Bovine arthrogryposis multiplex congenita (AMC) is a syndromic term for a congenital condition characterized by multiple joint contractures. Rare inherited forms of bovine AMC have been reported in different breeds. For AMC in Angus cattle a causative genomic deletion encompassing the agrin (AGRN) gene, encoding an essential neural regulator that induces the aggregation of acetylcholine receptors (AChRs), is known. In 2015, three genetically related cases of generalized AMC affecting Red dairy calves were diagnosed in Denmark.

Renal syndromes are occasionally reported in domestic animals. Two identical twin Tyrolean Grey calves exhibited weight loss, skeletal abnormalities and delayed development associated with kidney abnormalities and formation of uroliths. These signs resembled inherited renal tubular dysplasia found in Japanese Black cattle which is associated with mutations in the claudin 16 gene. Despite demonstrating striking phenotypic similarities, no obvious presence of pathogenic variants of this candidate gene were found. Therefore further analysis was required to decipher the genetic etiology of the condition.

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.

Ichthyoses are hereditary skin disorders characterized by the formation of scales and defects in the outermost layer of the epidermis. In dogs, at least six different breed-specific ichthyoses including a relatively common PNPLA1-related autosomal recessive ichthyosis in Golden Retrievers are known. In this study, we investigated 14 Golden Retrievers with scales that were not homozygous for the mutant PNPLA1 allele suggesting a genetically distinct new form of ichthyosis. Histopathological examinations showed lamellar, orthokeratotic hyperkeratosis, and mildly hyperplastic epidermis that led to the diagnosis of a nonepidermolytic ichthyosis. Combined linkage and homozygosity mapping in 14 cases and 30 nonaffected family members delimited a critical interval of ∼12.7 Mb on chromosome 23. Whole-genome sequencing of an affected dog revealed a single protein-changing variant within this region that was not present in 795 control genomes. The identified variant is a 14 bp deletion in the ABHD5 gene (c.1006_1019del), leading to a frameshift and altering the last 14 codons p.(Asp336Serfs*6). The genotypes at this variant showed perfect cosegregation with the ichthyosis phenotype in a large family comprising 14 cases and 72 controls. ABHD5 encodes an acyltransferase required for lipid metabolism. In humans, variants in ABHD5 cause Chanarin-Dorfman syndrome, a neutral lipid storage disease with ichthyosis. Our data in dogs together with the knowledge on the effects of ABHD5 variants in humans strongly suggest ABHD5:c.1006_1019del as candidate causative genetic variant for a new canine form of ichthyosis, which we propose to designate as Golden Retriever ichthyosis type 2 (ICH2).

Investigations of hereditary phenotypes in spontaneous mutants may help to better understand the physiological functions of the altered genes. We investigated two unrelated domestic shorthair cats with bulbous swellings of the hair shafts. The clinical, histopathological, and ultrastructural features were similar to those in mice with lanceolate hair phenotype caused by loss-of-function variants in Dsg4 encoding desmoglein 4. We sequenced the genomes from both affected cats and compared the data of each affected cat to 61 control genomes. A search for private homozygous variants in the DSG4 candidate gene revealed independent frameshift variants in each case, c.76del or p.Ile26fsLeu*4 in case no. 1 and c.1777del or p.His593Thrfs*23 in case no. 2. DSG4 is a transmembrane glycoprotein located primarily in the extracellular part of desmosomes, a complex of adhesion molecules responsible for connecting the keratin intermediate filaments of neighbouring epithelial cells. Desmosomes are essential for normal hair shaft formation. Both identified DSG4 variants in the affected cats lead to premature stop codons and truncate major parts of the open-reading frame. We assume that this leads to a complete loss of DSG4 function, resulting in an incorrect formation of the desmosomes and causing the development of defective hair shafts. Together with the knowledge on the effects of DSG4 variants in other species, our data suggest that the identified DSG4 variants cause the hair shaft dystrophy. To the best of our knowledge, this study represents the first report of pathogenic DSG4 variants in domestic animals.

Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia in humans and several domestic animal species. Typical clinical findings are chronic recurrent infections of the respiratory tract and fertility problems. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by PCD. The parents were unaffected suggesting autosomal recessive inheritance. Linkage and homozygosity mapping defined critical intervals comprising ~118 Mb. Whole genome sequencing of one case and comparison to 601 control genomes identified a disease associated frameshift variant, c.43delA, in the NME5 gene encoding a sparsely characterized protein associated with ciliary function. Nme5-/- knockout mice exhibit doming of the skull, hydrocephalus and sperm flagellar defects. The genotypes at NME5:c.43delA showed the expected co-segregation with the phenotype in the Alaskan Malamute family. An additional unrelated Alaskan Malamute with PCD and hydrocephalus that became available later in the study was also homozygous mutant at the NME5:c.43delA variant. The mutant allele was not present in more than 1000 control dogs from different breeds. Immunohistochemistry demonstrated absence of the NME5 protein from nasal epithelia of an affected dog. We therefore propose NME5:c.43delA as the most likely candidate causative variant for PCD in Alaskan Malamutes. These findings enable genetic testing to avoid the unintentional breeding of affected dogs in the future. Furthermore, the results of this study identify NME5 as a novel candidate gene for unsolved human PCD and/or hydrocephalus cases.

Familial cerebellar ataxia with hydrocephalus in Bullmastiffs was described almost 40 years ago as a monogenic autosomal recessive trait. We investigated two young Bullmastiffs showing similar clinical signs. They developed progressive gait and behavioural abnormalities with an onset at around 6 months of age. Neurological assessment was consistent with a multifocal brain disease. Magnetic resonance imaging of the brain showed intra-axial bilateral symmetrical focal lesions localised to the cerebellar nuclei. Based on the juvenile age, nature of neurological deficits and imaging findings, an inherited disorder of the brain was suspected. We sequenced the genome of one affected Bullmastiff. The data were compared with 782 control genomes of dogs from diverse breeds. This search revealed a private homozygous frameshift variant in the MFF gene in the affected dog, XM_038574000.1:c.471_475delinsCGCTCT, that is predicted to truncate 55% of the wild type MFF open reading frame, XP_038429928.1: p.(Glu158Alafs*14). Human patients with pathogenic MFF variants suffer from 'encephalopathy due to defective mitochondrial and peroxisomal fission 2'. Archived samples from two additional affected Bullmastiffs related to the originally described cases were obtained. Genotypes in a cohort of four affected and 70 unaffected Bullmastiffs showed perfect segregation with the disease phenotype. The available data together with information from previous disease reports allow classification of the investigated MFF frameshift variant as pathogenic and probably causative defect of the observed neurological phenotype. In analogy to the human phenotype, we propose to rename this disease 'mitochondrial fission encephalopathy (MFE)'.

A single male Rottweiler dog with severe footpad hyperkeratosis starting at an age of eight weeks was investigated. The hyperkeratosis was initially restricted to the footpads. The footpad lesions caused severe discomfort to the dog and had to be trimmed under anesthesia every 8-10 weeks. Histologically, the epidermis showed papillated villous projections of dense keratin in the stratum corneum. Starting at eight months of age, the patient additionally developed signs consistent with atopic dermatitis and recurrent bacterial skin and ear infections. Crusted hyperkeratotic plaques developed at sites of infection. We sequenced the genome of the affected dog and compared the data to 655 control genomes. A search for variants in 32 candidate genes associated with human palmoplantar keratoderma (PPK) revealed a single private protein-changing variant in the affected dog. This was located in the DSG1 gene encoding desmoglein 1. Heterozygous monoallelic DSG1 variants have been reported in human patients with striate palmoplantar keratoderma I (SPPK1), while biallelic DSG1 loss of function variants in humans lead to a more pronounced condition termed severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome. The identified canine variant, DSG1:c.2541_2545delGGGCT, leads to a frameshift and truncates about 20% of the coding sequence. The affected dog was homozygous for the mutant allele. The comparative data on desmoglein 1 function in humans suggest that the identified DSG1 variant may have caused the footpad hyperkeratosis and predisposition for allergies and skin infections in the affected dog.

We investigated three related Rough Collies with recurrent inflammatory pulmonary disease. The clinical symptoms were similar to primary ciliary dyskinesia (PCD). However, the affected dogs did not carry any known pathogenic PCD variants. Pedigree analysis suggested a recessive mode of inheritance. Combined linkage and homozygosity mapping in three cases and seven non-affected family members delineated 19 critical intervals on 10 chromosomes comprising a total of 99 Mb. The genome of one affected dog was sequenced and compared to 601 control genomes. We detected only a single private homozygous protein-changing variant in the critical intervals. The detected variant was a 4 bp deletion, c.2717_2720delACAG, in the AKNA gene encoding the AT-hook transcription factor. It causes a frame-shift introducing a premature stop codon and truncates 37% of the open reading frame, p.(Asp906Alafs*173). We genotyped 88 Rough Collies consisting of family members and unrelated individuals. All three available cases were homozygous for the mutant allele and all 85 non-affected dogs were either homozygous wildtype (n = 67) or heterozygous (n = 18). AKNA modulates inflammatory immune responses. Akna-/- knockout mice die shortly after birth due to systemic autoimmune inflammatory processes including lung inflammation that is accompanied by enhanced leukocyte infiltration and alveolar destruction. The perfect genotype-phenotype association and the comparative functional data strongly suggest that the detected AKNA:c.2717_2720delACAG variant caused the observed severe airway inflammation in the investigated dogs. Our findings enable genetic testing, which can be used to avoid the unintentional breeding of affected puppies.

Loss-of-function variants in the NSDHL gene have been associated with epidermal nevi in humans with congenital hemidysplasia, ichthyosiform nevi, and limb defects (CHILD) syndrome and in companion animals. The NSDHL gene codes for the NAD(P)-dependent steroid dehydrogenase-like protein, which is involved in cholesterol biosynthesis. In this study, a female Chihuahua cross with a clinical and histological phenotype consistent with progressive epidermal nevi is presented. All exons of the NSDHL candidate gene were amplified by PCR and analyzed by Sanger sequencing. A heterozygous frameshift variant, c.718_722delGAACA, was identified in the affected dog. In lesional skin, the vast majority of NSDHL transcripts lacked the five deleted bases. The variant is predicted to produce a premature stop codon truncating 34% of the encoded protein, p.Glu240Profs*17. The mutant allele was absent from 22 additionally genotyped Chihuahuas, as well as from 647 control dogs of diverse breeds and eight wolves. The available experimental data together with current knowledge about NSDHL variants and their functional impact in humans, dogs, and other species prompted us to classify this variant as pathogenic according to the ACMG guidelines that were previously established for human sequence variants. Therefore, we propose the c.718_722delGAACA variant as causative variant for the observed skin lesions in this dog.

A 2-year-old male domestic shorthair cat was presented for a progressive history of abnormal posture, behavior, and mentation. Menace response was absent bilaterally, and generalized tremors were identified on neurological examination. A neuroanatomical diagnosis of diffuse brain dysfunction was made. A neurodegenerative disorder was suspected. Magnetic resonance imaging findings further supported the clinical suspicion. Whole-genome sequencing of the affected cat with filtering of variants against a database of unaffected cats was performed. Candidate variants were confirmed by Sanger sequencing followed by genotyping of a control population. Two homozygous private (unique to individual or families and therefore absent from the breed-matched controlled population) protein-changing variants in the major facilitator superfamily domain 8 (MFSD8) gene, a known candidate gene for neuronal ceroid lipofuscinosis type 7 (CLN7), were identified. The affected cat was homozygous for the alternative allele at both variants. This is the first report of a pathogenic alteration of the MFSD8 gene in a cat strongly suspected to have CLN7.

A 1-month-old, female, smooth-haired miniature Dachshund with dilute color and neurological defects was investigated. The aim of this study was to characterize the clinical signs, histopathological changes and underlying genetic defect. The puppy had visible coat color dilution and was unable to hold its head on its own or to remain in a stable prone position for an extended period. Histopathological examination revealed an accumulation of clumped melanin and deposition of accumulated keratin within the hair follicles, accompanied by dermal pigmentary incontinence. These dermatological changes were compatible with the histopathology described in dogs with an MLPH-related dilute coat color. We sequenced the genome of the affected dog and compared the data to 795 control genomes. MYO5A, coding for myosin VA, was investigated as the top functional candidate gene. This search revealed a private homozygous frameshift variant in MYO5A, XM_022412522.1:c.4973_4974insA, predicted to truncate 269 amino acids (13.8%) of the wild type myosin VA protein, XP_022268230.1:p.(Asn1658Lysfs*28). The genotypes of the index family showed the expected co-segregation with the phenotype and the mutant allele was absent from 142 additionally genotyped, unrelated Dachshund dogs. MYO5A loss of function variants cause Griscelli type 1 syndrome in humans, lavender foal in horses and the phenotype of the dilute mouse mutant. Based on the available data, together with current knowledge on other species, we propose the identified MYO5A frameshift insertion as a candidate causative variant for the observed dermatological and neurological signs in the investigated dog.

Craniomandibular osteopathy (CMO) and calvarial hyperostotic syndrome (CHS) are proliferative, non-neoplastic disorders affecting the skull bones in young dogs. Different forms of these hyperostotic disorders have been described in many dog breeds. However, an incompletely dominant causative variant for CMO affecting splicing of SLC37A2 has been reported so far only in three Terrier breeds. The purpose of this study was to identify further possible causative genetic variants associated with CHS in an American Staffordshire Terrier, as well as CMO in seven affected dogs of different breeds. We investigated their whole-genome sequences (WGS) and filtered variants using 584 unrelated genomes, which revealed no variants shared across all affected dogs. However, filtering for private variants of each case separately yielded plausible dominantly inherited candidate variants in three of the eight cases. In an Australian Terrier, a heterozygous missense variant in the COL1A1 gene (c.1786G>A; p.(Val596Ile)) was discovered. A pathogenic missense variant in COL1A1 was previously reported in humans with infantile cortical hyperostosis, or Caffey disease, resembling canine CMO. Furthermore, in a Basset Hound, a heterozygous most likely pathogenic splice site variant was found in SLC37A2 (c.1446+1G>A), predicted to lead to exon skipping as shown before in SLC37A2-associated canine CMO of Terriers. Lastly, in a Weimaraner, a heterozygous frameshift variant in SLC35D1 (c.1021_1024delTCAG; p.(Ser341ArgfsTer22)) might cause CMO due to the critical role of SLC35D1 in chondrogenesis and skeletal development. Our study indicates allelic and locus heterogeneity for canine CMO and illustrates the current possibilities and limitations of WGS-based precision medicine in dogs.

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

We investigated three neonatal Basset Hound littermates with lesions consistent with epidermolysis bullosa (EB), a group of genetic blistering diseases. A clinically normal bitch was bred to her grandfather by artificial insemination. Out of a litter of seven puppies, two affected puppies died and one was euthanized, with these puppies being submitted for diagnostic necropsy. All had multiple bullae and ulcers involving the nasal planum and paw pads, as well as sloughing claws; one puppy also had oral and esophageal ulcers. The complete genome of one affected puppy was sequenced, and 37 known EB candidate genes were assessed. We found a candidate causative variant in COL7A1, which encodes the collagen VII alpha 1 chain. The variant is a complex rearrangement involving duplication of a 107 bp region harboring a frameshift deletion of 7 bp. The variant is predicted to truncate more than 75% of the open reading frame, p.(Val677Serfs*11). Targeted genotyping of this duplication confirmed that all three affected puppies were homozygous for the duplication, whereas 12 unaffected Basset Hounds did not carry the duplication. This variant was also not seen in the genomes of more than 600 dogs of other breeds. COL7A1 variants have been identified in humans and dogs with dystrophic epidermolysis bullosa (DEB). The identified COL7A1 variant therefore most likely represents the causative variant and allows the refinement of the preliminary EB diagnosis to DEB.