Independent COL5A1 Variants in Cats with Ehlers-Danlos Syndrome.

We investigated four cats with similar clinical skin-related signs strongly suggestive of Ehlers-Danlos syndrome (EDS). Cases no. 1 and 4 were unrelated and the remaining two cases, no. 2 and 3, were reportedly siblings. Histopathological changes were characterized by severely altered dermal collagen fibers. Transmission electron microscopy in one case demonstrated abnormalities in the collagen fibril organization and structure. The genomes of the two unrelated affected cats and one of the affected siblings were sequenced and individually compared to 54 feline control genomes. We searched for private protein changing variants in known human EDS candidate genes and identified three independent heterozygous COL5A1 variants. COL5A1 is a well-characterized candidate gene for classical EDS. It encodes the proα1 chain of type V collagen, which is needed for correct collagen fibril formation and the integrity of the skin. The identified variants in COL5A1 are c.112_118+15del or r.spl?, c.3514A>T or p.(Lys1172*), and c.3066del or p.(Gly1023Valfs*50) for cases no. 1, 2&3, and 4, respectively. They presumably all lead to nonsense-mediated mRNA decay, which results in haploinsufficiency of COL5A1 and causes the alterations of the connective tissue. The whole genome sequencing approach used in this study enables a refinement of the diagnosis for the affected cats as classical EDS. It further illustrates the potential of such experiments as a precision medicine approach in animals with inherited diseases.

The Effects of FGF4 Retrogenes on Canine Morphology.

Two FGF4 retrogenes (FGF4L1 on chromosome 18 and FGF4L2 on chromosome 12) have been identified to cause dwarfism across many dog breeds. Some breeds are nearly homozygous for both retrogenes (e.g., Dachshunds) and others are homozygous for just one (e.g., Beagles and Scottish Terriers). Since most breeds do not segregate both of these retrogenes, it is challenging to evaluate their individual effects on long bone length and body size. We identified two dog breeds selected for hunting ability, the Alpine Dachsbracke and the Schweizer Niederlaufhund, that segregate both of these retrogenes. Using individual measurements of height at the shoulder, back length, head width, thorax depth and width, and thoracic limb measurements, we evaluated the combined effects of FGF4 retrogenes within these breeds. We applied multivariable linear regression analysis to determine the effects of retrogene copy numbers on the measurements. Copy numbers of both retrogenes had significant effects reducing height at the shoulders and antebrachial length, with FGF4L1 having a much greater effect than FGF4L2. FGF4L1 alone influenced the degree of carpal valgus and FGF4L2 alone increased head width. Neither retrogene had an effect on thorax width or depth. Selectively breeding dogs with FGF4L1 and without FGF4L2 would likely lead to a reduction in the FGF4L2-related risk of intervertebral disc herniation while maintaining the reduction in leg length resulting from FGF4L1.

X-Linked Hypohidrotic Ectodermal Dysplasia in Crossbred Beef Cattle Due to a Large Deletion in EDA.

X-linked hypohidrotic ectodermal dysplasia-1 (ECTD1) in people results in a spectrum of abnormalities, most importantly hypotrichosis, anodontia/oligodontia, and absent or defective ectodermally derived glands. Five Red Angus-Simmental calves born over a 6-year period demonstrated severe hypotrichosis and were diagnosed as affected with ECTD1-like syndrome. Two died of severe pneumonia within a week of birth. The skin of three affected calves revealed a predominance of histologically unremarkable small-caliber hair follicles. Larger follicles (>50 µm) containing medullated hairs (including guard and tactile hairs) were largely restricted to the muzzle, chin, tail, eyelids, tragus and distal portions of the limbs and tail. The mean histological density of hair follicles in flank skin of two affected calves was slightly greater than that in two unaffected calves. One affected calf was examined postmortem at 10 days of age to better characterize systemic lesions. Nasolabial, intranasal and tracheobronchial mucosal glands were absent, whereas olfactory glands were unaffected. Mandibular incisor teeth were absent. Premolar teeth were unerupted and widely spaced. Other than oligodontia, histological changes in teeth were modest, featuring multifocal disorganization of ameloblasts, new bone formation in dental alveoli, and small aggregates of osteodentin and cementum at the margins of the enamel organ. A 52,780 base pair deletion spanning six out of eight coding exons of EDA and all of AWAT2 was identified. Partial deletion of the EDA gene is the presumed basis for the reported X-chromosomal recessive inherited genodermatosis.

ATP2A2 SINE Insertion in an Irish Terrier with Darier Disease and Associated Infundibular Cyst Formation.

A 4-month-old female Irish Terrier presented with a well demarcated ulcerative and crusting lesion in the right ear canal. Histological analysis revealed epidermal hyperplasia with severe acantholysis affecting all suprabasal layers of the epidermis, which prompted a presumptive diagnosis of canine Darier disease. The lesion was successfully treated by repeated laser ablation of the affected epidermis. Over the course of three years, the dog additionally developed three dermal nodules of up to 4 cm in diameter that were excised and healed without complications. Histology of the excised tissue revealed multiple infundibular cysts extending from the upper dermis to the subcutis. The cysts were lined by squamous epithelium, which presented with abundant acantholysis of suprabasal keratinocytes. Infundibular cysts represent a novel finding not previously reported in Darier patients. Whole genome sequencing of the affected dog was performed, and the functional candidate genes for Darier disease (ATP2A2) and Hailey-Hailey disease (ATP2C1) were investigated. The analysis revealed a heterozygous SINE insertion into the ATP2A2 gene, at the end of intron 14, close to the boundary of exon 15. Analysis of the ATP2A2 mRNA from skin of the affected dog demonstrated a splicing defect and marked allelic imbalance, suggesting nonsense-mediated decay of the resulting aberrant transcripts. As Darier disease in humans is caused by haploinsufficiency of ATP2A2, our genetic findings are in agreement with the clinical and histopathological data and support the diagnosis of canine Darier disease.

A Missense Variant Affecting the C-Terminal Tail of UNC93B1 in Dogs with Exfoliative Cutaneous Lupus Erythematosus (ECLE).

Cutaneous lupus erythematosus (CLE) in humans encompasses multiple subtypes that exhibit a wide array of skin lesions and, in some cases, are associated with the development of systemic lupus erythematosus (SLE). We investigated dogs with exfoliative cutaneous lupus erythematosus (ECLE), a dog-specific form of chronic CLE that is inherited as a monogenic autosomal recessive trait. A genome-wide association study (GWAS) with 14 cases and 29 controls confirmed a previously published result that the causative variant maps to chromosome 18. Autozygosity mapping refined the ECLE locus to a 493 kb critical interval. Filtering of whole genome sequence data from two cases against 654 controls revealed a single private protein-changing variant in this critical interval, UNC93B1:c.1438C>A or p.Pro480Thr. The homozygous mutant genotype was exclusively observed in 23 ECLE affected German Shorthaired Pointers and an ECLE affected Vizsla, but absent from 845 controls. UNC93B1 is a transmembrane protein located in the endoplasmic reticulum and endolysosomes, which is required for correct trafficking of several Toll-like receptors (TLRs). The p.Pro480Thr variant is predicted to affect the C-terminal tail of the UNC93B1 that has recently been shown to restrict TLR7 mediated autoimmunity via an interaction with syndecan binding protein (SDCBP). The functional knowledge on UNC93B1 strongly suggests that p.Pro480Thr is causing ECLE in dogs. These dogs therefore represent an interesting spontaneous model for human lupus erythematosus. Our results warrant further investigations of whether genetic variants affecting the C-terminus of UNC93B1 might be involved in specific subsets of CLE or SLE cases in humans and other species.

Whole Genome Sequencing Indicates Heterogeneity of Hyperostotic Disorders in Dogs.

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.

NME5 frameshift variant in Alaskan Malamutes with primary ciliary dyskinesia.

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.