Increased Genetic Risk Or Protection for Canine Autoimmune Lymphocytic Thyroiditis in Giant Schnauzers Depends on DLA Class II Genotype M

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Tissue Antigens ISSN 0001-2815

Increased genetic risk or protection for canine autoimmune lymphocytic thyroiditis in Giant Schnauzers depends on DLA class II genotype M. Wilbe1, K. Sundberg1,I.R.Hansen1*, E. Strandberg1,R.F.Nachreiner2, A.˚ Hedhammar3, L. J. Kennedy4, G. Andersson1 &S.Bj¨ornerfeldt1†

1 Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden 2 Animal Health Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA 3 Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden 4 Centre for Integrated Genomic Medical Research (CIGMR), University of Manchester, Manchester, UK * Present address: The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden † Present address: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Key words Abstract canine lymphocytic thyroiditis; canis familiaris; disease association; dog; Dogs represent an excellent comparative model for autoimmune thyroiditis as several hypothyroidism; major histocompatibility dog breeds develop canine lymphocytic thyroiditis (CLT), which is clinically simi- complex class II lar to Hashimoto’s thyroiditis in human. We obtained evidence that dog leukocyte antigen (DLA) class II genotype function as either genetic risk factor that pre- Correspondence disposes for CLT or as protective factor against the disease. Genetic diversity at Susanne Bjornerfeldt¨ their DLA-DRB1, -DQA1, and -DQB1 loci were deﬁned and potential association Department of Medical Biochemistry and Microbiology to major histocompatibility complex II haplotypes and alleles was analyzed. Giant Uppsala University Schnauzers carrying the DLA-DRB1*01201/DQA1*00101/DQB1*00201 haplotype BMC showed an increased risk (odds ratio of 6.5) for developing CLT. The same risk Box 582 haplotype has, to date, been observed in three different breeds affected by this dis- SE-751 23 Uppsala ease, Giant Schnauzer, Dobermann, and Labrador Retriever, indicating that it is a Sweden common genetic risk factor in a variety of breeds affected by this disease. Impor- Tel: +46 18 471 4525 tantly, protection for development of the disease was found in dogs carrying the Fax: +46 18 471 4673 e-mail: [email protected] DLA-DRB1*01301/DQA1*00301/DQB1*00501 haplotype (odds ratio of 0.3).

Received 25 May 2009; revised 7 December 2009; accepted 4 January 2010 doi: 10.1111/j.1399-0039.2010.01449.x

Introduction CLT is characterized by progressively deteriorating thyroid function with increased levels of thyroid-stimulating hormone Purebred dogs are particularly prone to develop autoim- (TSH) and the presence of circulating autoantibodies against mune diseases. The selective breeding practice has caused a thyroglobulin (TgAA) (10, 11). Lymphocytic thyroiditis in reduced genetic variation and an increase in homozygosity (1). beagles was shown to be accompanied by destruction of thy- This inbreeding effect may explain the observed increased roid follicles and subsequent hypothyroidism (12). Epidemio- frequency of different autoimmune diseases in various dog logical studies have identiﬁed two working group dog breeds, breeds. Canine breed populations therefore offer an advantage the Giant Schnauzer and Hovawart as high-risk breeds in compared with human populations to identify genes underly- Sweden for hypothyroid disease. The odds ratios for CLT in ing the observed increased risks for developing autoimmune Swedish Giant Schnauzer and Hovawart populations were as diseases (2–4). Autoimmune canine lymphocytic thyroiditis high as 7 and 4, respectively (13). (CLT) is the most common endocrinopathy in dogs with Predisposition to develop CLT is strongly associated with unknown etiology affecting several dog breeds (5). It is a com- major histocompatibility complex (MHC) class II genes. In a plex disease caused by yet unknown predisposing genetic and recent study, DLA-DQ was shown to be a genetic risk factor environmental risk factors (6). The clinical disease phenotype for CLT in Dobermann dogs (3). In human, HLA class II is in is similar to Hashimoto’s thyroiditis (HT) in humans (7–9). fact the most importantly known susceptibility locus for HT

712 © 2010 John Wiley & Sons A/S · Tissue Antigens 75, 712–719 M. Wilbe et al. Susceptibility for or protection against CLT is associated with DLA class II with increased frequency of HLA-DR5 or DR3 in affected Diagnostic procedures family members (14, 15). Furthermore, HLA-DRB1*03 was Diagnostic information concerning the levels of TSH, thy- shown to be linked to the development of HT (16). Functional roxine (T4), and TgAA was available for all dogs included evidence for thyroglobulin as an autoantigen in the context in this study. Enzyme-linked immunosorbent assay (ELISA) of HLA-DRB1*0301 or DRB1*1502 expression and devel- was used to detect TgAA (10, 24). None of the controls but opment of thyroiditis was obtained in transgenic mice (17). a large number of cases were on thyroid hormone replace- Autoantibodies to several thyroid antigens besides Tg have ® ® ment (Levaxin or Forthyrone )atadosageof10–20μg/kg been described (18), and thyroid peroxidase has been impli- bodyweight. cated as a major autoantigen in HT and in a fraction of Diagnostic procedures were performed at the Department hypothyroid dogs (19, 20). of Biomedical Sciences and Veterinary Public Health, division The dog leukocyte antigen (DLA) class II genes DLA- of diagnostic imaging and clinical pathology at SLU, Sweden DRB1, -DQB1, and -DQA1 are highly polymorphic with and at Animal Health Diagnostic Laboratory at the Michigan 106, 62, and 26 alleles, respectively, identified in Canids, State University, MI (23). whereas like in most other mammals, except horse, the Dogs with age or TSH, TgAA, and fT4 levels outside the DLA-DRA gene is indicated to be monomorphic (21). To predetermined diagnostic criteria (cases, controls, and bor- evaluate whether MHC class II genotype is a predisposing derlines; Table 1) were excluded from the study population. genetic risk factor for the development of CLT in Giant Dogs classified as cases or controls were not affected by other Schnauzers, we conducted a comparative sequence analysis polyendocrine diseases, but a few dogs had other presumed of DLA-DRB1, -DQA1, and -DQB1 in affected and con- autoimmune diseases. trol individuals. The dog DLA-DRB1, -DQB1, and -DQA1 genes are considered orthologous to the corresponding human Number of available samples DRB1, DQB1, and DQA1 loci (22). In our study, the DLA-DRB1*01201 allele carried on the DLA-DRB1*01201/ From a total number of 242 Giant Schnauzers, 132 dogs were DQA1*00101/DQB1*00201 haplotype was shown to be a identified as cases, borderline dogs or controls (Table S1) and significant risk allele for developing CLT. A significant could therefore be used in this study. Among these samples, protective effect against development of CLT was also 30 dogs were identified as controls, 74 as CLT-positive cases, found in Giant Schnauzers carrying the DLA-DRB1*01301/ and 28 as borderline dogs, defined by strict inclusion and DQA1*00301/DQB1*00501 haplotype. exclusion criteria (Table 1). Among 49 Hovawarts, 20 dogs were identified as controls, 23 as CLT-positive cases, and 5 as borderline dogs. One dog could not be classified because Materials and methods of missing data (Table 1). Study population PCR amplification Giant Schnauzers born between 1992 and 2007 were sampled μ from a Swedish population, indicated as a high-risk popula- Genomic DNA was extracted from 200 l ethylenediaminete- tion for CLT by cooperation with the breed club and several traacetic acid-blood by using a standard salt extraction veterinary clinics throughout Sweden. All dogs were regis- protocol or the Qiagen QIAamp DNA Blood Mini Kit (Qia- tered in the Swedish Kennel Club (www.skk.se), where all gen, Valencia, CA). information about the dog and their relatives were accessi- PCR fragments containing DLA-DRB1, -DQA1, and -DQB1 ble. Some of the Giant Schnauzers used in this study were exon 2 sequences were amplified using three different primer μ also included in a preceding study of two birth cohorts (23). pairs (Table S2) and 25 ng DNA in a 20 l reaction. This × We also sampled dogs from another high-risk breed, the Hov- included 1 PCR buffer II (Applied Biosystems, Foster City, μ awart, for comparative purposes. CA), 1.5 mM of MgCl2,0.5 M of each primer, 0.2 mM

Table 1 Diagnostic criteria and status of the Giant Schnauzer and Hovawart samples

Diagnostic Number of samples Number of samples code Diagnostic criteria (Giant Schnauzer) (Hovawart) Status

1 TgAA ≥ 200 and TSH ≥ 40 12 6 Case 2 TgAA ≥ 200 20 2 Case 3TSH≥ 40 24 13 Case 4 CLT prior diagnosed 18 2 Case 5 TgAA ≥ 150 and TSH ≥ 30 28 5 Borderline 6TSH≤ 25, TgAA ≤ 130, fT4 ≥ 5, age ≥ 7 years when analyzed 30 20 Control

CLT, canine lymphocytic thyroiditis; fT4, pmol/l; TgAA, thyroglobulin autoantibodies (% compared with a negative control serum); TSH, thyroid- stimulating hormone (mU/l).

© 2010 John Wiley & Sons A/S · Tissue Antigens 75, 712–719 713 Susceptibility for or protection against CLT is associated with DLA class II M. Wilbe et al. dNTP, 0.7 U of AmpliTaq Gold™ (ABI PRISM® BigDye™ heterozygotes, and finally additional haplotypes not found in Terminator Cycle Sequencing Ready Reaction Kits, Original homozygotes were identified. and Version 2.0 Protocol; Applied Biosystems). The PCR pro- ◦ file included an initial denaturation step at 95 C for 15 min, DLA-DRB1, -DQA1, and -DQB1 allele designations followed by 14 touchdown-cycles [30 s of denaturation at ◦ ◦ ◦ The nomenclature for the DLA-DRB1, -DQA1, and -DQB1 95 C, 1 min annealing starting at 62 C (DLA-DRB1), 54 C ◦ ◦ alleles was according to the International Society for Ani- (DLA-DQA1), 73 C (DLA-DQB1) and decreasing 0.5 C each ◦ mal Genetics (ISAG) and as defined by the IPD-MHC cycle, followed by extension at 72 C for 1 min], 20 cycles ◦ database (21, 25, 26). of amplification (denaturation at 95 C for 30 s, annealing at ◦ ◦ ◦ 55 C (DLA-DRB1), 47 C (DLA-DQA1), 66 C (DLA-DQB1) ◦ Ethical considerations for 1 min and extension at 72 C for 1 min), and an additional ◦ extension step of 10 min at 72 C. The final product sizes were Ethical approval for performing this study has been granted 303 bp for DLA-DRB1, 345 bp for DLA-DQA1, and 300 bp by the Ethical board for experimental animals in Uppsala, for DLA-DQB1. Sweden (Dnr C138/6).

Statistical analyses DNA sequencing Statistical analyses were performed using VassarStats (http:// The purified PCR products were sequenced using capillary faculty.vassar.edu/lowry/VassarStats.html) and with the SAS/ electrophoresis on an Applied Biosystems 3100 Genetic ® STAT Software. Haplotypes with frequencies <10% in the Analyzer with Big Dye Terminator V3, used for sequence total material were joined into one haplotype. This led to reactions (Applied Biosystems). The sequencing procedure four haplotypes apart from the joined genotype (consisting was only made in one direction, reverse for DLA-DRB1 and of five rare haplotypes) (Table 2). A logistic regression (Proc DQA1 and forward for DLA-DQB1. Logistic in SAS) was used to test the overall effect of the MHC haplotypes. A 2 × 2 contingency table was used to calculate Data analysis odds ratios, risk ratios, confidence interval, and P-values for each haplotype in Giant Schnauzers. A 99% confidence All nucleotide sequences for DLA-DRB1, -DQA1, and interval was used, which is corrected for the number of -DQB1 were analyzed using MATCHTOOLS and MATCHTOOLS- haplotypes studied. For the χ2 test, Yates values were used, NAVI GATOR (Applied Biosystems). The sequences were first which is corrected for continuity. analyzed using the program MATCHTOOLSNAVI GATOR ,bycom- paring each sequence to an already prepared consensus sequence containing all known polymorphic sites. Each Results polymorphic site was analyzed manually. The corrected sequence was then matched to a reference sequence library Genetic polymorphism at the DLA-DRB1, -DQA1, and DQB1 loci (http://www.ebi.ac.uk/ipd/mhc/index.html) using MATCHTOOLS to provide the closest matching allele. Haplotypes were first The genetic complexity at exon 2 of the DLA-DRB1, -DQA1, identified in homozygous dogs, and then assigned in the and -DQB1 loci was assessed in both Giant Schnauzer and

Table 2 DLA-DRB1, -DQA1, and -DQB1 haplotype frequencies among all Giant Schnauzer samples, and for the study population (cases and controls)

Haplotype Cases and borderlinesc Controls (30 DRB1*DQA1*DQB1* All samples (132 dogs) % (na) Casesb (74 dogs) % (na) (102 dogs) % (na) dogs) % (na)

*00101*00101*00201 25.0 (66) 29.1 (43) 26.5 (54) 20.0 (12) *01201*00101*00201 15.2 (40) 18.2 (27) 18.6 (38) 3.3 (2) *00601*00401*01303 17.0 (45) 15.5 (23) 16.2 (33) 20.0 (12) *01301*00101*00201 7.2 (19) 8.8 (13) 7.4 (15) 6.7 (4) *01301*00301*00501 22.0 (58) 15.5 (23) 18.1 (37) 35.0 (21) *02301*00301*00501 8.7 (23) 8.1 (12) 8.8 (18) 8.3 (5) *00901*00101*008011 2.3 (6) 2.0 (3) 2.0 (4) 3.3 (2) *01501*00601*02201 1.9 (5) 1.4 (2) 1.5 (3) 3.3 (2) *02001*00401*01301 0.8 (2) 1.4 (2) 1.0 (2) 0 (0)

TgAA, thyroglobulin autoantibodies; TSH, thyroid-stimulating hormone. aTwo chromosomes per individual. bTgAA ≥ 200 and/or TSH ≥ 40. cTgAA ≥ 150 and/or TSH ≥ 30. Bold numbers indicate differences between cases and controls where statistical tests were performed.

Table 3 DLA-DRB1, -DQA1, and -DQB1 allele frequencies among all Evaluation of genetic predisposition for CLT Giant Schnauzer samples and the study population (cases and controls) To assess the potential genetic association between MHC Cases and class II genotype and CLT, the DLA-DRB1, -DQA1, and All samples (132 Casesb (74 borderlinesc (102 Controls (30 -DQB1 haplotypes and allele frequencies were calculated and a a a 3 Allele dogs) % (n ) dogs) % (n ) dogs) % (n ) dogs) % (n ) compared between cases and controls (Tables 2 and 3). No DRB1 significant difference between gender among the CLT-positive 00101 25.0 (66) 29.1 (43) 26.5 (54) 20.0 (12) and negative dogs was observed. Nor did haplotype and allele 01201 15.2 (40) 18.2 (27) 18.6 (38) 3.3 (2) frequencies diverge between males and females. 00601 17.0 (45) 15.5 (23) 16.2 (33) 20.0 (12) In Giant Schnauzer, the haplotype effect was significant 01301 29.2 (77) 24.3 (36) 25.5 (52) 41.7 (25) in the logistic regression (likelihood ratio test, P = 0.014 02301 8.7 (23) 8.1 (12) 8.8 (18) 8.3 (5) 00901 2.3 (6) 2.0 (3) 2.0 (4) 3.3 (2) and 0.027 when excluding or including borderline dogs, 01501 1.9 (5) 1.4 (2) 1.5 (3) 3.3 (2) respectively). The frequencies of two specific haplotypes dif- 02001 0.8 (2) 1.4 (2) 1.0 (2) 0 (0) fered markedly between cases and controls. The DLA-DRB1 DQA1 *01201/DQA1*00101/DQB1*00201 haplotype was found in 00101 49.6 (131) 58.1 (86) 54.4 (111) 33.3 (20) 3.3% of the controls and in 18.2% of the cases (Table 2), 00401 17.8 (47) 16.9 (25) 17.2 (35) 20.0 (12) and the DLA-DRB1*01301/DQA1*00301/DQB1*00501 hap- 00301 30.7 (81) 23.6 (35) 27.0 (55) 43.3 (26) 00601 1.9 (5) 1.4 (2) 1.5 (3) 3.3 (2) lotype was found in 35.0% of the controls and 15.5% of the DQB1 cases (Table 2). The odds ratio from the logistic regression 00201 47.3 (125) 56.1 (83) 52.2 (107) 30.0 (18) was 12.3 and 10.8 for the former versus the latter haplotype 01303 17.0 (45) 15.5 (23) 16.2 (33) 20.0 (12) (99.5% CI 1.3–114 and 1.2–95) when excluding or including 00501 30.7 (81) 23.6 (35) 27.0 (55) 43.3 (26) borderline dogs as cases, respectively. 008011 2.3 (6) 2.0 (3) 2.0 (4) 3.3 (2) From the contingency table, the risk for CLT development 02201 1.9 (5) 1.4 (2) 1.5 (3) 3.3 (2) associated with the DLA-DRB1*01201/DQA1*00101/DQB1 01301 0.8 (2) 1.4 (2) 1.0 (2) 0 (0) *00201 haplotype was estimated, showing an odds ratio of TgAA, thyroglobulin autoantibodies; TSH, thyroid-stimulating hormone. 6.5 (99% CI 1.1–136). Thus, the increased frequency of a Two chromosomes per individual. this haplotype in cases identifies MHC class II as a genetic b ≥ ≥ TgAA 200 and/or TSH 40. risk factor for CLT in Giant Schnauzer (Table 4) and also cTgAA ≥ 150 and/or TSH ≥ 30. shows an increased genetic predisposition in dogs carrying this Bold numbers indicate differences between cases and controls where statistical tests were performed. haplotype. By including borderline dogs, the haplotype was found in 18.6% of the population, giving an odds ratio of 6.6 (99% CI 1.2–137) (Table 4). Furthermore, the allele frequencies of DLA-DRB1*01201, DLA-DQA1*00101, and DLA- DQB1*00201, all found in the risk haplotype, also have higher Hovawart. A total of nine DLA-DRB1, -DQA1, and -DQB1 frequencies in dogs with CLT compared with the controls haplotypes were identified in Giant Schnauzer (Table 2). when comparing all allele frequencies separately (Table 3). Among these nine haplotypes, eight DLA-DRB1 alleles, DLA-DRB1*01201 was found in 3.3 % of the controls and four DLA-DQA1 alleles, and six DLA-DQB1 alleles were in 18.2% of the cases with an odds ratio of 6.5 (99.5% CI recognized (Table 3). The four most common haplotypes 1.0–194) (Table 5), whereas DLA-DQA1*00101 was found had frequencies between 15.2% and 25.0%, whereas the in 33.3% of the controls and 58.1% of cases with an odds ratio other five haplotypes were rare and ranged in frequencies of 2.8 (99.5% CI 1.1–6.8), and finally DLA-DQB1*00201 between 0.8% and 8.7%. Genetic polymorphism at the DLA- was found in 30.0% of the controls and in 56.1% of DRB1 locus was markedly different between Giant Schnauzer the cases with an odds ratio of 3.0 (99.5% CI 1.2–7.5) and Hovawart. Only six DLA-DRB1 alleles were found in (Table 5). However, DLA-DRB1*01201 was only found in Hovawart and DLA-DRB1*01201 had a total allele fre- the risk haplotype, whereas both DLA-DQA1*00101 and quency of 48% (Table S3), whereas the additional five alle- DLA-DQB1*00201 were found in multiple haplotypes. This les were rare or moderately frequent with allele frequencies indicates that allele DLA-DRB1*01201 may be critical for ranging between 1.0% and 18.4%. In contrast, four DLA- causing the increased genetic predisposition to develop CLT. DRB1 alleles with high allele frequencies were found in A significant protective effect of the DLA-DRB1*01301/ Giant Schnauzer. Three of the identified DLA-DRB1 alle- DQA1*00301/DQB1*00501 haplotype was shown in Giant les (DRB1*01201, DRB1*01301, and DRB1*01501), as well Schnauzers, by a 19.5% decrease in frequency between cases as three of the identified DLA-DQA1 alleles (DQA1*00101, and controls (controls: 35.0%; cases: 15.5%; Table 2), an DQA1*00401, and DQA1*00601), and only one DLA- odds ratio of 0.3 (99% CI 0.14–0.85) (Table 4). Including DQB1 allele (DQB1*00201) were shared between the two borderline dogs, this haplotype still showed significance breeds. (controls: 35.0%; cases: 18.1%; Table 2) with an odds ratio of

Table 4 Number of MHC class II risk haplotypes/protective haplotypes in casesa,b and controls for the Giant Schnauzer dogs, showing odds ratio, risk ratios, P-values, and conﬁdence intervals (CI) (99%)c

Haplotype DRB1* DQA1*DQB1* Absent Present χ2P-value adj Odds ratio (99% CI)

Risk haplotype *01201 *00101*00201 Casesa 121 27 Controls 58 2 0.048 1.1 < 6.5 > 136 Casesb 166 38 Controls 58 2 0.035 1.2 < 6.6 > 137 Protective haplotype *01301 *00301*00501 Casesa 125 23 Controls 39 21 0.017 0.14 < 0.34 > 0.85 Casesb 167 37 Controls 39 21 0.047 0.18 < 0.41 > 0.95

MHC, major histocompatibility complex; TgAA, thyroglobulin autoantibodies; TSH, thyroid-stimulating hormone. aTgAA ≥ 200 and/or TSH ≥ 40. bTgAA ≥ 150 and/or TSH ≥ 30. cAsymptotic tests were used except when few observations in a cell (<5), then Fisher’s exact test was used.

Table 5 Number of MHC class II risk alleles/protective alleles in casesa,b and controls for the Giant Schnauzer dogs, showing odds ratio, risk ratios, P-values, and conﬁdence intervals (CI) (99.5%)c

Haplotype DRB1* DQA1*DQB1* Absent Present χ2 P-value Odds ratio (99.5% CI)

DRB1*01201 Casesa 121 27 Controls 58 2 0.11.0< 6.5 > 194 Casesb 166 38 Controls 58 2 0.07 1.08 < 6.6 > 195 DRB1*01301 Casesa 112 36 Controls 35 25 0.20.18< 0.45 > 1.1 Casesb 152 52 Controls 35 25 0.20.20< 0.48 > 1.13 DQA1*00101 Casesa 62 86 Controls 40 20 0.02 1.13 < 2.8 > 6.8 Casesb 93 111 Controls 40 20 0.06 1.01 < 2.4 > 5.67 DQA1*00301 Casesa 113 35 Controls 34 26 0.08 0.16 < 0.40 > 1.01 Casesb 149 55 Controls 34 26 0.20.21< 0.48 > 1.14 DQB1*00201 Casesa 65 83 Controls 42 18 0.01 1.2 < 3.0 > 7.5 Casesb 97 107 Controls 42 18 0.04 1.06 < 2.6 > 6.2 DQB1*00501 Casesa 113 35 Controls 34 26 0.08 0.16 < 0.40 > 1.01 Casesb 149 55 Controls 34 26 0.20.21< 0.48 > 1.14

0.4 (99% CI 0.18–0.95) (Table 4). In contrast, the protective 25% in the US population, has been reported to have a higher effect was not detected when comparing the alleles separately than expected incidence of hypothyroidism (32). (Table 5). The alleles, DLA-DRB1*01301, DQA1*00301, and The current study shows association between MHC class II DQB1*00501, in the protective haplotype do not show any and autoimmune thyroid disease in dogs, indicating that significant difference between cases and controls separately DLA-DRB1 is the crucial genetic risk factor. The risk and all are also present in other haplotypes with no obvious allele DLA-DRB1*01201 is frequently found among sev- protective effect. Combined, these data argue more strongly eral different dog breeds prone to develop CLT (Table S4). for a protective haplotypic effect rather than a protective allelic A further argument for being a single allelic effect, rather effect against development of CLT in dogs. than a haplotypic effect, is the fact that the DLA-DQA1 and DQB1 alleles found in the risk haplotype in Giant Schnauzers are also found in other haplotypes not giving Discussion an increased risk for CLT. In contrast, the protective haplo- CLT is a major problem in several dog breeds (27). The type DLA-DRB1*01301/DQA1*00301/DQB1*00501 found particularly high incidence in certain breeds, including Giant in Giant Schnauzers is consistent with a haplotypic effect, Schnauzer and Hovawart (23), strongly suggests a genetic because DLA-DRB1*01301 is found in other haplotype com- predisposition in these breeds. The mode of inheritance for binations with no protective effect. CLT is likely to be complex as in other autoimmune diseases In a recent study, Kennedy et al. reported that DLA- with strong but unknown environmental factors and several DQA1 was a risk factor for CLT in Dobermann (3). How- genetic risk factors. Evidence for DLA-DRB1 as a risk factor ever, in our study the association between MHC and CLT in was obtained at the genotype level. Furthermore, a particular Giant Schnauzer was evaluated by determining both haplotype DLA class II haplotype was shown to be a protective factor and allele frequencies. Odds ratios at significant levels were against the disease. obtained for DLA-DRB1*01201, implicating it as the most important MHC class II locus predisposing for CLT. With- out knowledge whether homozygotes versus heterozygotes Polymorphism at the DLA-DRB1 locus in Giant manifest with different disease severity, it is recommended Schnauzer versus Hovawart to perform both haplotype and allele analysis (2). The Swedish Giant Schnauzer population appears to be mod- The role of DR versus DQ or both as genetic risk erately genetically diverse with four DLA-DRB1 alleles in factors in CLT disease etiology in different dog breeds relatively high frequencies and with additional five rare alle- remains to be defined by functional and clinical studies. les, whereas Hovawart appears to exhibit lower genetic vari- The immunological mechanisms for the involvement of MHC ation at the MHC. This breed variation and the number class II in autoimmune thyroid disease have been extensively of DLA-DRB1 alleles are expected and within the range reviewed and clearly involve disturbances of self-tolerance of MHC polymorphisms reported earlier for different dog because of MHC class II-dependent antigen presentation. breeds (28–31). The major difference in MHC polymorphism Other genetic risk factors besides MHC class II may also between these two breeds was the fact that a CLT-risk hap- be crucial for developing CLT. Further identification of addi- lotype DLA-DRB1*01201/DQA1*00401/DQB1*013017 con- tional genetic risk factors for CLT will be performed by taining the same DLA-DRB1*01201 allele found in the Giant genome-wide association analysis using the recently devel- Schnauzer risk haplotype was present at a haplotype frequency oped dog SNP array (33). In summary, we have identi- of 48% among the Hovawart dogs analyzed. This high fre- fied DLA-DRB1*01201 as a major risk allele for CLT quency may explain the high prevalence (13%) of CLT in this development in Giant Schnauzers and we also identified breed. DLA-DRB1*01301/DQA1*00301/DQB1*00501 as a protec- The presence of this DLA-DRB1 allele has recently been tive haplotype. Furthermore, by including the 28 Giant correlated with the development of hypothyroid disease in Schnauzers that were classified as borderline dogs among the Dobermanns (3). In that report, the DLA-DRB1*01201 was cases (TSH levels between 30 and 39 mU/l and TgAA con- reported to be found on a rare haplotype. Available DLA geno- centration between 150% and 199% compared with a negative type data from the 14th International Histocompatiblity work- control serum), we achieved even stronger support for the shop show that haplotypes with DLA-DRB1*01201 are found risk allele. If these borderline dogs instead were included in 16 different breeds. In seven of these breeds, the number of among the controls, the distribution of the risk allele did dogs was sufficient to obtain a likely percentage of dogs with not differ significantly between cases and controls (data not DLA-DRB1*01201 haplotypes (Table S4) (31). The present shown). Therefore, inclusion of borderline dogs among the study adds two more breeds belonging to the working dog cases probably more correctly reflects the prevalence of CLT group, which contains the DLA-DRB1*01201 allele. This is in the Giant Schnauzer population. a quite common allele in Giant Schnauzer (15.2%). Moreover, Combined with future studies aiming at identifying addi- Golden Retriever, with a frequency of DLA-DRB1*01201 at tional genetic risk and protective factors for CLT in dogs,

© 2010 John Wiley & Sons A/S · Tissue Antigens 75, 712–719 717 Susceptibility for or protection against CLT is associated with DLA class II M. Wilbe et al. knowledge from the current study can assist in breeding prac- insured dogs in Sweden during 1995 and 1996. Vet Rec 2000: tices aiming to reduce the frequency of CLT in high-risk dog 146: 519–25. breeds and also be of comparative value in understanding 14. Roman SH, Greenberg D, Rubinstein P, Wallenstein S, human hypothyroiditis. Davies TS. Genetics of autoimmune thyroid disease: lack of evidence for linkage to HLA within families. J Clin Endocrinol Metab 1992: 74: 496–503. Acknowledgments 15. Ban Y, Davies TF, Greenberg DA, Concepcion ES, Tomer Y. The inﬂuence of human leucocyte antigen (HLA) genes on Funding for these studies was provided by FORMAS, AGRIA autoimmune thyroid disease (AITD): results of studies in Research fund, the European Commission (FP7-LUPA, GA- HLA-DR3 positive AITD families. 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29. Kennedy LJ, Barnes A, Happ GM et al. Extensive interbreed, Table S1 Diagnostic and clinical information for all Giant but minimal intrabreed, variation of DLA class II alleles and Schnauzer and Hovawarts in the study. haplotypes in dogs. Tissue Antigens 2002: 59: 194–204. Table S2 Primer pairs used for ampliﬁcation of DLA-DRB1/ 30. Seddon JM, Ellegren H. MHC class II genes in European DQA1/DQB1 (1). wolves: a comparison with dogs. Immunogenetics 2002: 54: Table S3 DLA-DRB1, -DQA1, and -DQB1 haplotype 490–500. frequencies for all sampled Hovawarts. 31. Angles JM, Kennedy LJ, Pedersen NC. Frequency and distribution of alleles of canine MHC-II DLA-DQB1, Table S4 The percentage of dog breeds with DRB1*01201 DLA-DQA1 and DLA-DRB1 in 25 representative American haplotypes or alleles (2). Kennel Club breeds. Tissue Antigens 2005: 66: 173–84. Please note: Wiley-Blackwell is not responsible for the 32. Milne KL, Hayes HM Jr. Epidemiologic features of canine content or functionality of any supporting materials supplied hypothyroidism. Cornell Vet 1981: 71: 3–14. by the authors. Any queries (other than missing material) 33. Karlsson EK, Baranowska I, Wade CM et al. Efﬁcient should be directed to the corresponding author for the article. mapping of mendelian traits in dogs through genome-wide association. Nat Genet 2007: 39: 1321–8.

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