21-OH Autoantibodies Research Articles

Identification of autoimmune type 1 diabetes and multiple organ-specific autoantibodies in adult-onset non-insulin-requiring diabetes in China: A population-based multicentre nationwide survey.

To investigate the prevalence of adult-onset autoimmune diabetes (ADM) and predisposition to autoimmune diseases by quantifying serum organ-specific autoantibodies in people with phenotype of type 2 diabetes (T2D). We included a nationally representative sample of 46 239 adults aged ≥20 years from 14 provinces, of whom 4671 had diabetes, plus 1000 control subjects with normal glucose tolerance (NGT). Participants were screened centrally for autoantibodies to glutamic acid decarboxylase (GAD), islet antigen 2 (IA2) and zinc transporter isoform-8 (Znt8) and were defined as having ADM where positive for these antibodies. We then assayed thyroid peroxidase (TPO), tissue transglutaminase (tTG) and 21-hydroxylase (21-OH) autoantibodies in randomly selected participants with ADM and in age-matched, sex-matched and non-ADM controls with T2D plus controls with NGT. Post-normalization, the standardized prevalence rate of ADM was 6.0% (95% confidence interval [CI] 5.3-6.8) in initially non-insulin-requiring participants with ADM, corresponding to six million adults in China, in whom adjusted antibody positivity was: TPO autoantibodies 16.3% (95% CI 10.8-21.8), tTG autoantibodies 2.1% (95% CI 0.0-4.2), and 21-OH autoantibodies 1.8% (95% CI -0.2 to 3.8). Those participants with ADM who were GAD autoantibody-positive had high risk of TPO autoantibody positivity (odds ratio [OR] 2.39, P = 0.0031) and tTG autoantibody positivity (OR 6.98, P = 0.027), while those positive for IA2 autoantibodies had a high risk of tTG autoantibody positivity (OR 19.05, P = 0.001). A proportion of people with phenotype of T2D in China have ADM, with diabetes-associated autoantibodies, and may be at risk of developing other organ-specific autoimmune diseases; therefore, it may be clinically relevant to consider screening such Chinese populations.

ATPase4A Autoreactivity and Its Association With Autoimmune Phenotypes in the Type 1 Diabetes Genetics Consortium Study.

Autoantibodies targeting the H+/K+-ATPase proton pump of the gastric parietal cell (parietal cell antibodies [PCA]) are diagnostic of atrophic body gastritis (ABG) leading to pernicious anemia (PA). PCA, ABG, and PA occur in increased frequency in patients with type 1 diabetes and their relatives and are considered “minor” components of forms of autoimmune polyglandular syndrome (APS). A customized radioimmunoprecipitation assay was applied to 6,749 samples from the Type 1 Diabetes Genetics Consortium to measure ATP4A autoreactivity. Autoantibody prevalence was correlated with variants in HLA class II, PTPN22, and CTLA4 genes. With an ATP4A radioimmunoprecipitation assay, PCA were detected in sera from 20.9% of affected individuals. PCA prevalence increased with age and was greater in females (25.3%) than males (16.5%) and among Hispanics (36.3%) and blacks (26.2%) compared with non-Hispanic whites (20.8%) and Asians (16.7%). PCA and other organ-specific autoantibodies GAD65, IA-2, thyroid peroxidase (TPO), 21-hydroxylase (21-OH), and transglutaminase (TG) clustered within families with heritability estimates from 71 to 95%. PCA clustered with TPO, 21-OH, and persistent GAD65 autoantibodies but not with celiac (TG) or IA-2 autoantibodies. PCA-positive subjects showed an increased frequency of DRB1*0404, DPB1*0201, and PTPN22 R620W (rs2476601-T) and a decreased frequency of DRB1*0101, DPB1*0301, and CTLA4 CT60 (rs3087243-T). Genetic variants accounted for 4–5% of the heritable risk for PCA. The same alleles were associated with other autoantibody phenotypes in a consistent pattern. Whereas most of the heritable risk for PCA and other antibodies reflects genetic effects that are tissue specific, parietal cell autoimmunity is a major pathogenetic contributor in APS2.

The PTPN22 1858T allele but not variants in the proximal promoter region ofIL-21gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The -448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5'-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.

T Cell Responses to Steroid Cytochrome P450 21-Hydroxylase in Patients with Autoimmune Primary Adrenal Insufficiency

Autoimmune Addison's disease is thought to result from T cell mediated autoimmunity. Autoantibodies against the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are found in most patients, and 21OH is therefore a likely target for antigen-specific T cells. The aim was to study cellular immunity to 21OH and its associations with 21OH autoantibodies and human leukocyte antigen alleles in autoimmune Addison's disease. Peripheral blood mononuclear cells were collected from 33 patients with autoimmune Addison's disease and 21 controls. Cellular proliferation and production of cytokines in response to stimulation with 21OH or 21OH-derived peptides were tested. Cellular proliferation (P = 0.0009) and secretion of interferon-gamma (P < 0.0001) in response to 21OH was significantly higher in patients compared to healthy controls and associated with the presence of 21OH autoantibodies (P = 0.0052). Furthermore, the 21OH-specific production of interferon-gamma was enhanced in the presence of 21OH autoantibodies. This effect was partially inhibited by antibodies against the Fc receptor for IgG, CD32. Moreover, mature dendritic cells proved superior to the other antigen-presenting cells in invoking cellular responses to 21OH. An association between cellular immunity to 21OH and the high-risk HLA genotype for Addison's disease, DRB1*0301-DQ2/DRB1*0404-DQ8, was observed (P = 0.0089). Finally, a significant association between the DRB1*0404-DQ8 haplotype and cellular responses to a 21OH-derived peptide predicted to bind to DRB1*0404 was detected (P = 0.0055). Patients with autoimmune Addison's disease have circulating 21OH-specific T cells, with amino acids 342-361 of 21OH possibly constituting a disease-specific epitope presented by HLA-DRB1*0404.

Homozygosity of the polymorphism MICA5.1 identifies extreme risk of progression to overt adrenal insufficiency among 21-hydroxylase antibody-positive patients with type 1 diabetes.

Autoimmunity associated with Addison's disease (AD) can be detected by measuring 21-hydroxylase (21OH) autoantibodies. Subjects with type 1 diabetes (T1D) are at increased risk for AD. Genetic factors including HLA-DRB1*0404 and MICA have been associated with AD in populations with and without T1D. The objective of the study was to examine the effect of the MICA5.1 allele in subjects with 21OH autoantibodies on progression to AD. Two components were used: 1) a cross-sectional study with subjects with AD identified and enrolled from September 1993 to November 2008 and 2) a cohort study prospectively following up patients with T1D who screened positive for 21OH autoantibodies. Subjects were identified from the Barbara Davis Center and through the National Adrenal Diseases Foundation. Sixty-three subjects with AD were referred through the National Adrenal Diseases Foundation (AD referrals). Sixty-three subjects with positive 21OH antibodies from the Barbara Davis Center were followed up for progression to AD, and 11 were diagnosed with AD (progressors). Seventy-three percent of progressors (eight of 11) and 57% of AD referrals (36 of 63) were MICA5.1 homozygous (P = ns). Overall, 59% of patients with AD (44 of 74) were MICA5.1/5.1 compared with 17% of nonprogressors (nine of 52) (P < 0.0001) and 19% of normal DR3/4-DQB1*0302 controls (64 of 336) (P < 0.0001). Identifying extreme risk should facilitate monitoring of progression from 21OH antibody positivity to overt AD. The HLA-DR3/0404 genotype defines high-risk subjects for adrenal autoimmunity. MICA5.1/5.1 may define those at highest risk for progression to overt AD, a feature unique to AD and distinct from T1D.

Idiopathic central diabetes insipidus in children and young adults is commonly associated with vasopressin‐cell antibodies and markers of autoimmunity

Autoimmune targeting of hypothalamic-neurohypophyseal structures in children and young adults with posterior pituitary and anterior pituitary dysfunction, as well as pituitary stalk involvement, are not yet completely understood. We aimed to (1) evaluate the presence of circulating vasopressin-cell autoantibodies (AVPc-Abs) in young patients with central diabetes insipidus (CDI), (2) detect organ-specific autoantibodies as markers of autoimmunity, and (3) define the relationship between immune markers and neuroimaging findings. Twenty patients were evaluated at a median age of 16.3 years. Twelve patients had idiopathic CDI, six had Langerhans cell histiocytosis (LCH) and two had germinoma. AVPc-Abs were evaluated in 40 healthy children. Magnetic resonance imaging (MRI) of the hypothalamic-pituitary region was performed longitudinally in all subjects. Circulating arginine vasopressin (AVP), protein tyrosine phosphatase (IA2), glutamic acid decarboxylase (GAD), 21-hydroxylase (21-OH), endomysium antibodies (EMA), parietal cell (PCA), thyroid peroxidase (TPO), thyroglobulin (TG) and TSH-receptor (TSHr) autoantibodies were evaluated. Circulating AVPc-Abs were found in 15 patients (75%), nine with idiopathic CDI, four with LCH and two with germinoma; the pituitary stalk was involved in most of them. Five patients with idiopathic CDI showed a persistence of AVPc-Abs during follow-up and one became positive subsequently. Serum IA2 autoantibodies were demonstrated in 14 patients (70%) and 21-OH autoantibodies in three of them. In idiopathic CDI, circulating AVPc-Abs suggest an autoimmune involvement of the neurohypophyseal system. The identification of AVPc-Abs in subjects who could have either idiopathic CDI or LCH or germinoma, however, indicates that AVPc-Abs cannot be considered a completely reliable marker of autoimmune CDI. Thus, close clinical and MRI follow-up are needed because AVPc-Abs may mask germinoma or LCH.

Estimated Risk for Developing Autoimmune Addison’s Disease in Patients with Adrenal Cortex Autoantibodies

Patients with adrenal cortex autoantibodies (ACA) without overt autoimmune Addison's disease (AAD) are at risk of adrenal failure. To assess the contribution of different clinical, immunological, genetic, and functional factors in the progression to AAD, we followed up 100 ACA-positive and 63 ACA-negative patients without AAD for a maximum of 21 yr (mean 6.0 yr, median 4.8). ACA were measured by immunofluorescence and 21-OH autoantibodies (Abs) by RIA. Adrenal function was assessed by measuring basal levels of cortisol, aldosterone, ACTH, renin activity, and cortisol response to ACTH. The risk of developing AAD was calculated using survival and multivariate analyses. AAD developed in 31 ACA-positive patients and one ACA-negative patient. The cumulative risk of disease in ACA-positive patients was 48.5% [95% confidence interval (CI) 40.8-56.1]. The cumulative risk was higher in children than adults (100 vs. 31.9%; P < 0.0001), males than females (68.6 vs. 42.7%; P = 0.006), patients with subclinical rather than normal adrenal function at entry (87.4 vs. 30.1%; P < 0.0001), patients with hypoparathyroidism and/or candidiasis than patients with other autoimmune or nonautoimmune diseases (100 vs. 29.7%; P < 0.0001), and patients with high rather than low-medium ACA titers (62.8 vs. 41.2%; P = 0.12). The presence of human leukocyte antigen (HLA)-DRB1 did not appear to contribute to the prediction of AAD. Adjusted hazard ratios by Cox model for the development of AAD were 3.37 for males (CI 1.38-8.24), 5.23 for hypoparathyroidism and/or candidiasis (CI 1.53-17.92), 3.33 for high antibody titers (CI 1.43-7.78), and 6.15 for impaired adrenal function at entry (CI 2.79-13.57). These results were used to construct a risk algorithm for estimating the probability of developing AAD from the combination of gender, age, adrenal function, antibody titer, and associated autoimmune disorders at entry. The values of estimated risk could be used to decide appropriate follow-up intervals and future immunointervention strategies.

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling

To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

A study of the epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison's disease.

Steroid 21-hydroxylase (21-OH) autoantibodies are found in patients with autoimmune Addison's disease (AAD), either isolated or associated with autoimmune polyglandular syndrome (APS) type I and II and in adrenal-cortex autoantibody (ACA)-positive patients without AAD. In order to assess any differences in the 21-OH autoantibodies in these different patient groups, we have studied their reactivity with different epitopes on 21-OH using full length and modified 35S-labelled 21-OH proteins produced in an in vitro transcription/translation system. There were no major differences in the pattern of autoantibody reactivity with the different modified 21-OH proteins in patients with isolated AAD or with APS types I and II, and in 21-OH autoantibody-positive patients with clinical AAD, subclinical AAD and those maintaining a normal adrenal function. Our studies also indicate that the main epitopes for 21-OH autoantibodies in patients with different forms of autoimmune adrenal disease are located in the C-terminal end and in a central region of 21-OH.

A Conformation-Dependent Epitope in Addison’s Disease and Other Endocrinological Autoimmune Diseases Maps to a Carboxyl-Terminal Functional Domain of Human Steroid 21-Hydroxylase

Idiopathic Addison's disease develops as a consequence of autoimmune destruction of steroid-producing cells in the adrenal gland. A major autoantigen is 21-hydroxylase (21OH; P450c21), which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. We selected a number of functionally important 21OH amino acid substitutions, found in patients with congenital adrenal hyperplasia, to study their effects on the binding of 21OH autoantibodies (21OHAb) to 21OH. The ability of 21OHAb to bind in vitro transcribed and translated wild-type 21OH and five different 21OH mutant proteins was quantified by liquid-phase assays. Sera from 21OHAb-positive patients with idiopathic Addison's disease (n = 24), Graves' disease (n = 3), and insulin-dependent diabetes mellitus (n = 1) were used. While the P105L, delE196, and G291S mutations had no effect on autoantibody binding, the P453S mutation had a considerable effect, and the R483P mutation almost completely abolished binding. Synthetic peptides corresponding to linear epitopes defined by amino acids 447-461 and 477-491 were unable to compete with wild-type 21OH for binding to autoantibodies. Direct 21OH DNA sequencing could not reveal any specific genetic variation in alleles found in 21OHAb-positive patients. We conclude that the region involving R483 plays a key role in the formation of a three-dimensional epitope in a functionally important C-terminal domain of the enzyme.

Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies.

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.

Autoantibody Binding to Steroid 21-hydroxylase – Effect of Five Mutations

Steroid 21-hydroxylase (21-OH) is a key haem containing steroidogenic enzyme and a major adrenal specific autoantigen. Cys 428 in 21-OH is thought to have an important role in haem binding and we now describe the effects of mutations at Cys 428 (to Ser, Arg and Phe) on 21-OH autoantibody binding. Expression of wild type and mutated 21-OH was carried out using an in vitro transcription/translation (TnT) system and reactivity of 21-OH autoantibodies with mutated 21-OH analysed by western blotting (in the case of unlabelled proteins) or immunoprecipitation assay (IPA) (in the case of 35S-labelled proteins). All 3 substitutions at Cys 428 had similar effects on 21-OH autoantibody binding and each one caused a reduction in autoantibody binding to about 50% of wild type in the case of IPA and to about 70% of wild type in the case of western blotting analysis. In addition to mutations at Cys 428, we studied 2 naturally occurring mutations at Pro 30 to Leu and He 172 to Asn which are associated with diminished 21-OH enzyme activity. The Pro 30 mutation had no effect, but the He 172 mutation caused a reduction in 21-OH autoantibody binding in the IPA to about 80% of wild type.Overall, our studies emphasise the close relationship between the 21-OH aminoacid sequences important for 21-OH enzyme activity and 21-OH autoantibody binding.

Hormonal responses during various phases of autoimmune adrenal failure: no evidence for 21-hydroxylase enzyme activity inhibition in vivo.

Adrenal autoantibodies (ACA) are markers of adrenal cortex involvement in idiopathic Addison's disease. Recently the 21-hydroxylase (21-OH) enzyme has been discovered to be the major autoantigen of the ACA. A potential role of these antibodies in determining adrenal failure by inhibition of the 21-OH has been recently postulated. To test this hypothesis, cortisol and aldosterone (final products of adrenal steroid synthesis) and 17-hydroxyprogesterone (17-OH-progesterone) (as a marker of 21-OH impairment) have been investigated in baseline conditions and after ACTH (1-24) stimulation test in a group of 42 patients positive for both ACA and 21-OH autoantibodies. Patients were divided into five groups according to the stages (0-4) of adrenal failure. With progression toward overt Addison's disease, baseline 17-OH-progesterone, cortisol, and aldosterone remained almost unchanged but with impairment of their responses to ACTH (1-24) stimulation. The 17-OH-progesterone/cortisol ration remained normal both in basal conditions and after stimulation at stages 0-3. At stage 4 (overt Addison's disease), this ratio increased in baseline condition with no changes after ACTH (1-24), probably because of persistent 17-OH-progesterone gonadal production. In conclusion, there was a progressive and concomitant impairment of the synthesis of all steroids tested over various phases of adrenal failure. The pattern of response of the 17-OH-progesterone/cortisol ratio to ACTH stimulation in patients with 21-OH autoantibodies was not consistent with the autoantibodies inhibiting the 21-OH activity. This suggests that the inhibiting effect of 21-OH autoantibodies on 21-OH activity is not usually evident in vivo.

Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison's disease, and premature ovarian failure.

Autoantibodies to steroidogenic enzymes, steroid 17 alpha-hydroxylase (17 alpha-OH), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroid 21-hydroxylase (21-OH), were measured using specific and sensitive immunoprecipitation assays (IPAs) in patients with various forms of autoimmune adrenal disease. Autoantibodies to 17 alpha-OH were detected in 6 of 11 (55%) patients with autoimmune polyglandular syndrome (APS) type I, 8 of 24 (33%) patients with APS type II, 11 of 56 (20%) patients with adrenal cortex antibody (ACA; measured by immunofluorescence)-positive patients without Addison's disease, and only 3 of 64 (5%) patients with Addison's disease. Autoantibodies to P450scc were found at a prevalence similar to those to 17 alpha-OH: in 5 of 11 (45%) APS type I patients, 10 of 24 (42%) APS type II patients, 11 of 56 (20%) ACA-positive patients without Addison's disease, and only 6 of 64 (9%) patients of the Addison disease group. Autoantibodies to 21-OH were found in a majority of patients with APS type I (7 of 11;64%), APS type II (23 of 24; 96%), Addison's disease (41 of 64; 64%), and ACA-positive patients without Addison's disease (48 of 56; 86%). All sera that were positive for 17 alpha-OH or P450scc were also positive for 21-OH autoantibodies, except in 1 case. There was good agreement between the presence of ACA measured by immunofluorescence and 21-OH antibodies measured by IPA in all patient groups studied, and this indicates that 21-OH is a major autoantigen in adrenal autoimmune disease regardless of whether the disease presents as isolated Addison's disease or APS type I or type II. Autoantibodies to 17 alpha-OH and P450scc appeared to be the major components of the steroid-producing cell antibodies measured by immunofluorescence. No autoantibodies to 21-OH, 17 alpha-OH, or P450scc were detected in 17 sera from patients with premature ovarian failure without evidence of adrenal autoimmunity (as judged by immunofluorescence studies), except for 1 serum in which low levels of 17 alpha-OH antibodies were found. Overall, our studies indicate that 35S-labeled 17 alpha-OH, P450scc, and 21-OH can be used successfully in IPAs for their respective autoantibodies. Assays such as these may well be valuable in the immunological assessment of patients at risk for or suspected of adrenal autoimmunity.

Immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in autoimmune adrenal diseases

Adrenal autoantibodies characteristic of autoimmune Addison disease are directed towards steroid 21-hydroxylase (21-OH; EC 1.14.99.10). We describe a new assay to measure 21-OH autoantibodies (21-OH Abs), based on immunoprecipitation by the antibodies of 35S-labeled human 21-OH. Using this immunoprecipitation assay (IPA), we detected 21-OH Abs in 42 of 64 (66%) patients with Addison disease and in 14 of 19 (74%) patients with autoimmune polyendocrine syndromes type I and type II. No 21-OH Abs were detected by the IPA in any patients with Addison disease attributable to tuberculosis (n = 9) or adrenoleukodystrophy (n = 9) or in patients with autoimmune thyroid disease (n = 28), systemic lupus erythematosus (n = 10), myasthenia gravis (n = 10), rheumatoid arthritis (n = 10), or insulin-dependent diabetes mellitus (n = 12). None of the 26 sera from healthy normal blood donors was positive for 21-OH Abs by the assay. We found good agreement between 21-OH Abs measured by IPA and by Western blotting (r = 0.83, n = 123, P < 0.001). The inter- and intraassay CVs for IPA were well < 10% at high, medium, and low concentrations of 21-OH Abs. Overall, our studies indicate that the IPA provides a specific, sensitive, and convenient system for measuring 21-OH Abs.

Autoimmune Addison's disease--evidence for a role of steroid 21-hydroxylase autoantibodies in adrenal insufficiency.

Autoantibodies to steroid 21-hydroxylase (21-OH) are characteristic of adult onset Addison's disease and we have investigated the effects of these autoantibodies on recombinant human 21-OH enzyme activity. Antibody preparations from 11/11 Addison sera inhibited the ability of 21-OH to convert progesterone to deoxycorticosterone with 8 IgGs showing almost complete inhibition, 2 partial inhibition and 1 weak inhibition. Control IgGs from patients with autoimmune thyroid disease and normal blood donors had little or no effect on 21-OH activity. Our results suggest that 21-OH autoantibodies have the potential to contribute to adrenal failure in Addison's disease by inhibiting the 21-OH enzyme.

Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding.

Human 21-hydroxylase (21-OH) genes containing various mutations, truncations, and deletions were expressed in yeast, and autoantibody binding was studied by Western blotting using patient sera and rabbit antibodies to 21-OH. 21-OH autoantibodies in 13 Addisonian sera showed a marked reduction in their ability to recognize 21-OH mutated at Pro453-->Ser (mean +/- SD, 31 +/- 9% of binding to wild type), whereas the effect on rabbit antibody binding was small (88 +/- 11% of binding to wild type; n = 7). Mutation at Arg339-->His had a less pronounced effect on autoantibody binding (85 +/- 11% of binding to wild type; n = 13) and caused a small enhancement of rabbit antibody binding (124 +/- 16% of binding to wild type; n = 7). These studies indicate that Pro453 has a key role in forming an autoantigenic epitope on 21-OH. It is important to note, however, that the Pro453 mutation caused only partial loss of autoantibody binding, i.e. all Addisonian sera studied still reacted with the mutated protein. This may indicate that each serum sample contains at least two different populations of 21-OH autoantibodies, only one of which recognizes a site dependent on Pro453. A series of more extensive modifications of the 21-OH sequence, including truncations (amino acids 460-494, 448-494, and 418-494) and deletions (amino acids 165-379, 142-240, and 142-280) indicated that most of the sequence of amino acids from 241-494 is important for autoantibody binding. The involvement of such an extensive region of the molecule suggests that the binding sites are generated by three-dimensional folding, with Pro453 having a critical role in forming at least one major autoantigenic epitope.