Dog leucocyte antigen (DLA) class II haplotypes and risk of canine diabetes mellitus in specific dog breeds.

BACKGROUND: Canine diabetes mellitus (DM) is a common endocrine disease in domestic dogs. A number of pathological mechanisms are thought to contribute to the aetiopathogenesis of relative or absolute insulin deficiency, including immune-mediated destruction of pancreatic beta cells. DM risk varies considerably between different dog breeds, suggesting that genetic factors are involved and contribute susceptibility or protection. Associations of particular dog leucocyte antigen (DLA) class II haplotypes with DM have been identified, but investigations to date have only considered all breeds pooled together. The aim of this study was to analyse an expanded data set so as to identify breed-specific diabetes-associated DLA haplotypes. METHODS: The 12 most highly represented breeds in the UK Canine Diabetes Register were selected for study. DLA-typing data from 646 diabetic dogs and 912 breed-matched non-diabetic controls were analysed to enable breed-specific analysis of the DLA. Dogs were genotyped for allelic variation at DLA-DRB1, -DQA1, -DQB1 loci using DNA sequence-based typing. Genotypes from all three loci were combined to reveal three-locus DLA class II haplotypes, which were evaluated for statistical associations with DM. This was performed for each breed individually and for all breeds pooled together. RESULTS: Five dog breeds were identified as having one or more DLA haplotype associated with DM susceptibility or protection. Four DM-associated haplotypes were identified in the Cocker Spaniel breed, of which one haplotype was shared with Border Terriers. In the three breeds known to be at highest risk of DM included in the study (Samoyed, Tibetan Terrier and Cairn Terrier), no DLA haplotypes were found to be associated with DM. CONCLUSIONS: Novel DLA associations with DM in specific dog breeds provide further evidence that immune response genes contribute susceptibility to this disease in some cases. It is also apparent that DLA may not be contributing obvious or strong risk for DM in some breeds, including the seven breeds analysed for which no associations were identified.

Thirteen novel canine dog leukocyte antigen-88 alleles identified by sequence-based typing.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Major histocompatibility complex class II alleles and haplotypes associated with non-suppurative meningoencephalitis in greyhounds.

Non-suppurative meningoencephalitis is a breed-restricted canine neuroinflammatory disorder affecting young greyhounds in Ireland. A genetic risk factor is suspected because of the development of disease in multiple siblings and an inability to identify a causative infectious agent. The aim of this study was to examine potential associations between dog leucocyte antigen (DLA) class II haplotype and the presence of the disease. DLA three locus haplotypes were determined in 31 dogs with non-suppurative meningoencephalitis and in 115 healthy control dogs using sequence-based typing (SBT) methods. All dogs were unrelated at the parental level. Two haplotypes (DRB1*01802/DQA1*00101/DQB1*00802 and DRB1*01501/DQA1*00601/DQB1*02201) were significantly (P = 0.0099 and 0.037) associated with the presence of meningoencephalitis, with odds ratios (95% confidence interval) of 5.531 (1.168-26.19) and 3.736 (1.446-9.652), respectively. These results confirm that there is an association between DLA class II haplotype and greyhound meningoencephalitis, suggesting an immunogenetic risk factor for the development of the disease. Greyhound meningoencephalitis may be a suitable model for human neuroinflammatory diseases with an immunogenetic component.

Balancing selection and heterozygote advantage in major histocompatibility complex loci of the bottlenecked Finnish wolf population.

Maintaining effective immune response is an essential factor in the survival of small populations. One of the most important immune gene regions is the highly polymorphic major histocompatibility complex (MHC). We investigated how a population bottleneck and recovery have influenced the diversity and selection in three MHC class II loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1, in the Finnish wolf population. We studied the larger Russian Karelian wolf population for comparison and used 17 microsatellite markers as reference loci. The Finnish and Karelian wolf populations did not differ substantially in their MHC diversities (GST″ = 0.047, P = 0.377), but differed in neutral microsatellite diversities (GST″ = 0.148, P = 0.008). MHC allele frequency distributions in the Finnish population were more even than expected under neutrality, implying balancing selection. In addition, an excess of nonsynonymous compared to synonymous polymorphisms indicated historical balancing selection. We also studied association between helminth (Trichinella spp. and Echinococcus canadensis) prevalence and MHC diversity at allele and SNP level. MHC-heterozygous wolves were less often infected by Trichinella spp. and carriers of specific MHC alleles, SNP haplotypes and SNP alleles had less helminth infections. The associated SNP haplotypes and alleles were shared by different MHC alleles, which emphasizes the necessity of single-nucleotide-level association studies also in MHC. Here, we show that strong balancing selection has had similar effect on MHC diversities in the Finnish and Russian Karelian wolf populations despite significant genetic differentiation at neutral markers and small population size in the Finnish population.

Evaluation of DLA promoters in Doberman hepatitis.

Doberman hepatitis (DH) is associated with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 indicating a role for the immune system in the development of the disease. The dog leucocyte antigen (DLA) class II expression is controlled at the transcriptional level with proximal promoters. Differential expression of DLA class II molecules of antigen-presenting cells is reported to affect susceptibility to or protection from different immune-mediated diseases. The aim of this study was to evaluate, whether the variation in promoter areas of homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans could explain why some dogs become afflicted with DH and others do not. Our findings suggest that promoter variants are not associated as risk modifiers in homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans, but additional factors are needed. Nevertheless, our study indicates that the whole DLA block is associated to the disease.

Risk of canine cranial cruciate ligament rupture is not associated with the major histocompatibility complex.

OBJECTIVES: To investigate the association of the major histocompatability (MHC) class II allele haplotype frequencies with the diagnosis of cranial cruciate ligament (CCL) rupture in two breeds of dog. METHODS: DNA samples from populations of Labrador Retrievers and Golden Retrievers with CCL rupture and general populations of the same breeds were characterised for three DLA class II loci (DRB1*, DQA1* and DQB1*) alleles using sequence-based typing or reference strand-mediated conformation analysis. RESULTS: Although distinct differences in haplotype types, frequencies and homozygozity were observed between the two breeds, no disease specific association could be identified for the development of the CCL rupture within either population. CLINICAL SIGNIFICANCE: The risk for developing CCL rupture was not associated with DLA haplotype group(s) in Labrador Retrievers or Golden Retrievers, thus the hypothesis that there is an autoimmune basis to CCL rupture was not supported.

Major histocompatibility complex diversity in the endangered Ethiopian wolf (Canis simensis).

The major histocompatibility complex (MHC) influences immune response to infection and vaccination. In most species, MHC genes are highly polymorphic, but few wild canid populations have been investigated. In Ethiopian wolves, we identified four DLA (dog leucocyte antigen)-DRB1, two DLA-DQA1 and five DQB1 alleles. Ethiopian wolves, the world's rarest canids with fewer than 500 animals worldwide, are further endangered and threatened by rabies. Major rabies outbreaks in the Bale Mountains of southern Ethiopia (where over half of the Ethiopian wolf population is located) have killed over 75% of wolves in the affected sub-populations. In 2004, following a rabies outbreak, 77 wolves were vaccinated, and 19 were subsequently recaptured to monitor the effectiveness of the intervention. Pre- and post-vaccination rabies antibody titres were available for 18 animals, and all of the animals sero-converted after vaccination. We compared the haplotype frequencies of this group of 18 with the post-vaccination antibody titre, and showed that one haplotype was associated with a lower response (uncorrected P < 0.03). In general, Ethiopian wolves probably have an adequate amount of MHC variation to ensure the survival of the species. However, we sampled only the largest Ethiopian wolf population in Bale, and did not take the smaller populations further north into consideration.

Association of Doberman hepatitis to canine major histocompatibility complex II.

Doberman hepatitis (DH) is a chronic and progressive inflammatory liver disease that mainly affects female dogs. The high incidence of chronic hepatitis in Dobermans is suggestive of a genetic predisposition. DH is characterized by mononuclear cell infiltration and copper accumulation in the liver and major histocompatibility complex (MHC) class II antigen expression in the hepatocytes. In dogs, the MHC is referred to as the dog leukocyte antigen (DLA) system. In this study, the potential role of DLA genes in DH was investigated by sequence-based typing in the exon 2 of DLA-DRB1, -DQA1 and -DQB1. The case group comprised 37 Dobermans with subclinical or clinical DH. The control group consisted of 37 healthy Dobermans, with normal liver enzyme values and without immunosuppressive medication. The control dogs were over 10 years old to include dogs with the lowest genetic risk of DH. Our results indicate that Dobermans with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 [odds ratio (OR) = 14.9, confidence limit (CL) = 3.1-71.7, P < 0.00005], especially with homozygosity for DLA-DRB1*00601 (P < 0.0005), are susceptible to DH. The DQ heterodimer DLA-DQA1*00901/DQB1*00101 and the allele DLA-DRB1*01501 appear to confer protection against DH (P < 0.001). Allele and haplotype frequencies were compared using chi-squared statistics. The disease shows a complex pattern of inheritance, but the observed DLA class II association with DH suggests a role for the immune system in the development of the disease.

Genetic diversity of the major histocompatibility complex class II in Alaskan caribou herds.

We have sampled five different herds of caribou in Alaska to ascertain their major histocompatibility complex (MHC) class II diversity, and to assess whether the herds were significantly different in their MHC class II allele profiles. We complemented the MHC results with data from nine neutral microsatellite markers. The results indicate that while the microsatellites are diverse, there are no significant differences between the herds. However, for the MHC, we have shown that there is diversity at three of the four loci studied, the different herds have significantly different MHC class II allele profiles. It is also clear that although some of the herds have overlapping ranges, they are still different for their MHC class II alleles.

Assessment of the functionality of genome-wide canine SNP arrays and implications for canine disease association studies.

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome-wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large-scale assessment of the functionalities (LD and SNP tagging performance) of canine genome-wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi-marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome-wide association studies (GWAS).

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.

CTLA4 promoter polymorphisms are associated with canine diabetes mellitus.

Canine diabetes mellitus (DM) shares many similarities with human type 1 diabetes (T1D). It is a complex genetic disorder, which shows marked differences in breed susceptibility, with Samoyed dogs being highly susceptible, whereas the Boxer breed is relatively resistant. A number of immune response genes, which have been associated with human T1D, have also been implicated in determining susceptibility to canine DM, suggesting an immune-mediated component to the disease pathogenesis. Single nucleotide polymorphisms (SNPs) in the CTLA4 gene have consistently and reproducibly been associated with human T1D and other autoimmune diseases but the canine CTLA4 gene has not previously been investigated for involvement in canine DM. SNPs of particular interest in the human association studies are those in the promoter region which affect CTLA4 expression levels, and that of exon 1 which results in a non-synonymous amino acid change. We performed a canine SNP discovery investigation of CTLA4 on a region of DNA containing exon 1 and 1.5 kb upstream sequence in order to identify promoter region SNPs. Confirmed SNPs were used in a genetic association study of a canine diabetic cohort showing that CTLA4 promoter polymorphisms were associated with diabetes in crossbreed dogs and in five Pedigree breeds-Samoyed, Miniature Schnauzer, West Highland White Terrier, Border Terrier and Labrador. Meta-analysis of these breeds showed 9 out of 15 SNPs were associated with DM and genotype and haplotype analyses also confirmed the allelic associations in these breeds.

Increased genetic risk or protection for canine autoimmune lymphocytic thyroiditis in Giant Schnauzers depends on DLA class II genotype.

Dogs represent an excellent comparative model for autoimmune thyroiditis as several dog breeds develop canine lymphocytic thyroiditis (CLT), which is clinically similar to Hashimoto's thyroiditis in human. We obtained evidence that dog leukocyte antigen (DLA) class II genotype function as either genetic risk factor that predisposes for CLT or as protective factor against the disease. Genetic diversity at their DLA-DRB1, -DQA1, and -DQB1 loci were defined and potential association to major histocompatibility complex II haplotypes and alleles was analyzed. Giant Schnauzers carrying the DLA-DRB1*01201/DQA1*00101/DQB1*00201 haplotype showed an increased risk (odds ratio of 6.5) for developing CLT. The same risk haplotype has, to date, been observed in three different breeds affected by this disease, Giant Schnauzer, Dobermann, and Labrador Retriever, indicating that it is a common genetic risk factor in a variety of breeds affected by this disease. Importantly, protection for development of the disease was found in dogs carrying the DLA-DRB1*01301/DQA1*00301/DQB1*00501 haplotype (odds ratio of 0.3).

Association of canine anal furunculosis with TNFA is secondary to linkage disequilibrium with DLA-DRB1*.

Anal furunculosis (AF) is a chronic inflammatory disease of perianal tissues that particularly affects German Shepherd dogs (GSD). An immune-mediated aetiopathogenesis is suggested by T-cell infiltration, upregulated cytokine gene expression, clinical response to ciclosporin therapy and a strong genetic association with the DLA-DRB1*00101 allele. Given the close proximity of TNFA and DLA-DRB1 in the canine major histocompatibility complex (MHC), together with the strong linkage disequilibrium (LD) observed across this region, the primary disease association could be with either locus. We have investigated whether there may be an association of AF with TNFA gene polymorphism in GSDs. Cohorts of AF-affected and AF-unaffected GSDs of known dog leucocyte antigen (DLA) class II profile were genotyped for 10 single nucleotide polymorphisms (SNPs) in the canine TNFA locus using Sequenom iPLEX technology. Seven discrete TNFA haplotypes were identified in GSDs for combinations of these SNPs. TNFA haplotype frequencies were compared in cases and controls. The TNFA haplotype 3 (ATCGTTACGG), was at significantly increased frequency in cases (29% vs 15%, OR 2.5, 95% CI 1.4-4.8; P = 0.003). All seven discrete TNFA SNP haplotypes were examined for their association with DLA-DRB1/DQA1/DQB1 established haplotypes. TNFA haplotype 3 was preferentially associated with both DLA-DRB1*00101(3A)- and DLA-DRB1*00102(3B)-positive haplotypes. The DLA-DRB1* 00101/TNFA-3A haplotype was significantly associated with AF (19.3% vs 5.8%; OR 3.7, 95% CI: 1.5-8.9; P = 0.003), whereas the DLA-DRB1*00102/TNFA-3B haplotype was not (P = NS). These findings suggest that susceptibility to AF in GSDs is primarily associated with DLA-DRB1*00101 and any association with the TNFA locus is secondary and is likely to be because of LD.

Single locus typing of MHC class I and class II B loci in a population of red jungle fowl.

In species with duplicated major histocompatibility complex (MHC) genes, estimates of genetic variation often rely on multilocus measures of diversity. It is possible that such measures might not always detect more detailed patterns of selection at individual loci. Here, we describe a method that allows us to investigate classical MHC diversity in red jungle fowl (Gallus gallus), the wild ancestor of the domestic chicken, using a single locus approach. This is possible due to the well-characterised gene organisation of the 'minimal essential' MHC (BF/BL region) of the domestic chicken, which comprises two differentially expressed duplicated class I (BF) and two class II B (BLB) genes. Using a combination of reference strand-mediated conformation analysis, cloning and sequencing, we identify nine BF and ten BLB alleles in a captive population of jungle fowl. We show that six BF and five BLB alleles are from the more highly expressed locus of each gene, BF2 and BLB2, respectively. An excess of non-synonymous substitutions across the jungle fowl BF/BL region suggests that diversifying selection has acted on this population. Importantly, single locus screening reveals that the strength of selection is greatest on the highly expressed BF2 locus. This is the first time that a population of red jungle fowl has been typed at the MHC region, laying the basis for further research into the underlying processes acting to maintain MHC diversity in this and other species.

Canine diabetes mellitus: from phenotype to genotype.

Breed differences in susceptibility to diabetes mellitus in dogs suggest an underlying genetic component to the pathogenesis of the disease. There is little evidence for an equivalent of human type 2 diabetes in dogs, and it has been proposed that canine diabetes is more comparable to the type 1 form of the disease. Certain immune response genes, particularly those encoding major histocompatibility complex molecules involved in antigen presentation, are important in determining susceptibility to human type 1 diabetes. We tested the hypothesis that canine major histocompatibility complex genes (known as the dog leucocyte antigen) are associated with diabetes in dogs. A total of 530 diabetic dogs and more than 1000 controls were typed for dog leucocyte antigen, and associations were found with three specific haplotypes. The DLA-DRB1*009/DQA1*001/DQB1*008 haplotype shows the strongest association with diabetes in the UK dog population. This haplotype is common in diabetes-prone breeds (Samoyed, cairn terrier and Tibetan terrier) but rare in diabetes-resistant breeds (boxer, German shepherd dog and golden retriever), which could explain differences in the prevalence of diabetes in these different breeds. There is evidence that the DLA-DQA1*001 allele is also associated with hypothyroidism, suggesting that this could represent a common susceptibility allele for canine immune-mediated endocrinopathies.

Risk of anal furunculosis in German shepherd dogs is associated with the major histocompatibility complex.

Anal furunculosis (AF) is a chronic, progressive inflammatory disease of the perianal tissues most frequently affecting middle-aged or older German Shepherd dogs (GSD). Because this breed accounts for over 80% of all reported cases, there is likely to be a genetic association with disease susceptibility. Although there are some similarities with perianal fistulation that occurs in human Crohn's disease, the aetiology and pathogenesis of AF are still poorly understood. Recent research has suggested an immune-mediated aetiology, and evidence for this has been further provided by clinical responses to the immunosuppressive drug cyclosporin. The aim of the current study was to investigate canine major histocompatibility complex immune response genes. Dog leucocyte antigen class II alleles and haplotypes were characterised by sequence-based typing of 107 GSD affected with AF and 196 breed-matched controls collected in the UK. A highly significant association of DLA-DRB1*00101 with the presence of AF was observed (OR = 5.01, CI = 2.7-9.3, P < 0.00000001). This association was confirmed in a second cohort of GSD collected in Finland. Homozygosity for this allele is associated with an earlier disease onset.

Major histocompatibility complex typing of dogs from Russia shows further dog leukocyte antigen diversity.

Blood samples from 85 Russian dogs and wolves were collected as dried blood spots on paper and transported to the UK by mail. We obtained partial or complete three-locus canine major histocompatibility complex [dog leukocyte antigen (DLA)] class II haplotypes on 81 of these samples. Six new alleles were identified: three DLA-DRB1 and three DLA-DQB1. These alleles occurred in haplotypic combinations not previously seen in other European dogs. One haplotype appeared to lack a DQB1 allele. Two of the new haplotypes segregated through a family of dogs that was investigated.

14th International HLA and Immunogenetics Workshop: report on joint study on canine DLA diversity.

The canine DLA diversity component of the 14th International HLA and Immunogenetics (IHI) Workshop had three main aims: i) to establish the range of DLA class II (DRBI, DQA1, DQB1) diversity and their distribution in dog breeds, ii) to identify DLA class II haplotypes by segregation in families and iii) to examine DLA class II associations with disease susceptibility. Over 1600 dogs from seven participating laboratories were typed for three DLA class II loci as part of the workshop. Standard sequence-based typing methods were used, and all the data were of high quality.