Bergithe E. Oftedal

Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Radioimmunoassay for autoantibodies against interferon omega; its use in the diagnosis of autoimmune polyendocrine syndrome type I

Patients with the autoimmune polyendocrine syndrome I (APS I) have high titers of neutralizing IgG autoantibodies against type I interferons (IFNs), in particular IFN-omega. Until now, the most specific assay has been the antiviral interferon neutralizing assay (AVINA), which has the drawbacks of requiring a cytolytic virus, being cumbersome and difficult to standardise. We have developed a fast and reliable immunoassay based on radiolabelled IFN-omega for quantifying anti-IFN-omega antibodies. Sera from 48 APS I patients were analysed together with those from 5 control groups. All sera from APS I patients were positive for anti-IFN-omega, while, except one serum, all sera from the controls were negative. This method has the advantage over bioassays that it is readily adapted to high throughput. It provides an alternative, sensitive and specific diagnostic test for APS I, and an ideal screening tool to precede mutational analyses of the AIRE gene in suspected APS I cases.

ARMC5 Mutations Are Common in Familial Bilateral Macronodular Adrenal Hyperplasia

CONTEXT Bilateral macronodular adrenal hyperplasia (BMAH) is a rare form of adrenal Cushings syndrome. Familial cases have been reported, but at the time we conducted this study, the genetic basis of BMAH was unknown. Recently, germline variants of armadillo repeat containing 5 (ARMC5) in patients with isolated BMAH and somatic, second-hit mutations in tumor nodules, were identified. OBJECTIVE Our objective was to identify the genetic basis of familial BMAH. DESIGN We performed whole exome capture and sequencing of 2 affected individuals from each of 4 BMAH families (BMAH-01, BMAH-02, BMAH-03, and BMAH-05). Based on clinical evaluation, there were 7, 3, 3, and 4 affected individuals in these families, respectively. Sanger sequencing of ARMC5 was performed in 1 other BMAH kindred, BMAH-06. RESULTS Exome sequencing identified novel variants Chr16:g.31477540, c.2139delT, p.(Thr715Leufs*1) (BMAH-02) and Chr16:g.31473811, c.943C→T, p.(Arg315Trp) (BMAH-03) in ARMC5 (GRch37/hg19), validated by Sanger sequencing. BMAH-01 had a recently reported mutation Chr16:g.31476121, c.1777C→T, p.(Arg593Trp). Sanger sequencing of ARMC5 in BMAH-06 identified a previously reported mutation, Chr16:g. 31473688; c.799C→T, p.(Arg267*). The genetic basis of BMAH in BMAH-05 was not identified. CONCLUSIONS Our studies have detected ARMC5 mutations in 4 of 5 BMAH families tested, confirming that these mutations are a frequent cause of BMAH. Two of the 4 families had novel mutations, indicating allelic heterogeneity. Preclinical evaluation did not predict mutation status. The ARMC5-negative family had unusual prominent hyperaldosteronism. Further studies are needed to determine the penetrance of BMAH in ARMC5 mutation-positive relatives of affected patients, the practical utility of genetic screening and genotype-phenotype correlations.

Flow cytometry study of blood cell subtypes reflects autoimmune and inflammatory processes in autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS I) is a recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is expressed in medullary epithelial cells where it activates transcription of organ‐specific proteins in thymus, thereby regulating autoimmunity. Patients with APS I have, in addition to autoimmune manifestations in endocrine organs, also often ectodermal dystrophies and chronic mucocutaneous candidiasis. The aim of this study was to characterize immune cell subpopulations in patients with APS I and their close relatives. Extensive blood mononuclear cell immunophenotyping was carried out on 19 patients with APS I, 18 first grade relatives and corresponding sex‐ and age‐matched healthy controls using flow cytometry. We found a significant relative reduction in T helper cells coexpressing CCR6 and CXCR3 in patients with APS I compared to controls (mean = 4.10% versus 5.94% respectively, P = 0.035). The pools of CD16+ monocytes and regulatory T cells (Tregs) were also lower in patients compared with healthy individuals (mean = 15.75% versus 26.78%, P = 0.028 and mean = 4.12% versus 6.73%, P = 0.029, respectively). This is the first report describing reduced numbers of CCR6+CXCR3+ T helper cells and CD16+ monocytes in patients with APS I We further confirm previous findings of reduced numbers of Tregs in these patients.

AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I

Autoimmune Addisons disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed.

Clinical and Serologic Parallels to APS-I in Patients with Thymomas and Autoantigen Transcripts in Their Tumors

Patients with the autoimmune polyendocrine syndrome type I (APS-I), caused by mutations in the autoimmune regulator (AIRE) gene, and myasthenia gravis (MG) with thymoma, show intriguing but unexplained parallels. They include uncommon manifestations like autoimmune adrenal insufficiency (AI), hypoparathyroidism, and chronic mucocutaneous candidiasis plus autoantibodies neutralizing IL-17, IL-22, and type I IFNs. Thymopoiesis in the absence of AIRE is implicated in both syndromes. To test whether these parallels extend further, we screened 247 patients with MG, thymoma, or both for clinical features and organ-specific autoantibodies characteristic of APS-I patients, and we assayed 26 thymoma samples for transcripts for AIRE and 16 peripheral tissue-specific autoantigens (TSAgs) by quantitative PCR. We found APS-I–typical autoantibodies and clinical manifestations, including chronic mucocutaneous candidiasis, AI, and asplenia, respectively, in 49 of 121 (40%) and 10 of 121 (8%) thymoma patients, but clinical features seldom occurred together with the corresponding autoantibodies. Both were rare in other MG subgroups (n = 126). In 38 patients with APS-I, by contrast, we observed neither autoantibodies against muscle Ags nor any neuromuscular disorders. Whereas relative transcript levels for AIRE and 7 of 16 TSAgs showed the expected underexpression in thymomas, levels were increased for four of the five TSAgs most frequently targeted by these patients’ autoantibodies. Therefore, the clinical and serologic parallels to APS-I in patients with thymomas are not explained purely by deficient TSAg transcription in these aberrant AIRE-deficient tumors. We therefore propose additional explanations for the unusual autoimmune biases they provoke. Thymoma patients should be monitored for potentially life-threatening APS-I manifestations such as AI and hypoparathyroidism.

A longitudinal follow-up of autoimmune polyendocrine syndrome type 1

Context: Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disease defined by the presence of two of the three major components: hypoparathyroidism, primary adrenocortical insufficiency, and chronic mucocutaneous candidiasis (CMC). Information on longitudinal follow-up of APS1 is sparse. Objective: To describe the phenotypes of APS1 and correlate the clinical features with autoantibody profiles and autoimmune regulator (AIRE) mutations during extended follow-up (1996–2016). Patients: All known Norwegian patients with APS1. Results: Fifty-two patients from 34 families were identified. The majority presented with one of the major disease components during childhood. Enamel hypoplasia, hypoparathyroidism, and CMC were the most frequent components. With age, most patients presented three to five disease manifestations, although some had milder phenotypes diagnosed in adulthood. Fifteen of the patients died during follow-up (median age at death, 34 years) or were deceased siblings with a high probability of undisclosed APS1. All except three had interferon-ω) autoantibodies, and all had organ-specific autoantibodies. The most common AIRE mutation was c.967_979del13, found in homozygosity in 15 patients. A mild phenotype was associated with the splice mutation c.879+1G>A. Primary adrenocortical insufficiency and type 1 diabetes were associated with protective human leucocyte antigen genotypes. Conclusions: Multiple presumable autoimmune manifestations, in particular hypoparathyroidism, CMC, and enamel hypoplasia, should prompt further diagnostic workup using autoantibody analyses (eg, interferon-ω) and AIRE sequencing to reveal APS1, even in adults. Treatment is complicated, and mortality is high. Structured follow-up should be performed in a specialized center.

AIRE-mutations and autoimmune disease

The gene causing the severe organ-specific autoimmune disease autoimmune polyendocrine syndrome type-1 (APS-1) was identified in 1997 and named autoimmune regulator (AIRE). AIRE plays a key role in shaping central immunological tolerance by facilitating negative selection of T cells in the thymus, building the thymic microarchitecture, and inducing a specific subset of regulatory T cells. So far, about 100 mutations have been identified. Recent advances suggest that certain mutations located in the SAND and PHD1 domains exert a dominant negative effect on wild type AIRE resulting in milder seemingly common forms of autoimmune diseases, including pernicious anemia, vitiligo and autoimmune thyroid disease. These findings indicate that AIRE also contribute to autoimmunity in more common organ-specific autoimmune disorders.

Measuring Autoantibodies against IL-17F and IL-22 in Autoimmune Polyendocrine Syndrome Type I by Radioligand Binding Assay Using Fusion Proteins

Autoantibodies against interleukin (IL)‐17A, IL‐17F and IL‐22 have recently been described in patients with autoimmune polyendocrine syndrome type I (APS I), and their presence is reported to be highly correlated with chronic mucocutaneous candidiasis (CMC). The aim of this study was to develop a robust high‐throughput radioligand binding assays (RLBA) measuring IL‐17F and IL‐22 antibodies, to compare them with current enzyme‐linked immunosorbent assays (ELISA) of IL‐17F and IL‐22 and, moreover, to correlate the presence of these antibodies with the presence of CMC. Interleukins are small molecules, which makes them difficult to express in vitro. To overcome this problem, they were fused as dimers, which proved to increase the efficiency of expression. A total of five RLBAs were developed based on IL‐17F and IL‐22 monomers and homo‐ or heterodimers. Analysing the presence of these autoantibodies in 25 Norwegian APS I patients revealed that the different RLBAs detected anti‐IL‐17F and anti‐IL‐22 with high specificity, using both homo‐ and heterodimers. The RLBAs based on dimer proteins are highly reproducible with low inter‐ and intravariation and have the advantages of high throughput and easy standardization compared to ELISA, thus proving excellent choices for the screening of IL‐17F and IL‐22 autoantibodies.

Radioligand-Binding Assay Reveals Distinct Autoantibody Preferences for Type I Interferons in APS I and Myasthenia Gravis Subgroups

Patients with autoimmune polyendocrine syndrome type I (APS I) or acquired thymoma-associated myasthenia gravis (MG) surprisingly share several common features, including defective expression of the transcription factor AIRE and autoantibodies against type I interferons. Here, we have adapted and validated the radioligand-binding assay we recently developed against 35S-Met-interferon-ω, for rapid and specific screening for autoantibodies against interferons-α2 and -α8. We then investigated their potential for diagnosis and for predicting clinical manifestations in patients with APS I and different subgroups of MG. Autoantibodies against interferons-ω, -α2, and -α8 occurred more often in patients with APS I (100%) and MG with thymoma (73%) than in late-onset MG (39%) and early-onset MG (5%). These autoantibodies showed preferences for interferon-ω in APS I and for the interferon-αs in MG, hinting at thymic aberrations in both groups. The exact profile of type I interferon antibodies may indicate MG subtype and may hint at thymoma recurrence.