Linkage Analysis in Autoimmune Addison's Disease: NFATC1 as a Potential Novel Susceptibility Locus.

BACKGROUND: Autoimmune Addison's disease (AAD) is a rare, highly heritable autoimmune endocrinopathy. It is possible that there may be some highly penetrant variants which confer disease susceptibility that have yet to be discovered. METHODS: DNA samples from 23 multiplex AAD pedigrees from the UK and Norway (50 cases, 67 controls) were genotyped on the Affymetrix SNP 6.0 array. Linkage analysis was performed using Merlin. EMMAX was used to carry out a genome-wide association analysis comparing the familial AAD cases to 2706 UK WTCCC controls. To explore some of the linkage findings further, a replication study was performed by genotyping 64 SNPs in two of the four linked regions (chromosomes 7 and 18), on the Sequenom iPlex platform in three European AAD case-control cohorts (1097 cases, 1117 controls). The data were analysed using a meta-analysis approach. RESULTS: In a parametric analysis, applying a rare dominant model, loci on chromosomes 7, 9 and 18 had LOD scores >2.8. In a non-parametric analysis, a locus corresponding to the HLA region on chromosome 6, known to be associated with AAD, had a LOD score >3.0. In the genome-wide association analysis, a SNP cluster on chromosome 2 and a pair of SNPs on chromosome 6 were associated with AAD (P <5x10-7). A meta-analysis of the replication study data demonstrated that three chromosome 18 SNPs were associated with AAD, including a non-synonymous variant in the NFATC1 gene. CONCLUSION: This linkage study has implicated a number of novel chromosomal regions in the pathogenesis of AAD in multiplex AAD families and adds further support to the role of HLA in AAD. The genome-wide association analysis has also identified a region of interest on chromosome 2. A replication study has demonstrated that the NFATC1 gene is worthy of future investigation, however each of the regions identified require further, systematic analysis.

A novel cell-based assay for measuring neutralizing autoantibodies against type I interferons in patients with autoimmune polyendocrine syndrome type 1.

An important characteristic of autoimmune polyendocrine syndrome type 1 (APS 1) is the existence of neutralizing autoantibodies (nAbs) against the type I interferons (IFN) -α2 and -ω at frequencies close to 100%. Type 1 IFN autoantibodies are detected by antiviral neutralizing assays (AVA), binding assays with radiolabelled antigens (RLBA), enzyme-linked immunosorbent assay (ELISA), or by reporter-based cell assays. We here present a simple and reliable version of the latter utilizing a commercially available cell line (HEK-Blue IFN-α/ß). All 67 APS 1 patients were positive for IFN-ω nAbs, while 90% were positive for IFN-α2 nAbs, a 100% and 96% correlation with RLBA, respectively. All blood donors and non-APS 1 patients were negative. The dilution titer required to reduce the effect of IFN-ω nAbs correlated with the RLBA index. This cell-based autoantibody assay (CBAA) is easy to perform, suitable for high throughput, while providing high specificity and sensitivity.

Autoantibodies against aromatic amino acid hydroxylases in patients with autoimmune polyendocrine syndrome type 1 target multiple antigenic determinants and reveal regulatory regions crucial for enzymatic activity.

Patients with autoimmune polyendocrine syndrome type 1 (APS-1) frequently have autoantibodies directed against the aromatic amino acid hydroxylases tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). We aimed to characterize these autoantibodies with regard to their antigenic determinants, their influence on enzymatic activity and their clinical associations. In particular, we wanted to compare autoantibodies against the two different isoforms of TPH, which display different tissue distribution. Using sera from 48 Scandinavian APS-1 patients we identified 36 patients (75%) with antibodies against one or more of these three enzymes. Antibodies against TPH1, but not TPH2, were associated with malabsorption in the whole Scandinavian cohort, while TH antibodies were associated with dental enamel hypoplasia in Norwegian patients. Subsequent experiments with selected patient sera indicated that while the C-terminal domain was the immunodominant part of TPH1, the epitopes of TPH2 and TH were mainly located in the N-terminal regulatory domains. We also identified a TPH1 specific epitope involved in antibody mediated inhibition of enzyme activity, a finding that provides new insight into the enzymatic mechanisms of the aromatic amino acid hydroxylases and knowledge about structural determinants of enzyme autoantigens. In conclusion, TPH1, TPH2 and TH all have unique antigenic properties in spite of their structural similarity.

Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines.

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A-producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis.

Autoantibodies against type I interferons as an additional diagnostic criterion for autoimmune polyendocrine syndrome type I.

CONTEXT: In autoimmune polyendocrinopathy syndrome type I (APS-I), mutations in the autoimmune regulator gene (AIRE) impair thymic self-tolerance induction in developing T cells. The ensuing autoimmunity particularly targets ectodermal and endocrine tissues, but chronic candidiasis usually comes first. We recently reported apparently APS-I-specific high-titer neutralizing autoantibodies against type I interferons in 100% of Finnish and Norwegian patients, mainly with two prevalent AIRE truncations. OBJECTIVES: Because variability in clinical features and age at onset in APS-I frequently results in unusual presentations, we prospectively checked the diagnostic potential of anti-interferon antibodies in additional APS-I panels with other truncations or rare missense mutations and in disease controls with chronic mucocutaneous candidiasis (CMC) but without either common AIRE mutation. DESIGN: The study was designed to detect autoantibodies against interferon-alpha2 and interferon-omega in antiviral neutralization assays. SETTING AND PATIENTS: Patients included 14 British/Irish, 15 Sardinian, and 10 Southern Italian AIRE-mutant patients with APS-I; also 19 other patients with CMC, including four families with cosegregating thyroid autoimmunity. OUTCOME: The diagnostic value of anti-interferon autoantibodies was assessed. RESULTS: We found antibodies against interferon-alpha2 and/or interferon-omega in all 39 APS-I patients vs. zero of 48 unaffected relatives and zero of 19 British/Irish CMC patients. Especially against interferon-omega, titers were nearly always high, regardless of the exact APS-I phenotype/duration or AIRE genotype, including 12 different AIRE length variants or 10 point substitutions overall (n=174 total). Strikingly, in one family with few typical APS-I features, these antibodies cosegregated over three generations with autoimmune hypothyroidism plus a dominant-negative G228W AIRE substitution. CONCLUSIONS: Otherwise restricted to patients with thymoma and/or myasthenia gravis, these precocious persistent antibodies show 98% or higher sensitivity and APS-I specificity and are thus a simpler diagnostic option than detecting AIRE mutations.

Autoimmune polyendocrine syndrome type I in Slovakia: relevance of screening patients with autoimmune Addison's disease.

BACKGROUND: Autoimmune polyendocrine syndrome type I (APS I) is a monogenic disease affecting endocrine glands and other organs due to mutations of the autoimmune regulator (AIRE) gene. There is a wide variability in clinical phenotypes in patients with APS I, which makes the diagnosis a challenge. OBJECTIVE: To screen for APS I among Slovakian patients with sporadic Addison's disease and clinical features that raised the suspicion of APS I. METHODS: All 14 exons and exon-intron boundaries of the AIRE gene were sequenced. In addition, autoantibodies specific for Addison's disease and polyendocrine syndromes were assayed. RESULTS: Using clinical criteria we identified four patients with APS I in three families. Two patients had a novel missense mutation in exon 2 (c.274C>T, p.R92W) and either the Finnish major mutation (c.769C>T) or the common 13 bp deletion (c.967-979del13bp). APS I was diagnosed in a brother of the latter after his death due to an adrenal crisis. A fourth patient had primary adrenal failure and hypoparathyroidism without AIRE mutations or APS-I specific autoantibodies. CONCLUSIONS: Four patients with APS I were found in a Slovakian cohort of Addison patients, although the lack of detectable AIRE mutations and APS I-specific autoantibodies raises uncertainty regarding the pathogenesis in one of the patients. This study demonstrates the merits of screening patients with phenotypic features or autoantibody findings that could indicate APS I, even in adult patients. It is necessary to identify APS I patients in order to provide appropriate treatment and follow-up of the various components of APS I.