Quality of DNA extracted from formalin-fixed, paraffin-embedded canine tissues.

Veterinary pathology tissue banks are valuable resources for genetic studies. However, limited data exist as to whether quality DNA can be extracted from these tissues for use in canine genotyping studies. We extracted DNA from 44 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from dogs; 9 of these dogs had DNA available from whole blood samples that had been banked. We genotyped DNA from 30 of 44 tissue blocks and 9 whole blood samples on the Illumina CanineHD BeadChip; DNA quality was insufficient in 14 of 44 samples from tissue blocks. There was significant correlation between the 260/280 ratio and single-nucleotide variation (SNV) call rate (p = 0.0276; r2 = 0.162); 23 of 30 samples from FFPE were genotyped with > 65% call rates. Median pairwise identical-by-state (IBS) analysis was 0.99 in 8 pairs of dogs with call rates > 65%. Neither age of tissue block nor specific tissue types were associated with significant differences in DNA concentration, 260/280 ratio, or SNV call rate. DNA extracted from tissue blocks can have variable quality, although comparable levels of homozygosity suggest that extracts from FFPE with call rates > 65% might provide similar results to samples from whole blood when analyzed on the Illumina CanineHD BeadChip

Canine fibroblast growth factor receptor 3 sequence is conserved across dogs of divergent skeletal size

<p>Abstract</p> <p>Background</p> <p>Fibroblast growth factor receptor 3 (FGFR3) is expressed in the growth plate of endochondral bones and serves as a negative regulator of linear bone elongation. Activating mutations severely limit bone growth, resulting in dwarfism, while inactivating mutations significantly enhance bone elongation and overall skeletal size. Domesticated dogs exhibit the greatest skeletal size diversity of any species and, given the regulatory role of FGFR3 on growth plate proliferation, we asked whether sequence differences in FGFR3 could account for some of the size differences.</p> <p>Methods</p> <p>All exons, the promoter region, and 60 bp of the 3' flanking region of the canine FGFR3 gene were sequenced for nine different dog breeds representing a spectrum of skeletal size. The resultant sequences were compared to the reference Boxer genome sequence.</p> <p>Results</p> <p>There was no variation in sequence for any FGFR3 exons, promoter region, or 3' flanking sequence across all breeds evaluated.</p> <p>Conclusion</p> <p>The results suggest that, regardless of domestication selection pressure to develop breeds having extreme differences in skeletal size, the FGFR3 gene is conserved. This implies a critical role for this gene in normal skeletal integrity and indicates that other genes account for size variability in dogs.</p

FGF4 retrogene on CFA12 is responsible for chondrodystrophy and intervertebral disc disease in dogs.

Chondrodystrophy in dogs is defined by dysplastic, shortened long bones and premature degeneration and calcification of intervertebral discs. Independent genome-wide association analyses for skeletal dysplasia (short limbs) within a single breed (PBonferroni = 0.01) and intervertebral disc disease (IVDD) across breeds (PBonferroni = 4.0 × 10-10) both identified a significant association to the same region on CFA12. Whole genome sequencing identified a highly expressed FGF4 retrogene within this shared region. The FGF4 retrogene segregated with limb length and had an odds ratio of 51.23 (95% CI = 46.69, 56.20) for IVDD. Long bone length in dogs is a unique example of multiple disease-causing retrocopies of the same parental gene in a mammalian species. FGF signaling abnormalities have been associated with skeletal dysplasia in humans, and our findings present opportunities for both selective elimination of a medically and financially devastating disease in dogs and further understanding of the ever-growing complexity of retrogene biology

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

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