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. 2015 Nov 30;10(11):e0143458.
doi: 10.1371/journal.pone.0143458. eCollection 2015.

Autoantibodies Against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis Lupus Familiaris) Affected With Hypoadrenocorticism (Addison's Disease)

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Autoantibodies Against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis Lupus Familiaris) Affected With Hypoadrenocorticism (Addison's Disease)

Alisdair M Boag et al. PLoS One. .
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Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

Conflict of interest statement

Competing Interests: This study was supported by the Biotechnology and Biological Sciences Research Council as an industrial CASE PhD studentship (Grant number: BB/G0169921), with Dechra Ltd. as the industrial partner. Dr Peter Graham was a paid employee of Dechra Ltd. and a shareholder of Dechra Pharmaceuticals plc, and served as an Industrial Co-supervisor during this BBSRC-CASE funded project. Dr Peter Graham and Dechra Ltd. assisted in the process of sample acquisition resulting from its activities in commercial diagnostic laboratory operations. Dechra Ltd. no longer has a business interest in diagnostic laboratory operations. Dr Peter Graham reviewed study design and manuscript drafts in his role as co-supervisor. There are no other potential competing interests relating to this study. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.


Fig 1
Fig 1. Amplification and expression of canine adrenal autoantigens.
(A) Coding regions of selected canine adrenal steroid synthesis enzymes were amplified and cloned. Shown are PCR results of (1) screening primers and (2) cloning primers for canine CYP17A1 and (3) screening primers and (4) cloning primers for canine CYP11A1. M, molecular weight ladder (Hyperladder I, Bioline, London, UK). Image has been cropped for ease of interpretation; the original image, with extra cDNA samples represented by extra lanes, is available as S1 Fig. (B) Autoradiograph of radiolabelled recombinant canine adrenal autoantigens. The coding regions of (1) canine CYP21A2 (21-hydroxylase; 55 kDa), (2) CYP17A1 (17-hydroxylase; 57 kDa), (3) HSD3B2 (3β hydroxysteroid dehydrogenase; 42 kDa) and (4) CYP11A1 (P450 side-chain cleavage enzyme; 60 kDa) were cloned into the pVAX1 vector and 35S-methionine radiolabelled recombinant protein expressed in an in vitro transcription and translation assay. Translates were subjected to SDS-PAGE and exposed to X-ray film for 24 h.
Fig 2
Fig 2. Radioimmunoprecipitation assays with canine sera against recombinant radiolabelled adrenal antigens.
Radioactivity (counts per minute; CPM) for hypoadrenocorticism cases (n = 20; filled boxes) and control (n = 4; open boxes) serum samples after immunoprecipitation with recombinant canine (A) 21-OH; (B) 17-OH; (C) 3βHSD and (D) P450scc. Data are shown as mean + SEM. Hatched line represents positive threshold (mean + 3SD of control samples), with values above this indicated (*).
Fig 3
Fig 3. Serum antibodies to canine P450scc in radioimmunoprecipitation assay.
(A) Scatter plot from a representative 96-well plate showing P450scc immunoreactivity (counts per minute; CPM, mean of triplicate wells) in serum samples from hospital control dogs (Control; n = 11) and dogs affected with hypoadrenocorticism (HypoAd; n = 19). Negative standard (triangle) and positive standard (square) serum samples were identified from initial autoantibody screening experiments and were used on every plate for normalisation purposes. (B) Relative P450scc autoantibody reactivity in control dogs (n = 80), dogs affected with spontaneous hypoadrenocorticism (HypoAd; n = 213), dogs affected with hyperadrenocorticism (HAC; n = 58) and dogs with iatrogenic hypoadrenocorticism (iHAd; n = 4). Circles represent normalised values for each individual serum sample in the P450scc radioimmunoprecipitation assay. Mean + 3SD line represents the threshold positive value established from a reference population of 30 canine control patients.

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