Deafness in Australian Cattle Dogs associated to QTL on chromosome 20 in genome-wide association study analyses.

Pigment-associated deafness is a common hereditary condition in a range of dog breeds. The aim of this study was to perform a genome-wide association analysis to investigate the genetic architecture of deafness in Australian Cattle Dogs. Genotypes for 104 757 polymorphisms in 216 dogs were available for analyses after quality control. A genomic relationship matrix was used in the mixed model analyses to account for polygenic effects, as we tested each polymorphism for its association with deafness, in a case/control experimental design. Three approaches were used to code the genotypes and test for additive, recessive and dominant SNP effects. The genome-wide association study analyses identified a clear association peak on CFA20, with the most significant SNPs on this chromosome (1.29 × 10-4 ) in the vicinity of MITF. Variants in MITF have been associated with white pigmentation in dogs and with deafness in humans and other species, supporting the premise that canine deafness is associated with variants in or near this gene. A recessive inheritance for the peak in CFA20 is possible given the significant results in the recessive model; however, the estimated heritability was low (4.54 × 10-5 ). Further validation, identification of variants and testing in other dog breeds are needed.

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 (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.

Estimated frequency of the canine hyperuricosuria mutation in different dog breeds.

BACKGROUND: Hyperuricosuria is a condition that predisposes dogs to urate urolithiasis. A mutation that causes canine hyperuricosuria was previously identified in 3 unrelated dog breeds. The occurrence of the mutation in additional breeds was not determined. HYPOTHESIS/OBJECTIVES: Identify additional breeds that have the hyperuricosuria mutation and estimate the mutant allele frequency in those breeds. ANIMALS: Three thousand five hundred and thirty dogs from 127 different breeds were screened for the hyperuricosuria mutation. METHODS: DNA samples were genotyped by pyrosequencing and allele-specific polymerase chain reaction methods. RESULTS: Mutant allele frequencies that range from 0.001 to 0.15 were identified in the American Staffordshire Terrier, Australian Shepherd, German Shepherd Dog, Giant Schnauzer, Parson (Jack) Russell Terrier, Labrador Retriever, Large Munsterlander, Pomeranian, South African Boerboel, and Weimaraner breeds. CONCLUSIONS AND CLINICAL IMPORTANCE: The hyperuricosuria mutation has been identified in several unrelated dog breeds. The mutant allele frequencies vary among breeds and can be used to determine an appropriate breeding plan for each breed. A DNA test is available and may be used by breeders to decrease the mutant allele frequency in breeds that carry the mutation. In addition, veterinarians may use the test as a diagnostic tool to identify the cause of urate urolithiasis.

Changes in mutation frequency of eight Mendelian inherited disorders in eight pedigree dog populations following introduction of a commercial DNA test.

INTRODUCTION: DNA testing for autosomal recessive disease mutations in many dog breeds is now relatively commonplace. There have, however, been few efforts made to determine changes in the frequency of disease causing mutations as a result of probable selection based on the results of DNA testing. This study makes use of genotype data from both DNA test results reported to the UK Kennel Club and where known from a 'hereditary status' (where a definitive genotype may be inferred and ascribed based on known parental genotypes) to do so. RESULTS: The results, using all known genotype data, show a general and sizeable decline in disease causing mutation frequency across eight diseases in eight breeds (by between 12-86% in dogs born 2-4 years after publication of the mutation, and by nearly 90% or more in those born 8-10 years after). In contrast, data from test results only, while revealing an almost complete and immediate end to the production of affected individuals, show little general decline in either the derived mutation frequency or the proportion of heterozygote carriers. It appears that the numerical size of the breed is an important determinant on the rate of uptake of a DNA test (as judged by the proportion of a breed born four years after publication of the disease-causing mutation with a known genotype). CONCLUSION: These results show that dog breeders appear to be incorporating the results of DNA testing into their selection strategies to successfully decrease the frequency of the mutation. It is shown that use of DNA test result data alone does not reveal such trends, possibly as some breeders undertake testing to determine clear stock which can then be used to produce future disease-free generations in the knowledge they are not carrying the disease causing mutation.

Gonioscopy in the dog: inter-examiner variability and the search for a grading scheme.

OBJECTIVES: To investigate inter-examiner variability in gonioscopic evaluation of pectinate ligament abnormality in dogs and to assess level of inter-examiner agreement for four different gonioscopy grading schemes. MATERIALS AND METHODS: Two examiners performed gonioscopy in 98 eyes of 49 Welsh springer spaniel dogs and estimated the percentage circumference of iridocorneal angle affected by pectinate ligament abnormality to the nearest 5%. Percentage scores assigned to each eye by the two examiners were compared. Inter-examiner agreement was assessed following assignment of the percentage scores to each of four grading schemes by Cohen's kappa statistic. RESULTS: There was a strong positive correlation between the results of the two examiners (R=0·91). In general, Examiner 1 scored individual eyes higher than Examiner 2, especially for eyes in which both examiners diagnosed pectinate ligament abnormality. A "good" level of agreement could only be achieved with a gonioscopy grading scheme of no more than three categories and with a relatively large intermediate bandwidth (κ=0·68). CLINICAL SIGNIFICANCE: A three-tiered grading scheme might represent an improvement on hereditary eye disease schemes which simply classify dogs to be either "affected" or "unaffected" for pectinate ligament abnormality. However, the large intermediate bandwidth of this scheme would only allow for the additional detection of those dogs with marked progression of pectinate ligament abnormality which would be considered most at risk of primary closed-angle glaucoma.

Prevalence and progression of pectinate ligament dysplasia in the Welsh springer spaniel.

OBJECTIVES: To determine the prevalence of pectinate ligament dysplasia in a large group of Welsh springer spaniels; to investigate associations between pectinate ligament dysplasia and age, sex and intraocular pressure and between intraocular pressure and age and sex; and to investigate progression of pectinate ligament dysplasia in individual dogs. METHODS: In a prospective study, gonioscopy was performed in both eyes of 227 Welsh springer spaniels and intraocular pressure measured by rebound tonometry. Eyes were classified as "unaffected" if 0% of the iridocorneal angle was affected with pectinate ligament dysplasia (grade 0), "mildly affected" if <20% was affected (grade 1), "moderately affected" if 20 to 90% was affected (grade 2) and "severely affected" if >90% was affected (grade 3). In a retrospective study, progression of pectinate ligament dysplasia over time was investigated for 65 dogs. RESULTS: One hundred and thirty-nine of 227 dogs (61·2%) were affected by pectinate ligament dysplasia (grades 1 to 3) and 82/227 (36·2%) were moderately or severely affected. There was a significant association between pectinate ligament dysplasia and age. There were no associations between pectinate ligament dysplasia and intraocular pressure or pectinate ligament dysplasia and sex. Thirty-five of 65 dogs (53·8%) demonstrated progression of pectinate ligament dysplasia. CLINICAL SIGNIFICANCE: Prevalence of pectinate ligament dysplasia was high despite widespread screening and selection against the condition. Our data indicate that gonioscopic features of pectinate ligament dysplasia can progress in the Welsh springer spaniel. Dogs deemed unaffected at an early age may subsequently be diagnosed with pectinate ligament dysplasia.

A second-generation genetic linkage map of the domestic dog, Canis familiaris.

Purebred strains, pronounced phenotypic variation, and a high incidence of heritable disease make the domestic dog uniquely suited to complement genetic analyses in humans and mice. A comprehensive genetic linkage map would afford many opportunities in dogs, ranging from the positional cloning of disease genes to the dissection of quantitative differences in size, shape, and behavior. Here we report a canine linkage map with the number of mapped loci expanded to 276 and 10-cM coverage extended to 75-90% of the genome. Most of the 38 canine autosomes are likely represented in the collection of 39 autosomal linkage groups. Eight markers were sufficiently informative to detect linkage at distances of 10-13 cM, yet remained unlinked to any other marker. Taken together, the results suggested a genome size of about 27 M. As in other species, the genetic length varied between sexes, with the female autosomal distance being approximately 1.4-fold greater than that of male meioses. Fifteen markers anchored well-described genes on the map, thereby serving as landmarks for comparative mapping in dogs. We discuss the utility of the current map and outline steps necessary for future map improvement.

Molecular characterization and mapping of canine cGMP-phosphodiesterase delta subunit (PDE6D).

cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.

Characterization of a minimal screening set of 172 microsatellite markers for genome-wide screens of the canine genome.

We have characterized a subset of 172 microsatellite markers from the canine map, termed 'Minimal Screening Set 1' (Canine MSS-1), which we propose be used for initial genome-wide genetic linkage studies. Three hierarchical criteria were used to select markers from the current meiotic linkage and radiation hybrid maps for MSS-1. Markers were selected that (1) provided as complete coverage as possible of the canine genome, (2) were highly informative, and (3) have been ordered in linkage groups with a high degree of statistical support. This resulting screening set spans all reported meiotic linkage and RH groups, leaving only 10 known gaps > or = 20 cM. The average polymorphic information content (PIC) value of markers tested is 0.74. Coverage estimates suggest 42% of the genome is within 5 cM of at least one marker in the minimal screening set, 77% of the genome is within 10 cM. This minimal mapping set therefore provides an efficient and cost effective way to begin screening pedigrees of interest for genetic linkage.

A new method of paternity testing for dogs, based on microsatellite sequences.

Microsatellite sequences, like minisatellites, belong to a class of polymorphic DNA that is commonly found in mammalian DNA. Although they vary significantly less in a population of animals than minisatellites, they have potential for use in paternity disputes. However, their inherently lower variability together with the more genetically homogeneous nature of pedigree dogs due to inbreeding (line breeding), raised doubts about their effectiveness for paternity tests. This paper demonstrates that canine microsatellites provide an adequate basis for assigning paternity in pedigree breeds. The system presented is more straightforward to perform and interpret than that based on canine minisatellites (DNA 'fingerprinting') and requires as little as 0.1 ml of blood.

Primary lens instability in ten related cats: clinical and genetic considerations.

OBJECTIVES: To describe bilateral lens instability in 10 related domestic shorthair cats over three generations. METHODS: Complete ophthalmic examinations were performed. Lentectomies were carried out. Sections of affected lenses focused on the equatorial area were examined by transmission electron microscopy. The potential involvement of several candidate genes (ADAMTS17, ADAMTSL4, ADAMTS10 and FBN1) known to be associated with lens luxation in other species was investigated. RESULTS: The group of animals included 10 related cats, nine of them being affected by lens instability over three generations. Transmission electron microscopy showed the presence of zonular material at the lens equator. Signs of lens instability were not associated with other ocular disease. Analysis of the pedigree suggests a dominantly inherited condition. A mutation in ADAMTS17 was excluded, but a possible association between the condition and a microsatellite flanking FBN1 indicates this gene should be considered a strong candidate responsible for primary lens luxation in this pedigree. CLINICAL SIGNIFICANCE: These observations suggest an inherent zonular defect unrelated to extraneous factors. The family relationship is compatible with a possible genetic basis, and the pedigree suggests that the condition could be dominant. Data also suggest the mutation in the FBN1 gene could be responsible for primary lens luxation in this pedigree of cats.

Canine limbal melanoma: 30 cases (1992-2004). Part 1. Signalment, clinical and histological features and pedigree analysis.

OBJECTIVES: (1) To review the signalment, clinical, and histological features of canine limbal melanoma; (2) to perform pedigree analysis on breeds predisposed to limbal melanoma to establish if common ancestry exists; and (3) to investigate if any ancestral relationship exists between canine limbal melanoma and canine anterior uveal melanoma (CAUM). DESIGN: Retrospective study. ANIMALS STUDIED: Thirty dogs with limbal melanoma. METHODS: Medical records of patients were reviewed. Follow-up information was obtained by re-examination of patients or telecommunications with the referring veterinary surgeons or the owners. Pedigrees were analyzed for common ancestry amongst affected dogs. RESULTS The mean age (+/- SD) at diagnosis was 6.2 (+/- 2.75) years with a range from 1 to 11 years. There was a bimodal distribution of ages with a peak at 3-4 years and a peak at 7-10 years. There was no eye predilection or predisposition for sex or coat color. Twenty-five (83%) of the limbal melanomas occurred within a dorsal arc from the dorsomedial to the ventrolateral limbus. Golden retrievers were four times more common in the melanoma group compared to the Animal Health Trust population (P < 0.0001). Labrador retrievers were three times more common in the melanoma group (P=0.01). Pedigree analysis on eight Golden retrievers [limbal melanoma (n=5), CAUM (n=2) and diffuse ocular melanosis (n=1)], revealed a pattern of inter-relatedness consistent with the condition(s) being caused, at least in part, by a genetic mutation(s). A similar level of inter relatedness was evident in six Labrador retrievers (limbal melanoma (n=2) and CAUM (n=4)). In 5/22 cases (23%), histological features suggestive of malignancy were present including intratumor necrosis in 4/22 cases (18%) and cellular atypia in 1/22 cases (5%). CONCLUSIONS: In Golden and Labrador retrievers there is evidence that limbal melanomas, CAUM and ocular melanosis are at least in part heritable and that the same genetic mutation(s) may be causally associated with melanocytic disease at different ocular sites. The same genetic mutation(s) may be present in these two breeds. Histology should be performed on all cases to identify those with greater malignant potential.

Canine RPGRIP1 mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human Leber congenital amaurosis.

Cone-rod dystrophy 1 (cord1) is a recessive condition that occurs naturally in miniature longhaired dachshunds (MLHDs). We mapped the cord1 locus to a region of canine chromosome CFA15 that is syntenic with a region of human chromosome 14 (HSA14q11.2) containing the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) gene. Mutations in RPGRIP1 have been shown to cause Leber congenital amaurosis, a group of retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. Using the newly available canine genome sequence we sequenced RPGRIP1 in affected and carrier MLHDs and identified a 44-nucleotide insertion in exon 2 that alters the reading frame and introduces a premature stop codon. All affected and carrier dogs within an extended inbred pedigree were homozygous and heterozygous, respectively, for the mutation. We conclude the mutation is responsible for cord1 and demonstrate that this canine disease is a valuable model for exploring disease mechanisms and potential therapies for human Leber congenital amaurosis.

Variability of canine microsatellites within and between different dog breeds.

Polymorphic animal microsatellites have proved valuable genetic markers. For this project, the variability of 19 canine microsatellite loci was examined within and between three pure breeds of dog: Greyhounds, Labradors, and German Shepherds. The number of alleles, absolute and relative frequencies, and the statistics that express polymorphism within a breed were determined. The evolutionary relationships among these closely related dog breeds were estimated by genetic distance measures developed for use with microsatellite loci. According to the pairwise genetic distances, Greyhounds and German Shepherds had longer diverse evolutionary histories than Greyhounds and Labradors or Labradors and German Shepherds. Although a few breed-specific alleles were observed, the significant differences between breeds are in their relative frequencies and distribution of the alleles across a locus. None of the three pure dog breeds corresponds to Hardy-Weinberg equilibrium. A considerable reduction in intrapopulation variation was observed within three pure breeds, compared with the population of individuals belonging to 15 dog breeds. This reduction was especially pronounced in the Greyhound breed, which expressed the lowest degree of variation. Intrapopulation variations of Labradors and German Shepherds did not differ significantly, that of Labradors being only slightly higher. The intra-species variation of dogs is lower than in humans, mouse, or rat, but similar to that in domestic animals, probably reflecting similarly high inbreeding coefficients. However, some highly informative loci were common to all dog breeds tested so far. Such population data are necessary for mapping studies and linkage analysis in dogs.

Establishing appropriate genome-wide significance levels for canine linkage analyses.

A threshold of 3.3 for a genome-wide maximum LOD score (MAXLOD) has been demonstrated in human linkage studies as corresponding to a type I error rate of 5%. Generalization of this work to other species assumes the presence of an infinitely dense marker map. While this assumption is increasingly realistic for the human genome, it may be unrealistic for the dog genome. In this study we establish the analytic and empirical thresholds for MAXLOD in canine linkage studies corresponding to type I error rates of 5% and 1% for autosomal traits. Empirical thresholds are computed via simulation assuming a 10 cM map with no fine mapping performed. Pedigree structures for simulations were drawn from two canine disease studies. Five thousand replicates of genome-wide null genotype data were simulated and analyzed for each disease. We determined that MAXLOD thresholds of 3.2 and 2.7 correspond to analytic and empirical type I error rates of 5%, respectively. In all cases, the MAXLOD thresholds from simulations were always at least 0.5 LOD units below the corresponding analytic thresholds. We therefore recommend that a threshold of 3.2 be used for canine linkage studies when fine mapping is performed, and that researchers perform their own simulation studies to assess genome-wide empirical significance levels when no fine mapping is performed.

Hereditary canine spinal muscular atrophy is phenotypically similar but molecularly distinct from human spinal muscular atrophy.

Hereditary canine spinal muscular atrophy (HCSMA) is an autosomal dominant motor neuron disease that is similar in pathology and clinical presentation to various forms of human motor neuron disease. We have tested the hypothesis that the canine survival motor neuron (SMN) gene is responsible for HCSMA by genetic and molecular analysis of a colony of mixed breed dogs, all descended from a single affected individual. We cloned the canine SMN gene and determined the DNA sequence in an affected and an unaffected dog. We found no germline mutations in the SMN gene of the affected individual. Using conventional linkage analysis with canine-specific microsatellite repeat markers we screened the canine genome and identified a single linkage group likely to contain the HCSMA gene. Analysis with a panel of canine/rodent hybrid cell lines revealed that the SMN gene did not map to the same chromosome as the HCSMA linkage group. Collectively these results suggest that the molecular basis for HCSMA is distinct from that of phenotypically similar human disorders caused by inherited mutations in the SMN gene. This further suggests that additional studies on the molecular nature of HCSMA may reveal an unknown element of the molecular pathway leading to motor neuron disease.

Isolation and characterization of microsatellites from the canine genome.

Microsatellite sequences comprising (dC-dA)n.(dG-dT)n repeats have been isolated from canine libraries and sequenced. Oligonucleotide primers have been synthesized to the microsatellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The degree of polymorphism of each microsatellite was estimated in a set of unrelated dogs. It is concluded that of the 10 loci studied, nine are sufficiently polymorphic to be useful in genetic studies.