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Bald thigh syndrome is a common hair loss disorder in sighthounds. Numerous possible causes, including environmental conditions, trauma, stress, endocrinopathies and genetic components have been proposed, but only endocrinopathies have been ruled out scientifically. The overall goal of our study was to identify the cause of bald thigh syndrome and the pathological changes associated with it. We approached this aim by comparing skin biopsies and hair shafts of affected and control dogs microscopically as well as by applying high-throughput technologies such as genomics, transcriptomics and proteomics. While the histology is rather unspecific in most cases, trichogram analysis and scanning electron microscopy revealed severe structural abnormalities in hair shafts of affected dogs. This finding is supported by the results of the transcriptomic and proteomic profiling where genes and proteins important for differentiation of the inner root sheath and the assembly of a proper hair shaft were downregulated. Transcriptome profiling revealed a downregulation of genes encoding 23 hair shaft keratins and 51 keratin associated proteins, as well as desmosomal cadherins and several actors of the BMP signaling pathway which is important for hair shaft differentiation. The lower expression of keratin 71 and desmocollin 2 on the mRNA level in skin biopsies corresponded with a decreased protein expression in the hair shafts of affected dogs. The genetic analysis revealed a missense variant in the IGFBP5 gene homozygous in all available Greyhounds and other sighthounds. Further research is required to clarify whether the IGFBP5 variant represents a predisposing genetic risk factor. We conclude from our results that structural defects in the hair shafts are the cause for this well-known disease and these defects are associated with a downregulation of genes and proteins essential for hair shaft formation. Our data add important knowledge to further understand the molecular mechanisms of HF morphogenesis and alopecia in dogs. [ABSTRACT FROM AUTHOR]

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of ~1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN1:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo. [ABSTRACT FROM AUTHOR]

Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (CTNNB1, LEF1, TCF3, WNT10B) and Shh (SHH, GLI1, SMO, PTCH2) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers SOX9, LHX2, LGR5, TCF7L1 and GLI1 whereas NFATc1, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (CYP1A1, CYP1B1, HSD17B14) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease. [ABSTRACT FROM AUTHOR]

Ichthyoses are a heterogeneous group of inherited cornification disorders characterized by generalized dry skin, scaling and/or hyperkeratosis. Ichthyosis vulgaris is the most common form of ichthyosis in humans and caused by genetic variants in the FLG gene encoding filaggrin. Filaggrin is a key player in the formation of the stratum corneum, the uppermost layer of the epidermis and therefore crucial for barrier function. During terminal differentiation of keratinocytes, the precursor profilaggrin is cleaved by several proteases into filaggrin monomers and eventually processed into free amino acids contributing to the hydration of the cornified layer. We studied a German Shepherd dog with a novel form of ichthyosis. Comparing the genome sequence of the affected dog with 288 genomes from genetically diverse non-affected dogs we identified a private heterozygous variant in the ASPRV1 gene encoding “aspartic peptidase, retroviral-like 1”, which is also known as skin aspartic protease (SASPase). The variant was absent in both parents and therefore due to a de novo mutation event. It was a missense variant, c.1052T>C, affecting a conserved residue close to an autoprocessing cleavage site, p.(Leu351Pro). ASPRV1 encodes a retroviral-like protease involved in profilaggrin-to-filaggrin processing. By immunofluorescence staining we showed that the filaggrin expression pattern was altered in the affected dog. Thus, our findings provide strong evidence that the identified de novo variant is causative for the ichthyosis in the affected dog and that ASPRV1 plays an essential role in skin barrier formation. ASPRV1 is thus a novel candidate gene for unexplained human forms of ichthyoses. [ABSTRACT FROM AUTHOR]

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients. [ABSTRACT FROM AUTHOR]

Congenital hepatic fibrosis has been described as a lethal disease with monogenic autosomal recessive inheritance in the Swiss Franches-Montagnes horse breed. We performed a genome-wide association study with 5 cases and 12 controls and detected an association on chromosome 20. Subsequent homozygosity mapping defined a critical interval of 952 kb harboring 10 annotated genes and loci including the polycystic kidney and hepatic disease 1 (autosomal recessive) gene (PKHD1). PKHD1 represents an excellent functional candidate as variants in this gene were identified in human patients with autosomal recessive polycystic kidney and hepatic disease (ARPKD) as well as several mouse and rat mutants. Whereas most pathogenic PKHD1 variants lead to polycystic defects in kidney and liver, a small subset of the human ARPKD patients have only liver symptoms, similar to our horses with congenital hepatic fibrosis. The PKHD1 gene is one of the largest genes in the genome with multiple alternative transcripts that have not yet been fully characterized. We sequenced the genomes of an affected foal and 46 control horses to establish a comprehensive list of variants in the critical interval. We identified two missense variants in the PKHD1 gene which were strongly, but not perfectly associated with congenital hepatic fibrosis. We speculate that reduced penetrance and/or potential epistatic interactions with hypothetical modifier genes may explain the imperfect association of the detected PKHD1 variants. Our data thus indicate that horses with congenital hepatic fibrosis represent an interesting large animal model for the liver-restricted subtype of human ARPKD. [ABSTRACT FROM AUTHOR]

Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on chromosome 5 (praw = 1.0×10−13) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (praw = 6.9×10−10). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis. [ABSTRACT FROM AUTHOR]

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (praw = 4.4×10−14). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis. [ABSTRACT FROM AUTHOR]