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

Chondrodysplastic and normal Norwegian Elkhounds and Karelian Bear Dogs.

<p>(A) A 5-year-old affected female Norwegian Elkhound with chondrodysplasia (left) and a 3-year-old unaffected female Norwegian Elkhound (right). The height at withers was 42 cm for the affected and 48 cm for the unaffected dog. (B) A 7-year-old affected male Elkhound with a height at withers of 38 cm. (C) A normal 5-month-old male Karelian Bear Dog together with its severely affected and significantly smaller male littermate. (D) An adult, less severely affected Karelian Bear Dog that is actively used in hunting. (E) The 5-month-old affected male puppy has prominent bilateral carpal valgus (arrows) and knock knees (genu valgus) (arrowheads). The muscles of the pelvis and thigh are underdeveloped due to severe hip dysplasia. (F) The left forepaw of the 5-month-old affected puppy. Outer digits are abnormally short (arrows). (G) The left forepaw (left) and the left hind paw (right) of an adult affected Elkhound. Similarly to the Karelian Bear Dog, the outer digits are abnormally short in this affected dog (arrows).</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

Body length measurements in affected and healthy Norwegian Elkhounds.

*<p>Measured only in two affected females.</p

Chondrodysplasia pedigrees are consistent with autosomal recessive inheritance.

<p>(A) A pedigree established around the affected Norwegian Elkhounds from Finland. Samples and phenotype information were obtained from all siblings in one litter only, otherwise the phenotypes of full siblings of affected dogs were not known. Denoted are the nine cases and controls that were genotyped using the canine SNP-chip. (B) A pedigree drawn around four affected Norwegian Elkhounds from the United States. (C) A pedigree of the chondrodysplasia phenotype in Karelian Bear Dogs. All affected Karelian Bear Dogs have a single popular sire as a common ancestor (arrow). In all three pedigrees, the recessive c.2083C>T mutation shows full segregation with the chondrodysplasia phenotype. Genotypes are marked with red (T/T), blue (C/T) and black (C/C).</p

Genetic studies.

<p>(A) A Manhattan plot of case-control genome-wide association test performed using 13 cases and 7 unaffected sibling-controls. (B) Results of the family-based testing on the disease associated chromosome 8. Plotted are single-point linkage analysis LOD scores, association test p-values and joint analysis p-values. (C) Genotypes at the disease associated locus on CFA8. All cases share a 1.5 Mb homozygous block, and within this block BICF2P948919 shows complete segregation with the disease. (D) A schematic representation of the seven genes found on the 1.5 Mb block and of the <i>SEL1L</i> gene structure. <i>SEL1L</i> exons are marked with black boxes and red denotes the untranslated regions (UTRs). BICF2P948919 is located on second <i>SEL1L</i> intron and the c.1972T>C mutation on exon 19. (E) Chromatograms of the c.1972T>C mutation in an affected, a carrier and a wild-type dog.</p

Finnish Hound ataxia pedigree.

<p>Pedigree shows those affected litters that were used in the study. Disease segregation is consistent with autosomal recessive mode of inheritance as all affected dogs are born from healthy parents and both sexes are affected. The proportion of affected puppies is 29%, which is close to expected 25%. Denoted are the clinically and pathologically examined cases and the dogs that were genotyped for genome-wide analyses. The numbering of litters and puppies refers to the numbering used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002759#pgen-1002759-t001" target="_blank">Table 1</a>. *In this one litter, the total number of offspring was unknown.</p