Late‐onset progressive retinal atrophy in the Gordon and Irish Setter breeds is associated with a frameshift mutation in C2orf71

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 (P_raw = 2.22 × 10^?8, P_genome = 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.     

Canine models of ocular disease: outcross breedings define a dominant disorder present in the English mastiff and bull mastiff dog breeds

Progressive retinal atrophies (PRA) are a heterogeneous group of inherited eye diseases common to both dogs and man. Over 100 individual canine breeds display some sort of retinal degeneration, making the dog an extremely valuable resource both for finding the genetic determinants of inherited blindness and for developing naturally occurring animal models that mimic human disease. Progressive retinal atrophies within the English mastiff displayed an ambiguous mode of inheritance. By conducting outcross matings between affected English mastiffs and normal animals from other breeds, the mode of inheritance was confirmed as dominant. This directed candidate gene analysis and led to identification of two synonymous mutations and one nonsynonymous mutation within the canine rhodopsin gene. The nonsynonymous mutation (T4R) is the cause of PRA in the English mastiff, and a test was developed to investigate its presence in 17 additional breeds. Testing of PRA-affected animals from 16 breeds revealed that none carry the T4R mutation, indicating a different cause of PRA. Analysis of two affected bull mastiffs revealed one heterozygote (+/T4R) and one homozygous normal individual (+/+). These findings suggest that the genetic origin of PRA is often breed specific and underline the value of outcross mating to circumvent problems that act to mask the mode of inheritance.     

CapsID: a web-based tool for developing parsimonious sets of CAPS molecular markers for genotyping

Background

The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL.

Results

Two full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL.

Conclusion

For the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.     

Compound heterozygosity for TNXB genetic variants in a mixed‐breed dog with Ehlers‐Danlos syndrome

The Ehlers‐Danlos syndromes (EDSs) are a heterogeneous group of inherited connective tissue disorders characterized by skin hyperextensibility, joint hypermobility and tissue fragility. Inherited disorders similar to human EDS have been reported in different mammalian species. In the present study, we investigated a female mixed‐breed dog with clinical signs of EDS. Whole‐genome sequencing of the affected dog revealed two missense variants in the TNXB gene, encoding the extracellular matrix protein tenascin XB. In humans, TNXB genetic variants cause classical‐like EDS or the milder hypermobile EDS. The affected dog was heterozygous at both identified variants. Each variant allele was transmitted from one of the case's parents, consistent with compound heterozygosity. Although one of the variant alleles, XM_003431680.3:c.2012G>A, p.(Ser671Asn), was private to the family of the affected dog and absent from whole‐genome sequencing data of 599 control dogs, the second variant allele, XM_003431680.3:c.2900G>A, p.(Gly967Asp), is present at a low frequency in the Chihuahua and Poodle population. Given that TNXB is a functional candidate gene for EDS, we suggest that compound heterozygosity for the identified TNXB variants may have caused the EDS‐like phenotype in the affected dog. Chihuahuas and Poodles should be monitored for EDS cases, which might confirm the hypothesized pathogenic effect of the segregating TNXB variant.     

Prediction of tandem repeat polymorphisms in the coding region of dog genome

After the dog genome was sequenced, an increasing number of studies involving genetic research of dogs have been conducted to understand gene functions and mammalian evolution. To study the genetic diversity in dogs and other mammals, genetic markers linked to function and conserved in wide lineages are necessary. Thus far, few polymorphic markers have been used in dogs. In this study, we surveyed the entire dog genome and predicted a total of 109 tandem repeats (TRs) located on the protein coding region that may be polymorphic by our prediction model. We selected 10 TRs that may be related to neurophysiology and neural developments, and tested them in 167 individuals of 8 dog breeds: 5 European dog breeds (Beagle, Golden Retriever, Labrador Retriever, German Shepherd, and Toy Poodle) and 3 Japanese dog breeds (Japanese Spitz, Shiba, and Shikoku). Among the tested TRs, nine were polymorphic indicating that 90% of the TRs were successfully predicted to be polymorphic. PCR fragments of the TRs were amplified from dog brain cDNA, showing their expression in the dog brain. Our results provide abundant opportunities for the study of phenotypic variations in dogs, and our prediction method for variable number of tandem repeats (VNTRs) can be applied to any other animal genome sequences for the survey of functional and polymorphic markers.     

An Intronic SINE Insertion in FAM161A that Causes Exon-Skipping Is Associated with Progressive Retinal Atrophy in Tibetan Spaniels and Tibetan Terriers

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 19 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 22 Tibetan Spaniel PRA cases and 10 controls, we identified a novel PRA locus, PRA3, on CFA10 (p_raw = 2.01×10⁻⁵, p_genome = 0.014), where a 3.8 Mb region was homozygous within 12 cases. Using targeted next generation sequencing, a short interspersed nuclear element insertion was identified near a splice acceptor site in an intron of a provocative gene, FAM161A. Analysis of mRNA from an affected dog revealed that the SINE causes exon skipping, resulting in a frame shift, leading to a downstream premature termination codon and possibly a truncated protein product. This mutation segregates with the disease in 22 out of 35 cases tested (63%). Of the PRA controls, none are homozygous for the mutation, 15% carry the mutation and 85% are homozygous wildtype. This mutation was also identified in Tibetan Terriers, although our results indicate that PRA is genetically heterogeneous in both Tibetan Spaniels and Tibetan Terriers.     

MC1R studies in dogs with melanistic mask or brindle patterns

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.     

Investigation of the role of opsin gene polymorphism in generalized progressive retinal atrophies in dogs

The generalized progressive retinal atrophies (gPRAs) form a group of retinal degenerations of pedigree dogs and cats, which have a variety of genetic origins (mostly unknown). We have examined the opsin gene for polymorphisms in several breeds of pedigree dog suffering from distinct forms of gPRA, by methods including single-strand conformation polymorphism analysis, microsatellite analysis and direct sequencing. The breeds examined included the Tibetan terrier, the miniature schnauzer, the Irish setter, the miniature poodle, the Labrador retriever and the English cocker spaniel, as well as individuals from breeds in which PRA has not been described and of mixed breed. Individuals from each of the named breeds suffering from PRA were compared with clinically normal dogs. Two polymorphisms were found. One, segregating within the Tibetan terrier population, but not seen in other breeds, was a synonymous transition at nucleotide position 780 in exon 3. Inheritance of this polymorphism suggests that opsin is unlikely to contain mutations causative of gPRA in this breed. The other polymorphism occurred between all miniature schnauzers examined and dogs of other breeds. It consisted of a single base insertion in intron 2. No polymorphisms in the opsin sequence were detected in any other breed. DNA sequencing allowed rigorous exclusion of mutations in opsin as a cause of gPRA in miniature poodles, English cocker spaniels or Labrador retrievers.     

Evaluation of RDS/Peripherin and ROM1 as candidate genes in generalised progressive retinal atrophy and exclusion of digenic inheritance

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.     

A CNGB1 Frameshift Mutation in Papillon and Phalène Dogs with Progressive Retinal Atrophy

Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA) or degeneration resembles human retinitis pigmentosa (RP) and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD) of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (P_Fisher = 1.4×10⁻⁸) with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.     

The genetic contribution to canine personality

The domestic dog may be exceptionally well suited for behavioral genetic studies owing to its population history and the striking behavior differences among breeds. To explore to what extent and how behavioral traits are transmitted between generations, heritabilities and genetic correlations for behavioral traits were estimated in a cohort containing over 10,000 behaviorally tested German shepherd and Rottweiler dogs. In both breeds, the pattern of co-inheritance was found to be similar for the 16 examined behavioral traits. Furthermore, over 50% of the additive genetic variation of the behavioral traits could be explained by one underlying principal component, indicating a shared genetic component behind most of the examined behavioral traits. Only aggression appears to be inherited independently of the other traits. The results support a genetic basis for a broad personality trait previously named shyness-boldness dimension, and heritability was estimated to be 0.25 in the two breeds. Therefore, breeds of dogs appear to constitute a valuable resource for behavioral genetic research on the normal behavioral differences in broad personality traits.     

Identification of a mutation in the CHAT gene of Old Danish Pointing Dogs affected with congenital myasthenic syndrome

The presence of a recessive inherited muscle disease in Old Danish Pointing Dogs has been well known for years. Comparisons of this disease with myasthenic diseases of other dog breeds and humans have pointed toward a defect in the synthesis of the neurotransmitter acetylcholine possibly due to decreased activity of the enzyme choline acetyltransferase. We sequenced exons 5-18 of the gene encoding choline acetyltransferase (CHAT) in 2 affected and 2 unaffected dogs and identified a G to A missense mutation in exon 6. The mutation causes a valine to methionine substitution and segregates in agreement with the inheritance of the disease. The mutation was not detected in 50 dogs representing 25 other dog breeds. A DNA test has been developed and is now available to the breeders of Old Danish Pointing Dogs.     

A single base deletion in the SLC45A2 gene in a Bullmastiff with oculocutaneous albinism

Oculocutaneous albinism type 4 (OCA4) in humans and similar phenotypes in many animal species are caused by variants in the SLC45A2 gene, encoding a putative sugar transporter. In dog, two independent SLC45A2 variants are known that cause oculocutaneous albinism in Doberman Pinschers and several small dog breeds respectively. For the present study, we investigated a Bullmastiff with oculocutaneous albinism. The affected dog was highly inbred and resulted from the mating of a sire to its own grandmother. We obtained whole genome sequence data from the affected dog and searched specifically for variants in candidate genes known to cause albinism. We detected a single base deletion in exon 6 of the SLC45A2 gene (NM_001037947.1:c.1287delC) that has not been reported thus far. This deletion is predicted to result in an early premature stop codon. It was confirmed by Sanger sequencing and perfectly co‐segregated with the phenotype in the available family members. We genotyped 174 unrelated dogs from diverse breeds, all of which were homozygous wildtype. We therefore suggest that SLC45A2:c.1287delC causes the observed oculocutaneous albinism in the affected Bullmastiff.     

Progressive retinal atrophy in Shetland sheepdog is associated with a mutation in the CNGA1 gene

Progressive retinal atrophy (PRA) is the collective name of a class of hereditary retinal dystrophies in the dog and is often described as the equivalent of retinitis pigmentosa in humans. PRA is characterized by visual impairment due to degeneration of the photoreceptors in the retina, usually leading to blindness. PRA has been reported in dogs from more than 100 breeds and can be genetically heterogeneous both between and within breeds. The disease can be subdivided by age at onset and rate of progression. Using genome‐wide association with 15 Shetland Sheepdog (Sheltie) cases and 14 controls, we identified a novel PRA locus on CFA13 (P_raw = 8.55 × 10^?7, P_genome = 1.7 × 10^?4). CNGA1, which is known to be involved in human cases of retinitis pigmentosa, was located within the associated region and was considered a likely candidate gene. Sequencing of this gene identified a 4‐bp deletion in exon 9 (c.1752_1755delAACT), leading to a frameshift and a premature stop codon. The study indicated genetic heterogeneity as the mutation was present in all PRA‐affected individuals in one large family of Shelties, whereas some other cases in the studied Sheltie population were not associated with this CNGA1 mutation. To our knowledge, this is the first report of a mutation in CNGA1 causing PRA in dogs.     

A comprehensive biomedical variant catalogue based on whole genome sequences of 582 dogs and eight wolves

The domestic dog serves as an excellent model to investigate the genetic basis of disease. More than 400 heritable traits analogous to human diseases have been described in dogs. To further canine medical genetics research, we established the Dog Biomedical Variant Database Consortium (DBVDC) and present a comprehensive list of functionally annotated genome variants that were identified with whole genome sequencing of 582 dogs from 126 breeds and eight wolves. The genomes used in the study have a minimum coverage of 10× and an average coverage of ~24×. In total, we identified 23 133 692 single‐nucleotide variants (SNVs) and 10 048 038 short indels, including 93% undescribed variants. On average, each individual dog genome carried ～4.1 million single‐nucleotide and ~1.4 million short‐indel variants with respect to the reference genome assembly. About 2% of the variants were located in coding regions of annotated genes and loci. Variant effect classification showed 247 141 SNVs and 99 562 short indels having moderate or high impact on 11 267 protein‐coding genes. On average, each genome contained heterozygous loss‐of‐function variants in 30 potentially embryonic lethal genes and 97 genes associated with developmental disorders. More than 50 inherited disorders and traits have been unravelled using the DBVDC variant catalogue, enabling genetic testing for breeding and diagnostics. This resource of annotated variants and their corresponding genotype frequencies constitutes a highly useful tool for the identification of potential variants causative for rare inherited disorders in dogs.     

Unequal contribution of sexes in the origin of dog breeds

Dogs (Canis familiaris) were domesticated from the gray wolf (Canis lupus) at least 14,000 years ago, and there is evidence of dogs with phenotypes similar to those in modern breeds 4000 years ago. However, recent genetic analyses have suggested that modern dog breeds have a much more recent origin, probably <200 years ago. To study the origin of contemporaneous breeds we combined the analysis of paternally inherited Y chromosome markers with maternally inherited mitochondrial DNA and biparentally inherited autosomal microsatellite markers in both domestic dogs and their wild ancestor, the gray wolf. Our results show a sex bias in the origin of breeds, with fewer males than females contributing genetically, which clearly differs from the breeding patterns in wild gray wolf populations where both sexes have similar contributions. Furthermore, a comparison of mitochondrial DNA and Y chromosome diversity in dog groups recognized by the World Canine Organization, as well as in groups defined by the breeds' genetic composition, shows that paternal lineages are more differentiated among groups than maternal lineages. This demonstrates a lower exchange of males than of females between breeds belonging to different groups, which illustrates how breed founders may have been chosen.     

A second KRT71 allele in curly coated dogs

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant ¹⁵¹Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.     

Distribution of extrahepatic congenital portosystemic shunt morphology in predisposed dog breeds

ABSTRACT: BACKGROUND: An inherited basis for congenital extrahepatic portosystemic shunts (EHPSS) has been demonstrated in several small dog breeds. If in general both portocaval and porto-azygous shunts occur in breeds predisposed to portosystemic shunts then this could indicate a common genetic background. This study was performed to determine the distribution of extrahepatic portocaval and porto-azygous shunts in purebred dog populations. RESULTS: Data of 135 client owned dogs diagnosed with EHPSS at the Faculty of Veterinary Medicine of Utrecht University from 2001 - 2010 were retrospectively analyzed. The correlation between shunt localization, sex, age, dog size and breed were studied. The study group consisted of 54 males and 81 females from 24 breeds. Twenty-five percent of dogs had porto-azygous shunts and 75 % had portocaval shunts. Of the dogs with porto-azygous shunts only 27 % was male (P = 0.006). No significant sex difference was detected in dogs with a portocaval shunt. Both phenotypes were present in almost all breeds represented with more than six cases. Small dogs are mostly diagnosed with portocaval shunts (79 %) whereas both types are detected. The age at diagnosis in dogs with porto-azygous shunts was significantly higher than that of dogs with portocaval shunts (P < 0.001). CONCLUSION: The remarkable similarity of phenotypic variation in many dog breeds may indicate common underlying genes responsible for EHPSS across breeds. The subtype of EHPSS could be determined by a minor genetic component or modulating factors during embryonic development.