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Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10

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Canine Chondrodysplasia Caused by a Truncating Mutation in Collagen-Binding Integrin Alpha Subunit 10

Kaisa Kyöstilä et al. PLoS One.

Abstract

The skeletal dysplasias are disorders of the bone and cartilage tissues. Similarly to humans, several dog breeds have been reported to suffer from different types of genetic skeletal disorders. We have studied the molecular genetic background of an autosomal recessive chondrodysplasia that affects the Norwegian Elkhound and Karelian Bear Dog breeds. The affected dogs suffer from disproportionate short stature dwarfism of varying severity. Through a genome-wide approach, we mapped the chondrodysplasia locus to a 2-Mb region on canine chromosome 17 in nine affected and nine healthy Elkhounds (praw = 7.42×10(-6), pgenome-wide = 0.013). The associated locus contained a promising candidate gene, cartilage specific integrin alpha 10 (ITGA10), and mutation screening of its 30 exons revealed a nonsense mutation in exon 16 (c.2083C>T; p.Arg695*) that segregated fully with the disease in both breeds (p = 2.5×10(-23)). A 24% mutation carrier frequency was indicated in NEs and an 8% frequency in KBDs. The ITGA10 gene product, integrin receptor α10-subunit combines into a collagen-binding α10β1 integrin receptor, which is expressed in cartilage chondrocytes and mediates chondrocyte-matrix interactions during endochondral ossification. As a consequence of the nonsense mutation, the α10-protein was not detected in the affected cartilage tissue. The canine phenotype highlights the importance of the α10β1 integrin in bone growth, and the large animal model could be utilized to further delineate its specific functions. Finally, this study revealed a candidate gene for human chondrodysplasias and enabled the development of a genetic test for breeding purposes to eradicate the disease from the two dog breeds.

Conflict of interest statement

Competing Interests: A DNA test is commercially available for the Norwegian Elkhound and Karelian Bear Dog breeds through Genoscoper Oy (Ltd), which is partly owned by HL. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Chondrodysplastic and normal Norwegian Elkhounds and Karelian Bear Dogs.
(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).
Figure 2
Figure 2. Size differences between chondrodysplastic and normal Norwegian Elkhounds.
Bar plots show the difference of means concerning (A) height at withers, (B) forearm length and (C) wrist to paw length. Error bars represent the standard error of the mean. Measurements were taken from 14 unaffected and 6 affected males, and from 11 unaffected and 3 affected females. The wrist to paw length was measured in two affected females only. *p≤0.01, **p≤0.001.
Figure 3
Figure 3. Chondrodysplasia pedigrees are consistent with autosomal recessive inheritance.
(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).
Figure 4
Figure 4. Radiographic findings in affected dogs.
(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.
Figure 5
Figure 5. Results of genome wide association analysis.
(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 ITGA10, 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.
Figure 6
Figure 6. A homozygous nonsense mutation in ITGA10.
(A) Chromatograms of the mutation position in a wild-type, a carrier and an affected dog. (B) A schematic representation of ITGA10 gene structure and of α10 protein domains. The protein coding sequence of the canine ITGA10 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.
Figure 7
Figure 7. ITGA10 expression on the RNA and protein level.
(A) Semi-quantitative analysis of ITGA10 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.

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Publication types

Grant support

This study was supported partly by the Academy of Finland, the Sigrid Juselius Foundation, Biocentrum Helsinki, the Jane and Aatos Erkko Foundation and the University of Helsinki Research Funds. HL is a member of Biocentrum Helsinki and KK is a student in the Helsinki Graduate Program in Biotechnology and Molecular Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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