Acute adrenal failure is a potentially fatal condition if overlooked. Occasionally, acute adrenal insufficiency may ensue from bilateral adrenal haemorrhage in patients with known antiphospholipid syndrome (APS). APS is characterized by recurrent arterial and venous thrombosis, pregnancy complications and detection of autoantibodies to phospholipids. This syndrome may be associated with non-organ specific diseases (e.g. connective tissue disorders) or with malignancies, but it may also appear in isolated form (primary APS). In a very few cases the heralding manifestation is given by adrenal failure. We report here a 63-year-old man presenting with acute adrenal insufficiency as the opening clinical manifestation of an APS. We also carried out a computer-aided search of the literature to identify all cases of primary adrenal failure as the first-recognized expression of a primary APS, a condition that not so infrequently may be tackled by endocrinologists. 20 patients fulfilled the inclusion criteria. The great majority of them were males (75%) with a mean age of 42 years. Abdominal pain was present in 14 patients, followed by fever (13 patients) and hypotension (12 patients). The main morphological findings by computed tomography or magnetic resonance were consistent with bilateral adrenal haemorrhage in 11 patients. Lupus anticoagulant was present in all of the 19 tested patients. Our observations emphasize the importance in the assessment of clotting times, and possibly of antiphospholipid antibodies, in all patients with diagnosis of rapidly progressive adrenal failure and concurrent abdominal pain.
Fabio Presotto, Francesca Fornasini, Corrado Betterle, Giovanni Federspil, and Marco Rossato
Corrado Betterle, Francesca Lazzarotto, Aglaura Cinzia Spadaccino, Daniela Basso, Mario Plebani, Beniamino Pedini, Silvia Chiarelli, and Mariapaola Albergoni
Objective: Patients with autoimmune Addison’s disease (AAD) are prone to develop other autoimmune manifestations. An increased prevalence of celiac disease (CD) has recently been demonstrated in Northern European patients with AAD. IgA deficiency is the most frequent type of immunodeficiency among humans and is present in about one in every 600 individuals in the population. IgA deficiency is frequent in patients with other autoimmune diseases, but data concerning AAD are still unavailable.
Design: The aim was to define the prevalence of CD and of IgA deficiency in a group of Italian patients with AAD.
Methods: One hundred and nine patients with AAD were enrolled and examined for tissue transglutaminase autoantibodies of the IgA class, circulating levels of IgA and adrenal cortex antibodies.
Results: Two (1.8%) of the patients were affected by already diagnosed CD and were already on a gluten-free diet. Out of the remaining 107 patients, four (3.7%) were found to be positive for IgA antibodies to human tissue transglutaminase. Three of the four patients who were positive for tissue transglutaminase autoantibodies agreed to undergo endoscopy and duodenal biopsies and, in one, a latent form of CD was identified. The clinical, silent or latent form of CD was present in six out of 109 (5.4%). This prevalence was significantly higher (P = 0.0001) than that reported for the Northern Italian population which was equal to 0.063%. Specifically, CD was present in 12.5% of the autoimmune polyglandular syndrome (APS) type 1 cases, in four out of 60 (6.7%) of the APS type 2 cases and in one out of 40 (2.5%) of the isolated AAD cases. IgA deficiency was present in two out of 109 patients (1.8%), all of whom had normal IgG anti-gliadin. Autoantibodies to the adrenal cortex were detected in 81 out of 109 patients (74.3%).
Conclusions: In patients with AAD there is a high prevalence of both CD and IgA deficiency. Consequently, it is important to screen for CD with tissue transglutaminase autoantibodies of the IgA class and for IgA levels.
Annalisa Brozzetti, Stefania Marzotti, Daria La Torre, Maria Luisa Bacosi, Silvia Morelli, Vittorio Bini, Bruno Ambrosi, Roberta Giordano, Roberto Perniola, Annamaria De Bellis, Corrado Betterle, and Alberto Falorni
Steroid-producing cell autoantibodies (SCAs) directed against 21-hydroxylase autoantibodies (21OHAbs), 17α-hydroxylase autoantibodies (17OHAb), and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies, P450sccAb) characterize autoimmune primary ovarian insufficiency (SCA-POI). The aim of the study was to analyze IgG subclass specificity of autoantibodies related to adrenal and ovarian autoimmunity.
We studied 29 women with SCA-POI, 30 women with autoimmune Addison's disease (AAD) without POI, and 14 patients with autoimmune polyendocrine syndrome type 1 (APS1). 21OHAb isotypes were also analyzed in 14 subjects with preclinical AAD. Samples from 30 healthy women served as control group to determine the upper level of normality in the isotype assays.
Immunoradiometric assays with IgG subclass-specific secondary antibodies.
In 21OHAb-positive sera, IgG1 isotype was detected in 90% SCA-POI and non-POI AAD sera and 67% APS1 patients. IgG1 isotype was found in 69% 17OHAb-positive SCA-POI and 100% 17OHAb-positive APS1 sera, and in 60% P450sccAb-positive SCA-POI and 80% P450sccAb-positive APS1 sera. For 21OHAb, IgG4 isotype was detected in 17% SCA-POI, 7% non-POI AAD, and 8% APS1 sera. None of the 17OHAb-positive sera was positive for IgG4. In P450sccAb-positive sera, 15% POI and 20% APS1 sera were positive for IgG4. Two 21OHAb-positive SCA-POI (7%), one 21OHAb-positive AAD (3%), three P450sccAb-positive SCA-POI (15%), and two P450sccAb-positive APS1 (20%) sera were positive for IgG4, in the absence of IgG1. All preclinical AAD sera resulted as positive for IgG1-21OHAb, but not for IgG4-21OHAb.
The autoantibody responses in POI and AAD are IgG1 dominated, which suggests a predominant Th1 response. Selective IgG4 isotype specificity identified a small subset of patients with Th2-oriented response.
Filippo Ceccato, Mattia Barbot, Marialuisa Zilio, Sergio Ferasin, Gianluca Occhi, Andrea Daniele, Sara Mazzocut, Maurizio Iacobone, Corrado Betterle, Franco Mantero, and Carla Scaroni
Salivary cortisol has recently been suggested for studies on the hypothalamic–pituitary–adrenal (HPA) axis. The lack of circadian rhythm is a marker of Cushing's syndrome (CS), and some authors have reported that low salivary cortisol levels may be a marker of adrenal insufficiency. The aim of our study was to define the role of salivary cortisol in specific diagnostic settings of HPA axis disease.
Subjects and methods
We analyzed morning salivary cortisol (MSC) and late-night salivary cortisol (LNSC) levels in 406 subjects: 52 patients with Cushing's disease (CD), 13 with ectopic CS, 17 with adrenal CS, 27 with CD in remission (a mean follow-up of 66±39 months), 45 with adrenal incidentaloma, 73 assessed as having CS and then ruled out for endogenous hypercortisolism, 75 with adrenal insufficiency, and 104 healthy subjects.
A LNSC value above 5.24 ng/ml differentiated CS patients from controls with high sensitivity (96.3%) and specificity (97.1%); we found higher LNSC levels in ectopic CS patients than in CD patients. We found no difference in MSC and LNSC levels between patients with CD in remission and healthy subjects. Both MSC and LNSC levels were higher in patients with adrenal incidentaloma than in healthy controls. A MSC value below 2.65 ng/ml distinguished patients with adrenal insufficiency from controls with high sensitivity (97.1%) and specificity (93.3%).
Salivary cortisol is a useful tool to assess endogenous cortisol excess or adrenal insufficiency and to evaluate stable CD in remission.
Ricardo Núñez Miguel, Shu Chen, Laleh Nikfarjam, Shiro Kominami, Byron Carpenter, Chiara Dal Pra, Corrado Betterle, Renato Zanchetta, Takashi Nakamatsu, Michael Powell, Rachel Hewer, Tom L Blundell, Bernard Rees Smith, and Jadwiga Furmaniak
Objective: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison’s disease.
Design: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH–21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity.
Methods: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed.
Results: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% ± 24% (n = 6), 86.7% ± 7.6% (n = 6) and 86.5% ± 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 ± 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model.
Conclusions: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.
Corrado Betterle, Riccardo Scarpa, Silvia Garelli, Luca Morlin, Francesca Lazzarotto, Fabio Presotto, Graziella Coco, Stefano Masiero, Anna Parolo, Maria Paola Albergoni, Roberta Favero, Susi Barollo, Monica Salvà, Daniela Basso, Shu Chen, Bernard Rees Smith, Jadwiga Furmaniak, and Franco Mantero
Addison's disease (AD) is a rare endocrine condition.
We aimed to evaluate clinical, immunologic, adrenal imaging, and genetic features in 633 Italian patients with AD followed up since 1967.
Adrenal cortex autoantibodies, presence of other autoimmune and nonautoimmune diseases, nonadrenal autoantibodies, adrenal imaging, and genetic profile for HLA-DRB1 and AIRE were analyzed.
A total of 492 (77.7%) patients were found to be affected by autoimmune AD (A-AD), 57 (9%) tuberculous AD, 29 (4.6%) genetic-associated AD, 10 (1.6%) adrenal cancer, six (0.94%) post-surgical AD, four (0.6%) vascular disorder-related AD, three (0.5%) post-infectious AD, and 32 (5.1%) were defined as idiopathic. Adrenal cortex antibodies were detected in the vast majority (88–100%) of patients with recent onset A-AD, but in none of those with nonautoimmune AD. Adrenal imaging revealed normal/atrophic glands in all A-AD patients: 88% of patients with A-AD had other clinical or subclinical autoimmune diseases or were positive for nonadrenal autoantibodies.
Based on the coexistence of other autoimmune disorders, 65.6% of patients with A-AD were found to have type 2 autoimmune polyendocrine syndrome (APS2), 14.4% have APS1, and 8.5% have APS4. Class II HLA alleles DRB1*03 and DRB1*04 were increased, and DRB1*01, DRB1*07, DRB1*013 were reduced in APS2 patients when compared with controls. Of the patients with APS1, 96% were revealed to have AIRE gene mutations.
A-AD is the most prevalent form of adrenal insufficiency in Italy, and ∼90% of the patients are adrenal autoantibody-positive at the onset. Assessment of patients with A-AD for the presence of other autoimmune diseases should be helpful in monitoring and diagnosing APS types 1, 2, or 4 and improving patients' care.
Annalisa Brozzetti, Stefania Marzotti, Cristina Tortoioli, Vittorio Bini, Roberta Giordano, Francesco Dotta, Corrado Betterle, Annamaria De Bellis, Giorgio Arnaldi, Vincenzo Toscano, Emanuela Arvat, Antonio Bellastella, Franco Mantero, and Alberto Falorni
Cytotoxic T lymphocyte antigen-4 (CTLA4) gene polymorphism has been associated with human autoimmune diseases, but discordant data are available on its association with autoimmune Addison's disease (AAD). We tested the human leukocyte antigen (HLA)-independent association of CTLA4+49 (A/G) (Ala 17) and/or CTLA4 CT60 (A/G) polymorphism with AAD.
DNA samples from 180 AAD patients and 394 healthy control subjects from continental Italy were analyzed, and association statistical analyses and meta-analysis of published studies were performed.
TaqMan minor groove binder chemistry assays and PCR fragment length polymorphism assays were used.
Frequency of allele G of CTLA4+49 was significantly increased among AAD patients (40% alleles) than among healthy controls (27% alleles; P<0.0001). CTLA4 CT60 polymorphism was associated with AAD only in the heterozygous A/G individuals. The frequency of +49 AG+GG genotypes was significantly higher among AAD patients than among healthy control subjects, in both a co-dominant (P<0.0001) and G dominant model (P<0.0001). CTLA4+49 allele G was significantly associated with disease risk in both patients with isolated AAD and in patients with autoimmune polyendocrine syndrome. Multivariate logistic regression analysis showed that CTLA4+49 allele G was positively associated with AAD (P<0.0001, odds ratio (OR)=2.43, 95% confidence interval=1.54–3.86) also after correction for DRB1*03-DQA1*0501-DQB1*0201, DRB1*04-DQA1*0301-DQB1*0302, and sex. Meta-analysis of five studies revealed a significant association of CTLA4+49 allele G with AAD (P<0.0001) with an overall OR of 1.48 (1.28–1.71).
The CTLA4+49 polymorphism is strongly associated with genetic risk for AAD, independently from the well-known association with HLA class II genes.
Marissa Penna-Martinez, Gesine Meyer, Anette Bøe Wolff, Beate Skinningsrud, Corrado Betterle, Alberto Falorni, William Er Ollier, Dag Undlien, Eystein Sverre Husebye, Simon H S Pearce, Anna L Mitchell, and Klaus Badenhoop
While vitamin D regulates immune cells, little is known about it in autoimmune Addison´s disease (AAD). We investigated the vitamin D status in AAD patients from five European populations to assess its deficiency. In addition, we studied two case-control cohorts for vitamin D metabolism and pathway genes.
A total of 1028 patients with AAD from Germany (n=239), Italy (n=328), Norway (n=378), UK (n=44) and Poland (n=39) and 679 controls from Germany (n=301) and Norway (n=378) were studied for 25(OH)D3 (primary objective). Secondary objectives (1,25(OH)2D3 and pathway genes) were examined in case-controls from Germany and Norway correlating 25(OH)D3 and single nucleotide polymorphisms within genes encoding the vitamin D receptor (VDR), 1-α-hydroxylase (CYP27B1), 25-hydroxylase (CYP2R1); 24-hydroxylase (CYP24A1) and vitamin D binding protein (GC/DBP).
Vitamin D deficiency (25(OH)D3 10-20 ng/ml) was highly prevalent in AAD patients (34-57%), 5-22% were severely deficient (<10 ng/ml), 28-38% insufficient (20-30 ng/ml) and only 7-14% sufficient (>30 ng/ml). Lower 25(OH)D3 and 1,25(OH)2D3 levels were observed both in Norwegian and German AAD (p = 0.03/0.003 and p = 1 x 10-5/< 1 x 10-7, respectively) the former was associated with CYP2R1 (rs1553006) genotype G. Whereas controls achieved sufficient median 25(OH)D3 in summers (21.4 to 21.9 ng/ml), AAD patients remained largely deficient (18.0 to 21.2 ng/ml) and synthesize less 1,25(OH)2D3.
Vitamin D deficiency and insufficiency are highly prevalent in AAD patients. The vitamin D status of AAD may be influenced by genetic factors and suggests individual vitamin D requirements throughout the year.
Lara Naletto, Anna Chiara Frigo, Filippo Ceccato, Chiara Sabbadin, Riccardo Scarpa, Fabio Presotto, Miriam Dalla Costa, Diego Faggian, Mario Plebani, Simona Censi, Jacopo Manso, Jadwiga Furmaniak, Shu Chen, Bernard Rees Smith, Stefano Masiero, Francesca Pigliaru, Marco Boscaro, Carla Scaroni, and Corrado Betterle
Adrenal cortex autoantibodies (ACAs) and/or 21-hydroxylase (21OHAb) are markers of autoimmune Addison’s disease (AAD) and progression to overt AAD. The reported cumulative risk of developing AAD varies from 0 to 90% in different studies.
To assess the predictive value of different parameters in the progression toward AAD in patients with ACA and/or 21OHAb-positive patients with autoimmune polyendocrine syndromes (APS).
Materials and methods
Twenty-nine patients with APS-1 and 114 patients with APS-2 or APS-4 were followed up for a median of 10 years (range 6 months to 33 years) and were assessed using ACTH test. The risk of AAD was estimated according to age, gender, stage of adrenal dysfunction, associated diseases and antibody titer. Univariate and multivariate Cox proportional hazard models were used for statistical analysis.
The cumulative risk (CR) of developing AAD was higher in APS-1 patients (94.2%) than in patients with APS-2/APS-4 (38.7%). The CR was high in both male and female APS-1 patients, while in patients with APS-2/APS-4 it was high only in males. Stage 1 (increased plasma renin) for patients with APS-1 and Stage 2 (no response of cortisol to ACTH test) for patients with APS-2/APS-4 were established as the points of no return in the progression to AAD. Adjusted hazard ratio analyses by multivariate Cox model for AAD showed that gender, diseases and adrenal function were independent risk factors for developing clinical AAD. The risk of developing clinical AAD appears to subside after 19 years of follow-up.
A model for estimating the probability to survive free of AAD has been developed and should be a useful tool in designing appropriate follow-up intervals and future therapeutic strategies.