The domestic dog (Canis familiaris) segregates more naturally-occurring diseases and phenotypic variation than any other species and has become established as an unparalled model with which to study the genetics of inherited traits. We used a genome-wide association study (GWAS) and targeted resequencing of DNA from just five dogs to simultaneously map and identify mutations for two distinct inherited disorders that both affect a single breed, the Cavalier King Charles Spaniel. We investigated episodic falling (EF), a paroxysmal exertion-induced dyskinesia, alongside the phenotypically distinct condition congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID), commonly known as dry eye curly coat syndrome. EF is characterised by episodes of exercise-induced muscular hypertonicity and abnormal posturing, usually occurring after exercise or periods of excitement. CKCSID is a congenital disorder that manifests as a rough coat present at birth, with keratoconjunctivitis sicca apparent on eyelid opening at 10–14 days, followed by hyperkeratinisation of footpads and distortion of nails that develops over the next few months. We undertook a GWAS with 31 EF cases, 23 CKCSID cases, and a common set of 38 controls and identified statistically associated signals for EF and CKCSID on chromosome 7 (Praw 1.9×10−14; Pgenome = 1.0×10−5) and chromosome 13 (Praw 1.2×10−17; Pgenome = 1.0×10−5), respectively. We resequenced both the EF and CKCSID disease-associated regions in just five dogs and identified a 15,724 bp deletion spanning three exons of BCAN associated with EF and a single base-pair exonic deletion in FAM83H associated with CKCSID. Neither BCAN or FAM83H have been associated with equivalent disease phenotypes in any other species, thus demonstrating the ability to use the domestic dog to study the genetic basis of more than one disease simultaneously in a single breed and to identify multiple novel candidate genes in parallel.

Progressive retinal atrophy (PRA) in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 14 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s) responsible have yet to be identified. Using genome-wide association with 16 Gordon Setter PRA cases and 22 controls, we identified a novel PRA locus, termed rod-cone degeneration 4 (rcd4), on CFA17 (Praw  = 2.22 × 10(-8) , Pgenome  = 2.00 × 10(-5) ), where a 3.2-Mb region was homozygous within cases. A frameshift mutation was identified in C2orf71, a gene located within this region. This variant was homozygous in 19 of 21 PRA cases and was at a frequency of approximately 0.37 in the Gordon Setter population. Approximately 10% of cases in our study (2 of 21) are not associated with this C2orf71 mutation, indicating that PRA in this breed is genetically heterogeneous and caused by at least two mutations. This variant is also present in a number of Irish Setter dogs with PRA and has an estimated allele frequency of 0.26 in the breed. The function of C2orf71 remains unknown, but it is important for retinal development and function and has previously been associated with autosomal recessive retinitis pigmentosa in humans.

The authors acknowledge that variant-77_-76ins30, identified independently in DNA sequences in this study (1), was also published in a 2001 patent (2) and posted in Genbank under Accession AX082874. In addition, the authors note that a related study was not discussed or cited in the published PLOS ONE article. In an article titled "Allelic variation in the canine Cox-2 promoter causes hypermethylation of the canine Cox-2 promoter in clinical cases of renal dysplasia" (3), the author used DNA from 13 dogs to query methylation within short DNA sequences in the 5'UTR region of the Cox-2 gene. The study uncovered methylated sequence variants upstream to the Cox-2 gene, in a few clinical cases of canine renal dysplasia. However, unmethylated DNA was found regardless of sequence variations. The biological significance of these short DNA sequences was previously discussed in an article for which post-publication concerns were raised regarding the reliability of the findings (4, 5). Compared to these previous studies (3-5), we investigated the same region on CanFam3.1 CFA7: 19,664,240-19,664,662, and observed 11 DNA sequence variations in addition to the variants tested previously for methylation status (1). The marked sequence diversity totaled 17 different haplotypes in 68 dogs, and no statistically significant differences were noted between the different phenotypes. Our interpretation is that there is no relation between different sequence variations in the 5'UTR of canine Cox-2 and canine renal dysplasia. The random methylation pattern in (3) provides yet another piece of evidence to support a benign role for these DNA variants. In response to questions raised after publication, the authors would also like to clarify the method used in aligning sequences in our study. Different sequence alignments can be easily explained when the biological function of the sequence is considered. The regional DNA sequence is GC-rich typical of 5'UTR region, with multiple repeats and indel sequence polymorphisms. These qualities present computational challenges making an algorithm-based alignment less reliable (6). Therefore, we aligned the sequences based on the 'ATG' start codon. This method allows the alignment to be anchored by a known highly conserved sequence. The verification for 'correct' alignment is seen to the right of the ATG where many transcripts including Cox-2, are highly conserved across species.

The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272–422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.