New Potential Genetic Predictors of Autoimmune Adrenal Insufficiency

The active single-stage case-control study included patients with isolated AAI and as part of APS-2 and conditionally healthy individuals. Patients were recruited into groups based on compliance with the inclusion criteria and in the absence of exclusion criteria (Tables 1; 2).

The study included 86 participants who were divided into the groups:

-1: patients with manifest AAI as part of APS-2 and with isolated AAI (n = 54);

-2: conditionally healthy without AI and AIDs (n = 32).

The participants analyzed polymorphic markers in the IL28B (rs12979860 and rs8099917), TLR9 (rs5743836 and rs352140) and TLR2 (rs5743708) genes.

All participants were examined by a physician-researcher for compliance with the inclusion criteria and identification of possible exclusion criteria. The initial examination scheme included collection of anamnestic data, including the presence of acute and chronic diseases. The day, month and year of the start and end of the recruitment period for this study: 10.05.2018-10.02.2021.

Blood was collected into vacuum tubes with inert gel and ethylenediaminetetraacetic acid from the cubital vein 08-10 am in a fasting state. The obtained samples were centrifuged using an Eppendorf 5810R centrifuge at 4°C at 3000 rpm for 15 minutes and then put into operation. Biochemical blood testing was performed on an Architect plus C 4000 analyzer (Abbott Diagnostics, USA). Determination of the TSH level (using standard kits (Abbott Diagnostics, USA)) was carried out by the immunochemiluminescence method on the Architect 2000 analyzer. The levels of LH, FSH, estradiol, and testosterone were determined by the chemiluminescent immunoassay method on the Vitros ECi 3600 automatic analyzer (Ortho-Clinical Diagnostics). Aldosterone and renin levels were measured by the immunochemiluminescent method on the DiaSorin Liaison analyzer (DiaSorin SpA, Italy). ACTH and cortisol in the blood were determined by the immunochemical method on the automated Cobas 6000 system (Roche, Germany). Determination of DHEA-S, PTH, AT to rTSH and TG was carried out on the electrochemiluminescence analyzer Cobas 6000 (Roche, Germany), AB to TPO - by the chemiluminescence immunoassay method on the automatic analyzer Architect i2000 (Abbott). Determination of AB to P450c21, ZnT8, IA-2, GAD, ICA, IAA was carried out by the ELISA method using commercial kits: BioVendor, Czech Republic (AT to P450c21), Medipan, Germany (AT to ZnT8, IA-2, ICA), EUROIMMUN, Germany (AT to GAD), Orgentec Diagnostika, Germany (IAA). The reference intervals (RI) for blood parameters were: glucose – 3.1–6.1 mmol/l, total Ca – 2.15–2.55 mmol/l, ionized Ca – 1.03–1.29 mmol/l, P – 0.74–1.52 mmol/l, aldosterone - 69.8-1085.8 pmol/l, renin - 2.8-39.9 mIU/l, ACTH - 7.2-63.3 pg/ml, DHEA-S - 1.65-11 μmol/l (for women 18-39 years old), 0.26-6.68 μmol/l (for women ≥ 40 years old), 1.2-13.4 μmol/l (for men 18-54 years old), 0.44-6.76 μmol/l (for men ≥ 55 years old), TSH - 0.25-3.5 mIU/l, PTH - 15-65 pg/ml, LH - 2.6-12.1 U/l (for women), 2.5-11 U/L (for men), FSH – 1.9-11.7 U/L (for women), 1.6-9.7 U/L (for men), estradiol – 97-592 pmol/L (for women), testosterone – 11-28.2 mmol/L (for men), AB to P450c21 < 0.4 U/ml, TPO – 0- 5.6 IU/ml, TG – 0-115 IU/ml, rTSH 0-1.75 IU/l, GAD – 0-10 U/ml, IA2 – 0-10 U/ml, ZnT8 – 0-15 U/ml, ICA – 0-1 U/ml, IAA – 0-10 U/ml, alanine aminotransferase – 0.0-55.0 U/L, aspartate aminotransferase – 5.0-34.0 U/L, creatinine – 50-98 mmol/L, potassium – 3.5-5.1 mmol/L, sodium – 136-145 mmol/L, erythrocytes – 3,8-5,2*1012 cl/L (for women), 4,3-5,8*10¹² cl/l (for men), white blood cells – 3,4-10,8*10⁹ cl/L (for women), 3,9-10*10⁹ cl/L (for men), hemoglobin – 112-153 g/l (for women), 132-172 g/l (for men), platelets – 152-372*10⁹ cells/l. The laboratory-accepted RI for blood cortisol (outside the insulin hypoglycemia test) was 171-536 nmol/L. Basal blood cortisol level (outside the insulin hypoglycemia test) ≥ 500 nmol/l excluded manifest AI (both 1- and 2-). Basal cortisol level < 140 nmol/l in combination with ACTH > 126.6 pg/ml confirmed manifest primary glucocorticoid deficiency. A biochemical blood test was performed on an Architect plus C 4000 analyzer (Abbott Diagnostics, USA), and a general blood test (with mandatory assessment of the level of leukocytes and platelets) was performed on an automatic analyzer Sysmex XE-2100 D, Sysmex, Japan.

Determination of glycated hemoglobin was carried out in capillary blood using an automatic biochemical analyzer D10 (BioRad Laboratories, USA) and a kit from the same manufacturer according to the standard method. The HbA1c level of up to 6% was considered normal.

DNA was isolated using a commercial Ribosorb kit (InterLabService, Russia). Next, the following polymorphic markers were studied by real-time polymerase chain reaction (PCR-RT): rs12979860 and rs8099917 in the IL28B gene, rs5743708 in the TLR2 gene, rs5743836 and rs352140 in the TLR9 gene. The analysis of polymorphic markers in the IL28B gene was carried out using commercial kits, according to the attached instructions (Syntol, Russia). To evaluate the polymorphic markers rs5743836 in the TLR9 gene and rs5743708 in the TLR2 gene, an adapted technique was used with commercial kits from Litech (Russia) and a "Kit for PCR-RT in the presence of the intercalating dye SYBR Green I" (Syntol, Russia). The polymorphic marker rs352140 in the TLR9 gene was studied using reagents from the "Set of reagents for PCR-RT" (Syntol, Russia) and specially synthesized primers and probes (Syntol, Russia). PCR-RT was performed on devices manufactured by DNA Technology (RF): "DT-96", "DTprime 4" and "DTprime 5".