Different Founding Effects Underlie Dominant Blue Eyes (DBE) in the Domestic Cat.

During the last twenty years, minimal white spotting associated with blue eyes was selected by feline breeders to create the Altai, Topaz, and Celestial breeds. Additionally, certain breeders introduced this trait in their lineages of purebred cats. The trait has been called "dominant blue eyes (DBE)" and was confirmed to be autosomal dominant in all lineages. DBE was initially described in outbred cats from Kazakhstan and Russia and in two purebred lineages of British cats from Russia, as well as in Dutch Maine Coon cats, suggesting different founding effects. We have previously identified two variants in the Paired Box 3 (PAX3) gene associated with DBE in Maine Coon and Celestial cats; however, the presence of an underlying variant remains undetermined in other DBE breeding lines. Using a genome-wide association study, we identified a single region on chromosome C1 that was associated with DBE in British cats. Within that region, we identified PAX3 as the strongest candidate gene. Whole-genome sequencing of a DBE cat revealed an RD-114 retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 (namely NC_018730.3:g.206975776_206975777insN[433]) known to contain regulatory sequences. Using a panel of 117 DBE cats, we showed that this variant was fully associated with DBE in two British lineages, in Altai cats, and in some other DBE lineages. We propose that this NC_018730.3:g.206975776_206975777insN[433] variant represents the DBEALT (Altai Dominant Blue Eye) allele in the domestic cat. Finally, we genotyped DBE cats from 14 lineages for the three PAX3 variants and showed that they were not present in four lineages, confirming genetic heterogeneity of the DBE trait in the domestic cat.

PAX3 haploinsufficiency in Maine Coon cats with dominant blue eyes and hearing loss resembling the human Waardenburg syndrome.

This study investigated the dominant blue eyes (DBE) trait linked to hearing impairment and variable white spotting in Maine Coon cats. Fifty-eight animals descending from two different DBE lineages, the Dutch and the Topaz lines, were sampled. They comprised 48 cats from the Dutch bloodline, including 9 green-eyed and 31 blue-eyed cats, with some individuals exhibiting signs of deafness, and 8 stillborn kittens. Samples from the Topaz lineage included ten blue-eyed animals. A brainstem auditory evoked potential test (BAER) revealed a reduced to absent response to auditory stimuli and absent physiological waveforms in all of the eight examined DBE animals. We sequenced the genome of two affected cats from the Dutch line and searched for variants in 19 candidate genes for the human Waardenburg syndrome and pigmentary disorders. This search yielded nine private protein-changing candidate variants in the genes PAX3, EDN3, KIT, OCA2, SLC24A5, HERC2 and TYRP1. The genotype-phenotype co-segregation was observed for the PAX3 variant within all animals from the Dutch lineage. The mutant allele was absent from 461 control genomes and 241 additionally genotyped green-eyed Maine Coons. We considered the PAX3 variant as the most plausible candidate -a heterozygous nonsense single basepair substitution in exon 6 of PAX3 (NC_051841.1: g.205,787,310G>A, XM_019838731.3:c.937C>T, XP_019694290.1:p.Gln313*), predicted to result in a premature stop codon. PAX3 variants cause auditory-pigmentary syndrome in humans, horses, and mice. Together with the comparative data from other species, our findings strongly suggest PAX3:c.937C>T (OMIA:001688-9685) as the most likely candidate variant for the DBE, deafness and minimal white spotting in the Maine Coon Dutch line. Finally, we propose the designation of DBERE (Rociri Elvis Dominant Blue Eyes) allele in the domestic cat.

A PAX3 insertion in the Celestial breed and certain feline breeding lines with dominant blue eyes.

During the last 60 years many inherited traits in domestic outbred cats were selected and retained giving birth to new breeds characterised by singular coat or morphological phenotypes. Among them, minimal white spotting associated with blue eyes was selected by feline breeders to create the Altai, Topaz, and Celestial breeds. Various established breeds also introduced this trait in their lineages. The trait, that was confirmed as autosomal dominant by breeding data, was first described in domestic cats from Kazakhstan and Russia, in British shorthair and British longhair from Russia, and in Maine Coon cats from the Netherlands, suggesting different founding effects. Using a genome-wide association study we identified a single region on chromosome C1 that was associated with the minimal white spotting and blue eyes phenotype (also called DBE by breeders for dominant blue eyes) in the French Celestial breed. Within that region we identified Paired Box 3 (PAX3) as the strongest candidate gene, since PAX3 is a key regulator of MITF (Melanocyte-Inducing Transcription Factor) and PAX3 variants have been previously identified in various species showing white spotting with or without blue eyes including the mouse and the horse. Whole genome sequencing of a Celestial cat revealed an endogenous retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 known to contain regulatory sequences (conserved non-coding element [CNE]) involved in PAX3 expression. The insertion is in the vicinity of CNE2 and CNE3. All 52 Celestial and Celestial-mixed cats with a DBE phenotype presented the insertion, that was absent in their 22 non-DBE littermates and in 87 non-DBE cats from various breeds. The outbred Celestial founder was also heterozygous for the insertion. Additionally, the variant was found in nine DBE Maine Coon cats related to the Celestial founder and four DBE Siberian cats with an uncertain origin. Segregation of the variant in the Celestial breed is consistent with dominant inheritance and does not appear to be associated with deafness. We propose that this NC_018730.3:g.206974029_206974030insN[395] variant represents the DBECEL (Celestial Dominant Blue Eyes) allele in the domestic cat.

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.

A CDH23 missense variant in Beauceron dogs with non-syndromic deafness.

Congenital coat-colour-related deafness is common among certain canine breeds especially those exhibiting extreme white spotting or merle patterning. We identified a non-syndromic deafness in Beauceron dogs characterised by a bilateral hearing loss in puppies that is not linked to coat colour. Pedigree analysis suggested an autosomal recessive transmission. By combining homozygosity mapping with whole genome sequencing and variant filtering in affected dogs we identified a CDH23:c.700C>T variant. The variant, located in the CHD23 (cadherin related 23) gene, was predicted to induce a CDH23:p.(Pro234Ser) change in the protein. Proline-234 of CDH23 protein is highly conserved across different vertebrate species. In silico tools predicted the CDH23:p.(Pro234Ser) change to be deleterious. CDH23 encodes a calcium-dependent transmembrane glycoprotein localised near the tips of hair-cell stereocilia in the mammalian inner ear. Intact function of these cilia is mandatory for the transformation of the acoustical wave into a neurological signal, leading to sensorineural deafness when impaired. By genotyping a cohort of 90 control Beauceron dogs sampled in France, we found a 3.3% carrier frequency. The CDH23:c.[700C>T] allele is easily detectable with a genetic test to avoid at-risk matings.

A PNPLA8 frameshift variant in Australian shepherd dogs with hereditary ataxia.

Hereditary ataxias are common among canine breeds with various molecular etiology. We identified a hereditary ataxia in young-adult Australian Shepherd dogs characterized by uncoordinated movements and spasticity, worsening progressively and leading to inability to walk. Pedigree analysis suggested an autosomal recessive transmission. By whole genome sequencing and variant filtering of an affected dog we identified a PNPLA8:c.1169_1170dupTT variant. This variant, located in PNPLA8 (Patatin Like Phospholipase Domain Containing 8), was predicted to induce a PNPLA8:p.(His391PhefsTer394) frameshift, leading to a premature stop codon in the protein. The truncated protein was predicted to lack the functional patatin catalytic domain of PNPLA8, a calcium-independent phospholipase. PNPLA8 is known to be essential for maintaining mitochondrial energy production through tailoring mitochondrial membrane lipid metabolism and composition. The Australian Shepherd ataxia shares molecular and clinical features with Weaver syndrome in cattle and the mitochondrial-related neurodegeneration associated with PNPLA8 loss-of-function variants in humans. By genotyping a cohort of 85 control Australian Shepherd dogs sampled in France, we found a 4.7% carrier frequency. The PNPLA8:c.[1169_1170dupTT] allele is easily detectable with a genetic test to avoid at-risk matings.

Golden cats: A never-ending story!

In the British feline breed a golden coat modification, called light-gold, akita or copper, was reported by breeders during the 2010s. This modification restricted eumelanin to the tip of the tail and hairs showed a wideband modification. Pedigree analyses revealed an autosomal recessive inheritance pattern. A single candidate region was identified using a genome-wide association study. Within that region, we identified CORIN (Corin, serine peptidase) as the strongest candidate gene, since two CORIN variants have previously been identified in Siberian cats with a golden phenotype. A homozygous CORIN:c.2425C>T nonsense variant was identified in copper British cats. Segregation of the variant was consistent with recessive inheritance. This nonsense CORIN:c.2425C>T variant, located in CORIN exon 19, was predicted to produce a truncated CORIN protein - CORIN:p.(Arg809Ter) - that would lack part of the scavenger receptor domain and the trypsine-like serine protease catalytic domain. All 30 copper cats were T/T homozygous for the variant, which was also found in 20 C/T heterozygous British control cats but was absent in 340 cats from the 99 Lives dataset. Finally, genotyping of 218 cats from 12 breeds failed to identify carriers in cats from other breeds. We propose that this third CORIN:c.2425C>T variant represents the wbBSH (British recessive wideband) allele in the domestic cat.

MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.

Donskoy cats as a new model of oculocutaneous albinism with the identification of a splice-site variant in Hermansky-Pudlak Syndrome 5 gene.

In the feline Donskoy breed, a phenotype that breeders call "pink-eye," with associated light-brown skin, yellow irises and red-eye effect, has been described. Genealogical data indicated an autosomal recessive inheritance pattern. A single candidate region was identified by genome-wide association study and SNP-based homozygosity mapping. Within that region, we further identified HPS5 (HPS5 Biogenesis Of Lysosomal Organelles Complex 2 Subunit 2) as a strong candidate gene, since HPS5 variants have been identified in humans and animals with Hermansky-Pudlak syndrome 5 or oculocutaneous albinism. A homozygous c.2571-1G>A acceptor splice-site variant located in intron 16 of HPS5 was identified in pink-eye cats. Segregation of the variant was 100% consistent with the inheritance pattern. Genotyping of 170 cats from 19 breeds failed to identify a single carrier in non-Donskoy cats. The c.2571-1G>A variant leads to HPS5 exon-16 splicing that is predicted to produce a 52 amino acids in-frame deletion in the protein. These results support an association of the pink-eye phenotype with the c.2571-1G>A variant. The pink-eye Donskoy cat extends the panel of reported HPS5 variants and offers an opportunity for in-depth exploration of the phenotypic consequences of a new HPS5 variant.

Werewolf, There Wolf: Variants in Hairless Associated with Hypotrichia and Roaning in the Lykoi Cat Breed.

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 (a.k.a. werewolf cat), 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 a 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) (HR lysine demethylase and nuclear receptor corepressor) as candidate causal gene 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.

Feline chimerism revealed by DNA profiling.

In dogs and cats, unusual coat colour phenotypes may result from various phenomena, including chimerism. In the domestic cat, the tortoiseshell coat colour that combines red and non-red hairs is the most obvious way to identify chimeras in males. Several cases of tortoiseshell males have been reported, some of which were diagnosed as chimeras without any molecular confirmation. Here, we report the case of a female feline chimera identified thanks to its coat colour and confirmed through DNA profiling and a coat colour test. We ruled out the hypothesis of mosaicism and aneuploidy. All the data were consistent with a natural case of female chimerism.

Genetic heterogeneity of polydactyly in Maine Coon cats.

OBJECTIVES: Polydactyly has been described in two breeds of domestic cats (Maine Coon and Pixie Bob) and in some outbred domestic cats (eg, Hemingway cats). In most cases, feline polydactyly is a non-syndromic preaxial polydactyly. Three variants located in a regulatory sequence involved in limb development, named ZRS (zone of polarising activity regulatory sequence), have been identified to be responsible for feline polydactyly. These variants have been found in outbred domestic cats in the UK (UK1 and UK2 variants) and in Hemingway cats in the USA (Hw variant). The aim of this study was to characterise the genetic features of polydactyly in Maine Coon cats. METHODS: Genotyping assay was used to identify the variant(s) segregating in a cohort of 75 polydactyl and non-polydactyl Maine Coon cats from different breeding lines from Europe, Canada and the USA. The authors performed a segregation analysis to identify the inheritance pattern of polydactyly in this cohort and analysed the population structure. RESULTS: The Hw allele was identified in a subset of polydactyl cats. Sequencing of two regulatory sequences involved in limb development did not reveal any other variant in polydactyl cats lacking the Hw allele. Additionally, genotype-phenotype and segregation analyses revealed the peculiar inheritance pattern of polydactyly in Maine Coon cats. The population structure analysis demonstrated a genetic distinction between Hw and Hw-free polydactyl cats. CONCLUSIONS AND RELEVANCE: Polydactyly in Maine Coon cats is inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity, and this trait is characterised by genetic heterogeneity in the Maine Coon breed. Maine Coon breeders should be aware of this situation and adapt their breeding practices accordingly.

Cataracts in a population of Bengal cats in France.

OBJECTIVE: To document the clinical appearance and prevalence of cataracts in a French population of Bengal cats. METHODS: Two distinct populations of Bengal cats were examined as follows: (i) 51 animals recruited for evaluation of national prevalence of ocular diseases in an observational study conducted between October 2014 and November 2016 at the Alfort ophthalmology unit; (ii) 12 patients referred for cataract diagnosis examined at a veterinary eye clinic located in central France, between December 2014 and February 2016. Buccal swabs or blood samples for DNA analysis were collected from all patients. The pedigrees of the examined Bengal cats were also investigated. RESULTS: Cataracts were diagnosed in 23 of 51 (45%) cats in the observational study and in all cats in the referral population, mostly bilaterally. Visual impairment was never reported. Age of subjects affected by cataracts ranged from 3 months to 9.6 years (median: 1.9 years). Cataracts were classified as nuclear cataracts (14 of 23 in the observational group and 12 of 12 in the referral group) with a focal, perinuclear, posterior, or complete nuclear pattern, or posterior polar subcapsular cataracts (10 of 23 only in the observational group). An inherited congenital origin appears to be the most likely hypothesis. The pedigree analysis suggests a hereditary component of cataract formation, but further analyses in a larger population or test matings are needed to determine the exact mode of inheritance. CONCLUSION: Presumed inherited cataracts appear to have a high prevalence in Bengal cats in France. The main manifestations are nuclear or subcapsular form, mostly bilateral, symmetrical, and apparently nonprogressive.

Clinical characterisation of polydactyly in Maine Coon cats.

Objectives Polydactyly has been reported in a number of vertebrate species, including the domestic cat. It is a common characteristic in some breeding lines of the Maine Coon. The aim of this study was to assess the limb phenotype of polydactyl cats using physical and radiographic examinations. Methods We used physical examination and radiography to characterise the polydactyly phenotype in a cohort of 70 Maine Coon cats, including 48 polydactyl cats from four different breeding lines from Europe, Canada and the USA. Results The phenotypic expression of polydactyly showed great variability, not only in digit number and conformation, but also in the structure of the carpus and tarsus. Comparison of the size of the radius in polydactyl and non-polydactyl 3-month-old kittens and adult females did not reveal any difference between polydactyl and non-polydactyl cats. Conclusions and relevance We conclude that polydactyly in Maine Coon cats is characterised by broad phenotypic diversity. Polydactyly not only affects digit number and conformation, but also carpus and tarsus conformation, with no apparent deleterious consequence on feline welfare.

Haplosufficiency of PAX3 for melanoma development in Tyr: NRASQ61K; Cdkn2a-/- mice allows identification and sorting of melanoma cells using a Pax3GFP reporter allele.

The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3(GFP/+) knock-in reporter system was combined with the Tyr::NRAS(Q61K); Cdkn2a(-/-) mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+) mice developed metastasizing melanoma as their Tyr::NRAS(Q61K); Cdkn2a(-/-); littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3(GFP/+) lesions were reduced by half. The Pax3(GFP) allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.

A COLQ Missense Mutation in Sphynx and Devon Rex Cats with Congenital Myasthenic Syndrome.

An autosomal recessive neuromuscular disorder characterized by skeletal muscle weakness, fatigability and variable electromyographic or muscular histopathological features has been described in the two related Sphynx and Devon Rex cat breeds (Felis catus). Collection of data from two affected Sphynx cats and their relatives pointed out a single disease candidate region on feline chromosome C2, identified following a genome-wide SNP-based homozygosity mapping strategy. In that region, we further identified COLQ (collagen-like tail subunit of asymmetric acetylcholinesterase) as a good candidate gene, since COLQ mutations were identified in affected humans and dogs with endplate acetylcholinesterase deficiency leading to a synaptic form of congenital myasthenic syndrome (CMS). A homozygous c.1190G>A missense variant located in exon 15 of COLQ, leading to a C397Y substitution, was identified in the two affected cats. C397 is a highly-conserved residue from the C-terminal domain of the protein; its mutation was previously shown to produce CMS in humans, and here we confirmed in an affected Sphynx cat that it induces a loss of acetylcholinesterase clustering at the neuromuscular junction. Segregation of the c.1190G>A variant was 100% consistent with the autosomal recessive mode of inheritance of the disorder in our cat pedigree; in addition, an affected, unrelated Devon Rex cat recruited thereafter was also homozygous for the variant. Genotyping of a panel of 333 cats from 14 breeds failed to identify a single carrier in non-Sphynx and non-Devon Rex cats. Finally, the percentage of healthy carriers in a European subpanel of 81 genotyped Sphynx cats was estimated to be low (3.7%) and 14 control Devon Rex cats were genotyped as wild-type individuals. Altogether, these results strongly support that the neuromuscular disorder reported in Sphynx and Devon Rex breeds is a CMS caused by a unique c.1190G>A missense mutation, presumably transmitted through a founder effect, which strictly and slightly disseminated in these two breeds. The presently available DNA test will help owners avoid matings at risk.

A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys.

BACKGROUND: Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP). FINDINGS: We used a panel of 127 donkeys to identify a recessive missense c.349 T > C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype. CONCLUSIONS: Thus, we propose to name the c.[349 T > C] allele in donkeys, the a(nlp) allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.