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.     

Occurrence and Population Size of <Emphasis Type="Italic">Malassezia</Emphasis> spp. in the External Ear Canal of Dogs and Cats Both Healthy and with Otitis

Malassezia yeasts are commensal organisms of human and animal skin that occasionally act as pathogens; lipid-dependent species are associated with human skin disorders. The aim of this work was to evaluate the occurrence, risk factors and population size of different species of Malassezia in the external ear canal of dogs and cats with and without otitis externa. Between 2001 and 2003, 107 healthy animals (25 cats and 82 dogs) and 123 animals with chronic otitis exteirna (48 cats and 75 dogs) were clinically examined. Sterile cotton swabs were used to collect specimens from the external ear canal and culture tests and cytological examinations were performed to detect the presence and population size of Malassezia yeasts. Malassezia yeasts were isolated from 72.9% and 40% of the cats and from 57.3% and 28.0% of the dogs, with and without otitis externa, respectively. Malassezia was frequently isolated from animals under 5 years of age. The highest prevalence of positive samples was reported in winter for cats and in autumn for dogs. Dogs with pendulous ears showed a higher incidence of infection than dogs with erect ears. More Malassezia yeasts were recovered from animals with otitis (i.e. 59.6 colony forming units – CFUs – for cats and 66.0 CFUs for dogs) than healthy animals. Out of the 413 isolates obtained from animals with and without otitis, 403 (97.6%) were identified as M. pachydermatis and 10 (2.4%) as M. globosa. A statistical evaluation of the occurrence of Malassezia yeasts in dogs and cats revealed that predisposing factors for Malassezia infections are sampling period for cats, and type of ear for dogs. The largest population of Malassezia yeasts was detected in animals with otitis, suggesting a role in the occurrence of lesions.     

Missense mutation G296S in GATA4 is not responsible for cardiac septal defects

BACKGROUND:

The most common type of congenital heart disease is the cardiac septal defects, which has reported to be caused by a missense mutation (G296S) in exon 3 of the GATA4 gene.

AIMS:

The present study was undertaken to find out whether GATA4 gene is the prime cause of the septal defects in Mysore population.

MATERIALS AND METHODS:

GATA4 gene analyses were undertaken on 21 confirmed CHD cases by PCR and DNA sequencing.

RESULTS AND CONCLUSION:

Analysis of this particular mutation in 21 septal defect patients revealed that none of the patients had the mutation, indicating that this mutation is population specific or septal defect in Mysore population is caused due to mutations in other regions of the GATA4 gene.     

Genetic Screening of the Canine Connexin 40 Gene in Dogs with Inherited Cardiac Conduction Defects

Connexin 40 (Cx40) is a gap-junction protein expressed in the heart where it mediates the coordinated electrical activation of the atria and ventricular conduction tissues, facilitates cell-to-cell adhesion, and provides pathways for direct intercellular communication. Recent studies have shown that Cx40 null mice have cardiac conduction abnormalities with a very high incidence of cardiac malformations in heterozygous (18%) and homozygous (33%) animals, indicating that Cx40 plays a vital role in cardiomorphogenesis. Since several inherited cardiac conduction defects have also been found in dogs, we hypothesized that the clinical findings are genetically linked to a tissue-specific mutation or mutations in the canine Cx40 gene. We therefore screened the Cx40 gene in dogs with inherited cardiac conduction defects for mutations. In this study, we have identified three heterozygous base changes (C384G, C402T, C837T) in the dogs screened and determined them to be synonymous mutations. These mutations, however, have recently been found in an unrelated group of normal dogs.     

<Emphasis Type="Italic">Tie-dyed1</Emphasis> and <Emphasis Type="Italic">Sucrose export defective1</Emphasis> act independently to promote carbohydrate export from maize leaves

tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) are recessive maize (Zea mays) mutants with nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars. In addition, both mutants display similar growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaves. As tdy1 and sxd1 are the only variegated leaf mutants known to accumulate carbohydrates in any plant, we investigated whether Tdy1 and Sxd1 function in the same pathway. Using aniline blue staining for callose and transmission electron microscopy to inspect plasmodesmatal ultrastructure, we determined that tdy1 does not have any physical blockage or alteration along the symplastic transport pathway as found in sxd1 mutants. To test whether the two genes function in the same genetic pathway, we constructed F₂ families segregating both mutations. Double mutant plants showed an additive interaction for growth related phenotypes and soluble sugar accumulation, and expressed the leaf variegation pattern of both single mutants indicating that Tdy1 and Sxd1 act in separate genetic pathways. Although sxd1 mutants lack tocopherols, we determined that tdy1 mutants have wild-type tocopherol levels, indicating that Tdy1 does not function in the same biochemical pathway as Sxd1. From these and other data we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Our genetic and cytological studies implicate Tdy1 functioning in veins, and a model discussing possible functions of TDY1 is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High prevalence of <Emphasis Type="Italic">Yersinia pestis</Emphasis> in black-tailed prairie dog colonies during an apparent enzootic phase of sylvatic plague

Sylvatic plague (Yersinia pestis) was introduced into North America over 100 years ago. The disease causes high mortality and extirpations in black-tailed prairie dogs (Cynomys ludovicianus), which is of conservation concern because prairie dogs provide habitat for the critically endangered black-footed ferret (Mustela nigripes). Our goal was to help elucidate the mechanism Y. pestis uses to persist in prairie ecosystems during enzootic and epizootic phases. We used a nested PCR protocol to assay for plague genomes in fleas collected from prairie dog burrows potentially exposed to plague in 1999 and 2000. No active plague epizootic was apparent in the 55 prairie dog colonies sampled in 2002 and 2003. However, 63% of the colonies contained plague-positive burrows in 2002, and 57% contained plague-positive burrows in 2003. Within plague-positive colonies, 23% of sampled burrows contained plague-positive fleas in 2002, and 26% contained plague-positive fleas in 2003. Of 15 intensively sampled colonies, there was no relationship between change in colony area and percentage of plague-positive burrows over the two years of the study. Some seasonality in plague prevalence was apparent because the highest percentages of plague-positive colonies were recorded in May and June. The surprisingly high prevalence of plague on study area colonies without any obvious epizootic suggested that the pathogen existed in an enzootic state in black-tailed prairie dogs. These findings have important implications for the management of prairie dogs and other species that are purported to be enzootic reservoir species.     

Does Physical Contact with a Dog or Person Affect Performance of a Working Memory Task?

ABSTRACT

Working memory (WM) plays a critical role in the execution of a wide variety of cognitive tasks and predicts academic success. This study was designed to compare the impact of the presence of a dog or a person, and physical contact with them, on the performance of a WM task. It also exam- ined whether the impact differed for two dogs, and whether these factors im- pacted arousal during the WM task. College students (n=31, aged 18–23 years) performed a WM task in five counterbalanced conditions; dog-touch, dog-no-touch, person-touch, person-no-touch, and alone. Participants were randomly assigned to one of two dogs; Miniature Poodle (n=16) or Border Collie (n=15). The WM task involved replicating increasingly complicated se- quences of colored lights by touching them on an iPad^®. Linear mixed model analyses revealed there was a significant interaction between collaborator and touch (p=0.05); best WM scores occurred without touch with either the per- son or the dog present, and worst WM scores occurred when the participant was touching a dog. Analyzing WM test during the dog conditions, touch (p=0.027) and dog breed (p=0.042) contributed independently to it; task completion was worse when the poodle was present and better without touch. Physiological measures [heart rate (HR) and heart rate variability] during the ex- periment indicated that the WM task was physiologically arousing (p<0.001) compared with listening, and HR was higher when touching a person than a dog during the task (p<0.046). These results are consistent with facilitation of performance by the presence of an observer. If there is a beneficial effect on cognition from a dog, physical contact with the dog might not be a necessary component. Aspects of the dog (e.g., breed) are also likely factors in WM task performance. This study highlights the importance of situational characteristics in studies evaluating the impact of companion animals.     

Genetic characterisation of a domestic dog<Emphasis Type="Italic">Canis familiaris</Emphasis> breed endemic to South African rural areas

Allozyme electrophoresis (horizontal starch gel and PAGE) and histochemical staining techniques were used to study the genetic composition of an endemic southern African domestic dogCanis familiaris Linnaeus, 1758, the Africanis breed. Genetic differentiation was analysed at 21 protein-coding loci. The results were compared to those for three other populations/breeds: representatives of established Western breeds, crossbred dogs of Western descent from rural areas in South Africa, and indigenous Saluki dogs from the Middle East. Nine polymorphic loci were found (Ak-1,-2, Ck, Per, Hb, Po-A-1 to-3 andPo-Tf). Two unique alleles at theCk andPo-A-2 loci separated the Africanis breed from the other groups. There were also significant differences between Africanis and the other breeds in pair-wise comparisons of allelic frequencies at polymorphic loci. An assignment test, fixation index values, gene flow and genetic distance values indicated a closer genetic association between the Africanis and Saluki breeds than with dogs of Western origin. This finding supports archaeological evidence that the endemic Africanis breed was introduced from the Middle East into Africa thousands of years ago, and not through later western influences. The average heterozygosity ranged from 0.106–0.15, with least heterozygosity in the Africanis and most in the rural crossbred group. The percentage of polymorphic loci, the mean number of alleles per locus (biologically more significant than heterozygosity), and conformation of genotypes to Hardy-Weinberg proportions showed no evidence of recent loss of genetic diversity in Africanis. Genetic differentiation and support of archaeological evidence by genetics indicate that the endemic southern African domestic dog breed is unique.     

The <Emphasis Type="Italic">Medicago truncatula SUNN</Emphasis> Gene Encodes a <Emphasis Type="Italic">CLV1</Emphasis>-like Leucine-rich Repeat Receptor Kinase that Regulates Nodule Number and Root Length

Four Medicago truncatula sunn mutants displayed shortened roots and hypernodulation under all conditions examined. The mutants, recovered in three independent genetic screens, all contained lesions in a leucine-rich repeat (LRR) receptor kinase. Although the molecular defects among alleles varied, root length and the extent of nodulation were not significantly different between the mutants. SUNN is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, expression levels of SUNN mRNA were reduced in sunn-1 roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated clv phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. A novel truncated copy of the SUNN gene lacking a kinase domain, RLP1, is found immediately upstream of SUNN and like SUNN is expressed at a reduced level in sunn-1 roots.     

Naturally occurring melanomas in dogs as models for non‐UV pathways of human melanomas

Spontaneously occurring melanomas are frequent in dogs. They appear at the same localizations as in humans, i.e. skin, mucosal sites, nail matrix and eyes. They display variable behaviors: tumors at oral localizations are more frequent and aggressive than at other anatomical sites. Interestingly, dog melanomas are associated with strong breed predispositions and overrepresentation of black‐coated dogs. Epidemiological analysis of 2350 affected dogs showed that poodles are at high risk of developing oral melanoma, while schnauzers or Beauce shepherds mostly developped cutaneous melanoma. Clinical and histopathological analyses were performed on a cohort of 153 cases with a 4‐yr follow‐up. Histopathological characterization showed that most canine tumors are intradermal and homologous to human rare morphological melanomas types – ‘nevocytoid type’ and ‘animal type’‐. Tumor cDNA sequencing data, obtained from 95 dogs for six genes, relevant to human melanoma classification, detected somatic mutations in oral melanoma, in NRAS and PTEN genes, at human hotspot sites, but not in BRAF. Altogether, these findings support the relevance of the dog model for comparative oncology of melanomas, especially for the elucidation of non‐UV induced pathways.     

The Shepherds’ Tale: A Genome-Wide Study across 9 Dog Breeds Implicates Two Loci in the Regulation of Fructosamine Serum Concentration in Belgian Shepherds

Diabetes mellitus is a serious health problem in both dogs and humans. Certain dog breeds show high prevalence of the disease, whereas other breeds are at low risk. Fructosamine and glycated haemoglobin (HbA1c) are two major biomarkers of glycaemia, where serum concentrations reflect glucose turnover over the past few weeks to months. In this study, we searched for genetic factors influencing variation in serum fructosamine concentration in healthy dogs using data from nine dog breeds. Considering all breeds together, we did not find any genome-wide significant associations to fructosamine serum concentration. However, by performing breed-specific analyses we revealed an association on chromosome 3 (p_corrected ≈ 1:68 × 10^-6) in Belgian shepherd dogs of the Malinois subtype. The associated region and its close neighbourhood harbours interesting candidate genes such as LETM1 and GAPDH that are important in glucose metabolism and have previously been implicated in the aetiology of diabetes mellitus. To further explore the genetics of this breed specificity, we screened the genome for reduced heterozygosity stretches private to the Belgian shepherd breed. This revealed a region with reduced heterozygosity that shows a statistically significant interaction (p = 0.025) with the association region on chromosome 3. This region also harbours some interesting candidate genes and regulatory regions but the exact mechanisms underlying the interaction are still unknown. Nevertheless, this finding provides a plausible explanation for breed-specific genetic effects for complex traits in dogs. Shepherd breeds are at low risk of developing diabetes mellitus. The findings in Belgian shepherds could be connected to a protective mechanism against the disease. Further insight into the regulation of glucose metabolism could improve diagnostic and therapeutic methods for diabetes mellitus.     

Allelic Combinations of Promoter and Exon 2 in <Emphasis Type="Italic">DQB1</Emphasis> in Dogs and Wolves

Polymorphism of PBRs of the major histocompatibility complex (MHC) genes is well recognized, but the polymorphism also extends to proximal promoter regions. Examining DQB1 variability in dogs and wolves, we identified 7 promoter variants and 13 exon 2 alleles among 89 dogs, including a previously unknown DQB1 exon 2 allele, and 8 promoter variants and 9 exon 2 alleles among 85 wolves. As expected from previous studies and from a close chromosomal location, strong linkage disequilibrium was demonstrated in both wolves and dogs by having significantly fewer promoter/exon 2 combinations than expected from simulations of randomized data sets. Interestingly, we noticed weaker haplotypic associations in dogs than in wolves. Dogs had twice as many promoter/exon 2 combinations as wolves and an almost 2-fold difference in the number of exon 2 alleles per promoter variant. This difference was not caused by an admixture of breeds in our group of dogs because the high ratio of observed to expected number of haplotypes persisted within a single dog breed, the German Shepherd. Ewens-Watterson tests indicated that both the promoter and exon 2 are under the balancing selection, and both regions appear to be more recently derived in the dog than in the wolf. Hence, although reasons for the differences are unknown, they may relate to altered selection pressure on patterns of expression. Deviations from normal MHC expression patterns have been associated with autoimmune diseases, which occur frequently in several dog breeds. Further knowledge about these deviations may help us understand the source of such diseases.     

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication,diet and diabetes

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch‐rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed‐dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.     

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.