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Special Feature |
Department of Medical Sciences (A.S., O.E., G.G.-M., H.H., E.L., F.R., O.K., T.N.) and Department of Women’s and Children’s Health (J.G.), University Hospital, SE-751 85 Uppsala, Sweden; Department of Bacteriology and Immunology, Haartman Institute, University Hospital and University of Helsinki (A.M., M.G.), FIN-00014 Helsinki, Finland; Department of Human Molecular Genetics, National Public Health Institute, and The Hospital for Children and Adolescents (P.E., M.H., J.P.), University of Helsinki, FIN-00290, Helsinki, Finland; Department of Medicine (T.T.), University Hospital, FIN-00290, Helsinki, Finland; Department of Medicine (E.S.H., A.G.M.), Haukeland University Hospital, N-5021 Bergen, Norway; Department of Pediatrics (A.G.M.), Akershus University Hospital, N-1474 Nordbyhagen, Norway; and Department of Gastroenterology and Hepatology (M.P.M.), Hannover Medical School, D-30625 Hannover, Germany
Address all correspondence and requests for reprints to: Thomas Nilsson, M.D., Ph.D., Department of Medical Sciences, University Hospital, Uppsala University, SE-751 85 Uppsala, Sweden. E-mail: thomas.nilsson{at}medsci.uu.se.
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Abstract |
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-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. |
Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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APS I is characterized by multiple organ-specific autoimmunity as well as ectodermal manifestations (1, 9). The disease usually begins in childhood with chronic mucocutaneous candidiasis, and later the patients contract autoimmune destruction of endocrine as well as nonendocrine organs resulting in a variable phenotype. Typically, the patients display a variety of autoantibodies against intracellular key enzymes present in the affected organs. The steroidogenic enzymes 21-hydroxylase (21-OH) (10, 11), side-chain cleavage enzyme (SCC) (10), and 17-hydroxylase (17-OH) (12) are all present in the adrenal cortex, and the latter two are also present in the gonads. Autoantibodies against glutamic acid decarboxylase 65 (GAD65), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (13), are common in APS I (14). Another pancreatic autoantigen in IDDM is the tyrosine phosphatase-like protein IA-2 (IA-2), whose function is still unknown. Whereas IA-2 autoantibodies are prevalent in idiopathic IDDM (15, 16), they are only detected in a minority of APS I patients with IDDM (17). The cytochromes P450 1A2 (CYP1A2) and 2A6 (CYP2A6) are enzymes that have been reported as autoantigens in patients with autoimmune hepatitis (18, 19). However, in a recent study CYP2A6 showed no correlation with hepatitis, whereas CYP1A2 was a highly specific, but insensitive, marker for disease (20). Aromatic L-amino acid decarboxylase (AADC) is also considered a hepatic autoantigen in patients with APS I (19, 21). Previously, we have identified the biopterine-dependent enzymes tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) as autoantigens associated with intestinal dysfunction and alopecia, respectively, in APS I (22, 23). Hypoparathyroidism has in one study been suggested to result from an autoimmune reaction directed against the extracellular domain of the calcium-sensing receptor (CaSR) on parathyroid cells (24). Unlike the other identified autoantigens in APS I, it is not an intracellular enzyme.
To clarify the significance of each of 10 different autoantibodies as markers for the various disease manifestations of APS I, we have used a multiple logistic regression analysis on the largest cohort of APS I patients analyzed to date (a total of 90 patients) from Finland, Norway, and Sweden.
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Patients and Methods |
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Sera were obtained from 62 Finnish (1, 25), 9 Swedish, and 19 Norwegian APS I patients (45 males and 45 females). The age range was from 3–59 yr with a mean of 26 yr. Sera from 100 healthy blood donors were used as controls for each autoantigen. The study was approved by local ethics committees.
The vectors
Human cDNA clones corresponding to 21-OH and GAD65 in pcDNA II and pSP64 poly A vectors, respectively, were kindly provided by Dr. A. Falorni (Department of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy) (26, 27). The intracellular portion of tyrosine phosphatase-like/IA-2, which contains the major IA-2 epitope in a pGEM 7 vector, was the kind gift by Dr. E. Bonifacio (Instituto Scientifico H. San Raffael, Milan, Italy) (15). A human SCC clone in a pET-vector (pET-scc) was obtained from Dr. B. C. Chung (Institute of Molecular Biology, Academia, Sinica, Nankang, Taipei, Taiwan) (28), and a human 17-OH clone (pCWmod17) was obtained from Dr. M. Waterman (Department of Biochemistry, University of Texas, Dallas, Texas) (29). The human CaSR cDNA was kindly donated by Dr. M. Freichel (Pharmakologisches Institut, Universität Heidelberg, Mannheim, Germany) (30). Its extracellular domains and the first membrane-spanning domain, 1949 bp, were cut out and extracted from low-melting agarose gel by JET Sorb kit (Genomed, Research Triangle Park, NC) and subsequently ligated into the HindIII/SacI site of the pSP64 PolyA Vector (Promega, Madison, WI). The ligated vector was propagated in Escherichia coli JM 109 and purified by the Plasmid Midi Kit (QIAGEN GmbH, Hilden, Germany). cDNA clones corresponding to AADC, TPH, and TH were subcloned into a pSP64-polyA vector as previously described (21, 22, 23) and a human cDNA clone coding for CYP 1A2 into a pCITE vector (20).
In vitro transcription and translation of the autoantigens
cDNA for each autoantigen was transcribed and translated in vitro with 35S-methionine in a TNT-coupled reticulocyte-lysate system (Promega). The correct sizes of the radioactive products were checked by SDS-PAGE (Bio-Rad, Richmond, CA). Immunoprecipitation was performed as previously described (21). After addition of scintillation fluid, the plates were counted in a MicroBeta counter (Wallac Oy, Turku, Finland), and the results were expressed as an index (cpm sample - cpm negative control)/(cpm positive control - cpm negative control) x 100. All patient and control sera were run in triplicate. Patient sera with a high titer of antibodies against each autoantigen served as positive controls and serum from one of the blood donors as the negative control. When analyzing autoantibodies against the CaSR, a polyclonal rabbit antiserum produced by immunizing with a synthetic peptide corresponding to amino acid residues 12–27 of the rat CaSR (Affinity BioReagents, Inc., Golden, CO) was used as the positive control. The normal upper level for autoantibodies against the respective autoantigen was calculated as the mean + 3 SD for healthy blood donors. The analysis of GAD65 autoantibodies on the 62 Finnish patients was determined by a radiobinding assay by Tuomi et al. (14) as previously described.
Mutational analysis of the AIRE gene
Eight patients in whom no autoantibodies were detected were screened for AIRE gene mutations. Each of the 14 exons of the AIRE gene was separately amplified using specific primers as previously described (31).
Statistics
Both univariate and multivariate logistic regression analyses, adjusting for age and sex, were used to test the importance of the antibody reactivity against different autoantigens as predictors for different disease components using SAS software (SAS Institute Inc., Cary, NC). The variables analyzed, except age, were dichotomous. The results are presented as odds ratios (ORs), P value, sensitivity, and specificity. A P value < 0.05 was considered statistically significant in all analyses.
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Results |
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Mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency (Addison’s disease), constituting the classical triad in APS I, were the most prevalent components (Table 1
). One patient with mucocutaneous candidiasis, intestinal dysfunction, and autoimmune hepatitis and one patient with Addison’s disease as the only disease manifestation were diagnosed to have APS I because they each had siblings with the syndrome. Additional manifestations were present in the majority of patients, 75 (83%) of the patients having more than three (Fig. 1). Among the 28 patients with hypogonadism there was a female predominance, 21 of 45 (47%) females, whereas 7 of 45 (16%) males (P < 0.001) had clinical signs of hypogonadism. Otherwise no sex differences were observed.
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The number of autoantibodies in individual patients varied between zero and eight, the most common being three (Table 2 and Fig. 2). There was no correlation between the frequency of autoantibodies and sex or age. Autoantibodies were absent in eight patients presenting with both mucocutaneous candidiasis and hypoparathyroidism, and, for five of them, these were the only disease manifestations. Mutational analysis showed that six of these eight patients had mutations in the coding region of the AIRE gene, whereas no mutations were found in the coding region in the remaining two patients (Table 3). The diagnosis of APS I in these two patients was based on the presence of candidiasis, hypoparathyroidism, and a positive family history.
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Univariate and multivariate logistic regression analyses were used to identify autoantibodies as markers for the different disease components (Table 4). Autoantibodies against 21-OH, SCC, and 17-OH were present in 53 (75%), 43 (61%), and 37 (52%) of the 71 patients with Addison’s disease, respectively. Multivariate analysis revealed that 17-OH autoantibodies only gave redundant information when 21-OH and SCC autoantibodies were present.
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-OH have previously been implicated as gonadal autoantigens in APS I (32, 33, 34). In our analysis, autoantibodies against SCC and/or 17-OH were found in 24 (86%), SCC in 22 (79%), and 17-OH in 16 (57%) of the 28 patients with hypogonadism. All but two patients with 17-OH reactivity also reacted with SCC. In contrast, among the patients with SCC autoantibodies, 17-OH autoantibodies were absent in eight. In three men and one woman with hypogonadism, these autoantibodies could not be detected. Autoantibodies against SCC, but not 17-OH, were associated with hypogonadism. Autoantibodies against GAD65 and IA-2 were detected in 33 (37%) and 6 (7%) of the 90 patients, respectively. Only IA-2 autoantibodies alone, however, were associated with IDDM, although with a low sensitivity of 33%.
Forty-one (45%) of the patients had autoantibodies against TPH. In accordance with our previous finding (22), TPH autoantibodies were an independent marker for intestinal dysfunction. They were present in 15 of 20 (75%) of the affected patients, compared with 26 of 70 (37%) of the patients without this manifestation. Unexpectedly, autoantibodies against GAD65 were also found to independently correlate with intestinal dysfunction and were detected in 15 of 20 (75%) of the affected patients. Thirteen patients had both TPH and GAD65 autoantibodies.
CYP 1A2, AADC, and TPH autoantibodies were each strongly associated with autoimmune hepatitis, but multivariate analysis showed that only TPH autoantibodies were independently associated with this manifestation. Of the 14 patients with autoimmune hepatitis, 5 had autoantibodies against CYP1A2, 13 against AADC, and 13 against TPH.
We could not detect any reactivity against the CaSR in the 90 patients’ sera, of which 73 were from patients with hypoparathyroidism, or in the control sera. The CaSR was, however, readily precipitated with a specific rabbit antiserum (data not shown).
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Discussion |
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Addison’s disease was present in 79% of the patients, and autoantibodies against at least one of the antigens (21-OH, SCC, or 17-OH) were found in 84% of the patients with Addison’s disease, which is in agreement with previous findings (10, 11, 12, 31, 32, 33, 35, 36). All three autoantibodies were associated with Addison’s disease, but the 21-OH and SCC autoantibodies were the only independent markers and these enzymes thus constitute the major adrenal cortex autoantigens in APS I.
Twenty-eight (31%) of the patients had hypogonadism. In accordance with previous studies (1), we found a female predominance (female/male ratio, 3:1), possibly due to the blood-testis barrier making the testis an immunological privileged zone (37). Both SCC and 17-OH autoantibodies have been reported to be associated with gonadal insufficiency in APS I, but their importance is a matter of debate (10, 32, 33, 34, 38). In this series, autoantibodies against SCC, but not 17-OH, were exclusively associated with hypogonadism consistent with SCC as the major gonadal autoantigen in APS I.
We have previously identified TPH as an antigen associated with intestinal dysfunction in APS I (22), and the data presented in this paper support that finding. Surprisingly, reactivity against GAD65 showed a similar association with this manifestation. Notably, the ORs became lower in the multivariate analysis for both TPH and GAD65 autoantibodies as predictors, implying that these two autoantibodies are positively correlated to each other. GAD65 is highly expressed in the nervous system (39), including the neural plexi of the gut (40). One may speculate that an autoimmune attack against GAD65, in addition to TPH, could be involved in the pathogenesis of the intestinal dysfunction in APS I, although GAD65 autoimmunity is not associated with the gastrointestinal neuropathy seen in long-standing idiopathic IDDM (41, 42).
Apart from the nervous system, GAD65 is expressed in the pancreatic islets and is considered a major autoantigen in idiopathic IDDM. In the present study, autoantibodies against GAD65 were detected in 33% of the patients, consistent with previous reports (14), but these autoantibodies were not found to be specifically associated with IDDM. Nondiabetic patients with GAD65 autoantibodies display a reduced C-peptide and insulin response (14), which may progress to overt IDDM (17), suggestive of an association between GAD65 autoantibodies and subclinical insulitis. Autoantibodies against IA-2, on the other hand, found in four of the 12 patients with IDDM in this study, were associated with IDDM, which is in line with previous reports (17). In fact, IA-2 autoantibodies seem to be the best predictor for IDDM in APS I with no, or little, additive information by GAD65 autoantibodies (17), in contrast to idiopathic IDDM where their simultaneous presence is highly predictive for diabetes (43). The autoantibodies associated with idiopathic IDDM thus display different sensitivity, specificity, and predictive value for diabetes of APS I. This might reflect that different pathophysiological disease mechanisms are operating in the ß-cell destruction and may also imply the existence of a so far unknown ß-cell autoantigen in APS I.
Autoantibodies against the known hepatic autoantigens CYP1A2 and AADC were both highly associated with autoimmune hepatitis as previously reported (18, 19, 21). Unexpectedly, TPH autoantibodies showed an even stronger correlation with autoimmune hepatitis. In contrast to AADC and CYP1A2, expression of TPH has not been described in the liver, making TPH an unlikely liver autoantigen. All three autoantibodies were positively correlated with each other, which made TPH autoantibodies the only independent marker for autoimmune hepatitis. We believe that CYP1A2 and AADC autoantibodies are involved in the pathogenesis of hepatitis whereas TPH may be considered just a marker for autoimmune hepatitis without importance for the pathogenic process.
AADC autoantibodies were also found to be associated with vitiligo, confirming our previous results (21). The relevance of these autoantibodies in vitiligo is, however, uncertain. Recently, we have found reactivity against the transcription factor SOX10 in a majority of APS I patients with vitiligo, but also in a subgroup of patients with idiopathic vitiligo (44), giving credence to SOX10 as a more important autoantigen. Further studies are necessary to evaluate the importance of the different autoantigens in vitiligo.
Li et al. (24) have provided data that the extracellular portion of the CaSR acts as an autoantigen in autoimmune hypoparathyroidism, thus implying a cell surface autoantigen. In this study, we were unable to detect any reactivity in the sera from the 90 patients, of whom 73 had hypoparathyroidism. This does, however, not exclude the existence of such autoantibodies. It is not likely that the results are explained by the lack of posttranslational modifications of the CaSR because glycosylation was not required for antibody reactivity in the paper by Li et al. (24). Because all manifestations in APS I studied thus far have been associated with autoantibodies, it is however, reasonable to assume the existence of a parathyroid autoantigen. The majority of APS I-associated autoantibodies react against intracellular enzymes, and future research and validating experiments will determine whether the parathyroid autoantigen constitutes an intracellular or extracellular protein.
The autoantibodies thus found to be independent markers for various manifestations of APS I were those directed against 21-OH, SCC, IA-2, TPH, and GAD65. The presence of an autoantibody is in many cases associated with an increased risk for developing clinical disease, and future studies should aim at defining these risks by sequential analysis of autoantibodies. Nevertheless, autoantibodies against 21-OH, SCC, IA-2, and GAD65 exist in other more common diseases, and detection of these may be of particular value as indicators for additional disease manifestations in a patient already diagnosed with APS I. The assessment of AADC, CYP1A2, TH, and TPH autoantibodies is valuable in differentiating between APS I and other autoimmune diseases because these autoantibodies are almost exclusively found in APS I (19, 21, 22, 23). Mutational analysis of the AIRE gene allows up to 90% of APS I patients to be diagnosed; in the small group of patients without detectable autoantibodies presenting with hypoparathyroidism and candidiasis as their only disease manifestations, this is the method of choice. A combined analysis of 21-OH, SCC, and AADC identifies 89% of the APS I patients, and although the mutational and autoantibody analyses are complementary, the serological methods are presently faster, simpler, less expensive to perform, and may provide information about the clinical picture.
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Acknowledgments |
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Footnotes |
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Abbreviations: AADC, Aromatic L-amino acid decarboxylase; AIRE, autoimmune regulator; APECED, autoimmune polyendocrinpathy-candidiasis-ectodermal dystrophy; APS I, autoimmune polyendocrine syndrome type I; CaSR, calcium-sensing receptor; CYP1A2, cytochrome P450 1A2; CYP2A6, cytochrome P450 2A6; GAD65, glutamic acid decarboxylase 65; IA-2, protein IA-2; IDDM, insulin-dependent diabetes mellitus; 17-OH, 17-hydroxylase; 21-OH, 21-hydroxylase; OR, odds ratio; SCC, side-chain cleavage enzyme; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase.
Received February 20, 2003.
Accepted October 1, 2003.
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