Molecular genetic studies in atopic dermatitis in West Highland white terriers

Canine atopic dermatitis (AD) is an inflammatory allergic skin disease that is usually associated with the production of IgE antibodies against environmental allergens. The genetic predisposition in canine AD is inherited, and in West Highland white terriers (WHWTs) the disease is common and often severe. There are many similarities between canine and human AD, suggesting a similar genetic cause in both species. In humans, mutations in the filaggrin gene (FLG) are the most common predisposing genetic factors for AD. However, in dogs there are few genetic studies on this disease. Hence, this thesis investigated the molecular genetic factors causing AD in a population of atopic and non-atopic WHWTs. Fifty-three atopic and 31 non-atopic WHWTs were enrolled in this study. All dogs were examined by veterinary dermatologists, and owners of non-atopic dogs completed a questionnaire to screen for previous and/or current skin disease. Diagnosis of canine AD was made using current criteria, and by excluding other causes of pruritic skin disease. Non-atopic dogs were over 5 years of age, had no clinical signs or history of AD, and 24/31 were from one line of minimally-atopic WHWTs. Genomic DNA was extracted from blood from all dogs, and serum was collected in 30/53 atopic and 25/31 non-atopic WHWTs. Skin was collected from 5 atopic and 3 non-atopic WHWTs. An initial segregation analysis suggested that AD in this population was consistent with a common, fully penetrant major locus. However, a dominant (risk allele frequency PD = 0.5, model log likelihood LL = - 19.28) or recessive model (PD = 0.9, LL = - 18.91) fitted the data equally well. A linkage analysis in 49/53 atopic and 30/31 non-atopic WHWTs investigated the role of the canine orthologue of filaggrin (flg) in canine AD. A major genetic influence from eleven haplotypes near flg was excluded by allelic association (P = 0.29), and both non-parametric (affected-only analysis P = 0.95; general pairs method P = 0.45) and parametric linkage analyses (at c = 0, LOD = - 0.40). However, the study lacked the power to detect small to intermediate genetic effects from the flg gene. Hence, mRNA expression of flg was investigated in the lesional and non-lesional skin of 7 atopic and 5 non-atopic dogs from 5 breeds, using reverse-transcriptase Real Time quantitative PCR (RT qPCR). Overall, flg mRNA expression in non-lesional atopic skin was decreased (two fold) compared to non-lesional non-atopic skin; however this difference was only significant (P = 0.03) in the subgroup of WHWTs (n = 8). A genome-wide association study (GWAS), using the Affymetrix V2 Dog SNP array, was performed in 35/53 atopic and 25/31 non-atopic WHWTs. This study found a 1.3 Mb area of association with AD on CFA17 (best genome-wide P = 5x10-5). Genomic sequencing of a candidate gene near the area, PTPN22, in 3 atopic and 3 non-atopic WHWTs, identified five variants with bioinformatically predicted potential functional effects. One of these variants was subsequently excluded as the cause of the GWAS association peak signal by large-scale genotyping in 72/84 WHWTs (P = 0.01). Allergen-specific serum IgE was measured using the Allercept® ELISA, to investigate its role in the pathogenesis of AD in WHWTs. The most common positive results in atopic WHWTs were to the house-dust mite Dermatophagoides farinae (D. farinae) (11/30 dogs). In non-atopic WHWTs, high positive ELISA reactions were reported to 45/48 allergens; mainly D. farinae (20/25 dogs). Positive ELISA results in non-atopic dogs were significantly higher than those in atopic dogs for 44/48 allergens (best ANOVA adjusted P = 3.3x10-6), including D. farinae, a clinically relevant allergen in canine AD. A GWAS was performed in 31 WHWTs with positive (> 150 EA units) and 24 WHWTs with negative (≤ 150 EA units) reactions to D. farinae in the ELISA. Significant allelic association to D. farinae-IgE responsiveness was found on a 2.3 Mb area on CFA35 (best genome-wide P = 1x10-5). Genomic sequencing of CD83, a nearby candidate gene, in two high-IgE responders and two low IgE-responders, identified a genetic variant with a bioinformatically predicted functional effect. A small effect of this variant on the IgE responsiveness trait was confirmed by large-scale genotyping in 54/79 WHWTs (P = 1x10-3). In conclusion, the predisposition to develop AD in the population of WHWTs studied in this thesis is an inherited trait, and a major locus controlling its phenotype is likely to exist on CFA17. D. farinae-specific IgE responsiveness is also inherited in these WHWTs, but is likely to be controlled by a separate locus on CFA35. Interestingly, the line of non-atopic WHWTs studied in this thesis may be characterized by elevated levels of allergen-specific serum IgE. Thus, this thesis has set the stage for future studies into the genetic cause of AD in dogs and humans, and possibly to a better understanding of the pathogenesis of this common chronic disease