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Autoimmune Addison’s disease is the predominant cause of primary adrenal failure, and is highly heritable. The genetic background has remained poorly understood due to the low prevalence and complex inheritance of the disease. We performed a genome-wide association study, which identified nine independent risk loci (P < 5 × 10–8). In addition to novel and previous risk loci involved in lymphocyte functionality, we further associated autoimmune Addison’s disease with two independent protein-coding alterations in the gene Autoimmune Regulator (AIRE). The most striking is the amino-acid substitution p.R471C (rs74203920, OR = 3.4 (2.7–4.3), P = 9.0 × 10–25), which introduces an additional cysteine residue in the zinc-finger motif of the PHD2 domain of AIRE. This unbiased elucidation of the genetic contribution to development of autoimmune Addison’s disease points to the importance of central immunological tolerance, and explains 35–41 percent of heritability.

Primary ovarian insufficiency (POI) is defined as hypergonadotropic amenorrhea before the age of 40 yr. In 4-5% of patients with POI, an ovarian autoimmune process is present.Serum concentrations of antimüllerian hormone (AMH) have been determined in 26 women with POI due to steroidogenic cell autoimmunity (SCA-POI), 66 with nonautoimmune idiopathic POI (iPOI), 40 postmenopausal women (PMW), and 44 healthy fertile women (HW). SCA-POI was diagnosed according to presence of steroidogenic enzyme autoantibodies (17alpha-hydroxylase, side chain cleavage, and 21-hydroxylase autoantibodies).AMH concentrations were significantly higher in women with SCA-POI than women with iPOI (P = 0.018) or PMW (P = 0.03) but significantly lower than HW (P0.0001). AMH was detected in 11 of 26 women with SCA-POI (42%) and seven of 66 with iPOI (11%) (P = 0.002). Serum concentrations above the fifth percentile of the normal range (0.6 ng/ml) were detected in nine of 26 women with SCA-POI (35%) and four of 66 with iPOI (6%) (P = 0.001). Eight of 12 women with SCA-POI with less than 5 yr (67%) and one of 14 with longer disease duration (7%) had AMH concentrations within the normal range (P = 0.003). AMH concentrations correlated inversely with disease duration in women with SCA-POI (rho = -0.563, P = 0.003) but not women with iPOI. AMH correlated inversely with FSH serum concentrations in HW (rho = -0.584, P0.001) but not PMW or women with POI.Two thirds of women with recent-onset SCA-POI had normal AMH concentrations. Women with SCA-POI, differently from those with iPOI, present a preserved ovarian follicle pool for several years after diagnosis of ovarian insufficiency.

Primary ovarian insufficiency (POI) is defined by hypergonadotropic amenorrhea occurring before the age of 40 yr. In 4-5% of women with POI, an ovarian autoimmune process can be demonstrated.We have determined the serum concentrations of total inhibin and inhibin B by sensitive ELISAs in 22 women with autoimmune POI (aPOI), 71 women with non-autoimmune idiopathic POI (iPOI), 77 postmenopausal women, and 90 healthy, fertile women (HW). Diagnosis of aPOI was made according to the presence of steroid cell autoantibodies and/or 17alpha-hydroxylase autoantibodies and/or cytochrome P450 side-chain cleavage autoantibodies. All aPOI patients were also positive for adrenal autoantibodies.Total inhibin levels were significantly higher in women with aPOI (median, 281 pg/ml) than in women with iPOI (median, 74 pg/ml) or HW (median, 133.5 pg/ml) (P0.001). Levels of inhibin B were also significantly higher in women with aPOI (median, 109 pg/ml) than in women with iPOI (median, 18 pg/ml) (P0.001) or HW (median, 39 pg/ml) (P0.05). Serum concentrations of total inhibin and inhibin B were significantly higher in women with POI than in postmenopausal women (P0.001), irrespective of the presence/absence of autoantibodies. At receiver-operating characteristic analysis, cutoff values of 133 pg/ml for total inhibin and 60.5 pg/ml for inhibin B ensured 86.4% sensitivity and 81-84.5% specificity for aPOI vs. iPOI.We conclude that a variable degree of ovarian function is preserved in women with POI and that aPOI is characterized by increased inhibin production resulting from a selective theca cell destruction, with initial preservation of granulosa cells.

We describe the clinical course of three women with presumptive autoimmune oophoritis who developed multiple follicles but very low to undetectable estradiol levels. Multiple follicles developed spontaneously in all subjects and during pulsatile GnRH treatment for ovulation induction in subject 1. The development of multiple dominant follicles was accompanied by LH levels in the postmenopausal range and FSH levels at the upper limit for premenopausal women. Serum inhibin B levels were elevated appropriately in the setting of multifollicular development, but estradiol levels remained low. Measurement of estradiol precursors demonstrated androstenedione and estrone levels below the 95th percentile in normal women. Adrenal cortical antibodies, and antibodies to 21-hydroxylase and P450 side chain cleavage enzymes were identified in all subjects. All subjects met the criteria for premature ovarian failure during follow-up. Subject 1 later developed adrenal failure, whereas subject 3 had adrenal failure at the time of the study. These subjects elucidate the hormonal pattern in autoimmune oophoritis, before the full criteria for premature ovarian failure are met. The elevated inhibin A and B levels, which accompany the development of multiple small and dominant follicles in these women, suppress FSH relative to LH levels, virtually independent of estradiol. These data provide further evidence for an important role of inhibin B and inhibin A in the negative feedback control of FSH. In addition, the normal inhibin A and inhibin B production in the absence of estradiol precursors and estradiol provide insight into the selective dysfunction of the theca cells in autoimmune oophoritis.

Autoimmune polyglandular syndromes are fairly common diseases that are classified into four constellations based on the clinical clustering of the various component diseases. In types 1, 2, and 4, primary adrenal insufficiency due to an autoimmune process is usually present, but its diagnosis is often delayed because it is difficult to detect in a subclinical phase. It is widely accepted that the classical dose of 250 microg ACTH(1-24) is supramaximal, whereas 0.06 microg has been shown to be one of the lowest ACTH doses that is able to stimulate adrenal secretion in normal young subjects. The aim of this study was to clarify the sensitivity and maximal secretory response of the adrenal gland to ACTH in a group of patients with at least two autoimmune diseases, without clinical signs and symptoms of overt or subclinical hypocortisolism. Cortisol (F), aldosterone (A), and dehydroepiandrosterone (DHEA) responses to the sequential administration of very low and supramaximal ACTH(1-24) doses [0.06 microg followed by 250 microg ACTH(1-24) i.v. at 0 and +60 min] were studied in 18 patients with at least two autoimmune diseases (AP; age, 20-40 yr; body mass index, 22-26 kg/m(2)). The results in the patients were compared with the results recorded in 12 normal age-matched control subjects (CS; age, 22-34 yr; body mass index, 20-25 kg/m(2)). At baseline, ACTH levels in AP were within the normal range but higher (P0.05) than in CS, whereas F, A, DHEA, urinary-free F, and plasma renin activity were similar in both groups. F, A, and DHEA responses to ACTH were dose dependent in both groups. However, in AP, F, A, and DHEA levels showed no response to the 0.06- micro g ACTH dose, which, in turn, elicited clear responses (P0.01) in CS. On the other hand, F, A, and DHEA responses to 250 microg ACTH in AP were not different from those in CS. In conclusion, patients with autoimmune diseases who displayed a normal basal adrenal function showed a loss of F, A, and DHEA response to the very low ACTH dose, although they were normal responders to the high ACTH dose. These data are likely to indicate that a reduced sensitivity to ACTH in all adrenal zones occurs in patients with different types of autoimmune disease.

Some type 2 diabetic subjects develop secondary failure to sulphonylurea treatment and require insulin therapy. To test the diagnostic sensitivity and specificity of epitopes of GAD65 autoantibodies (GAD65Ab) for insulin requirement, in patients with latent autoimmune diabetes of the adult, we studied 569 adult subjects with a clinical diagnosis of type 2 diabetes mellitus. All the patients had been initially treated with hypoglycemic agents and/or diet for at least 1 yr. The presence of GAD65Ab (61/569, 10.7%) depended on insulin therapy (P

Some type 2 diabetic subjects develop secondary failure to sulphonylurea treatment and require insulin therapy. To test the diagnostic sensitivity and specificity of epitopes of GAD65 autoantibodies (GAD65Ab) for insulin requirement, in patients with latent autoimmune diabetes of the adult, we studied 569 adult subjects with a clinical diagnosis of type 2 diabetes mellitus. All the patients had been initially treated with hypoglycemic agents and/or diet for at least 1 yr. The presence of GAD65Ab (61/569, 10.7%) depended on insulin therapy (P0.0001), low BMI (P0.0001), and low basal C-peptide (P = 0.01). The majority of GAD65Ab-positive subjects (47/61, 77%) had antibodies directed to both middle (GAD65-MAb) and COOH-terminal (GAD65-CAb) epitopes. However, GAD65-CAb were more frequent in insulin-treated subjects (92% of GAD65Ab+ individuals) than in subjects treated with hypoglycemic agents and/or diet (18.2% of GAD65Ab+ individuals), while the exclusive presence of GAD65-MAb was more frequent in subjects treated with hypoglycemic agents and/or diet (81.8% vs. 8%) (P0.0001). The presence of GAD65-CAb had a diagnostic specificity for insulin requirement as high as 99.4% (compared with 96.9% of GAD65Ab as measured in the traditional radiobinding assay) and identified a subgroup of patients with low BMI, low basal C-peptide values, and a need for insulin therapy. Subjects carrying only GAD65-MAb were phenotypically indistinguishable from GAD65Ab-negative patients. Patients positive for GAD65-M+CAb, but not those positive for GAD65-MAb only, showed an increased risk for thyroid autoimmunity, as revealed by the presence of thyroid peroxidase autoantibodies. Our study demonstrates that the use of epitope-specific antibody assays improves the diagnostic specificity of GAD65Ab, and that the presence of GAD65Ab binding to COOH-terminal epitopes is strongly associated with a need for insulin requirement.

The major histocompatibility complex class I chain-related MIC-A and MIC-B genes are located on chromosome 6 between the histocompatibility leucocyte antigen (HLA)-B and the B-associated transcript genes. The presence of 21-hydroxylase autoantibodies is a sensitive and specific marker of autoimmune Addison’s disease. We studied the polymorphism of exon 5 of the MIC-A gene, of intron 1 of the MIC-B gene, and of HLA-DRB1, -DQA1, and -DQB1 genes in 28 autoimmune (21-hydroxylase autoantibody positive) Addison’s disease patients and in 75 healthy subjects from central Italy. The MIC-A5.1 allele was significantly more frequent in Addison’s disease patients (79%) than in healthy subjects (36%) [odds ratio (OR) = 6.52, corrected P (Pc) = 0.0015], whereas MIC-A6 was significantly reduced in affected subjects (15% vs. 56%, OR = 0.13, Pc = 0.002). The A5.1/A5.1 genotype had an OR for autoimmune Addison’s disease as high as 18.0 and an absolute risk of 1 per 1131. In the presence of MIC-A5.1, MICB-CA-25 was significantly increased in Addison’s disease patients (25% vs. 4%, OR = 8.0, P = 0.0039, Pc = 0.047). The MICB-CA-17 allele was absent in Addison’s disease patients, but present in more than 25% healthy individuals (OR = 0.10, P = 0.0025, Pc = 0.03). Among HLA-DR and -DQ haplotypes, only DRB1*03-DQA1*0501-DQB1*0201 (DR3/DQ2) was significantly more frequent in Addison’s disease patients than in healthy subjects, but only in the presence of MIC-A5.1. The frequency of MIC-A5.1 was significantly increased in Addison’s disease patients only in the presence of HLA-DR3-DQ2. Our study demonstrates that susceptibility to autoimmune Addison’s disease is linked to the MIC-A microsatellite allele 5.1 and that both MIC-A5.1 and HLA-DR3/DQ2 are necessary to confer increased genetic risk for Addison’s disease.

To test the hypothesis that levels of adrenal autoantibodies correlate with the degree of adrenal dysfunction, we followed up adrenal cortex autoantibody (ACA) titers and 21-hydroxylase (21OH) autoantibody (21OHAb) levels in 19 ACA-positive subjects with preclinical Addison’s disease. On enrollment, all the 19 ACA-positive subjects were positive for 21OHAb. At follow-up, the concordance rate for simultaneous presence/absence of both ACA and 21OHAb was as high as 91% and a strong, positive correlation between 21OHAb levels and ACA titers was observed (P < 0.0001). The levels of adrenal autoantibodies were positively associated with the severity of adrenal dysfunction (ANOVA, P < 0.0001 for both 21OHAb and ACA): the 21OH index was significantly lower at stage 0 or 1 than at stage 2+3 (corrected P < 0.001 and P < 0.05) or stage 4 (corrected P < 0.001 and

Approximately 70-80% of cases of primary adrenal insufficiency are classified as idiopathic. An effective protocol for the etiological diagnosis of primary adrenal insufficiency is needed to ensure correct patient management. With the aim of developing an algorithm for the etiological diagnosis of primary adrenal insufficiency, we studied 56 Italian patients with nonsurgical primary adrenal insufficiency and 24 French patients with X-linked adrenoleukodystrophy (ALD) for serum levels of adrenal cortex, steroid-21-hydroxylase (21OHAb), islet cell (ICA), glutamate decarboxylase (GAD65Ab), IA2/ICA512 (ICA512Ab), thyroid peroxidase (TPOAb) autoantibodies, and plasmatic concentrations of very long chain fatty acids (VLCFA). High levels of 21OH and adrenal cortex antibodies were found in 35/42 (83%) and 17/42 (40%) Italian patients with idiopathic adrenal insufficiency, respectively. Levels of adrenal autoantibodies correlated inversely with disease duration (P < 0.0001). Elevated VLCFA were found in 4/42 (10%) idiopathic patients. A total of 34/35 (97%) idiopathic patients with a disease duration of less than 20 yr was positive for either 21OHSAb or elevated levels of VLCFA. None of 14 patients with posttuberculosis adrenal insufficiency had elevated levels of either adrenal antibodies or VLCFA. ICA, GAD65Ab, ICA512Ab, and TPOAb were found in 6/56 (11%), 8/56 (14%), 4/56 (7%), and 23/56 (41%) patients, respectively. None of 24 French ALD patients with adrenal insufficiency was positive for organ-specific autoantibodies. The measuring of 21OH antibodies and plasma VLCFA levels enabled a correct diagnosis of autoimmune (89%) and ALD (8%) in 97% of patients with idiopathic primary adrenal insufficiency of less than 20 yr of duration. The results of our study have important therapeutic and prognostic implications.

Autoantibodies against 21-hydroxylase (P450c21) are common in idiopathic autoimmune Addison's disease. In the present work, we have developed a sensitive radiobinding assay using in vitro translated recombinant human 35S-P450c21. Levels of P450c21 antibodies (P450c21-Ab) were expressed as a relative index (P450c21 index) using a P450c21-Ab positive Addisonian serum and two antibody-negative healthy sera as positive and negative standards in healthy individuals. The upper level of normal was the mean + 3 SD. Positivity for P450c21-Ab was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated 35S-P450c21. In 38 Addisonian patients, P450c21-Ab were found in 24/28 (86%) idiopathic, 0/5 post-tuberculosis, 0/3 adrenoleukodystrophy, and 0/2 post-adrenalectomy sera. Among healthy individuals, 1/70 (1.4%) were positive. The P450c21 index, as an estimate of P450c21-Ab levels, correlated inversely with the duration of idiopathic Addison's disease (r = -0.527; P = 0.007): 16/16 (100%) positive in patients with less than 20 yr and 8/12 (67%) positive in patients with more than 20 yr disease duration. The availability of this simple and sensitive radiobinding assay to evaluate levels of P450c21-Ab will permit large clinical studies as well as screening subjects at risk. In addition, the general population can now be screened to evaluate the predictive value of P450c21-Ab for Addison's disease.