Generalized linear mixed model analysis results on the association between environmental factors and comorbid anxieties.

<p>a) Results of the C analysis estimating the association of environmental factors comparing dogs with all anxieties (fearfulness, noise sensitivity, separation anxiety) with dogs with no above-mentioned anxieties reported.</p><p>Both the breed and the questionnaire version were used as a random factor in both analyses, and variables were reduced from the model one by one.</p><p>Results of the type III analysis are presented.</p><p>¹Only variables with statistical significance <0.1 are presented, variables with significance <0.05 are in bold.</p><p>Generalized linear mixed model analysis results on the association between environmental factors and comorbid anxieties.</p

Generalized linear mixed model analysis results on the association between environmental factors and fearfulness.

<p>a) in A analysis, the variable Fear-status is used as a binary response variable. Fit statistics: Gener. Chi-Square / df = 0.96</p><p>b) In B analysis, the variable Fear-status is used as a binary response variable, however, we have excluded all dogs that show a fearful reaction towards loud noises, and also dogs reported to have separation anxiety. Fit statistics: Gener. Chi-Square / df = 0.85</p><p>Both the breed and the questionnaire version were used as a random factor in both analyses, and variables were reduced from the model one by one.</p><p>Results of the type III analysis are presented.</p><p>¹Only variables with statistical significance <0.1 are presented, variables with significance <0.05 are in bold.</p><p>Generalized linear mixed model analysis results on the association between environmental factors and fearfulness.</p

Behavioral and environmental variables derived from questionnaire data.

<p>Behavioral and environmental variables derived from questionnaire data.</p

Generalized linear mixed model analysis results on the association between environmental factors and separation anxiety.

<p>a) In A analysis, the variable Separation anxiety-status is used as a binary response variable. Fit statistics: Gener. Chi-Square / df = 0.92</p><p>Both the breed and the questionnaire version were used as a random factor in both analyses, and variables were reduced from the model one by one.</p><p>Results of the type III analysis are presented.</p><p>¹Only variables with statistical significance <0.1 are presented, variables with significance <0.05 are in bold.</p><p>Generalized linear mixed model analysis results on the association between environmental factors and separation anxiety.</p

A homozygous nonsense mutation in <i>ITGA10</i>.

<p>(A) Chromatograms of the mutation position in a wild-type, a carrier and an affected dog. (B) A schematic representation of <i>ITGA10</i> gene structure and of α10 protein domains. The protein coding sequence of the canine <i>ITGA10</i> gene is composed of 30 exons, and the c.2083C>T change is positioned on exon 16. The α10-subunit is a single pass transmembrane protein with a small cytosolic domain. The largest part of the protein is located in the extracellular space. The nonsense mutation p.Arg695* is positioned approximately in the middle of the α10-subunit. SP = signal peptide, TM = transmembrane segment.</p

A summary of the variants found in the mutation screening <i>ITGA10</i> exons in two affected and two healthy Norwegian Elkhounds.

<p>The following <i>ITGA10</i> reference sequences were used in naming variants: XM_845262.1 (mRNA), XP_850355.1 (protein) and NC_006599.2 (genomic).</p

Results of genome wide association analysis.

<p>(A) The chondrodysplasia locus maps to CFA17. The Manhattan plots show both nominal and permutated p-values of the Fisher’s exact test across all chromosomes. A close-up of CFA17 shows two SNPs, BICF2S23329094 and BICF2S23345973 that reach genome-wide significance after permutation testing. (B) Genotypes at the CFA17 associated locus reveal a shared 2-Mb haplotype block in the affected dogs. (C) The critical region contains 33 genes, including <i>ITGA10</i>, which was selected as a primary candidate gene due to its known expression in the growth plate chondrocytes and involvement in the endochondral ossification process.</p

Pedigree of PRA affected Papillons and Phalènes.

<p>Pedigree indicates the affected dogs that were used in the study. Samples from six affected dogs were available for genotyping. Disease segregation is consistent with autosomal recessive mode of inheritance as all affected dogs are born from healthy parents and both sexes are affected. Obligate carrier parents of affected dogs are marked in the pedigree. Obligate carriers genotyped as heterozygous for <i>CNGB1</i> mutation are marked with a yellow background.</p

Radiographic findings in affected dogs.

<p>(A) The forearm of an unaffected 5-month-old male Karelian Bear Dog has narrow and even growth plates (arrows). (B) The forearm of a severely affected 5-month-old male Karelian Bear Dog with markedly short and bowed radius and ulna. The growth plates are wide and irregular and metaphyseal flaring can be observed (arrows). (C) The forearm of a 3-year-old affected male Norwegian Elkhound. The radius is slightly bowed cranially (arrow). (D) Normal hip joints of an unaffected 5-month-old Karelian Bear Dog. The femoral head sits in its correct position (arrows). (E) Abnormal hip joints of a 5-month-old affected Karelian Bear Dog. The femoral heads are misshapen (white arrow), femoral necks are abnormally short (arrowhead) and the joints are subluxated (red arrow). (F) Normal hip joints of a less severely affected 3-year-old Norwegian Elkhound. (G) Normal metacarpal bones and digits of an unaffected 5-month-old Karelian Bear Dog. (H) Distal forelimb of an affected 5-month-old Karelian Bear Dog with a very short fifth metacarpal bone (arrow). (I) Distal hind limbs of an affected 5-month-old Karelian Bear Dog. Wide growth plates and metaphyseal flaring are apparent. The proximal phalanx of the third digit of the right hind limb (arrow) and the fifth metatarsal bone of the left hind limb (arrowhead) are abnormally short. Dogs in images (A) and (B), (D) and (E) and (G)–(I) are littermates.</p

<i>ITGA10</i> expression on the RNA and protein level.

<p>(A) Semi-quantitative analysis of <i>ITGA10</i> mRNA expression in bronchial and tracheal tissue samples of an affected NE and an unaffected Australian Kelpie dog. PCR reactions were performed using three cycle numbers, 27, 32 and 37. Amplification of mRNA fragments was roughly equal in both dogs, which indicated that the mutated transcript is stable and not targeted for nonsense mediated decay. (B) A western blot analysis of ITGA10 protein expression. A polyclonal anti-ITGA10 antibody was probed against the total protein lysates from tracheal tissue samples of the affected NE and the unaffected Australian Kelpie. The full-length ITGA10 protein was detected in the unaffected control dog but not in the affected dog. GAPDH was used as a loading control.</p