genetic characterization of healthy and sebaceous adenitis ... · some standard poo-dles also...

Tissue Antigens ISSN 0001-2815

Genetic characterization of healthy and sebaceous adenitisaffected Standard Poodles from the United Statesand the United KingdomN. C. Pedersen1,2,3, H. Liu1,3, B. McLaughlin4 & B. N. Sacks2,5,6

1 Center for Companion Animal Health, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA2 Veterinary Genetics Laboratory (VGL), School of Veterinary Medicine, University of California – Davis, Davis, CA, USA3 Koret Foundation Center for Veterinary Genetics, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA4 Animal Health Trust, Lanwades Park Kentford, Newmarket, Suffolk, UK5 Canid Diversity and Conservation Unit, VGL, University of California – Davis, Davis, CA, USA6 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California – Davis, Davis, CA, USA

Key words

dog leukocyte antigen; genetic association;genetic diversity; matrilines; patrilines;sebaceous adenitis; Standard Poodle

Correspondence

Niels C. PedersenCenter for Companion Animal HealthUniversity of California – DavisOne Shields AvenueDavisCA 95616USATel: +1 530 752 7402Fax: +1 530 752 7701e-mail: [email protected]

Received 11 January 2012; revised 8 March2012; accepted 26 March 2012

doi: 10.1111/j.1399-0039.2012.01876.x

Abstract

The degree of heterogeneity associated with geographic origin and sebaceous adenitis(SA) status in Standard Poodles from the United States (US) and the United Kingdom(UK) was assessed. Healthy and SA-affected Standard Poodles from the US andthe UK shared a major mitochondrial DNA (mtDNA) haplotype and a single Ychromosome haplotype. However, minor mtDNA haplotypes and frequencies weresomewhat different between US and UK dogs and were significantly less associatedwith SA than major haplotypes across both populations. The US and UK populationsexhibited recent divergence from a common gene pool, based on allele frequenciesof 24 highly polymorphic short tandem repeats and principle coordinates and clusteranalyses of genotype frequencies. However, there was no differentiation between SAaffected and unaffected dogs. Over 90% of US and UK Poodles shared a commondog leukocyte antigen (DLA) class II haplotype, but showed some differentiation inminor haplotype frequency. No difference was observed in haplotype heterozygositybetween SA affected and unaffected dogs from the same country and no diseaseassociation for SA was found within the DLA region by a high density singlenucleotide polymorphism (SNP) scan. Zygosity mapping in the DLA region ofPoodles indicated much lower site-specific diversity than in an outbred population ofstreet dogs from Bali, Indonesia, reflecting the degree that breed associated historicalbottlenecks have reduced diversity in a polymorphic region of the genome. This studyshows possible pitfalls in more extensive genome-wide association studies, such ascase and control numbers, population stratification, the involvement of multiple genes,and/or the possibility that SA susceptibility is fixed or nearly fixed within the breed,which can reduce power to detect genetic associations.

Introduction

The Kennel Club of United Kingdom registered its first Poodlein 1874, and the Poodle Club of United Kingdom was foundedin 1876. Poodles did not gain their current popularity in theUK until after World War II. The American Kennel Club(AKC) registered its first Standard Poodle in 1886 and thePoodle Club of America (PCA) was initially conceived in1896 and reorganized in its present form in 1934. The breedwas slow to gain popularity in the United States (US) and therewere only about 34 Poodles registered by the AKC in the early

1930s. In 1935, a white Standard Poodle won Best in Show atthe Westminster Kennel Club show in New York. This causedthe Poodle’s popularity to soar and it became the number onedog registered by the AKC for almost a quarter century.

The Standard Poodle is known for its temperament, intel-ligence, and outstanding coat. However, as with most purebreeds, it has its own set of health problems. The PoodleHealth Registry database lists over 50 major health disordersof Standard Poodle (http://www.poodlehealthregistry.org). Tenof these disorders are of an autoimmune nature, includingsebaceous adenitis (SA), hypoadrenocorticism (Addison’s

© 2012 John Wiley & Sons A/S 1

Sebaceous adenitis in Standard Poodles N. C. Pedersen et al.

disease), immune-mediated hemolytic anemia (IMHA),chronic active hepatitis, diabetes mellitus (type I), immune-mediated thrombocytopenia (IMTP), masticatory myositis,systemic lupus erythematosus (SLE), discoid lupus erythe-matosus, symmetrical lupoid onychodystrophy, and hypothy-roidism (thyroiditis). A survey conducted in 2010/2011 bythe PCA Foundation with over 800 respondents placed theprevalence of SA among Standard Poodles at 2.7%, hypoad-renocorticism 2.5%, hypothyroidism/thyroiditis 1.8%, IMHA1.0%, chronic active hepatitis 0.7%, and IMTP 0.3% (PCAFoundation, unpublished information). Some Standard Poo-dles also suffer more than one autoimmune disorder duringtheir lifetimes, e.g. SA and hypoadrenocorticism, or hypothy-roidism and either SA or hypoadrenocorticism. Assuming thatthe majority of Standard Poodles suffer from only one autoim-mune disease, the overall prevalence of autoimmune diseasebased on survey results from US Standard Poodles wouldbe approximately 9%, a significant health problem. SA andhypoadrenocorticism disease are the two autoimmune dis-eases of greatest concern to the breed – hypoadrenocorticismbecause of its life threatening nature and cost of long-termtreatment, and SA because of its potential effects on the coat,one of the most praised phenotypic features of the breed.

SA in dogs was first described in detail by Scott (1). Thedisease has been reportedly recognized in a number of purebreeds of dogs (2), but is most prevalent in Akitas (2, 3),Standard Poodles (3, 4), English Springer Spaniels (3), andHavanese (5). The disorder is characterized by hair loss begin-ning on the ears, head, and back and progressing to thetrunk. Detailed histopathologic and immunohistopathologicdescriptions of lesions of SA have been reported by Scott (1),Reichler et al. (2), Gross and colleagues (6), and Rybniceket al. (7). A mixed inflammatory response targets sebaceousglands, leading to their eventual disappearance. The inflamma-tory response often subsides at this point, leaving in its wakeperifollicular fibrosis, follicular plugging, and occasional indi-cations of sebaceous gland regeneration. Hair loss, excessivedandruff, and occasional secondary pyoderma are outwardmanifestations of the underlying disease. The disease andassociated hair loss can evolve slowly or rapidly, localizedor generalized in nature, and progressive or regressive overtime. A subclinical form also exists, wherein biopsies showcharacteristic inflammation centered on sebaceous glands butwithout outward signs of disease of the coat. Therefore, thereis not always a direct relationship to histologic lesions andoutward clinical signs (2).

Dunstan and Hargis (8) suggested that SA follows an auto-somal recessive mode of inheritance, but no heritability stud-ies have been published. Moreover, the patterns of diseaseoccurrence and variable age at onset in related individualsdo not follow the pattern of a simple recessive trait. Pre-liminary genome-wide association study (GWAS) carried outin the UK on 20 SA affected and 28 healthy Standard Poo-dles using moderately dense single nucleotide polymorphism

(SNP) arrays (22,362 SNPs) failed to show an associationbetween any region of the genome and the disease (9). How-ever, this study was admittedly underpowered.

Although whole genome scanning has so far failed to showa genetic basis for SA in Standard Poodles, breeders oftenassociate disease risk with certain matings and bloodlines.There is also a tendency to associate many disease problemsof pure breed dogs to excessive inbreeding, which is usedto create more phenotypically desirable specimens or blood-lines (10). Indeed, autoimmune disorders occur disproportion-ately in pure breeds and frequently associate with specific dogleukocyte antigen (DLA) class II haplotypes, especially whenthey are in the homozygous state (reviewed in Refs 11, 12).Human autoimmune disorders also tend to associate with classII genes of the human leukocyte antigen complex, as well aswith genes controlling T-cell regulation, and genes involvedwith the production of immunoglobulins (13).

As a prelude to more extensive GWAS, this study wasfocused on analyzing genetic diversity both within andbetween Standard Poodle populations in the US and the UK.Maternal mitochondrial DNA (mtDNA) and Y SNP/singletandem repeat (STR) haplotypes for the breed were deter-mined. Genetic diversity and population structure, as deter-mined from a panel of 24 highly polymorphic STR markersspread across 20 chromosomes, were then compared in SAaffected and healthy Standard Poodles from both countries. Asearch was also made for associations with SA in the DLAregion, including the DLA class II genes. A high density asso-ciation study was conducted for the entire DLA region as partof a larger collaborative GWAS using Illumina canine 172KSNP arrays.

Materials and methods

Standard Poodle case and control samples

The collection of case material was accomplished in collabora-tion with breeders and owners of healthy or biopsy confirmedSA-affected Standard Poodles. One hundred forty-nine Stan-dard Poodles from the US and 84 dogs from the UK enrolledin the study. Forty-nine dogs from the US and 23 dogs fromthe UK suffered from SA. Unaffected Standard Poodles fromboth the US and the UK ranged in age from 0.4–15 years,with a mean age of 6.9 years in US Poodles, and 6.4 years indogs from the UK. The female to male ratio was 1.4:1 (US)and 1.67:1 (UK). Standard Poodles from the US and the UKwith a known history of SA ranged in age from 1.7–15 years(mean 7.1 for US dogs and 4.9 for UK dogs) and the femaleto male ratio was 1.25 for US dogs and 0.91:1 for UK dogs.

DNA containing samples were collected as either 2–5 mlEDTA blood (US dogs) or air dried buccal swabs using cyto-logical brushes (UK dogs). Blood samples were sent imme-diately by priority mail and without refrigeration. Sampleswere accompanied by a questionnaire that included the dog’s

2 © 2012 John Wiley & Sons A/S

N. C. Pedersen et al. Sebaceous adenitis in Standard Poodles

registered name and number, and other vital statistics. Recordsor statements of SA biopsy results were included along withother pertinent health information.

Pedigrees of all dogs included in the study were screenedfor relatedness to three generations (i.e. to grandparents).Pedigrees were either submitted with the sample or down-loaded from the AKC and the Kennel Club UK websites.After removing dogs related to the level of grandparents,107/149 dogs remained from the US (36 SA affected and71 unaffected) and 52/84 from the UK (13 affected and 39unaffected). Therefore, 72% of dogs from the US and 62%from the UK, regardless of SA status, were unrelated throughthe level of grandparents.

Analyses were performed on randomly related dogs (notscreened for relatedness), and on the unrelated subset ofthese dogs (sharing no common relative through the levelof grandparents). All analyses were performed on both thefull set of randomly related dogs as well as unrelated dogs.However, qualitative results were the same regardless ofdegree of relatedness. Therefore, only analyses based on thefull data set of randomly related dogs were presented in mostcomputations.

Indigenous Bali street dog samples

DNA was extracted from buccal swabs from 26 randomlyselected indigenous dogs from the streets of Bali (14). DNAwas then tested using high density SNP arrays and SNPsfrom the DLA region mined from the whole genome dataas described below. Bali street dogs are ancient descendentsof dogs migrating from Southeast Asia (14) and maintain thebroad genetic diversity of their ancestors (14, 15).

DNA extraction

DNA was extracted from whole EDTA blood or cytologicalbrushes using Qiagen Gentra Puregene Blood Kit (Qiagen,Valencia, CA) according to the manufacturer’s instructions.

Determination of paternal and maternal haplotypes

Y chromosome haplotypes were determined for 17 SAaffected and 48 unaffected male Standard Poodles from theUnited States and 12 SA affected and 29 unaffected fromthe UK using a panel of 11 Y SNPs (16). The SNPs wereassayed using a Sequenom MassARRAY Compact 96 usingiPLEX Gold technology (Sequenom, San Diego, CA). Primersequences for Y SNPs were previously reported (16). Ninety-one male Standard Poodles from the United States, including30 SA affected and 61 unaffected dogs, were also tested witha panel of seven Y-STR markers, including MS34A, MS34B,MS41A, MS41B, 990.35.4, 650.79.2, and 650.79.3. Primersequences and allele sizes have been previously reported (17).

mtDNA haplotypes were determined for 28 SA affectedand 75 unaffected Standard Poodles from the US and 23 SA

affected and 58 unaffected dogs from the UK by sequencing655 bp of the mitochondrial control region (nt 15452–16107)as described by Vila et al. (18). Primer sequences, condi-tions for polymerase chain reaction (PCR), cleaning of PCRproducts, and sequencing were as described by Pedersenet al. (12). Sequencing was conducted in both directions foreach sample and analyzed using SeqMan software (DNAS-TAR Lasergene 8, DNASTAR, Madison, WI). Final sequenceswere compared with a large canine forensic database providedby Beth Wictum, Director of Veterinary Forensics, Veteri-nary Genetics Laboratory (VGL), UC Davis, and the GenBanknucleotide database.

Genetic diversity using STR markers

Twenty-four STRs located on 20 different autosomes wereused in the study. An additional marker for amelogenin(AMELX and AMELY ) was added for confirmation of gender.Primers, dye labels, and multiplexes for 22 of these mark-ers have been published as part of the 2005 InternationalSociety for Animal Genetics canine panel for parentage ver-ification (www.isag.us/Docs/2005ISAGPanelDOG.pdf). Thispanel was augmented by three additional STRs, FH2001,FH2328, and LE1004. Repeat motif, chromosome assign-ment, known allele numbers, and allele size range for thisset of markers have been previously reported (12). STR-basedgenotyping was conducted by the VGL, UC Davis, and dataanalyses conducted using GeneMapper v3.7 (Applied Biosys-tems, Carlsbad, CA).

DLA class II genotyping

Alleles of the DLA class II genes, DRB1, DQA1, and DQB1,were determined for 47 SA affected and 90 unaffected Stan-dard Poodles from the US and 23 affected and 61 unaffectedPoodles from the UK by sequence-based typing using pub-lished locus-specific intronic primers (19, 20). PCR reactions,purification of PCR products, and sequencing procedures havebeen previously described (12).

Vector NTI advance™ software (Invitrogen, Carlsbad, CA)was used for alignment of sequence data. DLA class II alleleswere determined by a subtractive approach (19, 21), whereinhomozygous alleles were identified at each locus, and usedalongside published allele sequences to identify heterozygousalleles. Haplotypes were identified by consistent patterns oflinkage within a number of individuals of the same breed.Allele nomenclature was determined from known sequencesin GenBank and in the Immune Polymorphism Database(http://www.ebi.ac.uk/ipd).

DLA-wide SNP typing

DNA from 34 SA affected and 24 unaffected dogs fromthe US, and 23 SA affected and 16 unaffected dogs fromthe UK were tested on CanineHD Genotyping BeadChips



(Illumina, San Diego, CA). Data from 150 SNP markersoverlapping the DLA region (base 3802975–5672682) ofCanis familiaris autosome 12 (CFA12) were extracted fromthe GWAS. Thirty five of these SNPs were discarded for beingmonomorphic, leaving usable data from 115 SNPs across theentire DLA region. Genome and DLA-wide SNP associationswere determined by PLINK analysis with MAF >0.05, callrate >90%, and 50,000 permutations (20).

Data analysis

Haplotype frequencies (mtDNA and DLA) between US andUK populations and between affected and unaffected StandardPoodles were compared using chi-square tests of indepen-dence, with rarer haplotypes pooled to ensure that <20%of expected cell frequencies were <5 cases (22). Calcula-tion of descriptive statistics, expected (He) and observed(Ho) heterozygosity, and tests of Hardy–Weinberg equilib-rium were performed using Arlequin v3.1 (23), as were coef-ficients of inbreeding (FIS) within populations and fixationindices (FST) between populations. Tests for gametic (link-age) disequilibrium were performed using Genepop on theWeb (v 4.0.10) (24). Sequential Bonferroni adjustments wereapplied to P values to avoid inflation of type I errors due torepeated performance of Hardy–Weinberg and gametic equi-librium tests (25). Because numbers of individuals differedbetween US and UK samples, a rarefaction procedure per-formed in program HP-Rare was used to effectively equalizesample sizes for these estimates based on the lowest num-bers of genes sampled from any population and locus (26).Statistical comparison of averages across loci was based on95% confidence intervals (CIs) calculated from the Z distribu-tion (22). Principle coordinate analysis (PCoA) was performedusing GenAlex v6.41 (27).

As a blind genetic clustering approach, a Bayesian model-based method that uses genotype frequencies but no priorinformation on population of origin was implemented in Struc-ture v. 2.0, to assess substructure within the data set (28, 29).The admixture model with correlated allele frequencies wasused. Runs of 20,000 MCMC cycles (first 10,000 discarded asburn-in) were conducted for numbers of clusters (K ) ranging1–6 to assess how many clusters explained most of the devi-ation from Hardy–Weinberg and gametic equilibrium in thedata set. This was indicated by the logarithm of the probabilityof the data given an assumed number of clusters.

Results

Seven distinct mtDNA haplotypes were identified among 103randomly related Standard Poodles (28 SA affected and 75unaffected) from the US and 81 Poodles (23 SA affected and58 unaffected) from the UK (Table 1). Heteroplasmy betweenmtDNA haplotype A and G was observed in three dogs andthey were excluded from the study. All of the matrilines,

except for F and G, were identified in the VGL forensic dogdatabase. GenBank accession numbers corresponding to theseven mtDNA haplotypes identified in this study are given inTable 1.

Mitochondrial haplotype diversity (1 – sum of squared fre-quencies) (30) was slightly higher in the US (0.47) than theUK (0.41) dogs, although haplotype frequencies were not sig-nificantly different between the two countries (FST = 0.019;χ2 = 2; df = 0.17; P = 0.92). Therefore, dogs from the twocountries were pooled for comparison. Haplotype frequen-cies differed significantly between unaffected and affecteddogs (FST = 0.160; χ2 = 2; df = 6.3; P = 0.04), includinga twofold difference in haplotype diversity between unaf-fected (0.51) and SA-affected (0.25) dogs. This difference waslargely characterized by a greater frequency of the most com-mon haplotype in the SA-affected dogs and minor haplotypesin unaffected dogs (Table 1).

The 106 SA affected and unaffected dogs from the US andthe UK shared a single Y-SNP haplotype (AGAAGACCTCC),which is found in village dog populations from across south-east Asia, a region to which most modern breeds trace theirancestry (15). Ninety one of the SA affected and unaffectedmale dogs from the US and the UK tested possessed the iden-tical Y-STR haplotype 6p (D1D5) which is the most commonY-STR haplotype among breed dogs (17) (Table 2).

All 24 autosomal STRs were polymorphic in both USand UK Standard Poodles, yielding 172 alleles. The aver-age (across loci) observed heterozygosity (Ho = 0.576) wassignificantly (P < 0.0001) lower than average expected het-erozygosity (He = 0.622), indicating substructure within thetotal sample (Table 3). Allele frequencies differed signif-icantly between US and UK Poodles (FST = 0.024; P <

0.0001) but not between affected vs unaffected dogs withineither the United States (FST = 0.001, P = 0.19) or the UK(FST = −0.010, P = 0.99).

After sequential Bonferroni corrections, six loci in theUS and two loci in the UK were significantly out ofHardy–Weinberg equilibrium, including one locus (INRA21)in both populations (Table 3). The heterozygote deficiencyassociated with INRA21 was within the range of observed inother loci, indicating no evidence for null alleles. Therefore,disequilibrium was likely attributable to inbreeding or anadditional level of population substructure. After sequentialBonferroni corrections, 13 locus pairs in the US and 9 differentlocus pairs in the UK (of 276 pairwise combinations ineach population) exhibited significant gametic disequilibrium,also consistent with inbreeding or population substructure ineach of these populations. The coefficient of inbreeding wasstatistically significant in these populations albeit low, withFIS estimated across loci at 0.07 (SE = 0.013) in the US and0.05 (SE = 0.023) in the UK. To obtain unbiased (by samplesize differences) comparisons of allelic richness betweenpopulations, we rarified estimates to 100 genes (i.e. 50 dogsper population), yielding allelic richness estimates averaged



Table 1 The incidence and frequency of mtDNA haplotypes in Standard Poodles

United States United Kingdom

mtDNA type (GenBank number)Percent in Veterinary GeneticsLaboratory forensic data set SA (%) Control (%) SA (%) Control (%)

A (AB622536) 0.7 26 (92.9) 56 (77.8) 19 (82.6) 37 (63.8)B (AB622568) 1.1 0 7 (9.7) 3 (13.0) 15 (25.9)C (AB622564) 1.6 0 8 (11.1) 0 0D (AB622557) 1.8 1 (3.6) 1 (1.4) 0 0F (AF531740) 0 1 (3.6) 0 0 2 (3.5)G (AY706505) 0 0 0 1 (4.4) 2 (3.5)H (AB622517) 5.4 0 0 0 2 (3.5)

mtDNA, mitochondrial DNA; SA, sebaceous adenitis.

Table 2 Y-STR haplotype of 91 male sebaceous adenitis affected and unaffected Standard Poodles from the United States. All of the dogs possessedidentical alleles at each Y-STR locus, corresponding to the common 6p (D1D5) Y haplotype (15)

Y-STR markers

MS34A MS34B MS41A MS41B 990.35.4 650.79.2 650.79.3

Allele size (Veterinary Genetics Laboratory) 172 176 206 219 127 120/134 122/124Allele namea G I C H E D/K

STR, single tandem repeat.aNomenclature used by US Fish and Wildlife Service Forensics Laboratory, Ashland, Oregon for allele designation; the 650.79.3 Y STR is not used bythem.

Table 3 Microsatellite locus-specific observed (Ho) and expected (He) heterozygosity, heterozygote deficit (FIS), and rarified (to 100 genes) estimatesof Allelic richness (RAR) for Standard Poodles from the United States and the United Kingdom


Locus Ho He FIS RAR Ho He FIS RAR

AHT121 0.73 0.78 0.06 9.4 0.65 0.76 0.16 9.1AHT137 0.76 0.78 0.03 6.9 0.65 0.73 0.11 7.0AHTH130 0.69 0.76 0.09 6.2 0.67 0.81 0.17 6.0AHTh171-A 0.73 0.71 −0.03 7.9 0.66 0.61 −0.08 5.0AHTh260 0.46 0.57 0.19a 6.6 0.45 0.52 0.14 6.6AHTk211 0.39 0.42 0.08 3.7 0.40 0.38 −0.05 3.4AHTk253 0.70 0.72 0.03 5.0 0.66 0.78 0.15 5.0C22.279 0.59 0.62 0.04 5.8 0.66 0.68 0.03 5.0FH2001 0.67 0.72 0.07a 6.3 0.58 0.57 −0.03 4.9FH2054 0.47 0.56 0.16 6.0 0.52 0.51 −0.03 4.9FH2328 0.66 0.77 0.15 5.3 0.53 0.79 0.33a 5.7FH2848 0.19 0.22 0.17a 4.8 0.38 0.40 0.05 6.4INRA21 0.53 0.62 0.15a 5.8 0.61 0.66 0.07a 4.9INU005 0.48 0.51 0.05a 3.7 0.53 0.59 0.10 3.8INU030 0.70 0.69 0.00 5.3 0.64 0.73 0.12 5.0INU055 0.70 0.69 −0.02 4.8 0.61 0.68 0.10 6.6LEI004 0.37 0.38 0.03 4.0 0.30 0.32 0.04 4.2REN105L03 0.49 0.56 0.13 4.6 0.61 0.59 −0.03 4.5REN162C04 0.47 0.48 0.01 5.8 0.58 0.65 0.11 6.7REN169D01 0.70 0.72 0.03 6.5 0.61 0.66 0.07 5.9REN169O18 0.44 0.49 0.10 5.1 0.43 0.42 −0.03 4.5REN247M23 0.66 0.66 0.01 4.3 0.57 0.53 −0.07 3.6REN54P11 0.63 0.71 0.12a 4.9 0.79 0.74 −0.07 4.0REN64E19 0.63 0.65 0.03 5.7 0.80 0.67 −0.19 3.0

aSignificant deviation from Hardy–Weinberg equilibrium after sequential Bonferroni correction.

across loci of 5.6 (95% CI: 5.1–6.1) vs 5.2 (95% CI: 4.7–5.8)alleles per locus in the US and the UK, respectively, which didnot differ significantly. Heterozygosity was similar within thetwo populations, regardless of SA-affected status (Table 4).

A PCoA plot comparing randomly related dogs from USand UK populations showed overlapping populations withsome differentiation by country of origin (Figure 1A). TheSA-affected dogs were indistinguishable from unaffected dogs



Table 4 Diversity metrics calculated for randomly related US and UKStandard Poodles based on 24 single tandem repeat markers


Affected,n = 49

Unaffected,n = 100

Affected,n = 23

Unaffected,n = 60

Ho 0.561 (0.032) 0.584 (0.030) 0.596 (0.029) 0.571 (0.025)He 0.606 (0.031) 0.613 (0.028) 0.596 (0.028) 0.608 (0.029)

within their country of origin (Figure 1B, C). A blind clus-ter analysis was performed in structure using all unrelatedPoodles in the US and the UK to investigate patterns of sub-structure using no prior information on population member-ship. An analysis using K = 2 was conducted for comparisonand the US and UK dogs segregated by geographic origin(Figure 2A). Use of four genetic clusters (K = 4) was indi-cated as the optimum based on the log probability of the data(Figure 2B), but the analysis at K = 4 did not further dif-ferentiate populations. Rather, two of the clusters (indicatedby orange and green) again reflected the geographic origins,while the other two clusters (blue and purple) did not segre-gate SA affected from unaffected dogs. Therefore, regardlessof K , blind analyses provided no evidence that SA affectedand unaffected dogs reflected distinct subpopulations amongeither US or UK Standard Poodles.

Exon 2 sequencing of DLA-DRB1, -DQA1 and -DQB1loci was conducted on Standard Poodles from both the US(n = 137) and the UK (n = 84) (Table 5). Twelve DRB1,seven DQA1, and nine DQB1 alleles were identified betweenUS and UK Poodles. One DRB1 allele was unique and wasnamed drb001v until it can be given an official designation.The new drb001v allele differed from DRB1*00101 by asingle nucleotide.

The known alleles formed 14 tri-locus haplotypes (Table 5).The proportion of heterozygous genotypes (Ho) did not differsignificantly between affected and unaffected dogs in the US(Ho = 0.50; Fisher Exact P = 0.16) or the UK (Ho = 0.44;Fisher Exact P = 0.25). Nor was there a significant deviationfrom Hardy–Weinberg equilibrium in the United States (He =0.50; χ2 = 2; df = 0.87; P = 0.65) or the UK (He = 0.47;χ2 = 2; df = 0.54; P = 0.76). There also were no significantdifferences between haplotype frequencies of SA affected vsunaffected dogs in the US (χ2 = 5; df = 1.64; P = 0.90) or inthe UK (χ2 = 5; df = 2.52; P = 0.77). However, haplotypefrequencies differed slightly (FST = 0.016) but significantlybetween US and UK dogs (χ2 = 5; df = 24.7; P = 0.0002).This was due mainly to the relative occurrence and frequenciesof minor haplotypes (Tables 5 and 6).

In order to avoid bias from closely related dogs, DLA classII haplotype zygosity was calculated using only dogs unrelatedto three generations. About one-half of all unrelated US andUK Standard Poodles were homozygous for various DLAclass II haplotypes (Table 6). These proportions were virtuallyidentical to those of SA affected vs unaffected dogs from the

Co

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. 2

Coord. 1

A

B

C

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Figure 1 Principle coordinate analysis plot based on single tandemrepeat alleles of randomly related Standard Poodles. (A) US (opendiamonds) vs UK (closed squares); (B) unaffected (open circles) vssebaceous adenitis (SA) affected dogs (closed circles) from the US;(C) unaffected (open triangles) vs SA-affected dogs (closed triangles)from the UK.

same countries. Although there were some difference in thetypes of low frequency haplotypes between US and UK dogs,the 01501*00601*02301 haplotype, either in a homozygousor heterozygous state, was found in about 94% of US and UKdogs (Table 6).

Zygosity mapping was performed within a region on CFA12from base 3802975 to 5672682, which includes the entireDLA, for SA affected and unaffected Standard Poodles fromboth the US and the UK (Figure 3). Forty to fifty percentageof both the US and UK Standard Poodles, respectively, were



Figure 2 Structure analysis using single tan-dem repeats (STRs) from unrelated dogs, SAaffected and unaffected, from the US andUK. The actual population to which each dogbelonged was not listed and the programwas ‘asked’ to place each animal into dis-tinct genetic clusters irrespective of countryof origin and disease status. (A) Bar graphsshowing apportionment of ancestry whenconstrained to K = 2 and K = 4 clusters.(B) plot of the log probability of the data asa function of K, indicating 4 as the optimalnumber of genetic clusters.

K = 4

K = 2

US control UK controlUS SAUKSA A

B

-9100

-9000

-8900

-8800

-8700

-8600 0 1 2 3 4 5 6

Ln p

roba

bilit

y of

the

data

K

Table 5 Dog leukocyte antigen class II haplotype frequency in all randomly related SA affected and control Standard Poodles

Haplotype United States United Kingdom

DRB1 DQA1 DQB1 SA, n = 94 (%) Control, n = 180 (%) SA, n = 46 (%) Control, n = 122 (%)

01501 00601 02301 68 (72.34) 124 (68.89) 36 (78.26) 83 (68.03)01502 00601 02301 9 (9.57) 17 (9.44) 1 (2.17) 2 (1.64)01501 00901 00101 6 (6.38) 12 (6.67) 5 (10.87) 22 (18.03)02001 00401 01303 6 (6.38) 12 (6.67) 2 (4.35) 5 (4.1)01503 00601 02301 1 (1.06) 5 (2.78) 1 (2.17) 7 (5.34)00901 00101 008011 1 (1.06) 3 (1.67) 1 (2.17) 001501 00601 04901 1 (1.06) 1 (0.56) 0 002301 00301 00501 1 (1.06) 0 0 000601 005011 00701 1 (1.06) 0 0 0001v 00101 00201 0 2 (1.11) 0 000101 00101 00201 0 1 (0.56) 0 2 (1.64)00201 00901 00101 0 1 (0.56) 0 001101 00201 01302 0 1 (0.56) 0 1 (0.82)010011 00201 01501 0 1 (0.56) 0 0

SA, sebaceous adenitis.

predominately heterozygous across this entire region. Anadditional one-half to one-third of the respective US andUK dogs possessed a large region of homozygosity fromnucleotide positions 4547874 to 5412195, but were relativelyheterozygous both upstream and downstream of this region.

The DLA-DRB1, -DQA1 and -DQB1 genes are found atapproximate positions 5,155,200 to 5,311,100. Seven dogsfrom the US, two SA affected and five unaffected, werehomozygous for either major or minor alleles across theentire DLA region (Figure 3). In contrast, four SA-affected



Table 6 Zygosity of dog leukocyte antigen class II haplotypes in unrelated Standard Poodles from the United States and the United Kingdom.Homozygous haplotypes are in bold lettering


Haplotype SA, n = 35 (%) Control, n = 69 (%) SA, n = 13 (%) Control, n = 39 (%)

01501*00601*02301/01501*00601*02301 17 (48.57) 29 (42.03) 7 (53.85) 19 (48.72)01501*00601*02301/01502*00601*02301 5 (14.29) 12 (17.39) 1 (7.69) 001501*00601*02301/02001*00401*01303 5 (14.29) 8 (11.59) 1 (7.69) 2 (5.13)01501*00601*02301/01501*00901*00101 4 (11.43) 6 (8.70) 3 (23.08) 11 (28.21)01501*00601*02301/01503*00601*02301 1 (2.86) 3 (4.35) 0 2 (5.13)01501*00601*02301/02301*00301*00501 1 (2.86) 0 0 001501*00601*02301/010011*00201*01501 0 1 (1.45) 0 001501*00601*02301/01501*00601*04901 0 1 (1.45) 0 001501*00601*02301/01101*00201*01302 0 1 (1.45) 0 1 (2.56)01501*00601*02301/00101*00101*00201 0 1 (1.45) 0 1 (2.56)01501*00601*02301/00201*00901*00101 0 1 (1.45) 0 001501*00601*02301/00901*00101*008011 0 2 (2.90) 1 (7.69) 0001v*00101*00201/001v*00101*00201 0 1 (1.45) 0 002001*00401*01303/02001*00401*01303 0 1 (1.45) 0 001502*00601*02301/01502*00601*02301 0 1 (1.45) 0 001502*00601*02301/00601*005011*00701 1 (2.86) 0 0 001502*00601*02301/00901*00101*008011 1 (2.86) 0 0 001503*00601*02301/01503*00601*02301 0 1 (1.45) 0 001502*00601*02301/01503*00601*02301 0 0 0 1 (2.56)01501*00901*00101/01501*00901*00101 0 0 0 1 (2.56)01501*00901*00101/02001*00401*01303 0 0 0 1 (2.56)

SA, sebaceous adenitis.

dogs from the UK were homozygous across the entireDLA compared with one unaffected dog (Figure 3). Identicalzygosity mapping was carried out using the same 115 DLASNPs on 26 randomly selected indigenous (street) dogs fromthe Island of Bali, Indonesia (Figure 3). Bali street dogsshowed a much greater level of heterozygosity across theentire DLA region than SA affected or unaffected StandardPoodles from either the US or the UK. Interestingly, the regioncoding for the DLA class II genes was more homozygous andconserved across all breeds than regions flanking the DLA.

Chromosome-wide association was conducted on CFA12using 57 affected and 40 unaffected dogs from the US andUK and results shown for the DLA region. A singular, butnon-significant, peak was observed at approximately 4.5 MBwithin the DLA on CFA12 for the US Poodles (Figure 4).This peak was near the large region of homozygosity startingat position 4.55 MB. This peak was not seen in associationstudies with UK dogs or in a combined population of US andUK dogs. No disease association was found within the regiondefining the DLA class II genes at 5.16–5.31 MB (Figure 4).

Discussion

This study resulted from a larger GWAS to identify geneticassociation(s) with SA in Standard Poodles. Difficulties inidentifying significant associations were encountered, lead-ing to a decision to obtain a better understanding of geneticdiversity and population structure between US and UK

Standard Poodles and between affected and unaffected indi-viduals. However, such an undertaking requires a historicalknowledge of the breed’s development. Although the StandardPoodle is of relatively ancient origin, certain kennels as well asindividuals with outstanding breed type have played an inor-dinate role in breed evolution over the last century (31–33).Dogs from the Meadoware, Hill Hurst, and Red Brook kennelsdominated the breed early in the century, but their contribu-tions were soon supplanted by other kennels and individualdogs (32). The most noteworthy of these later contributorswas the Labory Kennels of Switzerland and a dog known asAnderl von Hugelberg, who lived in the 1920s and has beenreferred to as the ‘Adam of modern Standard Poodles’ (32).His descendants subsequently dominated the breed around theworld and resulted in the loss or diminution of lines that werepopular at an earlier time. A further bottleneck occurred withthe Wycliffe line. The Wycliffe line traces its origins to the late1950s or early 1960s to five Standard Poodles mainly from thethen dominant Anderl von Hugelberg line with minor contri-butions from several other lines (33). Extensive inbreeding ofthis foundation stock led to an extremely popular line, whichwas exported to many foreign countries. The proportion ofWycliffe ancestry among Standard Poodles in the US, the UK,and Scandinavia progressively increased to levels approaching40%–50% by 1980 in dogs with the more common coat col-ors and has remained at that level to the present day (33). Itis noteworthy that two autoimmune disorders, SA and hypoa-drenocorticism, have appeared and increased in prevalence in



Figure 3 Zygosity mapping of 115 SNPs across the dog leukocyte antigen (DLA) region of unrelated sebaceous adenitis (SA) affected (panel I) andunaffected (panel II) Standard Poodles from the US, SA affected (panel III) and unaffected (panel IV) dogs from the UK. Panel V shows the zygositymap for 26 randomly selected indigenous (village) dogs from Bali, Indonesia. The SNP markers in the DLA class II region are colored gray. Majoridentical homozygous SNP alleles are colored black, minor homozygous alleles are colored gray, and all heterozygous alleles are colored white.Individuals possessing the major DLA class II haplotypes 01501*00601*02301/01501*00601*02301 are identified as ++.

parallel with the emergence and dominance of the Wycliffeline (33).

All Standard Poodles shared a single paternal (Y chro-mosome) haplotype based on a panel of 11 Y SNPs. Thisparticular Y haplotype, as would be expected from the slowevolution of SNPs, is rooted deep in the ancestry of village dogpopulations from across Southeast Asia and is common amongwestern dogs regardless of breed (15). This limits its useful-ness in inferring numbers of male founders. However, STRs

have much higher resolution (15, 17). Sixty-seven Y-STRhaplotypes have been identified among 50 modern breeds ofdogs, and the 6p (D1D5) Y-STR haplotype found in Stan-dard Poodles is the most common of these haplotypes (15).This was also one of the most common haplotypes in the VGLcanine forensic database and predominates in breeds that sharecharacteristics with the Standard Poodle, such as the AiredaleTerrier, Maltese Terrier, Bichon Frise, Borzoi, German Short-haired Pointer, Komondor, and Norfolk Terrier. Interestingly,



1.2

0.9

0.6

0.3

0.03.5 4 4.5

SNP marker Location (Mb)

-Log

10 (

EM

P2)

5 5.5 6

Figure 4 Dog leukocyte antigen (DLA)-wide association mapping ofsebaceous adenitis based on results of 34 affected and 24 unaffectedStandard Poodles from the United States and 115 SNP markers fromacross the DLA region of CFA12. Data extracted from a genome-wideassociation study. A singular, but not significant (EMP2 0.5247), peakwas observed within the DLA region at position 4418982 on CFA12upstream of the DLA class II loci.

Standard Poodles also share this Y-STR haplotype with Tai-wan and Philippine village dogs in the database, again show-ing the Southeast Asian origins of most modern breeds anda possible geographical origin of the male founder(s) of theStandard Poodle (15).

The relatively high resolution matrilineal marker (mito-chondrial hypervariable region I) used in this study waspolymorphic as has been found in other breeds (34), therebyallowing comparison of haplotype frequencies. Seven mtDNAhaplotypes were identified in this study, US and UK Poo-dles each possessed five mtDNA haplotypes, three of whichwere shared and two being unique to each population. Stan-dard Poodles with SA exhibited a higher frequency (88%)of mtDNA haplotype A than did unaffected Poodles (70%),which had a correspondingly higher frequency of rare hap-lotypes B–H (30%) and, consequently, higher mitochondrialdiversity. Although mtDNA polymorphisms have been asso-ciated with susceptibility to autoimmune disease in laboratorymice (35), the observed association in this study could alsobe the result of greater inbreeding in the SA-affected Poodles.

The predominance of mtDNA haplotype A in the US andUK Standard Poodles supports what is known about the recenthistory of the breed and a bottleneck occurring with the adventof the Wycliffe line in the 1950s. This line had a great impacton the subsequent genetic makeup of the breed in the US,the UK, Scandinavia and other countries and was probablyresponsible for the virtual disappearance of lines that werepopular at the turn of the century (31). The minor mtDNAhaplotypes observed in this study may be remnants of linesthat were more common prior to the 1950s. It is noteworthythat these minor maternal lines remain relatively free of SA,suggesting that SA entered the breed with what has nowbecome the dominant maternal lineage.

All approaches of data analysis, F statistics, PCoA, andadmixture analysis (structure), suggested that US and UK dogs

were closely related but not a single panmictic population, asto be expected given their independently selected breedingover the past 25–50 generations. The implication of thisdegree of population substructure on GWAS using a pool ofStandard Poodles from both the US and the UK is unknown,but it is one consideration in determining the minimumnumber of case and control dogs from each country and themanner in which data are analyzed and interpreted.

Interrogation of the DLA and DLA class II region wascarried out with two objectives in mind: (1) to look directlyfor DLA class II associations with SA because studies inother breeds involving a variety of autoimmune diseases havefound associations with specific haplotypes (reviewed in Refs11, 12) and (2) to study a small region of the genome as awindow into what may be happening in other regions of thegenome. Exon 2 sequencing of the three DLA class II genesdetected 12 DRB1, 7 DQA1, and 9 DQB1 alleles forming 14three loci haplotypes. These were among the 245 DRB1, 39DQA1, and 79 DQB1 alleles and over 200 three-locus hap-lotypes previously identified from purebred and indigenousdogs around the world (Kennedy LJ, personal communica-tion). The present findings for DLA class II alleles and hap-lotypes were similar to those reported by Kennedy (36) on81 Standard Poodles from the US and the UK. She reported9/12 of the same DRB1 alleles, 5/7 DQA1 alleles, and 4/9of the same DQB1 alleles. The frequency of the varioushaplotypes was also similar, with 01501/00601/02301 beingpresent in 105/162 (65%) chromosomes in her study. Differ-ences between US and UK dogs, when they occurred, wereseen mainly with minor alleles and their relative frequencies.Kennedy reported DLA class II haplotypes and zygosity ofStandard Poodles from the US, Canada, and the UK (37, 38).Eleven haplotypes were identified among 31 samples fromaround the world and 10 from among 50 samples submit-ted by the Animal Health Trust, UK. About one-half of thesedogs were homozygous for the DLA class II genes and 90%of these dogs were homozygous for the same major haplotype,DRB1*01501/DQA1*00601/DQB1*02301.

DLA class II haplotype diversity among Standard Poodleswas somewhat greater than in breeds such as the Italian Grey-hound (12) and Pug Dog (11). However, like the Pug Dog, thefrequency distributions of alleles and haplotypes were highlyskewed because of differences in the number and frequenciesof minor haplotypes. Nonetheless, DLA haplotype diversity inan outbred population of village dogs in Bali, Indonesia (39)displayed heterozygosity in a single locus, DQA1 (Heterozy-gosity = 0.825) that was 70% higher than observed in thisstudy for the entire tri-locus DLA haplotype of Standard Poo-dles (0.49) in this study .

There was no apparent association between susceptibil-ity to SA in Standard Poodles in this study and any DLAclass II haplotype. This is different from autoimmune dis-orders described in other breeds like the Pug Dog (11).However, it is possible a major disease association existed



with 01501/00601/02301, in which case it could have goneundetected due to a near fixation of this haplotype within thebreed and insufficient numbers of case and control dogs. Usingthe US SA case and control population in a genetic power cal-culation (http://pngu.mgh.harvard.edu/∼purcell/gpc/cc2.html;2 df test), and assuming that the major disease associationwas in the DLA class II region, the high risk allele (hap-lotype) frequency 0.701, the SA prevalence 2%, the relativerisk in the heterozygous state 0.88 and in the homozygousstate 1.14, the linkage disequilibrium (D′) 0.7, the markerallele frequency 0.701, the number of cases 35, and 2:1 con-trol to case ratio, 2629 cases would be required to yield analpha of 0.05 at 80% power. If genetic associations are nearlyfixed in the DLA class II region, it is likely that a signifi-cant degree of linkage disequilibrium (LD) may be presentin other regions of the genome. This would also affect thenumbers of case and control dogs required to identify othermajor genetic associations. There is also the potential prob-lem of population stratification, which was evident betweenUS and UK Standard Poodles based on analysis of mito-chondrial DNA, autosomal STRs, and DLA SNPs. Autosomalgenetic associations in human autoimmune diseases are alsolargely polygenic, a pattern that has also been seen in SLE-related disease in Nova Scotia Duck Tolling Retrievers (40),hypoadrenocorticism in Portuguese Water Spaniels (41), anda multiple autoimmune disease syndrome of Italian Grey-hounds (12). The genetics of autoimmune diseases in humanshas been highly elusive, as elegantly stated by Johannessonand colleagues (42) – ‘from disease to genes: the monogenicsuccess and the polygenic failure’. The discovery of simpleMendelian traits with surprisingly small numbers of case andcontrol dogs has been remarkably easy, but studies of complextraits such as autoimmunity or epilepsy will likely be as chal-lenging in dogs as they have been in humans (12, 40, 41, 43).The finding that US Standard Poodles were no less inbredthan UK Poodles is counter to an assumption that US purebreeds of dogs have less DLA class II diversity than theirEuropean counterparts (44). Although geographic separationhad a limited effect on genetic diversity within US and UKdogs, trafficking of Standard Poodles between the US, Canada,the UK, and Scandinavia has been quite extensive throughoutthe century. It may be important for Standard Poodle breed-ers to search for remnants of bloodlines, possibly based onmtDNA types, which may remain free of autoimmune disor-ders like SA. Such lines may exist in a few older kennels, ormore likely, in parts of the world less influenced by the bot-tlenecks of the 1920s and 1950s. In the absence of definitivegenetic markers for SA susceptibility, a recommendation hasbeen made to break this bottleneck by crossing Standard Poo-dles with Miniature and Toy Poodles (37), which have a muchlower prevalence of SA and hypoadrenocorticism. However,Toy, Minature, and Standard Poodles are not as geneticallyrelated as one might presume (45) and without tests to iden-tify individuals SA carriers, backcrossing to re-establish the

desired Standard Poodle phenotype could recreate the originalproblem.

Acknowledgments

Funding for this study was provided by the PCA Foundation.We are also grateful for partial matching funds from theCenter for Companion Animal Health, UC Davis. We wishto also thank the staff of the Veterinary Genetics Laboratory(VGL), UC Davis for running STR parentage panels and SRYhaplotyping and Angel Del Valle for assisting in mtDNA andDLA class II sequencing. Beth Wictum, head of the VeterinaryForensics Unit of the VGL kindly allowed us access to a largemtDNA sequence database of pure breed dogs. We are alsograteful for assistance rendered by the Standard Poodle ClubUK and hundreds of Standard Poodle breeders and owners.

Conflict of interest

The authors have declared no conflicting interests.

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