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The dog leukocyte antigen (DLA) system contains many of the functional genes of the immune system, thereby making it a candidate region for involvement in immune-mediated disorders. A number of studies have identified associations between specific DLA class II haplotypes and canine immune hemolytic anemia, thyroiditis, immune polyarthritis, type I diabetes mellitus, hypoadrenocorticism, systemic lupus erythematosus-related disease complex, necrotizing meningoencephalitis (NME) and anal furunculosis. These studies have relied on sequencing approximately 300 bases of exon 2 of each of the DLA class II genes: DLA-DRB1, DLA-DQA1 and DLA-DQB1. An association (odds ratio = 4.29) was identified by this method between Weimaraner dogs with hypertrophic osteodystrophy (HOD) and DLA-DRB1*01501.
In the present study, a genotyping assay of 126 coding single nucleotide polymorphisms (SNPs) from across the entire DLA, spanning a region of 2.5 Mb (3,320,000–5,830,000) on CFA12, was developed and tested on Weimaraners with HOD, as well as two additional breeds with diseases associated with DLA class II: Nova Scotia duck tolling retrievers with hypoadrenocorticism and Pug dogs with NME. No significant associations were found between Weimaraners with HOD or Nova Scotia duck tolling retrievers with hypoadrenocorticism and SNPs spanning the DLA region. In contrast, significant associations were found with NME in Pug dogs, although the associated region extended beyond the class II genes. By including a larger number of genes from a larger genomic region a SNP genotyping assay was generated that provides coverage of the extended DLA region and may be useful in identifying and fine mapping DLA associations in dogs.
A large number of autoimmune disorders in dogs have been associated with certain alleles and haplotypes of the dog leukocyte antigen (DLA), which includes the canine major histocompatibility complex (MHC) class II genes DRB1, DQA1 and DQB1. This diverse group of diseases includes type I diabetes mellitus of the Samoyed dog and Cairn and Tibetan terriers (Kennedy et al., 2006a, 2007c; Catchpole et al., 2008), immune-hemolytic anemia (Kennedy et al., 2006b), systemic lupus erythematosus-related disease complex (Wilbe et al., 2009) and hypoadrenocorticism (Hughes et al., 2010) in Nova Scotia duck tolling retrievers, anal furunculosis in German shepherd dogs (Kennedy et al., 2008), necrotizing meningoencephalitis (NME) of Pug dogs (Greer et al., 2010), autoimmune lymphocytic thyroiditis (Wilbe et al., 2010) and Vogt-Koyanagi-Harada-like syndrome of the Akita (Angles et al., 2005a).
These studies have taken a candidate gene approach to examine the classic DLA class II genes (DRB1, DQA1 and DQB1). The genes encode the molecules that associate to form the heterodimeric peptide binding cleft of the cells (macrophages, B cells and dendritic cells) that present exogenous antigens to CD4+ T cells (Klein, 1986). Canine MHC association studies have focused on typing a portion (~300 bp) of the coding sequence of the second exon (which encodes the functional peptide binding domain) of each of the three classic class II genes (DRB1, DQA1 and DQB1); a fourth gene (DRA) is non-polymorphic (Wagner et al., 1995). The direct sequencing of this region (approximately 900 bases) conventionally yields the characterized DRB1/DQA1/DQB1 ‘alleles’ and haplotypes (Kennedy, 2007; Kennedy et al., 2007a) and is utilized in the investigation of disease associations (Kennedy et al., 2007c).
Numerous reports have also described genetic associations between the human leukocyte antigen (HLA) classic class II genes (DRB1, DRA, DQA1 and DQB1) and rheumatoid arthritis (Fugger and Svejgaard, 2000; Holmdahl, 2000; Jawaheer et al., 2002; Coenen and Gregersen, 2009), type I diabetes (Lie et al., 1999a; Gorodezky et al., 2006; Ilonen and Hermann, 2010), multiple sclerosis (Svejgaard, 2008; Ramagopalan and Ebers, 2009; Dieudé et al., 2011) and autoimmune hypoadrenocorticism disease (Thomsen et al., 1975; Gombos et al., 2007; Falorni et al., 2008), as well as a range of other conditions.
One postulated mechanism for the large number of associations between autoimmune diseases and the class II genes is allele-specific presentation of autoantigens to T cells (Raymond et al., 2005; Thorsby and Lie, 2005; Ettinger et al., 2006). According to this theory, differential presentation of antigens (which allows presentation of autoantigens) is a consequence of the amino acid sequence in the peptide-binding cleft of the heterodimer (Holmdahl, 2000; Weyand and Goronzy, 2000; Zanelli et al., 2000; Undlien et al., 2001; Ettinger et al., 2006).
However, it is unlikely that the peptide sequence determines disease susceptibility, since in multiple sclerosis almost all common HLA-DRB1 haplotypes are associated with the disease (Ramagopalan and Ebers, 2009). So far, no mutations that can explain a mechanism for increased susceptibility have been identified in the classic MHC class II genes in humans (Holmdahl, 2000; Coenen and Gregersen, 2009; Ramagopalan and Ebers, 2009) or dogs (Kennedy et al., 2006a and b, 2007c, 2008; Barnes et al., 2009).
An association between DLA and hypertrophic osteodystrophy (HOD), an immune-mediated disorder in Weimaraner dogs, has been investigated by the authors since DLA involvement was implicated in many canine autoimmune diseases. HOD is a disease with specific breed predispositions that affects large breed dogs during the rapid growth stage (Bellah, 1993). Sick dogs exhibit swelling and pain in their legs, with reluctance to stand or walk. In addition to bone pain, there are variable general signs including fever, lethargy, depression and loss of appetite. The diagnosis of HOD is established by ruling out infectious osteomyelitis and by radiographic evidence of bone involvement (Watson et al., 1973; Grondalen, 1976; Woodard, 1982). The prognosis for severe cases is poor due to relapsing episodes and a low quality of life for affected puppies, often resulting in euthanasia.
The Weimaraner breed is susceptible to HOD and entire litters have been known to be affected (Woodard, 1982; Harrus et al., 2002). Although the cause of HOD is unknown, an inherited component to the disease is likely (Watson et al., 1973; Munjar et al., 1998; LaFond et al., 2002). An immune-mediated etiology has been suggested on the basis of a lack of response to conservative treatment with non-steroidal anti inflammatory drugs concurrent with a positive response to immunosuppressive doses of corticosteroid treatment (Abeles et al., 1999; Harrus et al., 2002). DLA was considered as a candidate region in HOD of the Weimaraner because of the autoinflammatory nature of the disease. Autoinflammatory disorders are characterized by recurrent episodes of inflammation in the absence of infection or circulating autoantibodies (Farasat et al., 2008).
When the three DLA class II genes (DRB1, DQA1 and DQB1), were typed in Weimaraner dogs, a significant association was identified between HOD and the DLA-DRB1*01501 allele (Kennedy et al., 2007c). Since no causative variants have been identified within the DRB1/DQA1/DQB1 genes so far (Fugger and Svejgaard, 2000; Ota et al., 2001; Gorodezky et al., 2006; Ramagopalan and Ebers, 2009; Eike et al., 2009), we ‘fine mapped’ the association between HOD and DLA in an attempt to uncover a causal variant.
To target the extended DLA region, we developed an expanded DLA-wide single nucleotide polymorphism (SNP) genotyping Sequenom assay with 126 SNPs across the 2.5 Mb DLA region. Using the DLA-wide assay, we then reanalyzed the association with Weimaraner HOD, as well as two previously published DRB1/DQA1/DQB1 disease associations: hypoadrenocorticism in the Nova Scotia duck tolling retriever (Hughes et al., 2010) and NME in Pug dogs (Greer et al., 2010). Differential results were obtained using the expanded DLA-wide panel, demonstrating the importance of confirming DLA sequenced haplotype associations.
NCBI database coding (missense, nonsense and frameshift) SNPs1 were mined across the extended DLA region (canine chromosome 12, CFA12:3.32-5.83 megabases) and the Assay Designer V.4.0 software (Sequenom) was used to develop an assay of 124 SNPs within 53 genes and two SNPs in non-coding sequence. To the best of our knowledge, these SNPs have not been used in previous DLA association studies. To represent the DLA class II genes that are conventionally represented in DLA association studies, six SNPs were selected from within the DRB1 gene, five SNPs from the DQA1 gene and two SNPs from the DQB1 gene. We also included two database SNPs representing missense mutations in DRA1, the fourth gene in the classic MHC II, which is non-polymorphic in dogs (Wagner et al., 1995) (Supplementary Table 1). The 126 SNPs were multiplexed into four Sequenom reaction wells of 37, 35, 33 and 21 SNPs, respectively (Supplementary Table 2).
Genotyping was performed using a Sequenom MassARRAY platform2 at the Veterinary Genetics Laboratory, University of California, Davis. Sample DNA was extracted from blood samples in ethylene diamine tetraacetic acid using a commercial kit (Puregene, Gentra Systems). Additional DNA was isolated from buccal swabs according to the method of Irion et al. (2003). PCR was performed in 5 μL volumes on an ABI 9700 Thermal Cycler (Applied Biosystems). Reactions and cycling conditions were performed according to the Sequenom iPLEX protocol for 96 well format using MassARRAY.
Sets of samples from two breeds with previously reported DRB1/DQA1/DQB1 exon 2 associations were reused for genotyping using the Sequenom DLA-wide SNP panel, along with a set of samples from Weimaraners:
The second exon of the DRB1, DQA1 and DQB1 genes were typed by direct sequencing of genomic DNA from 19 cases of HOD in Weimaraners and 11 unaffected Weimaraners using locus-specific intronic primers, according to Kennedy et al. (2006a).
DRB1/DQA1/DQB1 exon 2 haplotype determination was based on annotated sequences deposited in GenBank and the Immune Polymorphism Database (Robinson et al., 2005). Homozygous haplotypes identified at each locus were used to distinguish the heterozygous haplotypes. At least one parent of each animal was genotyped to allow verification of the assigned haplotype. Three-loci DRB1/DQA1/DQB1 super-haplotypes were identified according to Kennedy (2007) and Kennedy et al. (2007a and c). Haplotype frequencies in HOD cases and controls were compared by χ2 statistics. ORs with 95% CIs were calculated using a 2 × 2 contingency table (Bland and Altman, 2000).
Association analysis by χ2 statistics was performed using Haploview software V.4.2 (Barrett et al., 2005). The four-gamete rule was used to define haplotype blocks and 1,000 permutations were performed to correct for multiple-testing bias. The Pug dog NME P values were unresolved even with 10,000,000 permutations and the minimal obtainable (permuted) P value for the highest associated SNPs was <1 × 10−13 (Supplementary Table 3). ORs were generated using PLINK software (Purcell et al., 2007).
Nineteen Weimaraner HOD cases and 11 control Weimaraners were typed using the conventional method of sequencing exon 2 of DRB1/DQA1/DQB1. A significant association (OR 4.29; 95% CI 1.4–13.1; P = 0.01) between Weimaraner HOD and the DLA-DRB1*01501 allele was identified (Table 1). A protective allele, DLA-DRB1*01201, was also identified (OR 0.156; 95% CI 0.04–0.52; P = 0.002) (Table 1). These values are as high or higher than previously published class II associations (Kennedy et al., 2007c; Catchpole et al., 2008; Barnes et al., 2009). None of the seven other exon 2 alleles found in the Weimaraner sample set were associated with HOD. No significant association was observed between HOD and the three-locus super haplotypes in our data set (Table 2).
Fifty-one Weimaraner HOD cases and 67 control Weimaraner dogs were genotyped using the DLA-wide SNP panel. This larger sample set included the 19 HOD cases and 11 control Weimaraners that were studied by exon 2 sequencing. Of the 126 SNPs genotyped, 33 SNPs were not polymorphic in the Weimaraner data set and a total of 93 SNPs spanning the entire DLA region were analyzed (Supplementary Table 4). Even though the DRB1 gene was represented by five SNPs (Supplementary Table 4), no significant association was identified between the larger sample set of Weimaraner dogs with HOD and the DLA region (Supplementary Table 5).
Of the 126 SNPs assayed in the Nova Scotia duck tolling retriever sample set, 32 SNPs were not informative. A total of 94 SNPs spanning the entire DLA region were genotyped; the DRB1 gene was represented by six SNPs, DQB1 by two SNPs and DQA1 by five SNPs (Supplementary Table 4). No significant association (P =0.05 based on 1,000 permutations) was identified between Nova Scotia duck tolling retriever dogs with hypoadrenocorticism and the DLA region (Supplementary Table 6). As 10 additional cases and 17 additional controls became available, their genotypes were added to the analysis. No significant association was identified between the larger set of 44 hypoadrenocorticism cases and 51 control Nova Scotia duck tolling retrievers and DLA.
Of the 126 SNPs assayed, 26 SNPs were not polymorphic in the Pug dog data set. A total of 100 SNPs spanning the extended DLA region were genotyped; the DRB1 gene was represented by six SNPs, DQB1 by two SNPs and DQA1 by five SNPs. Gene names, genomic locations of the SNPs and minor allele frequencies (MAF) are presented in Supplementary Table 4. SNPs located across the extended DLA region cfa12:3.32-5.83 (2.5 Mb) had significant associations with NME in Pug dogs (Supplementary Table 3); χ2 values 12.69 (106 permutations; P = 0.0217) to 53.37 (106 permutations; P <1 × 10−13). The highest χ2 and lowest P values were assigned to SNP 22177967 (cfa12: 5.23 Mb), in the 3′ UTR of the DLA-DQA1 gene. The second highest χ2 value (52.2, 106 permutations, P <1 × 10−13) was assigned to SNP 22255769 (cfa12: 5.25 Mb), located near the 5′ UTR of the DLA-DQB1 gene, in agreement with the previously published DLA class II association (Greer et al., 2010).
When the significantly associated χ2 values were plotted by Mb position on CFA12 (Fig. 1), it was observed that highly significant χ2 values were assigned to SNPs located downstream of the DLA class II on cfa12. A χ2 value of 39.974 (106 permutations; P = 3 × 10−7) was assigned for SNP 8569523 located at the 3′ end of the typed region typed (cfa12: 5.76 Mb), within the DAXX gene.
We generated a DLA-wide SNP genotyping tool for fine mapping of an association between HOD in Weimaraners and DRB1 and tested two additional breeds with previously published DRB1/DQA1/DQB1 associations, Pug dogs with NME (Greer et al., 2010) and Nova Scotia duck tolling retrievers with hypoadrenocorticism (Hughes et al., 2010). Genotyping using the DLA-wide panel confirmed a strong association between NME in Pug dogs and the DLA; however, DLA associations in Nova Scotia duck tolling retrievers with hypoadrenocorticism and Weimaraners with HOD were not replicated using this expanded set of markers.
These results are in agreement with two previous publications: (1) a negative linkage analysis between Nova Scotia duck tolling retrievers with hypoadrenocorticism and the DLA class II region (Hughes et al., 2010); and (2) localization of NME of Pug dogs to the DLA region on cfa12 by a genome-wide association (GWA) study (Greer et al., 2010). Since the DLA-wide SNP panel assays the entire DLA region of 2.5 Mb containing 53 genes, including the MHC class II DRB1, DQA1 and DQB1 genes, for evidence of associations, it is unlikely that a true genetic association would be missed.
The reproducible association between DLA and NME of Pug dogs demonstrates that genetic associations can be identified using SNPs across the DLA. While SNPs from the entire DLA region were significantly associated with NME of Pug dogs, a plot of χ2 by physical position (Fig. 1) shows that the high χ2 values are not only observed around DLA class II, but remain high across the extended DLA region. This implies that the association with NME in the Pug dog might be located downstream of, or outside, the extended DLA region on CFA12.
Since population stratification (different degrees of kinship between the case and control groups) could lead to spurious associations (Hinds et al., 2004; Wang, 2009; Wu et al., 2011), this should be considered as a potential cofounding factor in canine association studies. However, it is unlikely that the inconsistency in the DLA association results could be explained by population structure, because identical samples were used for the analyses. A possible explanation for the observed spurious associations could be the small number of DRB1/DQA1/DQB1 alleles in the Nova Scotia duck tolling retriever and Weimaraner breeds.
Although the DRB1/DQA1/DQB1 genes are highly polymorphic across different breeds of dogs, only limited numbers of alleles (typically 4–7 for each locus) have been identified within a range of breeds tested (Ollier et al., 2001; Angles et al., 2005b; Kennedy, 2007; Kennedy et al., 2006a, b and c, 2007a, b and c, 2008; Catchpole et al., 2008; Wilbe et al., 2009; Hughes et al., 2010). In Nova Scotia duck tolling retrievers, Hughes et al. (2010) identified seven different DRB1/DQA1/DQB1 haplotypes, comprising five DRB1 alleles, four DQA1 alleles and six DQB1 alleles. In Weimaraners, we identified nine different DRB1/DQA1/DQB1 haplotypes comprising three DRB1 alleles, four DQA1 alleles and five DQB1 alleles (Table 2). The DRB1/DQA1/DQB1 haplotypes reported by Kennedy et al. (2007a) comprised 100 DRB1 alleles, 60 DQB1 alleles and 26 DQA1 alleles identified across 80 different dog breeds. Out of 360 dogs from 25 breeds typed for the DRB1/DQA1/DQB1 alleles, Angles et al. (2005b) found that 40% were homozygous at the DRB1 site, 52% at the DQA1 and 44% at the DQB1 site.
Considering these findings, a significant statistical association within a breed population with a limited number of haplotypes should be considered cautiously and proof of a biological effect or linkage analysis should be sought as part of the validation of the statistical result. To illustrate that a spurious association is attainable in a breed with a small number of DLA alleles (conventionally tested), an association analysis was performed for Weimaraner sex (a trait determined by the presence or absence of the Y chromosome) and the DLA class II DRB1/DQA1/DQB1 alleles. DRB1 alleles associated with a ‘high risk’ of being a male (DRB1*1501: OR 2.68; P = 0.025) as well as with a ‘protective effect’ (DRB1*1201: OR 0.34; P = 0.013) were identified in this example of a spurious association. When the same association was performed using the DLA-wide SNP panel, no significant association was identified.
It is likely that the restricted number of DRB1/DQA1/DQB1 alleles and haplotypes do not segregate equally within a breed population due to inbreeding practices, use of popular sires, and/or genetic drift. Since both linkage disequilibrium (LD) and allelic content vary for different HLA haplotypes (Horton et al., 2008), this may be true for the DLA. The exon 2 ‘alleles’ may represent different haplotypes that appear to be identical due to convergence (Seddon et al., 2010). When Seddon et al. (2010) sampled SNPs from the MHC class II region to evaluate canine cases and controls for type I diabetes, it was concluded that the published class II association with type I diabetes (Kennedy et al., 2006a), could be outside the DRB1/DQA1/DQB1 exon 2 region or within a region larger than just exon 2. It is also possible that associations are more complex than those involving a single haplotype (Seddon et al., 2010). Differentiation of each of these possibilities should be possible when genotyping SNPs across the extended DLA region for disease association studies.
We recognized that DRB1/DQA1/DQB1 exon 2 typing may give rise to spurious associations due to restricted numbers of haplotypes within breeds. This implies that exon 2 typing may not always be an informative tool when used to assess DLA diversity and that there is risk of misleading results due to focal low polymorphism. A DLA-wide SNP panel that spans the entire 2.5 Mb of the DLA region may present a more appropriate tool for the analysis of MHC diversity in pedigree dog populations.
The MHC region harbors the highest density of genes in the genome, yet most, if not all, of the genetic associations point to the class II classic genes in humans (Feder et al., 1996; Lie et al., 1999a and b; Undlien et al., 1999; Fugger and Svejgaard, 2000; Roudier, 2000; Weyand and Goronzy, 2000; Ota et al., 2001; Undlien et al., 2001; Turner and Colbert, 2002; Raymond et al., 2005; Thorsby and Lie, 2005; Vandiedonck et al., 2005; Ettinger et al., 2006; Gorodezky et al., 2006; Falorni et al., 2008; Eike et al., 2009) and dogs (Ollier et al., 2001; Kennedy et al., 2006a, b and c, 2007c, 2008; Catchpole et al., 2008; Wilbe et al., 2009, 2010). In humans, an obstacle to localizing disease-predisposing genetic variants within the HLA is the strong LD typical of this region (Malfroy et al., 1997), presumably due to selection (Raymond et al., 2005).
LD or non-random association of alleles at adjacent HLA loci presents a difficulty in trying to establish which HLA locus is the primary disease-predisposing gene and which only appears to be associated because of LD to the primary associated gene (Feder et al., 1996). LD in the MHC is considered to be complex in comparison with other genomic regions demonstrated by events of deletions, duplications, and unequal crossing-over that have been documented within an inbred mouse strain (Kumánovics et al., 2002).
LD in dog breeds extends across 0.4 to 3.2 megabases depending on the size of the breed population and breed-specific history (Sutter et al., 2004; Lindblad-Toh et al., 2005). The long regions in LD are homozygous for breed-specific phenotypes, and show a mosaic pattern of short ancestral haplotype blocks that are shared between different breeds (Karlsson et al., 2007). It is expected that disease association studies of DLA haplotypes will be affected by breed-specific long LD blocks as well as the restricted number of breed-specific DLA haplotypes, giving associations with linked but non-causative mutations (Seddon et al., 2010).
In humans, studies targeting the entire HLA region have led researchers to identify associations outside of the classic class II genes DRB1/DRA/DQA1/DQB1. For example, type 1 diabetes mellitus, an autoimmune disease with strong genetic associations to the HLA-DRB1/DQA1/DQB1 genes (Karlsson et al., 2007) has non-HLA risk loci identified by GWA (Wallace et al., 2010) and a meta-analysis of SNPs located across the extended HLA region enabled the identification of genetic associations to four potential causal loci (TCF19,POU5F1, CCHCR1 and PSORS1C1), all within a 56 kb region (Cheung et al., 2011). While it is presumed that strong LD within the HLA is a confounding factor in identifying associated genes outside DRB1/DRA/DQA1/DQB1, this could be overcome, as illustrated in this study, by utilizing novel analyzing algorithms for SNPs across the extended HLA region (Cheung et al., 2011).
While human MHC studies advance to investigate the numerous MHC genes outside of the classic class II, progressing beyond the antigen-presenting peptide-cleft hypothesis, our data suggest that canine MHC association studies would benefit from the same approach. We generated a tool that allows DLA-wide investigation and could be utilized to identify disease associations within and outside DLA-II as well as evaluate DLA diversity in tested populations.
This work was partially funded by grant 1R21AI090277-01/NIH/NIAID, awarded to D.L. Bannasch and by the Center for Companion Animal Health at the University of California, Davis.
Supplementary data associated with this article can be found in the online version at doi: …
1NCBI Data base SNPs: www.ncbi.nlm.nih.gov/projects/SNP/
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriate influence of bias the content of the paper.
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