New potential genetic predictors of autoimmune adrenal insufficiency

Rationale: In Russia, assessment of anti-P450c21 antibodies (AB) in the diagnosis of autoimmune adrenal insufficiency (AAI) has not been commonly used, and the disease screening has not been implemented.Aims: 1) To determine the sensitivity and specificity of anti-P450c21 AB determination in the AAI diagnosis; 2) To estimate the prevalence of anti-P450c21 AB carriage in patients without AAI.Materials and methods: Anti-P450c21 AB were assessed in 40 patients (group 1) with manifest AAI; 171 patients without established diagnosis of AAI, including 113 subjects with autoimmune thyroid disorders or type 1 diabetes mellitus (AID, group 2); 25 carriers of AB markers of thyroid AID and/or type 1 diabetes mellitus without any target organ dysfunctions (group 3); 33 patients with non-autoimmune endocrine disorders (group 4), and 25 healthy individuals (group 5).Results: Determination of anti-P450c21 AB for the diagnosis of AAI had 95% sensitivity, with specificity of 100%, predictive value of a positive result of 100%, and predictive value of a negative result 92.6%. Anti-P450c21 AB were inversely correlated with the duration of glucocorticoid replacement therapy (r=-0.222, p<0.05). High levels of anti-P450c21 AB were found in 4 (3.5%) patients of group 2; based on the results of additional hormonal testing, 50% cases were diagnosed with the latent stage of the disease and 50% cases with the potential stage.Discussion: The sensitivity of the anti-P450c21 AB determination for AAI diagnosis in our study was higher, than in the works by other authors. We have confirmed a time-related reduction of anti-P450c21 AB levels, whereby the strength of the correlation was higher in the subgroups with autoimmune polyendocrine syndrome type II and, to a greater extent, autoimmune polyendocrine syndrome type I. This might be related to their different pathogenesis, with an abnormality of central immune tolerance in autoimmune polyendocrine syndrome type I and that of peripheral immune tolerance in autoimmune polyendocrine syndrome type II. According to our data, in 50% of cases, the development of AAI was preceded by the manifestation of other AIDs (in 15% of cases being multiple). Among all patients with no AAI diagnosis at the study entry, increased anti-P450c21 AB levels were found exactly in those with pre-existing AID. Thus, we have confirmed the feasibility of AAI screening primarily in a cohort of patients with other AID (especially multiple) belonging to the risk group.Conclusion: The determination of blood anti-P450c21 AB is a highly sensitive and highly specific method to diagnose AAI. The frequency of anti-P450c21 AB detection might depend on the duration of glucocorticoid treatment. Screening for early AAI stages is relevant primarily in the risk groups with multiple autoimmune disorders.

Autoimmune adrenal insufficiency (AAI) is a rare disease. This research evaluates three patients with AAI, including autoimmune polyglandular syndrome (APS) type 2. Two patients had APS or AAI during childhood, and one had a history of endocrine autoimmune disease, indicating a possible hereditary basis of the condition. Trio-based exome sequencing and high-resolution HLA typing were employed to analyze patients and their parents. Benign or likely benign variants of the AIRE gene were identified in all participants of the study. These variants, coupled with clinical data and the results of antibody studies to type I interferons, helped to exclude APS-1. Patients with APS-2, in contrast to patient with AAI, inherited distinct variants of unknown significance in the CLEC16A gene, which is associated with autoimmune diseases, including AAI. Various risk alleles in other genes associated with autoimmunity were identified in all patients. HLA typing of class II loci revealed alleles related to APS. Nevertheless, the frequencies of the haplotypes identified are substantial in the healthy Russian population. Immunological tests can detect antibody carriers and assess the risk of autoimmune disease development. In the future, to identify genetic predictors of autoimmune endocrinopathies, it is recommended to analyze the whole genome of patients and their relatives, examining clinically relevant variants in non-coding regions.

Introduction: Currently, the need to expand knowledge about the mechanisms of impaired immune tolerance in autoimmune adrenal insufficiency (AAI) remains relevant. It is not excluded that in AAI there are disturbances in the system of regulatory B-lymphocytes (Breg).Objective: To assess the state of the B-regulatory cells system of immunity in AAI.Material and methods: the content of Breg was evaluated in patients with AAI, including isolated AAI and AAI as part of autoimmune polyglandular syndrome type 2 (APS-2) and as part of APS-1, in patients with primary adrenal insufficiency (1-AI) of non-autoimmune genesis, conditionally healthy individuals without AI and autoimmune diseases.Results: A decrease in the content of Breg in vivo was revealed in patients with isolated AAI and AAI in the composition of APS-2 compared with conditionally healthy participants. The content of Breg in vivo in patients with 1-AI of non-autoimmune genesis did not statistically differ from the indicators of conditionally healthy participants.Conclusion: for the first time in the world, a tendency to a decrease in the content of Breg in AAI, caused by a disturbance of peripheral immune tolerance, was found. Thus, these cells can be considered as promising markers for the prognosis, early diagnosis, and differential diagnosis of AAI.

Aseptic prosthetic loosening (APL) and prosthetic joint infections (PJI) are frequent complications of hip and knee implants. Polymorphisms of cytokines and nitric oxide (NO), key inflammatory molecules in APL and PJI pathogenesis, could explain individual susceptibility to these complications. Three cytokines (IL-1-a, IL-1-β, TNF-α) and two nitric oxide synthase (NOS2, NOS3) genes polymorphisms were genotyped in 77 APL and 117 PJI patients and 145 controls with aseptic hip or knee implants that were implanted for > 16 years. Plasma cytokines and nitrate-nitrite (NOx) levels also were measured. The TT genotype and T allele of (+3954 C/T, exon 5, rs1143634) IL-1β polymorphism were more frequent in APL patients compared to controls (P = 0.03 and P = 0.02, respectively). No genotypic associations in PJI patients were observed. Plasma IL-6, TNF-α and NOx were significantly different between APL and controls (P < 0.0001). Plasma IL-1β and IL-6 were significantly higher in APL T allele carriers vs. non-carriers (P < 0.03). Knee implant (HR 2.488, 95% CI 1.307–4.739, P = 0.005), male gender (HR 2.252, 95% CI 1.121–4.525, P = 0.023), carriages of the TT genotype of the (+3954 C/T) IL-1β polymorphism (HR 3.704, 95% CI 1.274–10.753, P = 0.016) and AA genotype of the (exon 22) NOS2 polymorphism (HR 3.509, 95% CI 1.266–9.709, P = 0.016) were independently associated with a shorter implant survival by Cox regression. No genotypic associations in PJI patients were observed. Genotyping of IL-1β (+3954 C/T, exon 5, rs1143634) and NOS2 (exon 22) polymorphisms could be useful as predictors of early hip or knee APL.

Recent progress in the understanding of autoimmune adrenal disease, including a detailed analysis of a group of patients with Addison's disease (AD), has been reviewed. Criteria for defining an autoimmune disease and the main features of autoimmune AD (history, prevalence, etiology, histopathology, clinical and laboratory findings, cell-mediated andhumoral immunity, autoantigens and their autoepitopes, genetics, animal models, associated autoimmune diseases, pathogenesis, natural history, therapy) have been described. Furthermore, the autoimmune polyglandular syndromes (APS) associated with AD (revised classification, animal models, genetics, natural history) have been discussed. Of Italian patients with primary AD (n = 317), 83% had autoimmune AD. At the onset, all patients with autoimmune AD (100%) had detectable adrenal cortex and/or steroid 21-hydroxylase autoantibodies. In the course of natural history of autoimmune AD, the presence of adrenal cortex and/or steroid 21-hydroxylase autoantibodies identified patients at risk to develop AD. Different risks of progression to clinical AD were found in children and adults, and three stages of subclinical hypoadrenalism have been defined. Normal or atrophic adrenal glands have been demonstrated by imaging in patients with clinical or subclinical AD. Autoimmune AD presented in four forms: as APS type 1 (13% of the patients), APS type 2 (41%), APS type 4 (5%), and isolated AD (41%). There were differences in genetics, age at onset, prevalence of adrenal cortex/21-hydroxylase autoantibodies, and associated autoimmune diseases in these groups. "Incomplete" forms of APS have been identified demonstrating that APS are more prevalent than previously reported. A varied prevalence of hypergonadotropic hypogonadism in patients with AD and value of steroid-producing cells autoantibodies reactive with steroid 17alpha-hydroxylase or P450 side-chain cleavage enzyme as markers of this disease has been discussed. In addition, the prevalence, characteristic autoantigens, and autoantibodies of minor autoimmune diseases associated with AD have been described. Imaging of adrenal glands, genetic tests, and biochemical analysis have been shown to contribute to early and correct diagnosis of primary non-autoimmune AD in the cases of hypoadrenalism with undetectable adrenal autoantibodies. An original flow chart for the diagnosis of AD has been proposed.

Disclosure: S. Amankwah: None. H.K. Driscoll: None. C. Muojieje: None. M. Sanati: None. Background: Approximately 50-65% of patients with autoimmune adrenal insufficiency have one or more autoimmune endocrine disorders. This case demonstrates the importance of considering autoimmune polyendocrine syndrome (APS) when a patient presents with more than one autoimmune endocrine disorder. Case: A 29-year-old woman with history of idiopathic intracranial hypertension, hypothyroidism on levothyroxine 50 mcg daily, endometriosis, and chronic migraines complained of fatigue, word finding difficulty, misspeaking of words, and staring spells. An MRI of the brain w+wo IV contrast revealed hypo-enhancing appearance of the pituitary gland. The patient was referred to endocrinology by neurology. Hormonal work up was performed checking for prolactin, ACTH, morning cortisol, TSH, Free T4, FSH, LH, IGF-1 and a serum chemistry panel. Thyroid antibodies were also ordered to help understand the etiology of her hypothyroidism. Pituitary hormones were normal except elevated TSH and borderline low serum cortisol. Subsequently a co-syntropin stimulation test resulted in inadequate cortisol rise. ACTH level measured inappropriately low normal. The patient then started steroid replacement therapy. Thereafter, 21-hydroxylase antibodies, serum renin and aldosterone were checked to screen for Addison Disease. Her 21-hydroxylase antibody was positive, aldosterone level low, and renin elevated pointing to autoimmune adrenalitis etiology. Her thyroid antibodies were also positive indicating autoimmune thyroid disease. Considering these results, the condition of APS came into question. Hence, the patient was screened for type 1 diabetes mellitus which was positive for glutamic acid decarboxylase 65 antibody but negative for insulin and pancreatic islet cell antibodies. Her hemoglobin A1c was 5.0%. Due to her history of gluten intolerance, she was screened for celiac disease which was negative for t-Transglutaminase antibody. The patient’s fatigue improved on steroid replacement therapy. She became euthyroid after increasing her levothyroxine dose to 75 mcg daily, and repeated MRI a year later did not show pituitary abnormalities. Discussion: APS type 2 is a polygenic inherited disorder associated with HLA-DR3 and DR4 that affects adult females more than males. The syndrome is characterized by type 1 diabetes mellitus, autoimmune thyroid disease and Addison disease. Associated nonendocrine autoimmune syndromes include celiac disease, pernicious anemia, and atrophic gastritis. These characteristics overlap with APS type 1 except for autoimmune thyroid disease that presents with type 2. It should be noted that thyroxine therapy can precipitate a life-threatening Addison crisis in a patient with untreated adrenal insufficiency and hypothyroidism. Therefore, it is necessary to evaluate adrenal function in all hypothyroid patients in whom APS is suspected. Presentation: Friday, June 16, 2023

The Impact of IL28B Gene Polymorphisms on Drug Responses

Background Aseptic prosthetic loosening (APL) and prosthetic joint infections (PJI) are frequent complications of hip and knee implants. Polymorphisms of cytokines and nitric oxide (NO), key inflammatory molecules in APL and PJI pathogenesis, could explain individual susceptibility to these complications. Methods Three cytokines (IL-1-a, IL-1-β, TNF-α) and two nitric oxide synthase (NOS2, NOS3) genes polymorphisms were genotyped in 77 APL and 117 PJI patients and 145 controls with aseptic hip or knee implants lasting &amp;gt; 16 years. Plasma cytokines and nitrate-nitrite (NOx) levels were measured. Results The TT genotype and T allele of the (+3954C/T, exon 5, rs1143634) IL-1β polymorphism were more frequent in APL patients compared to controls (p=0.03 and p=0.02, respectively). No genotypic associations in PJI patients were observed. S. epidermidis was their most frequently isolated microorganism (39.3%). Plasma IL-6, TNF-α and NOx were significantly different between APL and controls (p&amp;lt; 0.0001). Plasma IL-1β and IL-6 were significantly higher in APL T allele carriers vs. non-carriers (p&amp;lt; 0.03). Knee implant (HR 2.488, 95% CI 1.307-4.739, p=0.005), male gender (HR 2.252, 95% CI 1.121-4.525, p=0.023), carriages of the TT genotype of the (+3954C/T) IL-1β polymorphism (HR 3.704, 95% CI 1.274-10.753, p=0.016) and AA genotype of the (exon 22) NOS2 polymorphism (HR 3.509, 95% CI 1.266-9.709, p=0.016) were independently associated with a shorter implant survival by Cox regression. Conclusion Genotyping of IL-1β (+3954C/T, exon 5, rs1143634) and NOS2 (exon 22) polymorphisms could be useful as predictors of early hip or knee APL. Disclosures All Authors: No reported disclosures.

HCV genotype 4 dominates the HCV epidemic in Egypt. Drug resistance was the most serious side effect that reflects bad clinical outcome. Several studies had demonstrated that baseline serum interferon-γ-inducible-protein 10 (IP-10) levels and interleukin 28B polymorphisms were associated with the resistance to the standard of care pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy and development of post-treatment relapse. Our purpose was to assess the predictive value of combining IP-10 levels and IL28B genotypes to PEG-IFNα/RBV therapy response in Egyptian chronic HCV infection patients with genotype 4. Ninety Egyptian patients chronically infected by HCV genotype-4 treated with pegylated interferon alpha and ribavirin (PEG-IFNα/RBV) therapy were enrolled. Serum IP-10 levels were determined by enzyme linked immunosorbent assay pre- and post- treatment. IL-28B (rs12979860 and rs8099917) polymorphisms were performed by PCR-RFLP in all patients. Overall, 38 patients (42.2%) achieved sustained virologic response (SVR) and 52 (57.8%) patients have non-viral response (NVR). Pretreatment serum IP-10 mean levels were significantly lower in patients who achieved SVR than in NVR (P<0.05). CC genotype in IL28B polymorphism (rs12979860) was the favorable genotype as 65.8% achieved SVR, while TT genotype in IL-28B polymorphism (rs8099917) was the favorable genotype as 81.5% achieved SVR. Baseline IP-10 was significantly correlated to genotypes CC in rs12979860 and TT in rs8099917. Combined use of serum baseline IP-10 levels with IL-28B polymorphisms could improve the prediction of SVR to PEG-IFNα/RBV therapy in Egyptian chronic HCV infection patients with genotype 4.

POLYENDOCRINOPATHY, CANDIDIASIS, ECTODERMAL DYSTROPHY, KERATOCONJUNCTIVITIS, AUTOIMMUNE HEPATITIS, ADRENAL INSUFFICIENCY AND HYPOPARATHYROIDISM IN AN ADULT FEMALE.

Atrial fibrillation (AF) is one of the most common cardiac arrhythmias and a major predictor of morbidity and mortality. AF is a polygenic and polyetiological disease. In various ethnic groups, the strongest and most independent relationship with the development of AF was found with the 4q25 locus, where the ATFB5 gene is located. An analysis of the literature data showed that the carriage of the TT genotype of the rs2200733 ATFB5 gene polymorphism is the most unfavorable genotype for the development of AF. The purpose of the study was to identify the prevalence of genotypes and alleles of the rs2200733 polymorphism of the ATFB5 gene in Uzbek patients with AF. The study included 69 Uzbek patients with paroxysmal (n=20) and persistent AF (n=49). The control group (n=30) was composed of Uzbek patients without AF. Genotyping for the carriage of allelic variants of the rs2200733 polymorphism of the ATFB5 gene was performed using the Polymerase Chain Reaction-Restriction Length Polymorphism (PCR-RFLP) method. The distribution of the C and T alleles and the CC, CT, and TT genotypes of the rs2200733 polymorphism of the ATFB5 gene in patients with AF and controls were compared. After genotyping 69 patients with AF, the following distribution of the ATFB5 gene polymorphism rs2200733 was revealed: the CC genotype was detected in 35 (50.72%) patients, the CT genotype in 25 (36.23%) patients, and the TT genotype in 9 (13.05%) patients (p<0.001, χ²=22.435). Moreover, the C allele was detected in 95 (68.8%) patients, and the T allele was detected in 43 (31.2%) patients (p<0.001, χ²=37.696). The distribution of genotypes in the control group was as follows: the CC genotype was detected in 17 individuals (56.7%), the CT genotype was detected in 12 individuals (40%), and the TT genotype was detected in 1 individual (3.3%) (p<0.001, χ²=20.100). Moreover, the C allele was detected in 46 (76.7%) patients, and the T allele was detected in 14 (23.3%) patients (p<0.001, χ²=32.033). The TT genotype of the ATFB5 gene was found to be significantly more prevalent in patients with AF than in controls (13.1% vs 3.3%, p=0.0001). The TT genotype of the rs2200733 polymorphism of the ATFB5 gene was found to be significantly more prevalent in Uzbek patients with AF than in controls.

Background: Selecting patients for new direct acting antiviral treatment of HCV has prompted a conflicting matter worldwide because of its high cost and limited availability. Genotyping of IL28B polymorphisms will aid clinical decision making for identifying priorities of urgent treatment in resource-limited countries.&#x0D; Objectives: The aim of the present study was to design a simple tetra-primer amplification refractory mutation system–polymerase chain reaction (T-ARMS-PCR) for genotyping of the rs8099917 and rs12979860 IL28B gene polymorphisms. Furthermore, we identify the correlation of variables such as gender, serum ALT level, histology of liver and baseline viral load with these polymorphisms.&#x0D; Patients and Methods: We efficiently designed a T-ARMS-PCR for detection of rs12979860 and rs8099917 IL28B gene polymorphisms. Using this method, we genotyped 148 hepatitis C patients. To ensure T-ARMS genotyping quality, we, regenotyped samples with the PCR- sequencing method.&#x0D; Results: Results of genotyping of rs12979860 and rs8099917 by T-ARMS PCR method were 100% concordant with the sequencing results. Among these 148 patients with chronic hepatitis C, the frequency of the rs12979860 CT, TT and CC genotypes was 72.3%, 14.2% and 13.5%, respectively and the frequency of the rs8099917 TT, GT and GG genotypes was 58.1%, 38.5% and 3.4%. Low frequency (2.7%) of association of two unfavourable homozygous genotypes (TT rs12979860 / GG rs809917) as well as 56.7% of association of 3 or 4 favorable alleles could explain good response of Iranians to HCV treatment with interferon-based regimens. About correlation of polymorphisms with different variables, only high viral load showed a statistically significant correlation to unfavorable genotype of TT rs12979860 ( p value = 0/05 ) and there was no correlation of serum ALT level, gender and histology of liver to IL28B genotypes.&#x0D; Conclusions: We report that rs8099917 polymorphisms could predict outcomes better than rs12979860 in Iranian HCV patients.

Autoimmune polyglandular syndromes (APS) are rare disorders characterized by the coexistence of endocrine and non-endocrine dysfunctions mediated by autoimmune mechanisms. Autoimmune polyglandular syndrome type 1 is defined as coexistence of chronic mucocutaneous candidiasis, hypoparathyroidism, and autoimmune adrenal insufficiency. Addison's disease as the obligatory component is potentially life threatening.Herein, we demonstrate a case of a 44-year-old woman with APS-1 (hypoparathyroidism, adrenal insufficiency, hypergonadotropic hypogonadism) and SARS-CoV-2-induced adrenal crisis. The patient presented with the typical manifestations of hypotensive shock, electrolyte disturbances of hyponatremia and hyperkalemia, and hypoglycaemia.Our case report illustrates the increased risk of severe course of COVID-19 in APS-1 syndrome patients along with heightened exposure to medical complications. The case reinforced the significance of a timely diagnosis, appropriate treatment, and education of patients with such a rare condition like APS-1.

IL-28B polymorphisms are predictors of response to therapy in adults infected with hepatitis C. We do not know whether they are markers of response to therapy in children and adolescents. The aim of this study was to determine whether single-nucleotide polymorphisms (SNPs) in the IL-28B gene could influence the probability of response to therapy compared with other known baseline prognostic factors and correlate with clinical findings in pediatric patients infected with hepatitis C virus (HCV) genotypes 1 or 4. We determined three SNPs of IL-28B (rs12979860, rs12980275, and rs8099917) in 82 patients with chronic HCV infection treated with pegylated interferon alpha and ribavirin (peg-IFNα/RBV). Treatment response and clinical data were analyzed. Overall, sustained virological response (SVR) was achieved by 45 % of patients infected with difficult-to-treat HCV genotypes 1 and 4. Except for IL-28B polymorphisms, there was no association of SVR with any other clinical data. IL-28B rs12979860 CC [odds ratio (OR), 6.81; p = 0.001] and rs8099917 TT (OR, 3.14; p = 0.013) genotypes were associated with higher SVR rates. IL-28B rs12980275 was not significantly associated with SVR (p = 0.058). Only the distribution between CC and CT–TT genotypes of rs12979860 significantly differentiated patients achieving early virological response (EVR) (OR, 10.0; p = 0.011). Children with the rs12979860 CC genotype had significantly higher baseline viral load compared with CT–TT patients (p = 0.010). In children and adolescents chronically infected with HCV genotypes 1 and 4, IL-28B rs12979860 and rs8099917 polymorphisms were the only predictors of response to peg-IFN/RBV.

Purpose The goal of this study is to determine the association of L162V polymorphism of PPAR-alpha gene, A603G polymorphism of tissue factor gene and the risk of coronary heart disease development in Russian population. Materials and Methods A clinical and genetic study of 414 patients with CHD and 220 people of comparable age without CHD which amounted to a control group was performed. L162L and L162V genotypes of L162V polymorphism of PPAR-α gene, A603A, A603G and G603G genotypes of A603G polymorphism of tissue factor gene were determined by polymerase chain reaction followed by restriction analysis. Results A carriage of L162V genotype and V allele of PPAR-α gene was associated with an increase risk of CHD in 2,13 times (L162V genotype) and in 2,21 times (V allele), with an increase in risk of CHD before the age of 45 years in 4,68 times (L162V genotype) and in 3,88 times (V allele). Significantly higher in patients with CHD compared with the general population and in patients with a carriage of G603G genotype and G allele of tissue factor gene was associated with the increase of CHD risk in 2,68 times (G603G genotype) and in 4,37 times (G allele), occurred more frequently in patients with debut of disease at age of 45 years and younger. The level of tissue factor was significantly higher in patients with CHD – carriers G603G genotype compared with carriers A603A genotype (217,9±15,2 pg/ml and 152,6±30,4 pg/ml, respectively, p=0,04). A carriage of the combination of L162V and G603G genotypes was associated with an increased risk of CHD in 3,04 times. Conclusion A carriage of V allele of L162V polymorphism of PPAR-α gene and G allele of A603G polymorphism of tissue factor gene, as well as their pair combination are associated with an increased CHD risk, especially at age 45 years or less.