The genetics of the patient is a critical aspect of an individual’s health care for some diseases. The reduced cost of WGS and WES has made feasible the examination of entire genomes for individualized healthcare. Animals now have the same benefit since lower sequencing costs have allowed the development of DNA variant databases from normal control animals for comparison to individuals with diseases. The study of rare, simply inherited recessive diseases particularly benefit from the WGS approach. Although a large database of normal DNA variants within the genome of black-footed cats is not available, because these wild felids are phylogenetically closely related to domestic cats, the domestic cat genome variant database was used for comparison of polymorphisms to exclude variants in common between the species. Herein is the first cross-species comparison of WGS data to support the captive management of an endangered species.

Vision loss is a significant concern in the vulnerable African black-footed cat as poor eyesight could result in a poor quality of life and failure to thrive for individuals, impacting healthy captive breeding practices for the species as a whole. Clinical examination of the affected male proband and female sibling cat revealed findings consistent with PRA. The ERG confirmed that both cats retained minimal or no retinal function, consistent with end-stage PRA. Clinical abnormalities in affected cats were present as early as 3 months of age and rapidly progressed to end-stage PRA, supporting an early-onset retinal disorder affecting both rod and cone photoreceptors. The male proband and female cat are full-siblings, but from different litters. Pedigree analysis supported an autosomal recessive mode of inheritance since visually normal parents produced both affected (n = 2) and unaffected kittens (n = 3), the parents of affected kittens had consanguinity and the affected kittens were either male (n = 3) and female (n = 2) (Fig. 3).

Results of ophthalmic examination and testing, pedigree analysis, and lack of evidence for nutritional, toxic, and hypertensive etiology for the retinal degeneration in the affected African black-footed cats strongly suggested a genetic basis for early-onset PRA. The male proband and female sibling cat were identified at 7 and 3 months of age, respectively, however initial signs of compromised photoreceptor integrity and function may have occurred at an earlier time point. The phenotype and electrodiagnostic findings in PRA-affected black-footed cats are chronologically most consistent with early-onset PRAs in Persians and Bengals, and inconsistent with autosomal dominant early-onset PRA of CRX (rdy) and autosomal recessive late-onset PRA of CEP290 (rdAc)-mutant Abyssinians. Although the presence of a domestic cat variant for vision loss would be highly unlikely in these black-footed cats, genetic testing for cat PRAs is rapid and low cost. Genotyping excluded the Persian autosomal recessive early-onset AIPL1 variant as well as Abyssinian rdAc and rdy PRA variants. The WGS would allow more exhaustive consideration of these same genes for a species-specific variant.

To identify the causal gene, WGS of a trio of cats, comprising two normal obligate carrier parents, and one affected offspring, was conducted. This study of the single black-footed cat was assisted by comparison variants from the closely related domestic cat database, which included variants from 51 domestic cats and one Pallas cat (Otocolobus manul) at the time of the study. The Pallas cat was an opportunistic inclusion in the 99 Lives dataset, included as part of a separate study focusing on polycystic kidney disease. The Pallas cat is the next nearest feline lineage and did support the filtering of shared wild felid variants and shared variants with domestics that would not be causal. Forty-one percent of variants were eliminated by the cross-species comparison. One of the fifty high impact variants resided in a gene known to cause retinal degeneration (IQCB1) and listed on RetNet. A 2 bp deletion within the coding region of the black-footed cats IQCB1 was detected and segregated concordantly with the disease phenotype (Fig. 3). The mutation is predicted to cause a protein truncation at position 428 of the amino acid chain (p.L428*), deleting an IQ calmodulin-binding protein domain and the complete protein C-terminal51. Seven moderate impact missense variants were also detected in RetNet genes that were unique to the three sequenced black-footed cats and segregated with the disease. Mismatch genotype and phenotype in addition to the probability of an unrelated cat being affected excluded five of the missense variants as potential candidate genes. Additionally, the remaining two variants were most likely not associated with the retinal degeneration since they were present in individuals unrelated to the affected cats. Although more likely not associated, the variants within CDH23 and DTHD1_144 will be tested along with the IQCB1 2 bp deletion in all samples that will be received in the future, until when excluded or confirmed by the phenotype.

Variants in IQCB1 (IQ calmodulin-binding motif-containing protein-1) are known to historically cause Senior-Loken syndrome (SLSN) in humans, a rare autosomal recessive disease characterized by both nephronophthisis (NPHP) and retinal degeneration51,52. All affected human patients with SLSN exhibit Leber congenital amauorsis (LCA). Leber congenital amauorsis is generally inherited in an autosomal recessive manner and associated with over 21 gene variants in animals and humans related to retinal function, including RPE65, CRX, CEP290, AIPL1, and IQCB153. The IQCB1 gene encodes nephrocystin-5 (NPHP5), one of thirteen ciliary proteins collectively known as nephronophthisis (NPHP) proteins54. The NPHP5 protein is present in the photoreceptor connecting cilium, a zone that connects the photoreceptor cell body to the outer segments, where IQCB1 Interacts with CEP29051,55. The ciliary protein contains two calmodulin binding sites and co-localizes with nephrocystin-1, nephrocystin-451. The NPHP5 protein also interacts with nephrocystin-655 and retinal ciliopathy gene retinitis pigmentosa GTPase regulator (RPGR)51. Premature terminating mutations in NPHP5 lead to a truncated protein, which disrupts peptide transportation, compromising the structural and functional integrity of the photoreceptor, leading to cone and rod death and subsequent retinal degeneration51,55. Variants in NPHP proteins also cause a broad-spectrum of autosomal recessive kidney diseases that are the most frequent genetic cause of end-stage kidney disease in children and young adults56. Extra-renal manifestations occur in 10–15% of cases and retinal degeneration are the most common abnormality. Thus, kidney function of PRA-affected black-footed cats should be monitored.

IQCB1 variants have not been recognized in the domestic cat but an insertional mutation in NPHP5 (IQCB1) has been recently documented in American Pit Bull Terrier dogs with early-onset recessively inherited cone-rod dystrophy (crd2)57,58. In mutant dogs, the resulting ciliopathy causes early loss of rod photoreceptors and relative retention of central retinal cone photoreceptors that lack function58. In the affected dogs, non-ophthalmic problems were neither observed nor reported. Similarly, a variant in NPHP4 is associated with recessive cone-rod dystrophy in Standard Wire-haired Dachshunds. No kidney involvement has been reported in affected dachshunds59.

The pedigree reveals three unique lineages in the African black-footed cat captive population (Fig. 3). The IQCB1-mutant gene was isolated within one lineage, but has crossed to another lineage with a recent breeding of cats. Ideally, the IQCB1 variant should be eliminated within the breeding population of captive black-footed cats. This would require all living cats (<50) to be genotyped. Specifically cats with an increased risk for affected or carrier status based on the pedigree analysis and known genotypes (Fig. 3; 209, 260, 261, 262, 264, 265, and 266). IQCB1-carriers (or CDH23 and DTHD1 carriers) would need to be mated with wild type cats. If significant population changes occurred in African black-footed cats that necessitated breeding of IQCB1-mutant cats from a conservation standpoint, they should be bred to wild type individuals to maintain maximum genetic diversity within this species. Additionally, the black-footed cats related to the founder cats of the PRA lineage could be genotyped to prevent further influx of the variant into captive breeding programs worldwide.

This is the first report of early-onset recessively inherited PRA in the African black-footed cat. Genetic analysis revealed the etiology to likely be a frameshift mutation in the IQCB1 gene, or a missense mutation within CDH23 or DTHD1. IQCB1 represents the most likely variant associated with the phenotype, and the identified polymorphism is predicted to code for a truncated NPHP5 protein in the connecting cilium which is present in both rod and cone photoreceptors. Genetic testing all the candidate variants in more affected and sighted individuals will help confirm the causal variant associated with the PRA. The identification of the putative causal gene provides a DNA-based test for appropriate captive breeding practices and conservation of this vulnerable wild felid. Moreover, the WGS variant database comprised of 51 domestic cats and one Pallas cat at the time of the study has proven useful for cross-species comparisons for species that have recent common ancestors.