we present Dog10K_Boxer_Tasha_1.0, an improved chromosome-level highly contiguous genome assembly of Tasha created with long-read technologies that increases sequence contiguity &gt, 100-fold, closes &gt, 23,000 gaps of the CanFam3.1 reference assembly and improves gene annotation by identifying &gt, 1200 new protein-coding transcripts. The assembly and annotation are available at NCBI under the accession GCF_000002285.5.

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 dwarfism cats 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.Author summaryThe practice of genomic medicine is predicated on the availability of a high quality reference genome and an understanding of the impact of genome variation. Such resources have lead to countless discoveries in humans, however by working exclusively within the framework of human genetics, our potential for understanding diseases biology is limited, as similar analyses in other species have often lead to novel insights. The generation of Felis_catus_9.0, a new high quality reference genome for the domestic cat, helps facilitate the expansion of genomic medicine into the felis lineage. Using Felis_catus_9.0 we analyze the landscape of genomic variation from a collection of 54 cats within the context of human gene constraint. The distribution of variant impacts in cats is correlated with patterns of gene constraint in humans, indicating the utility of this reference for identifying novel mutations that cause phenotypes relevant to human and cat health. Moreover, structural variant analysis revealed a novel variant for feline dwarfism in UGDH, a gene that has not been associated with dwarfism in any other species, suggesting a role for UGDH in cases of undiagnosed dwarfism in humans.

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.

An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test, a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing, and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. Obligate carrier cats were heterozygous. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasizes the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.

A variety of cat breeds have been developed via novelty selection on aesthetic, dermatological traits, such as coat colors and fur types. A recently developed breed, the lykoi, was bred from cats with a sparse hair coat with roaning, implying full color and all white hairs. The lykoi phenotype is a form of hypotrichia, presenting as significant reduction in the average numbers of follicles per hair follicle group as compared to domestic shorthair cats, a mild to severe perifollicular to mural lymphocytic infiltration in 77% of observed hair follicle groups, and the follicles are often miniaturized, dilated, and dysplastic. Whole genome sequencing was conducted on a single lykoi cat that was a cross between two independently ascertained lineages. Comparison to the 99 Lives dataset of 194 non-lykoi cats suggested two variants in the cat homolog for Hairless (HR: lysine demethylase and nuclear receptor corepressor) as candidate causal variants. The lykoi cat was a compound heterozygote for two loss of function variants in HR, an exon 3 c.1255_1256dupGT (chrB1:36040783), which should produce a stop codon at amino acid 420 (p.Gln420Serfs*100) and, an exon 18 c.3389insGACA (chrB1:36051555), which should produce a stop codon at amino acid position 1130 (p.Ser1130Argfs*29). Ascertainment of 14 additional cats from founder lineages from Canada, France and different areas of the USA identified four additional loss of function HR variants likely causing the highly similar phenotypic hair coat across the diverse cats. The novel variants in HR for cat hypotrichia can now be established between minor differences in the phenotypic presentations.

Chediak-Higashi Syndrome (CHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). The feline model for CHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile, CHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. This study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat.

The factors guiding retrotransposon insertion site preference are not well understood. Different types of retrotransposons share common replication machinery and yet occupy distinct genomic domains. Autonomous long interspersed elements accumulate in gene-poor domains and their non-autonomous short interspersed elements accumulate in gene-rich domains. To determine genomic factors that contribute to this discrepancy we analysed the distribution of retrotransposons within the framework of chromosomal domains and regulatory elements. Using comparative genomics, we identified large-scale conserved patterns of retrotransposon accumulation across several mammalian genomes. Importantly, retrotransposons that were active after our sample-species diverged accumulated in orthologous regions. This suggested a similar evolutionary interaction between retrotransposon activity and conserved genome architecture across our species. In addition, we found that retrotransposons accumulated at regulatory element boundaries in open chromatin, where accumulation of particular retrotransposon types depended on insertion size and local regulatory element density. From our results, we propose a model where density and distribution of genes and regulatory elements canalise retrotransposon accumulation. Through conservation of synteny, gene regulation and nuclear organisation, mammalian genomes with dissimilar retrotransposons follow similar evolutionary trajectories.

Despite the contribution of a few major genes for disproportionate dwarfism in humans, many dwarf patients are yet genetically undiagnosed. In domestic cats, disproportionate dwarfism has led to the development of a defined breed, the Munchkin or Minuet. This study examined the genetic aspects of feline dwarfism to consider cats as a new biomedical model. DNA from dwarf cats was genetically analyzed using parentage, linkage, and genome-wide association studies as well as whole genome sequencing. Each genetic approach localized the dwarfism phenotype to a region on cat chromosome B1. No coding variants suspected as causal for the feline dwarfism were identified but a critical region of ∼5.7 Mb from B1:170,278,183-175,975,857 was defined, which implicates a novel gene controlling disproportionate dwarfism. A yet unidentified but novel gene variant, likely structural or regulatory, produces disproportionate dwarfism in cats, which may define undiagnosed human patients.