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Whole-exome sequencing and whole-genome sequencing data sets

Case summaryA 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 informationThis 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.

Supplemental material, sj-docx-1-jor-10.1177_20551169221137536 for CTSK variant implicated in suspected pyknodysostosis in a domestic cat by Maria Lyraki, Angie Hibbert, Sorrel Langley-Hobbs, Philippa Lait, Reuben M Buckley, Wesley C Warren, Leslie A Lyons and 99 Lives Consortium in Journal of Feline Medicine and Surgery Open Reports

Supplemental material, sj-docx-1-jor-10.1177_20551169221137536 for CTSK variant implicated in suspected pyknodysostosis in a domestic cat by Maria Lyraki, Angie Hibbert, Sorrel Langley-Hobbs, Philippa Lait, Reuben M Buckley, Wesley C Warren, Leslie A Lyons and 99 Lives Consortium in Journal of Feline Medicine and Surgery Open Reports

Supplemental material, sj-docx-2-jor-10.1177_20551169221137536 for CTSK variant implicated in suspected pyknodysostosis in a domestic cat by Maria Lyraki, Angie Hibbert, Sorrel Langley-Hobbs, Philippa Lait, Reuben M Buckley, Wesley C Warren, Leslie A Lyons and 99 Lives Consortium in Journal of Feline Medicine and Surgery Open Reports

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 invitro 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 invivo 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.

Supplementary Methods. Table S1. Correlation of CKD biomarkers and GFRs with total kidney volume. Table S2. Estimations of individual kidney volumes and TKVs for each ADPKD and control cat. Table S3. Correlation of kidney imaging parameters by imaging modalities. Table S4. Rank of kidney volumes estimated by different modalities. Figure S1.GFRs at different ages in 11 cats with ADPKD. Figure S2. Post-mortem kidneys of cats with ADPKD. (DOCX 1170 kb)

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random-bred domestic cats, focusing on individual and phenotypic identification. Seventy-eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot(®) ). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10(-19) across all Western cat populations and the likelihood ratio was 1.52 × 10(18) . These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.

AIPL1 Protein Sequence Alignments. Presented are sequences for Homo sapiens, Mus musculus, Felis silvestris catus and Felis silvestris catus with the identified mutation. In green, blue, purple and grey sequences of different domains inferred using the Homo sapiens domain protein annotation ( www.uniprot.org ). In yellow, residues that differ between the Homo sapiens and Mus musculus when compared to the feline protein sequence. The AIPL1 feline mutated sequence lacks partial TRP1 and TRP2 and TRP3. (DOCX 151Â kb)

AIPL1 c.577C > T and HES7 c.5A > G Genotypes in Domestic Cats. Table S2. Primer Sequences for the Analysis of AIPL1 in Domestic Cats. Table S3. Genome Coverage of Persian Cat PRA Trio. (DOCX 32 kb)

Extant populations of the European wildcat are fragmented across the continent, the likely consequence of recent extirpations due to habitat loss and over-hunting. However, their underlying phylogeographic history has never been reconstructed. For testing the hypothesis that the European wildcat survived the Ice Age fragmented in Mediterranean refuges, we assayed the genetic variation at 31 microsatellites in 668 presumptive European wildcats sampled in 15 European countries. Moreover, to evaluate the extent of subspecies/population divergence and identify eventual wild × domestic cat hybrids, we genotyped 26 African wildcats from Sardinia and North Africa and 294 random-bred domestic cats. Results of multivariate analyses and Bayesian clustering confirmed that the European wild and the domestic cats (plus the African wildcats) belong to two well-differentiated clusters (average Ф ST = 0.159, r st = 0.392, P > 0.001; Analysis of molecular variance [AMOVA]). We identified from c. 5% to 10% cryptic hybrids in southern and central European populations. In contrast, wild-living cats in Hungary and Scotland showed deep signatures of genetic admixture and introgression with domestic cats. The European wildcats are subdivided into five main genetic clusters (average Ф ST = 0.103, r st = 0.143, P > 0.001; AMOVA) corresponding to five biogeographic groups, respectively, distributed in the Iberian Peninsula, central Europe, central Germany, Italian Peninsula and the island of Sicily, and in north-eastern Italy and northern Balkan regions (Dinaric Alps). Approximate Bayesian Computation simulations supported late Pleistocene-early Holocene population splittings (from c. 60 k to 10 k years ago), contemporary to the last Ice Age climatic changes. These results provide evidences for wildcat Mediterranean refuges in southwestern Europe, but the evolution history of eastern wildcat populations remains to be clarified. Historical genetic subdivisions suggest conservation strategies aimed at enhancing gene flow through the restoration of ecological corridors within each biogeographic units. Concomitantly, the risk of hybridization with free-ranging domestic cats along corridor edges should be carefully monitored.

AIPL1 c.577C > T and HES7 c.5A > G Genotypes in Domestic Cats. Table S2. Primer Sequences for the Analysis of AIPL1 in Domestic Cats. Table S3. Genome Coverage of Persian Cat PRA Trio. (DOCX 32 kb)

AIPL1 Protein Sequence Alignments. Presented are sequences for Homo sapiens, Mus musculus, Felis silvestris catus and Felis silvestris catus with the identified mutation. In green, blue, purple and grey sequences of different domains inferred using the Homo sapiens domain protein annotation ( www.uniprot.org ). In yellow, residues that differ between the Homo sapiens and Mus musculus when compared to the feline protein sequence. The AIPL1 feline mutated sequence lacks partial TRP1 and TRP2 and TRP3. (DOCX 151Â kb)

Some Devon Rex and Sphynx cats have a variably progressive myopathy characterized by appendicular and axial muscle weakness, megaesophagus, pharyngeal weakness and fatigability with exercise. Muscle biopsies from affected cats demonstrated variable pathological changes ranging from dystrophic features to minimal abnormalities. Affected cats have exacerbation of weakness following anticholinesterase dosing, a clue that there is an underlying congenital myasthenic syndrome (CMS). A genome-wide association study and whole-genome sequencing suggested a causal variant for this entity was a c.1190G>A variant causing a cysteine to tyrosine substitution (p.Cys397Tyr) within the C-terminal domain of collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase (COLQ). Alpha-dystroglycan expression, which is associated with COLQ anchorage at the motor end-plate, has been shown to be deficient in affected cats. Eighteen affected cats were identified by genotyping, including cats from the original clinical descriptions in 1993 and subsequent publications. Eight Devon Rex and one Sphynx not associated with the study were identified as carriers, suggesting an allele frequency of ~2.0% in Devon Rex. Over 350 tested cats from other breeds did not have the variant. Characteristic clinical features and variant presence in all affected cats suggest a model for COLQ CMS. The association between the COLQ variant and this CMS affords clinicians the opportunity to confirm diagnosis via genetic testing and permits owners and breeders to identify carriers in the population. Moreover, accurate diagnosis increases available therapeutic options for affected cats based on an understanding of the pathophysiology and experience from human CMS associated with COLQ variants.