Carrier frequency of a common mutation of carnitine palmitoyltransferase 1A deficiency and long-term follow-up in Finland.

OBJECTIVE: To assess the long-term clinical course of carnitine palmitoyltransferase 1A (CPT1A) deficiency, caused by the c.1364A>C (p.K455T) mutation, and the carrier frequency of this mutation in Finland. STUDY DESIGN: This was a long-term follow-up of patients in whom the common mutation was detected. RESULTS: Between 1999 and 2010, 6 cases of CPT1A deficiency were diagnosed and treated with a high-carbohydrate, low-fat diet. The patients experienced their first symptoms during the first years of life, provoked by viral illness and/or fasting. The clinical features included hypoketotic hypoglycemia, hepatopathy, and loss of consciousness, ranging from transient unconsciousness to prolonged hyperlipidemic coma. Five cases carried a homozygous c.1364A>C (p.K455T) mutation, whereas 1 case had a compound c.1364A>C/c.1493A>C (p.Y498S) mutation. During dietary therapy, the patients had few transient decompensations. No carriers of mutation c.1364A>C were detected by minisequencing of 150 control samples. CONCLUSION: Even though CPT1A deficiency may be life-threatening and lead to prolonged coma, the long-term prognosis is good. A genotype-phenotype correlation implies that the mutations detected are disease-causing. Despite Finland's location close to the Arctic polar region, the carrier frequency of the c.1364A>C mutation in Finland is far lower than that of the variants found in Alaskan, Canadian, and Greenland native populations.

Elevated hydroxyacylcarnitines in a carrier of LCHAD deficiency during acute liver disease of pregnancy - a common feature of the pregnancy complication?

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is a mitochondrial fatty acid beta-oxidation defect characterized by accumulation of long-chain hydroxyacylcarnitine intermediates and female carriers of this disorder are in risk for pregnancy complications. We found elevated blood long-chain hydroxyacylcarnitine species in a carrier of LCHAD deficiency at 31weeks of pregnancy with a LCHAD deficient fetus during acute fatty liver of pregnancy-like liver involvement, but had been within the normal range at 25weeks of pregnancy. This finding supports the hypothesis of acylcarnitine accumulation in pathogenesis of AFLP in carriers of LCHAD and MTP deficiencies.

Functional interaction of AIRE with PIAS1 in transcriptional regulation.

AIRE (autoimmune regulator) promotes the establishment of self-tolerance by regulating gene expression in the thymus. Mutations in AIRE lead to an autoimmune disease, APECED. Here we have identified PIAS proteins as novel AIRE interaction partners. Although PIAS proteins function as E3 SUMO ligases, AIRE is not sumoylated. We expressed AIRE, wt PIAS1, and PIAS1 mutants with deleted SP-RING domain or SUMO interaction motif (SIM) in different cell lines and demonstrate that AIRE and PIAS1 localize to adjacent nuclear bodies (NBs). The expression of AIRE enhances the formation of PIAS1 NBs. The ability of PIAS1 to localize into NBs and interconnect with AIRE is neither dependent on the SP-RING domain nor the SIM. Further, we show that PIAS1 is able to attract AIRE into SUMO1-containing complexes and that the process is dependent on the SIM of PIAS1. PIAS1 and AIRE concurrently activate the human insulin promoter, a known target gene of AIRE, and the SP-RING is required for this activation. Moreover, AIRE represses and PIAS1 activates the CSTB promoter, used as a model for a housekeeping promoter, and both the SP-RING and SIM are needed for its activation by PIAS1. Collectively, our data suggest that AIRE and PIAS1 interact functionally to regulate the activities of the target genes of AIRE.

Anti-interferon autoantibodies in autoimmune polyendocrinopathy syndrome type 1.

BACKGROUND: The autoimmune regulator (AIRE) gene influences thymic self-tolerance induction. In autoimmune polyendocrinopathy syndrome type 1 (APS1; OMIM 240300), recessive AIRE mutations lead to autoimmunity targetting endocrine and other epithelial tissues, although chronic candidiasis usually appears first. Autoimmunity and chronic candidiasis can associate with thymomas as well. Patients with these tumours frequently also have high titre immunoglobulin G autoantibodies neutralising type I interferon (IFN)-alpha and IFN-omega, which are secreted signalling proteins of the cytokine superfamily involved in both innate and adaptive immunity. METHODS AND FINDINGS: We tested for serum autoantibodies to type I IFNs and other immunoregulatory cytokines using specific binding and neutralisation assays. Unexpectedly, in 60/60 Finnish and 16/16 Norwegian APS1 patients with both AIRE alleles mutated, we found high titre neutralising immunoglobulin G autoantibodies to most IFN-alpha subtypes and especially IFN-omega (60% homologous to IFN-alpha)-mostly in the earliest samples. We found lower titres against IFN-beta (30% homologous to IFN-alpha) in 23% of patients; two-thirds of these (from Finland only) also had low titres against the distantly related "type III IFN" (IFN-lambda1; alias interleukin-29). However, autoantibodies to the unrelated type II IFN, IFN-gamma, and other immunoregulatory cytokines, such as interleukin-10 and interleukin-12, were much rarer and did not neutralise. Neutralising titres against type I IFNs averaged even higher in patients with APS1 than in patients with thymomas. Anti-type I IFN autoantibodies preceded overt candidiasis (and several of the autoimmune disorders) in the informative patients, and persisted for decades thereafter. They were undetectable in unaffected heterozygous relatives of APS1 probands (except for low titres against IFN-lambda1), in APS2 patients, and in isolated cases of the endocrine diseases most typical of APS1, so they appear to be APS1-specific. Looking for potentially autoimmunising cell types, we found numerous IFN-alpha(+) antigen-presenting cells-plus strong evidence of local IFN secretion-in the normal thymic medulla (where AIRE expression is strongest), and also in normal germinal centres, where it could perpetuate these autoantibody responses once initiated. IFN-alpha2 and IFN-alpha8 transcripts were also more abundant in antigen-presenting cells cultured from an APS1 patient's blood than from age-matched healthy controls. CONCLUSIONS: These apparently spontaneous autoantibody responses to IFNs, particularly IFN-alpha and IFN-omega, segregate like a recessive trait; their high "penetrance" is especially remarkable for such a variable condition. Their apparent restriction to APS1 patients implies practical value in the clinic, e.g., in diagnosing unusual or prodromal AIRE-mutant patients with only single components of APS1, and possibly in prognosis if they prove to predict its onset. These autoantibody responses also raise numerous questions, e.g., about the rarity of other infections in APS1. Moreover, there must also be clues to autoimmunising mechanisms/cell types in the hierarchy of preferences for IFN-omega, IFN-alpha8, IFN-alpha2, and IFN-beta and IFN-lambda1.

The monopartite nuclear localization signal of autoimmune regulator mediates its nuclear import and interaction with multiple importin alpha molecules.

Autoimmune regulator (AIRE) is a transcriptional regulator involved in establishing immunological self-tolerance. Mutations in the AIRE gene lead to the development of the autosomal, recessively inherited, organ-specific autoimmune disease, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). The AIRE protein is mainly localized in the cell nucleus where it is associated with nuclear bodies. The N-terminal part of AIRE has been previously shown to mediate nuclear localization of the protein. However, the functional nuclear localization signal (NLS) and nuclear import mechanisms of AIRE have not been identified. We show that, although the amino-acid sequence of AIRE contains a potential bipartite NLS consisting of amino acids 110-114 and 131-133, only the latter part constitutes a functional NLS. Furthermore, we show by in vitro binding assays that AIRE interacts with multiple members of the nuclear transport receptor importin alpha family, mainly alpha1, alpha3, and alpha5, and that these interactions depend on the intactness of the Arg-Lys-rich NLS of AIRE. In addition, we found that AIRE binds to the 'minor' NLS-binding site of importin alpha3 and alpha5 proteins consisting of the C-terminal armadillo repeats 7-9. Our findings strongly suggest that the nuclear import of AIRE is mediated by the classical importin alpha/beta pathway through binding to several importin alpha family members.

Functional analysis of SAND mutations in AIRE supports dominant inheritance of the G228W mutation.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a rare disorder caused by mutations in the autoimmune regulator gene (AIRE) and characterized by a variable combination of organ-specific autoimmune diseases. Studies on AIRE-deficient mice suggest that AIRE is an important factor in the establishment and maintenance of self-tolerance. The AIRE protein contains several structural domains often found in transcriptional regulators and functions as a transcriptional transactivator in vitro. To date, more than 50 patient mutations have been identified in the coding region of the AIRE gene. So far, APECED has been reported to be inherited in an autosomal recessive manner. However, in contrast to all other AIRE mutations, a novel mutation c.682T>G (p.G228W) in the DNA-binding and/or multimerization domain SAND was recently described to be inherited in a dominant fashion. We analyzed the effects of mutant AIRE proteins containing the patient mutations c.682T>G (p.G228W) and c.755C>T (p.P252L) located in the SAND domain on the properties of the wild-type AIRE in a heterozygous situation in vitro. In addition to the patient mutations, we analyzed the effects of a double mutation [c.727A>G;c.728A>C;c.739C>G;c740G>C] (p.K243A;R247A) of positively charged amino acids in the SAND domain. Of the mutants studied, only c.682T>G (p.G228W) mutant changed the subcellular localization and in addition severely disrupted the transactivating capacity of the wild-type AIRE. Our results indicate that the c.682T>G (p.G228W) mutant AIRE protein acts with a dominant negative effect by binding to the wild-type AIRE, thus preventing the protein from forming the complexes needed for transactivation.

Prevalence and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine syndrome type I.

The prevalence of autoantibodies against nine intracellular enzyme autoantigens, namely 21-hydroxylase, side-chain cleavage enzyme (SCC), 17 alpha-hydroxylase, glutamic acid decarboxylase 65, aromatic L-amino acid decarboxylase, tyrosine phosphatase-like protein IA-2, tryptophan hydroxylase (TPH), tyrosine hydroxylase, cytochrome P450 1A2, and against the extracellular calcium-sensing receptor, was assessed in 90 patients with autoimmune polyendocrine syndrome type I. A multivariate logistic regression analysis was performed for the presence of autoantibodies as independent predictors for different disease manifestations. Reactivities against 21-hydroxylase and SCC were associated with Addison's disease with odds ratios (ORs) of 7.8 and 6.8, respectively. Hypogonadism was exclusively associated with autoantibodies against SCC with an OR of 12.5. Autoantibodies against tyrosine phosphatase-like protein IA-2 were associated with insulin-dependent diabetes mellitus with an OR of 14.9, but with low sensitivity. Reactivities against TPH and, surprisingly, glutamic acid decarboxylase 65, were associated with intestinal dysfunction, with ORs of 3.9 and 6.7, respectively. TPH reactivity was the best predictor for autoimmune hepatitis, with an OR of 27.0. Hypoparathyroidism was not associated with reactivity against any of the autoantigens tested. No reactivity against the calcium-sensing receptor was found. Analysis of autoantibodies in autoimmune polyendocrine syndrome type I patients is a useful tool for establishing autoimmune manifestations of the disease as well as providing diagnosis in patients with suspected disease.

APECED-causing mutations in AIRE reveal the functional domains of the protein.

A defective form of the AIRE protein causes autoimmune destruction of target organs by disturbing the immunological tolerance of patients with a rare monogenic disease, autoimmune polyendocrinopathy (APE)-candidiasis (C)-ectodermal dystrophy (ED), APECED. Recently, experiments on knockout mice revealed that AIRE controls autoimmunity by regulating the transcription of peripheral tissue-restricted antigens in thymic medullary epithelial cells. Thus, AIRE provides a unique model for molecular studies of organ-specific autoimmunity. In order to analyze the molecular and cellular consequences of 16 disease-causing mutations in vitro, we studied the subcellular localization, transactivation capacity, homomultimerization, and complex formation of several mutant AIRE polypeptides. Most of the mutations altered the nucleus-cytoplasm distribution of AIRE and disturbed its association with nuclear dots and cytoplasmic filaments. While the PHD zinc fingers were necessary for the transactivation capacity of AIRE, other regions of AIRE also modulated this function. Consequently, most of the mutations decreased transactivation. The HSR domain was responsible for the homomultimerization activity of AIRE; all the missense mutations of the HSR and the SAND domains decreased this activity, but those in other domains did not. The AIRE protein was present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturbed the formation of these complexes. In conclusion, we propose an in vitro model in which AIRE transactivates transcription through heteromeric molecular interactions that are regulated by homomultimerization and conditional localization of AIRE in the nucleus or in the cytoplasm.

AIRE mutations and human leukocyte antigen genotypes as determinants of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy phenotype.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, OMIM 240300) is a rare autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21q22.3. This monogenic disease provides an interesting model for studies of other common and more complex autoimmune diseases. The most common components of APECED are chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, but several other endocrine deficiencies and ectodermal dystrophies also occur and the phenotype varies widely. The AIRE genotype also varies; 42 different mutations have been reported so far. To understand the complexity of the phenotype, we studied the AIRE and human leukocyte antigen (HLA) class II genotypes in a series of patients with APECED. The only association between the phenotype and the AIRE genotype was the higher prevalence of candidiasis in the patients with the most common mutation, R257X, than in those with other mutations. Addison's disease was associated with HLA-DRB1*03 (P = 0.021), alopecia with HLA-DRB1*04- DQB1*0302 (P < 0.001), whereas type 1 diabetes correlated negatively with HLA-DRB1*15-DQB1*0602 (P = 0.036). The same HLA associations have previously been established for non-APECED patients. We conclude that mutation of AIRE per se has little influence on the APECED phenotype, whereas, in contrast to earlier reports, HLA class II is a significant determinant.

Aire deficient mice develop multiple features of APECED phenotype and show altered immune response.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal recessive disease caused by mutations in the AIRE gene. Here we have produced knock-out mice for the Aire gene. The Aire-/- mice develop normally; however, autoimmune features of APECED in Aire-/- mice are evident, including multiorgan lymphocytic infiltration, circulating autoantibodies and infertility. The distribution of B and T cells and thymic maturation as well as activation of T cells appear normal, while the TCR-Vbeta repertoire is altered in peripheral T cells of Aire-/- mice. When mice are challenged with immunization, the peripheral T cells of Aire-/- mice have a 3-5-fold increased proliferation. These findings suggest that the Aire gene is not necessary for normal T cell education and development, while a defect in immune response detected in challenged Aire-/- mice underlines the crucial role of AIRE/Aire in maintaining homeostatic regulation in the immune system.