Whole-Body Barometric Plethysmography Characterizes Upper Airway Obstruction in 3 Brachycephalic Breeds of Dogs.

BACKGROUND: A novel test using whole-body barometric plethysmography (WBBP) was developed recently to diagnose brachycephalic obstructive airway syndrome (BOAS) in unsedated French bulldogs. HYPOTHESIS/OBJECTIVES: The hypotheses of this study were: (1) respiratory characteristics are different between healthy nonbrachycephalic dogs and brachycephalic dogs; and among pugs, French bulldogs, and bulldogs; and (2) obesity and stenotic nares are risk factors for BOAS. The main objective was to establish a diagnostic test for BOAS in these 3 breeds. ANIMALS: A total of 266 brachycephalic dogs (100 pugs, 100 French bulldogs, and 66 bulldogs) and 28 nonbrachycephalic dogs. METHODS: Prospective study. Exercise tolerance tests with respiratory functional grading, and WBBP were performed on all dogs. Data from WBBP were associated with functional grades to train quadratic discriminant analysis tools to assign dogs to BOAS+ and BOAS- groups. A BOAS index (0-100%) was calculated for each dog. Receiver operating characteristic (ROC) curves were used to evaluate classification ability. RESULTS: Minute volume was decreased significantly in asymptomatic pugs (P = .009), French bulldogs (P = .026), and bulldogs (P < .0001) when compared to nonbrachycephalic controls. Respiratory characteristics were different among breeds and affected dogs had a significant increase in trace variation. The BOAS index predicted BOAS status for each breed with 94-97% (95% confidence interval [CI], 88.9-100%) accuracy (area under the ROC curve). Both obesity (P = .04) and stenotic nares (P = .004) were significantly associated with BOAS. CONCLUSIONS AND CLINICAL IMPORTANCE: The WBBP can be used as a clinical tool to diagnose BOAS noninvasively and objectively.

Association between anal sac gland carcinoma and dog leukocyte antigen-DQB1 in the English Cocker Spaniel.

Anal sac gland carcinomas occur frequently in English Cocker Spaniels and, to a lesser extent, in other spaniel breeds. The disease typically presents in dogs aged 8 years or older and frequently metastasises to the local lymph nodes. The association between anal sac gland carcinoma in English Cocker Spaniels and the major histocompatibility complex class II loci (the dog leukocyte antigen loci DLA-DRB1, -DQA1, -DQB1) was investigated in 42 cases and 75 controls. Based on a corrected error rate of 0.017 for each test, the allele distribution in DLA-DRB1 showed no significant difference between cases and controls (P value = 0.019), while a significant difference was obtained for DLA-DQA1 and -DQB1 alleles (P values are 0.010 and 3.3 × 10⁻5). The DLA-DQB1*00701 allele was the most common in both cases and controls, but it had a higher frequency among the former (0.89) than in the latter (0.61), while the second most common allele had a higher frequency in the controls (0.23) than in the cases (0.07). Haplotype distributions were also significantly different between the two groups (P value = 1.61 × 10⁻4). This is the second disease in English Cocker Spaniels for which the most common DLA-DQB1 allele in the breed has been shown to have a higher frequency in cases than controls, while the second most common allele in the breed (*02001) has a significantly higher frequency in the controls, compared with the cases.

Understanding the genetics of autoimmune disease: two loci that regulate late onset Addison's disease in Portuguese Water Dogs.

Addison's disease, an immune-mediated disorder caused by destruction of the adrenal glands, is a rare disorder of Western European populations. Studies indicate that the disorder is polygenic in nature, involving specific alleles of the CTLA-4, DRB1*04 and DQ, Cyp27B1, VDR and MIC-A and -B loci. A similar immune form of Addison's disease occurs in several breeds of domestic dog, with frequencies ranging from 1.5 to 9.0%. The high frequency of the disease in domestic dog breeds likely reflects the small number of founders associated with many breeds, subsequent inbreeding, and the frequent use of popular sires. The Portuguese Water Dog (PWD) is a significantly affected breed. An analysis of 11,384 PWDs surveyed between 1985 and 1996 suggests a breed-specific disease incidence of 1.5%. As with humans, the disease is typically of late onset. This study involves a genetic comparison of Addison's disease in the PWD to the analogous disease in humans. The study is facilitated by the existence of complete pedigrees and a relatively high degree of inbreeding among PWDs. The breed originated from 31 founders, with 10 animals responsible for 90% of the current gene pool. We describe, specifically, the identification of two disease-associated loci, on Canis familiaris (CFA) chromosomes CFA12 and 37, which are syntenic with the human DRB1 histocompatibility locus alleles HLA-DRB1*04 and DRB1*0301, and to a locus for immunosuppression syntenic with CTLA-4. Strong similarities exist therefore in the complex genetic background of Addison's disease in humans and in the PWD. With the completion of the canine and human genome sequence, the purebred dog is set to become an important comparative model for Addison's as well as other human immune disorders.

Chromosome rearrangements in canine fibrosarcomas.

We have previously reported the use of six- and seven-color paint sets in the analysis of canine soft tissue sarcomas. Here we combine this technique with flow sorting of translocation chromosomes, reverse painting, and polymerase chain reaction (PCR) analysis of the gene content of the reverse paint in order to provide a more detailed analysis of cytogenetic abnormalities in canine tumors. We examine two fibrosarcomas, both from female Labrador retrievers, and show abnormalities in chromosomes 11 and 30 in both cases. Evidence of involvement of TGFBR1 is presented for one tumor.

Evaluation of candidate genes in the absence of positional information: a poor bet on a blind dog!

More than 350 inherited diseases have been reported in dogs and at least 50% of them have human counterparts. To remove the diseases from dog breeds and to identify canine models for human diseases, it is necessary to find the mutations underlying them. To this end, two methods have been used: the functional candidate gene approach and linkage analysis. Here we present an evaluation of these in canine retinal diseases, which have been the subject of a large number of molecular genetic studies, and we show the contrasting outcomes of these approaches when dealing with genetically heterogeneous diseases. The candidate gene approach has led to 377 published results with 23 genes. Most of the results (66.6%) excluded the presence of a mutation in a gene or its coding region, while only 3.4% of the results identified the mutation causing the disease. On the other hand, five linkage analysis studies have been done on retinal diseases, resulting in three identified mutations and two mapped disease loci. Mapping studies have relied on dog research colonies. If this favorable application of linkage analysis can be extended to dogs in the pet population, success in identifying canine mutations could increase, with advantages to veterinary and human medicine.

Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina.

Recombinant adeno-associated virus (rAAV) vectors are attractive candidates for the treatment of inherited and acquired retinal disease. Although rAAV vectors are well characterized in rodent models, a prerequisite to their clinical application in human patients is the thorough evaluation of their efficacy and safety in intermediate animal models. In this study, we describe rAAV-2-mediated expression of GFP reporter gene in retinal cells following local vector delivery in dogs. Subretinal delivery of rAAV.CMV.GFP was performed unilaterally in eight normal dogs from 6 weeks of age. The area of retinal transduction was maximized by the optimization of surgical techniques for subretinal vector delivery by pars-plana vitrectomy and the use of fine-gauge subretinal cannulae to create multiple retinotomies. rAAV-2 vectors mediated efficient stable reporter gene expression in photoreceptors and retinal pigment epithelial cells. We found efficient transduction of cone photoreceptors in addition to rods in both the canine retina and after subretinal vector delivery in another intermediate animal model, the feline retina. GFP expression in dogs was confined to the area of the retinal bleb and was sustained in cells at this site for at least 18 months. Electroretinography demonstrated a modest reduction in global rod-mediated retinal function following subretinal delivery of rAAV.CMV.GFP. Three of the eight animals developed delayed-onset intraocular inflammation, in two cases associated with a serum antibody response to GFP protein. We conclude that rAAV-2 vectors mediate efficient sustained transgene expression in rod and cone photoreceptors following subretinal delivery in this intermediate animal model. The possibility of adverse effects including intraocular immune responses and reduced retinal function requires further investigation prior to clinical applications in patients.

Autosomal dominant retinal dystrophy (Rdy) in Abyssinian cats: exclusion of PDE6G and ROM1 and likely exclusion of Rhodopsin as candidate genes.

Retinal dystrophy (Rdy) is an autosomal dominant photoreceptor dysplasia of Abyssinian cats and a model for autosomal dominant retinitis pigmentosa (ADRP) in man. We have pursued a candidate gene approach in the search for the causal mutation in Rdy. The genes RHO (encoding rhodopsin), ROM1 (encoding the structural retinal outer-membrane protein-1) and PDE6G (encoding the gamma subunit of the visual transduction protein cyclic guanosine monophosphate-phosphodiesterase) were polymerase chain reaction-amplified from normal feline genomic DNA. Leader, coding and 3' untranslated regions of each gene, and parts of introns were sequenced. Single-stranded conformation polymorphism (SSCP) analysis of Rdy-affected and normal cats was used to identify intragenic polymorphisms within ROM1 and PDE6G. DNA sequencing of all three genes in Rdy-affected cats was used to confirm results from SSCP. For both ROM1 and PDE6G polymorphisms identified by SSCP and sequencing showed disconcordance between the polymorphism and the disease phenotype within an Rdy disease pedigree. SSCP analysis of RHO performed across the 5' untranslated region, the entire coding sequence and the intron/exon boundaries in Rdy-affected and control cats failed to identify any intragenic polymorphisms that could be used for linkage analysis. DNA sequencing of these regions showed no differences between Rdy-affected and control cats. Mutations in ROM1 or in PDE6G are not causative of feline Rdy. The absence of potentially pathogenic polymorphisms in sequenced portions of the RHO gene makes it unlikely that a mutation in this gene is the cause of Rdy.

Canine inherited retinal degenerations: update on molecular genetic research and its clinical application.

Inherited retinal degenerations in the dog include generalised progressive retinal atrophy, retinal pigment epithelial dystrophy, congenital stationary night blindness and day blindness (hemeralopia). The clinical phenotype and pathology of these diseases closely resemble some types of human inherited retinal degeneration, in particular retinitis pigmentosa, one of the most common inherited causes of blindness in man. Molecular genetic investigations aim to identify the genetic mutations underlying the canine inherited retinal degenerations. Two major research strategies, candidate gene analysis and linkage analysis, have been used. To date, candidate gene analysis has definitively identified the genetic mutations underlying nine inherited retinal degenerations, each in a different breed of dog, and linkage studies have identified genetic markers for a further retinal degeneration which is found in at least six different breeds. This review outlines the research strategy behind candidate gene and linkage studies and summarises recent results in the search for genetic causes of canine inherited retinal degenerations. The aim is to increase awareness of this rapidly changing field and to show how the research can be used to develop genetic tests for these diseases and thereby reduce the incidence of inherited eye disease in dogs.

A cryptic deletion of 2q35 including part of the PAX3 gene detected by breakpoint mapping in a child with autism and a de novo 2;8 translocation.

We report a de novo, apparently balanced (2;8)(q35;q21.2) translocation in a boy with developmental delay and autism. Cross species (colour) paint (Rx) and SKY FISH, forward and reverse chromosome painting, and FISH with subtelomeric probes were used to examine the patient's karyotype, but further rearrangements were not detected. FISH with region specific clones mapping near 2q35 and 8q21.2 breakpoints and STS mapping performed on the isolated derivative chromosomes were used to refine the location of the breakpoints further. A cryptic deletion of between 4.23 and 4.41 Mb in extent and involving at least 13 complete genes or transcription units was found at the breakpoint on 2q35. The deletion includes the promoter and 5' untranslated region of the paired box 3 (PAX3) gene. The child has very mild dystopia canthorum which may be associated with the PAX3 haploinsufficiency. The 8q21.2 breakpoint is within MMP16 which encodes matrix metalloproteinase 16. We postulate that the cryptic deletion and rearrangement are responsible for the patient's phenotype and that a gene (or genes) responsible for autism lies at 2q35 or 8q21.2. The results present a step towards identifying genes predisposing to autism.

Generation and analysis of canine retinal ESTs: isolation and expression of retina-specific gene transcripts.

Canine generalized progressive retinal atrophies (gPRA) are a group of degenerative retinal diseases that are a major cause of hereditary blindness in a number of dog breeds. The expressed sequence tag (EST) approach was used to identify and characterize potential candidate genes from canine retinal cDNA libraries. Both conventional and subtractive canine retinal cDNA libraries were constructed and analyzed. Differential hybridization was performed to identify abundantly retinal expressed cDNA clones. Sequences of both random and abundantly expressed clones were analyzed using GCG software and searched against GenEMBL databases. For genes of interest isolated from the libraries, Northern blotting and RT-PCR were performed to determine mRNA expression of the genes. DNA sequences from 85 differentially expressed clones and 100 random cDNAs were obtained and analyzed. A higher percentage of abundantly retina-expressed clones showed homology to database sequences compared with random clones (72 versus 43%). Five retinal genes and 2 anonymous retinal ESTs were selected to analyze mRNA expression. The five known genes, namely HRG4/unc119, cGMP-PDEA, transducin 1A, opsin, and sFRP2 showed retina-specific expression. In anonymous ESTs, clone p81 revealed retina-specific expression, while p3 showed expression in each of 14 canine tissues. Transcripts of the canine secreted frizzled related protein 2 (sFRP2) gene showed surprisingly high abundance in the canine retina. The isolated retinal ESTs here will be useful resources for further investigation of canine retinal function and canine genome mapping.

Use of flow-sorted canine chromosomes in the assignment of canine linkage, radiation hybrid, and syntenic groups to chromosomes: refinement and verification of the comparative chromosome map for dog and human.

The mapping of the canine genome has recently been accelerated by the availability of chromosome-specific reagents and publication of radiation hybrid (RH), genetic linkage, and dog/human comparative maps, but the assignment of mapping groups to chromosomes is incomplete. To assign published radiation hybrid, linkage, and "syntenic" groups to chromosomes, individual markers found within each group have been amplified from canine and vulpine flow-sorted, chromosome-specific DNAs as templates. Here a further 102 type I genetic markers (previously mapped in human) and 21 further type II markers are assigned to canine chromosomes using marker-specific PCR. We have assigned all linkage, RH, and syntenic groups in the two most recently published canine genome maps to chromosomes. This demonstrates directly that there is at least one published mapping group for each of the 38 canine autosomes and thus that the coverage of the canine chromosome map is approaching completion. The dog/human comparative map is one of the most complex so far described, with 90 separate segments of chromosomal homology previously seen in dog-on-human cross-species chromosome-painting studies. The total of 142 type I markers now placed on canine chromosomes using this method of marker mapping has allowed us to confirm the placement of the great majority (83) of the 90 homologous segments. The positions of the remaining homologous segments were confirmed in new cross-species chromosome-painting experiments (dog-on-human, fox-on-human).

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human.

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats.

Chromosome identification and assignment of DNA clones in the dog using a red fox and dog comparative map.

We have developed a novel method for identifying dog chromosomes and unambiguously mapping specific clones onto canine chromosomes. This method uses a previously established red fox/dog comparative chromosome map to guide the FISH mapping of cloned canine DNA. Mixing metaphase preparations of the red fox and dog enabled a single hybridization to be performed on both species. We used this approach to map the chromosomal locations of twenty-six canine cosmids. Each cosmid contains highly polymorphic microsatellite markers currently used by the DogMap project to compile the canine linkage map. All but two cosmids were successfully assigned to subchromosomal regions on red fox and dog chromosomes. For eight cosmids previously mapped on dog chromosomes, we confirmed and refined the canine chromosomal assignments of seven cosmids and corrected an erroneous assignment regarding cosmid CanBern1. These results demonstrate that the red fox and dog comparative chromosome map can greatly improve the accuracy and efficiency of chromosomal assignments of canine genetic markers by FISH.

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps.

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.

Primary ciliary dyskinesia in Newfoundland dogs.

Primary ciliary dyskinesia was diagnosed in three Newfoundland dogs with histories of chronic rhinitis and bronchopneumonia from an early age. Thoracic radiographs of two of them showed severe, dependent bronchopneumonia and right displacement of the cardiac apex but normal positioning of other organs. Histopathological examination of sections of lung from the other dog showed severe bronchopneumonia. A semen sample from one dog had a high percentage of spermatozoa with abnormal tails and poor progressive motility. Transmission electron microscopy of nasal brushings from all three dogs showed consistent ultrastructural defects in the cilia, including an absence of outer and inner dynein arms, disorganisation of peripheral doublets, occasional supernumerary doublets and singlets, and consistently disorganised basal bodies and foot processes; sections of trachea from one dog also had disorganised basal bodies. Pedigree analysis was consistent with a monogenic autosomal recessive pattern of inheritance for the defect. One dog is still alive, one dog died aged five years two months, and one dog was euthanased aged nine months. This is the first time primary ciliary dyskinesia has been reported in Newfoundland dogs.

cGMP phosphodiesterase-alpha mutation causes progressive retinal atrophy in the Cardigan Welsh corgi dog.

PURPOSE: To screen the alpha-subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6A) as a potential candidate gene for progressive retinal atrophy (PRA) in the Cardigan Welsh corgi dog. METHODS: Single-strand conformation polymorphism (SSCP) analysis was used to screen short introns of the canine PDE6A gene for informative polymorphisms in members of an extended pedigree of PRA-affected Cardigan Welsh corgis. After initial demonstration of linkage of a polymorphism in the PDE6A gene with the disease locus, the complete coding region of the PDE6A gene of a PRA-affected Cardigan Welsh corgi was cloned in overlapping fragments and sequenced. SSCP-based and direct DNA sequencing tests were developed to detect the presence of a PDE6A gene mutation that segregated with disease status in the extended pedigree of PRA-affected Cardigan Welsh corgis. Genomic DNA sequencing was developed as a diagnostic test to establish the genotype of Cardigan Welsh corgis in the pet population. RESULTS: A polymorphism within intron 18 of the canine PDE6A gene was invariably present in the homozygous state in PRA-affected Cardigan Welsh corgis. The entire PDE6A gene was cloned from one PRA-affected dog and the gene structure and intron sizes established and compared with those of an unaffected animal. Intron sizes were identical in affected and normal dogs. Sequencing of exons and splice junctions in the affected animal revealed a 1-bp deletion in codon 616. Analysis of PRA-affected anti obligate carrier Cardigan Welsh corgis showed that this mutation cosegregated with disease status. CONCLUSIONS: A single base deletion at codon 616 in the PDE6A gene cosegregated with PRA status with zero discordance in Cardigan Welsh corgis with PRA. A lod score of 4.816 with a recombination fraction (theta) of zero strongly suggests that this mutation is responsible for PRA in the breed. The mutation is predicted to lead to a frame shift resulting in a string of 28 altered codons followed by a premature stop codon. The authors suggest that this type of PRA be given the name rod-cone dysplasia 3 (rcd3).

Genomic screening for fucosidosis in English Springer Spaniels.

OBJECTIVE: To develop a robust molecular genetic test for alpha-L-fucosidosis in English Springer Spaniels and to screen dogs from the United Kingdom and United States for the mutant allele. ANIMALS: 35 English-bred English Springer Spaniels, 60 American-bred English Springer Spaniels, and 1 affected dog and its parents from a family of English Springer Spaniels in Colorado. PROCEDURE: Polymerase chain reaction analysis was used to amplify the mutated region in the gene encoding alpha-L-fucosidase. High guanine-cytosine (GC) content of the region required use of an amplification buffer with high pH. Mutant and normal alleles were separated by polyacrylamide gel electrophoresis. Molecular genetic test results were compared with enzyme data. RESULTS: A 262-bp PCR product was amplified from normal dogs and compared with a 248-bp product from affected dogs. Carriers had 1 copy of each allele, distinguishable by the 14-bp size difference. Two carriers among the English-bred dogs were identified by use of enzyme and genomic DNA analyses. The molecular defect in dogs from Colorado was proven to be the same as that in British and Australian dogs. None of the other 60 American-bred dogs carried the mutant allele. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR method that can be used to identify dogs affected with or carriers of the autosomal recessive disease fucosidosis was established. Amplification was achieved within a GC-rich region, using a method that may be useful in overcoming amplification problems in GC-rich areas within other genes. Using this test, fucosidosis can be controlled and ultimately eradicated from the English Springer Spaniel population.

The integration of canine genetic maps with the canine karyotype using specific gene amplification of chromosome-specific DNA.

We have used a rapid approach to place markers that are already represented in current genetic maps onto individual chromosomes in species for which chromosome paints exist. PCR-based techniques are used to look for the presence of individual marker genes within each chromosome-specific DNA pool. The presence of a given marker within a DNA pool allows assignment of the complete radiation hybrid group, or linkage group from which the marker is drawn, to an individual chromosome. We have used this method with a new set of canine chromosome paints (Yang et al., 1999). In this way, we have assigned 39 of 44 published RH or syntenic RH groups to canine chromosomes, together with 33 of 40 canine linkage groups in a recently published map (Neff et al., 1999).