Astyanax mexicanus surface and cavefish chromosome-scale assemblies for trait variation discovery.

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and non-coding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared to other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared to a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species.

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.

Elevated DNA Damage without signs of aging in the short-sleeping Mexican Cavefish.

Dysregulation of sleep has widespread health consequences and represents an enormous health burden. Short-sleeping individuals are predisposed to the effects of neurodegeneration, suggesting a critical role for sleep in the maintenance of neuronal health. While the effects of sleep on cellular function are not completely understood, growing evidence has identified an association between sleep loss and DNA damage, raising the possibility that sleep facilitates efficient DNA repair. The Mexican tetra fish, Astyanax mexicanus provides a model to investigate the evolutionary basis for changes in sleep and the consequences of sleep loss. Multiple cave-adapted populations of these fish have evolved to sleep for substantially less time compared to surface populations of the same species without identifiable impacts on healthspan or longevity. To investigate whether the evolved sleep loss is associated with DNA damage and cellular stress, we compared the DNA Damage Response (DDR) and oxidative stress levels between A. mexicanus populations. We measured markers of chronic sleep loss and discovered elevated levels of the DNA damage marker γH2AX in the brain, and increased oxidative stress in the gut of cavefish, consistent with chronic sleep deprivation. Notably, we found that acute UV-induced DNA damage elicited an increase in sleep in surface fish but not in cavefish. On a transcriptional level, only the surface fish activated the photoreactivation repair pathway following UV damage. These findings suggest a reduction of the DDR in cavefish compared to surface fish that coincides with elevated DNA damage in cavefish. To examine DDR pathways at a cellular level, we created an embryonic fibroblast cell line from the two populations of A. mexicanus. We observed that both the DDR and DNA repair were diminished in the cavefish cells, corroborating the in vivo findings and suggesting that the acute response to DNA damage is lost in cavefish. To investigate the long-term impact of these changes, we compared the transcriptome in the brain and gut of aged surface fish and cavefish. Strikingly, many genes that are differentially expressed between young and old surface fish do not transcriptionally vary by age in cavefish. Taken together, these findings suggest that have developed resilience to sleep loss, despite possessing cellular hallmarks of chronic sleep deprivation.

Identifying gene expression programs in single-cell RNA-seq data using linear correlation explanation.

OBJECTIVE: Gene expression analysis through single-cell RNA sequencing (scRNA-seq) has revolutionized our understanding of gene regulation in diverse cell types, tissues, and organisms. While existing methods primarily focus on identifying cell type-specific gene expression programs (GEPs), the characterization of GEPs associated with biological processes and stimuli responses remains limited. In this study, we aim to infer biologically meaningful GEPs that are associated with both cellular phenotypes and activity programs directly from scRNA-seq data. METHODS: We applied linear CorEx, a machine-learning-based approach, to infer GEPs by grouping genes based on total correlation optimization function in simulated and real-world scRNA-seq datasets. Additionally, we utilized a transfer learning approach to project CorEx-inferred GEPs to other scRNA-seq datasets. RESULTS: By leveraging total correlation optimization, linear CorEx groups genes and demonstrates superior performance in identifying cell types and activity programs compared to similar methods using simulated data. Furthermore, we apply this same approach to real-world scRNA-seq data from the mouse dentate gyrus and embryonic colon development, uncovering biologically relevant GEPs related to cell types, developmental ages, and cell cycle programs. We also demonstrate the potential for transfer learning by evaluating similar datasets, showcasing the cross-species sensitivity of linear CorEx. CONCLUSION: Our findings validate linear CorEx as a valuable tool for comprehensively analyzing complex signals in scRNA-seq data, leading to deeper insights into gene expression dynamics, cellular heterogeneity, and regulatory mechanisms.

Identification of Genomic Signatures for Colorectal Cancer Survival Using Exploratory Data Mining.

Clinicopathological presentations are critical for establishing a postoperative treatment regimen in Colorectal Cancer (CRC), although the prognostic value is low in Stage 2 CRC. We implemented a novel exploratory algorithm based on artificial intelligence (explainable artificial intelligence, XAI) that integrates mutational and clinical features to identify genomic signatures by repurposing the FoundationOne Companion Diagnostic (F1CDx) assay. The training data set (n = 378) consisted of subjects with recurrent and non-recurrent Stage 2 or 3 CRC retrieved from TCGA. Genomic signatures were built for identifying subgroups in Stage 2 and 3 CRC patients according to recurrence using genomic parameters and further associations with the clinical presentation. The summarization of the top-performing genomic signatures resulted in a 32-gene genomic signature that could predict tumor recurrence in CRC Stage 2 patients with high precision. The genomic signature was further validated using an independent dataset (n = 149), resulting in high-precision prognosis (AUC: 0.952; PPV = 0.974; NPV = 0.923). We anticipate that our genomic signatures and NCCN guidelines will improve recurrence predictions in CRC molecular stratification.

Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Phylogenomics analyses of all species of Swordtails (Genus Xiphophorus ) highlights hybridization precedes speciation.

Hybridization has been recognized as an important driving force for evolution, however studies of the genetic consequence and its cause are still lagging behind in vertebrates due to the lack of appropriate experimental systems. Fish of the central American genus Xiphophorus were proposed to have evolved with multiple ancient and ongoing hybridization events, and served as a valuable research model in evolutionary biology and in biomedical research on human disease for more than a century. Here, we provide the complete genome resource and its annotation of all 26 Xiphophorus species. On this dataset we resolved the so far conflicting phylogeny. Through comparative genomic analyses we investigated the molecular evolution of genes related to melanoma, for a main sexually selected trait and for the genetic control of puberty timing, which are predicted to be involved in pre-and postzygotic isolation and thus to influence the probability of interspecific hybridization in Xiphophorus . We demonstrate dramatic size-variation of some gene families across species, despite the reticulate evolution and short divergence time. Finally, we clarify the hybridization history in the genus Xiphophorus genus, settle the long dispute on the hybridization origin of two Southern swordtails, highlight hybridizations precedes speciation, and reveal the distribution of hybridization ancestry remaining in the fused genome.

Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

A second unveiling: Haplotig masking of the eastern oyster genome improves population-level inference.

Genome assembly can be challenging for species that are characterized by high amounts of polymorphism, heterozygosity, and large effective population sizes. High levels of heterozygosity can result in genome mis-assemblies and a larger than expected genome size due to the haplotig versions of a single locus being assembled as separate loci. Here, we describe the first chromosome-level genome for the eastern oyster, Crassostrea virginica. Publicly released and annotated in 2017, the assembly has a scaffold N50 of 54 mb and is over 97.3% complete based on BUSCO analysis. The genome assembly for the eastern oyster is a critical resource for foundational research into molluscan adaptation to a changing environment and for selective breeding for the aquaculture industry. Subsequent resequencing data suggested the presence of haplotigs in the original assembly, and we developed a post hoc method to break up chimeric contigs and mask haplotigs in published heterozygous genomes and evaluated improvements to the accuracy of downstream analysis. Masking haplotigs had a large impact on SNP discovery and estimates of nucleotide diversity and had more subtle and nuanced effects on estimates of heterozygosity, population structure analysis, and outlier detection. We show that haplotig masking can be a powerful tool for improving genomic inference, and we present an open, reproducible resource for the masking of haplotigs in any published genome.

Astyanax mexicanus surface and cavefish chromosome-scale assemblies for trait variation discovery.

The ability of organisms to adapt to sudden extreme environmental changes produces some of the most drastic examples of rapid phenotypic evolution. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, but repeated colonizations of cave environments have resulted in the independent evolution of troglomorphic phenotypes in several populations. Here, we present three chromosome-scale assemblies of this species, for one surface and two cave populations, enabling the first whole-genome comparisons between independently evolved cave populations to evaluate the genetic basis for the evolution of adaptation to the cave environment. Our assemblies represent the highest quality of sequence completeness with predicted protein-coding and non-coding gene metrics far surpassing prior resources and, to our knowledge, all long-read assembled teleost genomes, including zebrafish. Whole genome synteny alignments show highly conserved gene order among cave forms in contrast to a higher number of chromosomal rearrangements when compared to other phylogenetically close or distant teleost species. By phylogenetically assessing gene orthology across distant branches of amniotes, we discover gene orthogroups unique to A. mexicanus. When compared to a representative surface fish genome, we find a rich amount of structural sequence diversity, defined here as the number and size of insertions and deletions as well as expanding and contracting repeats across cave forms. These new more complete genomic resources ensure higher trait resolution for comparative, functional, developmental, and genetic studies of drastic trait differences within a species.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants.

BACKGROUND: The red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome. METHODS: We present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines. RESULTS: We demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference. CONCLUSIONS: We expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

Concurrent invasions of European starlings in Australia and North America reveal population-specific differentiation in shared genomic regions.

A species' success during the invasion of new areas hinges on an interplay between the demographic processes common to invasions and the specific ecological context of the novel environment. Evolutionary genetic studies of invasive species can investigate how genetic bottlenecks and ecological conditions shape genetic variation in invasions, and our study pairs two invasive populations that are hypothesized to be from the same source population to compare how each population evolved during and after introduction. Invasive European starlings (Sturnus vulgaris) established populations in both Australia and North America in the 19th century. Here, we compare whole-genome sequences among native and independently introduced European starling populations to determine how demographic processes interact with rapid evolution to generate similar genetic patterns in these recent and replicated invasions. Demographic models indicate that both invasive populations experienced genetic bottlenecks as expected based on invasion history, and we find that specific genomic regions have differentiated even on this short evolutionary timescale. Despite genetic bottlenecks, we suggest that genetic drift alone cannot explain differentiation in at least two of these regions. The demographic boom intrinsic to many invasions as well as potential inversions may have led to high population-specific differentiation, although the patterns of genetic variation are also consistent with the hypothesis that this infamous and highly mobile invader adapted to novel selection (e.g., extrinsic factors). We use targeted sampling of replicated invasions to identify and evaluate support for multiple, interacting evolutionary mechanisms that lead to differentiation during the invasion 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.

Genomic landscape and gene expression profiles of feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a cancer of the squamous cell lining in the oral cavity and represents up to 80% of all oral cancers in cats, with a poor prognosis. We have used whole exome sequencing (WES) and RNA sequencing of the tumor to discover somatic mutations and gene expression changes that may be associated with FOSCC occurrence. FOSCC offers a potential comparative model to study human head and neck squamous cell carcinoma (HNSCC) due to its similar spontaneous formation, and morphological and histological features. In this first study using WES to identify somatic mutations in feline cancer, we have identified tumor-associated gene mutations in six cats with FOSCC and found some overlap with identified recurrently mutated genes observed in HNSCC. Four samples each had mutations in TP53, a common mutation in all cancers, but each was unique. Mutations in other cellular growth control genes were also found such as KAT2B and ARID1A. Enrichment analysis of FOSCC gene expression profiles suggests a molecular similarity to human OSCC as well, including alterations in epithelial to mesenchymal transition and IL6/JAK/STAT pathways. In this preliminary study, we present exome and transcriptome results that further our understanding of FOSCC.

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.

High resolution genomes of multiple Xiphophorus species provide new insights into microevolution, hybrid incompatibility, and epistasis.

Because of diverged adaptative phenotypes, fish species of the genus Xiphophorus have contributed to a wide range of research for a century. Existing Xiphophorus genome assemblies are not at the chromosomal level and are prone to sequence gaps, thus hindering advancement of the intra- and inter-species differences for evolutionary, comparative, and translational biomedical studies. Herein, we assembled high-quality chromosome-level genome assemblies for three distantly related Xiphophorus species, namely, X. maculatus, X. couchianus, and X. hellerii Our overall goal is to precisely assess microevolutionary processes in the clade to ascertain molecular events that led to the divergence of the Xiphophorus species and to progress understanding of genetic incompatibility to disease. In particular, we measured intra- and inter-species divergence and assessed gene expression dysregulation in reciprocal interspecies hybrids among the three species. We found expanded gene families and positively selected genes associated with live bearing, a special mode of reproduction. We also found positively selected gene families are significantly enriched in nonpolymorphic transposable elements, suggesting the dispersal of these nonpolymorphic transposable elements has accompanied the evolution of the genes, possibly by incorporating new regulatory elements in support of the Britten-Davidson hypothesis. We characterized inter-specific polymorphisms, structural variants, and polymorphic transposable element insertions and assessed their association to interspecies hybridization-induced gene expression dysregulation related to specific disease states in humans.

The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus.

Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek's disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta.

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

Structurally divergent and recurrently mutated regions of primate genomes.

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

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.