Diagnostic Value of Autoantibodies against Steroidogenic Enzymes and Hormones in Infertile Women with Premature Ovarian Insufficiency.

The objective of the study was to evaluate the profile and diagnostic significance of serum autoantibodies in infertile patients with premature ovarian insufficiency (POI). The pilot study included 26 patients of reproductive age with POI and diminished ovarian reserve who received complex treatment using new surgical technologies (Group 1) and 18 patients without POI (Group 2). The profile of serum autoantibodies, including anti-ovarian antibodies, antibodies against thyroid peroxidase (TPO), steroidogenic enzymes, and steroid and gonadotropic hormones, was studied using modified ELISAs and human recombinant steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2). Patients in Group 1 had higher levels of IgG autoantibodies against steroidogenic enzymes, estradiol, progesterone, and TPO than those in Group 2. Tests for IgG antibodies against CYP11A1, CYP19A1, and CYP21A2 exhibited high sensitivity (65.4-76.9%), specificity (83.3-89.9%), and AUC values (0.842-0.910) for POI, the highest in the first test. Three-antibodies panel screening showed higher diagnostic accuracy (84.1% versus 75-79.6%). The levels of these antibodies correlated with menstrual irregularities and a decrease in the antral follicle count. Thus, antibodies against CYP11A1, CYP19A1, and CYP21A2 have a high diagnostic value for POI. Three-antibody panel screening may improve the accuracy of POI diagnosis and be useful for identifying high-risk groups, early stages of the disease, and predicting POI progression.

21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison's Disease Are Restricted by HLA-A2 and HLA-C7 Molecules.

Objectives: CD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison's disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD. Methods: Recombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH. Results: We found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated. Conclusion: We have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.

Reversible primary adrenal insufficiency related to anti-programmed cell-death 1 protein active immunotherapy: Insight into an unforeseen outcome of a rare immune-related adverse event.

The immune checkpoint inhibitors (ICPi) revolutionize the cancer therapeutics, though not being devoid of toxicity. The immune-related primary adrenal insufficiency (PAI) is a rare, yet potentially life-threatening, adverse event, posing diagnostic and therapeutic challenges. We report the first case of reversible PAI related to nivolumab (programmed cell-death 1 protein inhibitor) in a 42-year-old male with metastatic rectal adenocarcinoma. PAI manifested as profound fatigue, disorientation, hypotension, hyperpigmentation of palmar creases, and hyponatremia without hyperkalemia 16 weeks after initiation of nivolumab. Due to impending adrenal crisis, intravenous stress doses of hydrocortisone and hydration with normal saline were initiated. When the state of patient was stabilized, PAI was confirmed through 250 µg Synacthen test 24 h after temporary cessation of hydrocortisone. Hydrocortisone was fixed at maintenance dose, while mineralocorticoid substitution was not required. PAI was ascribed to nivolumab based on history, physical examination, and laboratory work-up with emphasis on positivity of anti-21-hydroxylase antibodies and exclusion of other causes of PAI by normal imaging of adrenal glands on computed tomography (CT). Reevaluation of adrenal function during follow up demonstrated complete recovery. A review of literature concerning the immune-related PAI indicated that the complete recovery of adrenal function, the normal CT imaging, and the positivity of anti-21-hydroxylase antibodies observed in our patient are exceptional findings of immune-related PAI. Finally, heightened suspicion of immune-related PAI in case of hyponatremia without hyperkalemia and constant vigilance for diagnosis of rare, but real, reversibility of immune-related PAI are of paramount importance.

Organ-specific autoantibodies in Chinese patients newly diagnosed with type 1 diabetes mellitus.

This study aims to investigate the prevalence of islet autoantibodies and other organ-specific autoantibodies in type 1 diabetes mellitus (T1DM) patients and characterize their clinical features. Glutamic acid decarboxylase antibody (GADA), insulinoma antigen 2 antibody (IA-2A), zinc transporter 8 antibody (ZnT8A) and tetraspanin7 antibody (TSPAN7A) were assayed by radioligand or luciferase immunoprecipitation system assays in 205 newly diagnosed acute-onset T1DM patients and 170 healthy controls. Other organ-specific autoantibodies, including thyroid peroxidase antibody (TPOA), thyroglobulin antibody (TGA), tissue transglutaminase antibody (tTGA) and 21-hydroxylase antibody (21-OHA), were also measured. The prevalence of GADA, IA-2A, ZnT8A, TSPAN7A, TPOA, TGA and 21-OHA was higher in T1DM patients than in healthy controls. The combinational assay of various islet autoantibodies could increase the frequency of autoantibody positivity in T1DM to 85.4%. GADA+ IA-2A+ T1DM patients preferentially had TPOA and TGA, while IA-2A+ patients often had tTGA. Patients positive for two or more islet autoantibodies often had TPOA and TGA. BMI of multiple islet autoantibody-positive patients was lower than that of patients with single or no islet autoantibodies, and there were no significant differences in C-peptide and glycated hemoglobin between patients positive for islet autoantibodies combined with other organ-specific antibodies and noncombined patients. Younger female patients who were islet autoantibody positive were more likely to have TPOA and TGA. The frequency of Graves' disease was much higher in T1DM patients than in healthy controls. T1DM usually occurs together with other organ-specific autoantibodies. Measuring of other organ-specific autoantibodies will be beneficial for T1DM patients.

Autoimmune Addison's disease.

Primary adrenal insufficiency (PAI) occurs in 1/5000-1/7000 individuals in the general population. Autoimmune Addison's disease (AAD) is the major cause of PAI and is a major component of autoimmune polyendocrine syndrome type 1 (APS1) and type 2 (APS2). Presence of 21-hydroxylase autoantibodies (21OHAb) identifies subjects with ongoing clinical or pre-clinical adrenal autoimmunity. AAD requires life-long substitutive therapy with two-three daily doses of hydrocortisone (HC) (15-25 mg/day) or one daily dose of dual-release HC and with fludrocortisone (0.5-2.0 mg/day). The lowest possible HC dose must be identified according to clinical and biochemical parameters to minimize long-term complications that include osteoporosis and cardiovascular and metabolic alterations. Women with AAD have lower fertility and parity as compared to age-matched healthy controls. Patients must be educated to double-triple HC dose in the case of fever or infections and to switch to parenteral HC in the case of vomiting, diarrhoea or acute hypotension.

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.

AIMS: 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). MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

21-hydroxylase autoantibodies are more prevalent in Turner syndrome but without an association to the autoimmune polyendocrine syndrome type I.

Turner syndrome (TS) is associated with an increased frequency of autoimmunity. Frequently observed autoimmune diseases in TS are also seen in the autoimmune polyendocrine syndrome type I (APS I), of which Addison disease is a key component. An overlapping antibody profile between TS and APS I could be considered. The aim of this work was to study women with TS regarding 21-hydroxylase (21-OH) antibodies and interferon omega (IFN-ω) antibodies, a highly specific marker for APS I, to determine if there are immunological overlaps between TS and APS I. Blood samples from 141 TS were assayed for 21-OH antibodies and IFN-ω antibodies using in-vitro-transcribed and translated autoantigen. Indices with a cut-off point of 57 and 200 for 21-OH antibody and IFN-ω antibody were used as reference. The median age of TS was 31·6 years (range = 11·2-62·2). Positive indices of 21-OH antibodies were present in six TS (4%), with a mean of 144·8 (range = 60-535). None had apparent adrenal insufficiency. There was no age difference comparing 21-OH antibody-positive TS (median age = 33·9 years, range = 17·7-44·7) and 21-OH antibody-negative TS (median age = 31·6 years, range = 11·2-62·2) (P = 0·8). No TS was positive for IFN-ω antibodies (mean = 42·4, range = -435-191). No overlapping autoimmune profile between TS and APS I was found. Autoimmunity against 21-OH among TS patients was more prevalent than previously identified, suggesting an increased risk of adrenal failure in TS. However, whether adrenal impairment will develop remains unknown.

The potential role for infections in the pathogenesis of autoimmune Addison's disease.

Autoimmune Addison's disease (AAD), or primary adrenocortical insufficiency, is a classical organ-specific autoimmune disease with 160 years of history. AAD is remarkably homogeneous with one major dominant self-antigen, the cytochrome P450 21-hydroxylase enzyme, which is targeted by both autoantibodies and autoreactive T cells. Like most autoimmune diseases, AAD is thought to be caused by an unfortunate combination of genetic and environmental factors. While the number of genetic associations with AAD is increasing, almost nothing is known about environmental factors. A major environmental factor commonly proposed for autoimmune diseases, based partly on experimental and clinical data and partly on shared pathways between anti-viral immunity and autoimmunity, is viral infections. However, there are few reports associating viral infections to AAD, and it has proved difficult to establish which immunological processes that could link any viral infection with the initiation or progression of AAD. In this review, we will summarize the current knowledge on the underlying mechanisms of AAD and take a closer look on the potential involvement of viruses.

The natural history of autoimmune Addison's disease with a non-classical presentation: a case report and review of literature.

Autoimmune Addison's disease (AAD) is the most frequent cause of adrenocortical insufficiency. The natural history of AAD usually comprises five consecutive stages with the first stage characterized by the increase of plasma renin consistent with the impairment of pars glomerulosa, which is usually the first affected layer of the adrenal cortex. We describe a 19-year-old female with Hashimoto's thyroiditis (HT) who underwent an autoantibody screening due to having the personal and family history of other autoimmune diseases in the absence of relevant clinical manifestations. She was positive for adrenal cortex autoantibodies (ACA) and steroid 21-hydroxylase autoantibodies (21-OH Ab) at high titers. She had increased basal levels of ACTH with normal basal cortisol not responding to ACTH stimulation, reduced levels of dehydroepiandrosterone-sulfate but normal levels of orthostatic renin and aldosterone. This scenario was consistent with a subclinical AAD presenting with first impairments in pars fasciculata and reticularis and conserved pars glomerulosa function. Only subsequently, progressive deficiency in pars glomerulosa function has become evident. Review of the literature showed that there was only one case, reported to date, with a similar atypical natural history of AAD. The strategies for screening for ACA/21-OH Ab in patients with HT are discussed.

A new ELISA for autoantibodies to steroid 21-hydroxylase.

BACKGROUND: A new ELISA for autoantibodies to steroid 21-hydroxylase (21-OH Ab) is described. METHODS: In the assay test sample autoantibodies form a bridge between 21-OH coated onto the plate well and liquid phase 21-OH-biotin. Bound 21-OH-biotin is detected by the addition of streptavidin peroxidase and colorogenic peroxidase substrate. RESULTS: Of 100 samples from patients with autoimmune Addison's disease, 86 (86%) were positive for 21-OH Ab ELISA whereas 84 (84%) were positive in an immunoprecipitation assay based on 125I-labeled 21-OH. Six (0.6%) of 928 healthy adult blood donors and 1 (2.0%) of 49 adult patients with type 1 diabetes mellitus (T1DM) were positive by ELISA. No samples from adult patients with Graves' disease (GD; n=50), celiac disease (n=29), systemic lupus erythematosis (n=9) or rheumatoid arthritis (n=20) were positive by ELISA. However, 2/51 (3.9%) children with GD, 3/69 (4.3%) children with Hashimoto's thyroiditis (HT) and 3/119 (2.5%) children with T1DM alone or associated with autoimmune thyroid disorders were ELISA positive. CONCLUSIONS: The new assay should be useful for screening patients known to be at increased risk of developing clinical autoimmune Addison's disease, in particular children with HT, GD and/or T1DM.

An Addison disease revealed with a serious hyponatremia.

We present the case of an Addison's disease revealed by a serious hyponatremia. The serum concentration of ACTH and 21-hydroxylase antibodies were increased and lead to the diagnosis. The cortisol blood level was lowered but required to take into account the stress induced by the hospitalisation of the patient. Addison's disease is characterized by the destruction of the adrenal cortex. Autoimmune adrenalitis is the main cause of adrenal insufficiency. Treatment involves normalisation of sodium concentration and corticosteroids replacement. With a good patient compliance, the survival rate of Addisonian patient is similar to that of the normal population. Management of patient requires vigilance because of the occurrence of others autoimmunes diseases during patient life.

Association of HLA alleles and haplotypes with CYP21A2 gene p. V282L mutation in the Croatian population.

The CYP21A2 mutations that are in linkage disequilibrium with particular HLA-A, -B, -DRB1 alleles/haplotypes, cause deficiency of the 21-hydroxylase enzyme (21-OHD) and account for the majority of congenital adrenal hyperplasia (CAH) cases. The aim of this study was to investigate those associations with the p.V282L mutation linked to the non-classical (NC) form of CAH among Croatians. The study included parents of patients with the NC form of CAH, positive for the p.V282L mutation (N = 55) and cadaveric donor samples (N = 231). All subjects were HLA-A, -B, and -DRB1 typed and tested for the presence of the p.V282L mutation. Among parents of patients, 92.73% of subjects were positive for the B*14:02 allele and almost half of them carried the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype. Among cadaveric samples 77 out of 96 subjects positive for the B*14:02 allele had the p.V282L mutation. Among them, 37 were positive for the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype, 23 had the HLA-A*33:01-B*14:02-DRB1*03:01 haplotype, 8 had the B*14:02-DRB1*01:02 combination and 5 were carrying the HLA-A*68:02-B*14:02-DRB1*13:03 haplotype. Only 4 of these subjects were positive for the B*14:02 allele. HLA-B*14:02 was the only single allele with association that reached statistically significant P value (RR = 12.00; P = 0.0024). Haplotypes B*14:02-DRB1*01:02 (P < 0.001) and HLA-A*68:02-B*14:02-DRB1*13:03 (P < 0.001) as well as HLA-A*33:01-B*14:02-DRB1*01:02 and HLA-A*33:01-B*14:02-DRB1*03:01 showed high relative risks (RR = 45.00, RR = 41.63 and RR = 36.96, respectively). Our data support the previously documented association of the HLA-A*33:01-B*14:02-DRB1*01:02 haplotype with the p.V282L mutation, but also point out a high frequency of the p.V282L mutation among Croatians with HLA-A*33:01-B*14:02-DRB1*03:01 and HLA-A*68:02-B*14:02-DRB1*13:03 haplotypes.

Organ-specific autoimmunity in relation to clinical characteristics in children with long-lasting type 1 diabetes.

BACKGROUND: The aim of this study was to assess the prevalence of diabetes and other organ-specific autoantibodies (Ab) associated with various autoimmune conditions, in Polish children with type 1 diabetes mellitus (T1DM). METHODS: In this study 114 patients, aged 13.4 years, with mean diabetes duration 5.2 years were included. Ab to islet cell antigens: glutamic acid decarboxylase (GAD), insulinoma antigen 2 (IA-2), zinc transporter 8 (ZnT8), together with thyroid peroxidase Ab (TPO Ab), thyroglobulin Ab (Tg Ab), tissue transglutaminase Ab (tTG Ab) and 21-hydroxylase Ab (21-OH Ab) were measured. RESULTS: The prevalence of at least one diabetes associated Ab was found in 87%, with the highest prevalence of 64% for ZnT8 Ab. In patients with disease duration <5 years, at least one antibody was present in 90%, the most prevalent was ZnT8 Ab (72%). In patients with duration >10 years, 50% had at least one antibody. The prevalence of other than islet cell autoimmunity was high (34%). Thyroid Ab were detected in 26% patients, 42% in girls vs. 8% in boys, p<0.001. tTG Ab were found in 11% patients, with a greater prevalence in children with early onset (p=0.01). 21-OH Ab were found in 2.6% T1DM patients. CONCLUSIONS: Islet Ab were found in most T1DM children and remained positive even 10 years after onset. ZnT8 Ab emerged as an important marker for the diagnosis of T1DM in the Polish children. Screening for non-diabetes Ab in T1DM may be helpful in identifying subclinical cases of autoimmune thyroid, celiac or Addison's disease (AD).

Endocrine autoimmunity in patients with Latent Autoimmune Diabetes in Adults (LADA) ­ association with HLA genotype.

INTRODUCTION: Latent autoimmune diabetes in adults (LADA) is a slow-developing form of autoimmune diabetes, characterised by the presence of type 1 diabetes-associated autoantibody and presentation at diagnosis similar to patients with type 2 diabetes. The aim of this study was to determine the prevalence of auto-antibodies related to endocrine autoimmune diseases in patients with LADA and to assess their association with HLA genotype. MATERIAL AND METHODS: We evaluated the presence of anti-thyroglobulin (ATG), anti-thyroid peroxidase (ATPO), anti-tissue transglutaminase IgA (ATTA), and anti 21 hydroxylase (A21H) in 70 patients with LADA, 69 with Type 2 diabetes, and in 50 healthy controls HLA genotype was assed in subpopulation of sluclied subjects. RESULTS: The presence of ATPO (28.6 vs. 10%); ATG (28.6 vs. 14%) was higher in patients with LADA in comparison to healthy controls and ATPO in comparison to patients with type 2 diabetes (38.6 vs. 17.4 %). In patients with LADA the presence of autoimmune thyroid autoantibodies was associated with newly diagnosed subclinical hypothyroidism; almost 7% of patients presented with high TSH. The presence of A21H (2.86 vs. 5.8 vs. 6.1%) and ATTA (2.86 vs. 4.3 vs. 6.0%) was not different between groups. Patients with high TSH level were positive for DQA1*0301 and DRB1*04 HLA genotype: DQB1*0201 and DQB1*02 were higher in patients positive for ATTA. CONCLUSIONS: Patients with LADA have higher prevalence of thyroid autoimmune diseases. In patients with LADA similarly to type 1 genotype DQA1*0301 seems to CONFER susceptibility to thyroid autoimmunity, and DQB1*0201 to celiac disease.

Fatigue in Primary Sjögren's Syndrome: Clinical, Laboratory, Psychometric, and Biologic Associations.

OBJECTIVE: To identify independent contributors of fatigue in primary Sjögren's syndrome (SS) patients, taking into account clinical, laboratory, and psychological features, and to explore the potential role of interferon (IFN)-induced gene indoleamine 2,3-dioxygenase (IDO-1), anti-21-hydroxylase (anti-21[OH]) antibodies, and soluble BAFF. METHODS: Detailed clinical and laboratory characteristics were recorded for 106 primary SS patients. The Functional Assessment of Chronic Illness Therapy-Fatigue, Zung Depression Scale, State-Trait Anxiety Inventory, Eysenck Personality Questionnaire Scale, and Athens Insomnia Scale were adopted to assess fatigue, depression, anxiety, and sleep disturbances, respectively. Peripheral whole blood expression levels of IDO-1, as well as type I and II IFN-induced genes were calculated using quantitative reverse transcriptase-polymerase chain reaction. Serum anti-21(OH) antibodies and soluble BAFF levels were determined by a radioimmunoassay and an enzyme-linked immunosorbent assay, respectively. Univariate and multivariate models were performed to identify determinants of fatigue. RESULTS: Fatigue was detected in 32 of 106 (30.2%) primary SS patients. In univariate analysis, fatigue was associated with arthralgias/myalgias, fibromyalgia hydroxychloroquine therapy, both state and trait anxiety scores, depression, and neuroticism, as well as impaired sleep patterns. Multivariate analysis revealed neuroticism (odds ratio [OR] 6.9, [95% confidence interval (95% CI) 1.7-28.0]), depression (OR 3.0 [95% CI 0.8-11.0]), and fibromyalgia (OR 5.5 [95% CI 1.1-27.7]) as independent fatigue contributors. Soluble BAFF levels, anti-21(OH) autoantibodies, and IDO-1 messenger RNA expression did not significantly differ between fatigued and nonfatigued primary SS patients. CONCLUSION: Depression, neuroticism, and fibromyalgia play a major role in primary SS-associated fatigue and should be addressed in clinical practice, with active collaboration between rheumatologists and mental health professionals. Further studies are warranted in order to explore underlying pathophysiologic pathways that might explain fatigue in the setting of primary SS.

Presence of 21-Hydroxylase Antibodies in a Boy with X-Linked Adrenal Hypoplasia Congenita.

BACKGROUND: X-linked adrenal hypoplasia congenita is a rare cause of primary adrenal insufficiency (PAI) in children due to mutations in NR0B1/DAX1 (nuclear receptor subfamily 0, group B, member 1/dosage-sensitive sex reversal-adrenal hypoplasia congenita at the critical region of the X chromosome, gene 1). Another rare cause of PAI in children is autoimmune adrenal disease (AAD) which could be either isolated or as part of autoimmune polyglandular syndrome. Antibody to major auto-antigen, 21-hydroxylase, is highly specific for AAD. METHODS: We report a now 19-month-old male with PAI due to NR0B1 gene mutation and positive adrenal antibodies. Initially, he presented at 15 days of life with isolated hypoaldosteronism which later unfolded into complete PAI. Data analysis was done via retrospective chart review. RESULTS: Genetic analysis of the NR0B1 gene revealed a known hemizygous mutation in c.1069C>T; p.Gln357X. Simultaneously, he was noted to have positive 21-hydroxylase antibodies. CONCLUSION: According to our knowledge, this is the first case in the literature with NR0B1 mutation causing adrenal insufficiency with coexistent positive adrenal antibodies. In addition to his already compromised adrenal function due to NR0B1 mutation, he is now at risk for the development of associated autoimmune conditions requiring close follow-up.

Design and Measurement of Nonislet-Specific Autoantibodies for the Type 1 Diabetes Genetics Consortium Autoantibody Workshop.

The Type 1 Diabetes Genetics Consortium (T1DGC) comprised groups of investigators from many countries throughout the world, with a common goal of identifying genes predisposing to type 1 diabetes. The T1DGC ascertained and collected samples from families with two or more affected siblings with type 1 diabetes and generated a broad array of clinical, genetic, and immunologic data. The T1DGC Autoantibody Workshop was designed to distribute data for analyses to discover genes associated with autoantibodies in those with type 1 diabetes. In the T1DGC-affected sibling pair families, three T1DGC Network laboratories measured antibodies to the islet autoantigens GAD65 and the intracellular portion of protein tyrosine phosphatase (IA-2A). The availability of extensive genetic data provided an opportunity to investigate the associations between type 1 diabetes and other autoimmune diseases for which autoantibodies could be measured. Measurements of additional nonislet autoantibodies, including thyroid peroxidase, tissue transglutaminase, 21-hydroxylase, and the potassium/hydrogen ion transporter H+/K+-ATPase, were performed by the T1DGC laboratory at the Barbara Davis Center for Childhood Diabetes, Aurora, CO. Measurements of all autoantibodies were transmitted to the T1DGC Coordinating Center, and the data were made available to members of the T1DGC Autoantibody Working Groups for analysis in conjunction with existing T1DGC genetic data. This article describes the design of the T1DGC Autoantibody Workshop and the quality-control procedures to maintain and monitor the performance of each laboratory and provides the quality-control results for the nonislet autoantibody measurements.

Determination of 21-hydroxylase autoantibodies: inter-laboratory concordance in the Euradrenal International Serum Exchange Program.

BACKGROUND: 21-Hydroxylase autoantibodies (21OHAb) are markers of an adrenal autoimmune process that identifies individuals with autoimmune Addison's disease (AAD). Quality and inter-laboratory agreement of various 21OHAb tests are incompletely known. The objective of the study was to determine inter-laboratory concordance for 21OHAb determinations. METHODS: Sixty-nine sera from 51 patients with AAD and 51 sera from 51 healthy subjects were blindly coded by a randomization center and distributed to 14 laboratories that determined 21OHAb, either by an "in-house" assay (n=9) using in vitro-translated (35)S-21OH or luciferase-labeled 21OH or a commercial kit with (125)I-21OH (n=5). Main outcome measures were diagnostic accuracy of each participating laboratory and inter-laboratory agreement of 21OHAb assays. RESULTS: Intra-assay coefficient of variation ranged from 2.6% to 5.3% for laboratories using the commercial kit and from 5.1% to 23% for laboratories using "in-house" assays. Diagnostic accuracy, expressed as area under ROC curve (AUC), varied from 0.625 to 0.947 with the commercial kit and from 0.562 to 0.978 with "in-house" methods. Cohen's κ of inter-rater agreement was 0.603 among all 14 laboratories, 0.691 among "in-house" laboratories, and 0.502 among commercial kit users. Optimized cutoff levels, calculated on the basis of AUCs, increased the diagnostic accuracy of every laboratory (AUC >0.9 for 11/14 laboratories) and increased the Cohen's κ of inter-rater agreement. Discrepancies in quantitation of 21OHAb levels among different laboratories increased with increasing autoantibody levels. CONCLUSIONS: The quality of 21OHAb analytical procedures is mainly influenced by selection of cutoff value and correct handling of assay materials. A standardization program is needed to identify common standard sera and common measuring units.

Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease--an association study and expression analysis.

Autoimmune Addison's disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561-0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

HLA similarities indicate shared genetic risk in 21-hydroxylase autoantibody positive South African and United States Addison's disease.

Genetic similarities between patients from the United States and South African (SA) Addison's Disease (AD) strengthen evidence for genetic association. SA-AD (n = 73), SA healthy controls (N = 78), and US-AD patients (N = 83) were genotyped for DQA1, DQB1, DRB1, and HLA-B alleles. Serum was tested for the quantity of 21OH-AA and IFNα-AA at the Barbara Davis Center. Although not as profound as in US-AD, in SA-AD 21OH-AA + subjects the predominantly associated risk haplotypes were DRB1*0301-DQB1*0201 (DR3), DRB1*04xx-DQB1*0302 (DR4), and the combined DR3/4 genotype. DQB1*0302 associated DRB1*04xx haplotypes conferred higher risk than those DRB1*04xx haplotypes associated with other DQB1 alleles. We found negative association in 21OH-AA + SA-AD for DQA1*0201-DQB1*0202 and DQA1*0101-DQB1*0501 vs SA controls, and positive association for DQA1*0401-DQB1*0402 vs US-AD. Apart from the class II DR3 haplotype, HLA-B8 did not have an independent effect; however together DR3 and HLA-B8 conferred the highest risk vs 21OH-AA negative SA-AD and SA-controls. HLA-B7 (often with DR4) conferred novel risk in 21OH-AA + SA-AD vs controls. This study represents the first comparison between South African and United States AD populations utilizing genotyping and serology performed at the same center. SA-AD and US-AD 21OH-AA + patients share common HLA risk haplotypes including DR4 (with HLA-B7) and DR3 (with HLA-B8), strengthening previously described HLA associations and implicating similar genetic etiology.