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Коморбідний ендокринологічний пацієнт

Международный эндокринологический журнал Том 19, №1, 2023

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Комбінований вплив поліморфізму генів BDNF (rs6265), VDR (rs2228570) та NMDA (rs4880213) на когнітивні порушення у хворих на автоімунний тиреоїдит та гіпотиреоз

Комбінований вплив поліморфізму генів BDNF (rs6265), VDR (rs2228570) та NMDA (rs4880213) на когнітивні порушення у хворих на автоімунний тиреоїдит та гіпотиреоз

Авторы: I. Kamyshna (1), L. Pavlovych (2), I. Pankiv (2), V. Pankiv (3), V. Maslyanko (2), N. Bytsko (2), A. Kamyshnyi (1)
(1) — I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine
(2) — Bukovinian State Medical University, Chernivtsi, Ukraine
(3) — Ukrainian Research and Practical Center for Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the Ministry of Health of Ukraine, Kyiv, Ukraine

Рубрики: Эндокринология

Разделы: Клинические исследования

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Резюме

Актуальність. Численні дослідження показали, що захворювання щитоподібної залози можуть впливати на когнітивні функції. Поліморфізм генів, які пов’язані з патологією ендокринної та нервової системи, має етнічну та популяційну специфічність, що визначає необхідність їх вивчення в певному регіоні. Метою дослідження було дослідити комбінований вплив поліморфізму генів BDNF (rs6265), VDR (rs2228570) та NMDA (rs4880213) на когнітивні порушення серед хворих на автоімунний тиреоїдит і гіпотиреоз у популяції західних регіонів України та спрогнозувати виникнення когнітивних розладів. Матеріали та методи. Обстежено 153 особи з автоімунним тиреоїдитом та гіпотиреозом. Генотипування поліморфізму генів VDR (rs2228570), BDNF (rs6265) і NMDA (rs4880213) за допомогою зондів TaqMan і TaqMan Genotyping Master Mix (4371355) виконували в системі CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., США). Полімеразну ланцюгову реакцію для генотипування TaqMan проводили згідно з інструкцією до набору (Applied Biosystems, США). Когнітивні функції досліджували за допомогою шкали Mini-Mental State Exami­nation. Результати. У носіїв комбінації генотипів CC/AG/CC значно знижений ризик розвитку когнітивних порушень (відношення шансів (ВШ) = 0,1410; 95% довірчий інтервал (ДI) 0,0181–1,0965; p = 0,0416). При цьому носійство комбінації генотипів CT/AG/CT підвищує ризик когнітивних порушень більше ніж у 5 разів (ВШ = 5,1915; 95% ДI 1,2471–21,6107; p = 0,0214), а комбінації генотипів CT/AG/TT (ВШ = 10,1224; 95% ДI 1,1037–92,8401; p = 0,0281) — у 10 разів. Носії комбінації генотипів CT/AA/CT мають в 6,4 раза вищий ризик когнітивних порушень (ВШ = 6,4062; 95% ДI 1,2019–34,1471; p = 0,0253). Висновки. Серед пацієнтів з авто­імунним тиреоїдитом і гіпотиреозом носії комбінації генотипів CT/AG/CT, CT/AG/TT і CT/AA/CT мають підвищений ризик розвитку когнітивних порушень, а в носіїв комбінації генотипів СС/АГ/СС генів BDNF (rs6265), VDR (rs2228570) і NMDA (rs4880213) цей ризик знижений.

Background. Numerous studies have demonstrated that thyroid conditions can affect cognitive function. Gene polymorphisms associated with pathology of the endocrine and nervous system have ethnic and population specificity, which determines the need to study them in a certain region. The purpose of the study was to investigate the combined impact of the BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) gene polymorphisms on cognitive impairment in patients with autoimmune thyroiditis and hypothyroidism among the population of Western regions of Ukraine, and to predict the onset of cognitive disorders. Materials and methods. The study involved a total of 153 patients with autoimmune thyroiditis and hypothyroidism. Genotyping of the VDR (rs2228570), BDNF (rs6265), and NMDA (rs4880213) gene polymorphism using TaqMan probes and TaqMan Genotyping Master Mix (4371355) was performed on CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., USA). Polymerase chain reaction for TaqMan genotyping was carried out according to the kit instructions (Applied Biosystems, USA). We detect a decline in cognitive function using the Mini-Mental State Examination. Results. Carrying a combination of CC/AG/CC genotypes significantly reduces the risk of developing cognitive impairment (odds ratio (OR) = 0.1410; 95% confidence interval (CI) 0.0181–1.0965; p = 0.0416). At the same time, carrying a combination of CT/AG/CT genotypes increases the risk of cognitive impairment by more than 5 times (OR = 5.1915; 95% CI 1.2471–21.6107; p = 0.0214) and a combination of CT/AG/TT genotypes — by 10 times (OR = 10.1224; 95% CI 1.1037–92.8401; p = 0.0281). Carriers of the CT/AA/CT genotype combination have a 6.4-fold increased risk of cognitive impairment (OR = 6.4062; 95% CI 1.2019–34.1471; p = 0.0253). Conclusions. Among patients with autoimmune thyroiditis and hypothyroidism, carriers of the CC/AG/CC genotype combination of the BDNF (rs6265), VDR (rs2228570) and NMDA (rs4880213) genes have a reduced risk of developing cognitive disorders, while carriers of the CT/AG/CT, CT/AG/TT and CT/AA/CT have an increased risk of cognitive impairment.


Ключевые слова

генотип; автоімунний тиреоїдит; гіпотиреоз; когнітивні порушення

genotype; autoimmune thyroiditis; hypothyroidism; cognitive disorders

doi: http://dx.doi.org/10.22141/2224-0721.19.1.2023.1235

Introduction

The influence of thyroid hormone (TH) on neurogenesis, neuronal migration, neuronal and glial differentiation, myelination, and synaptogenesis has been found to regulate the differentiation of the nervous system [1]. Problems with cognition, visual attention, visual processing, motor abilities, language and memory might result from TH deficiency [2]. However, little is known about the molecular processes that underlie TH-mediated control of neuronal cells of these disorders. Additionally, a higher level of thyroid-stimulating hormone (TSH) has been linked to an increased risk of dementia [3].
The investigation of the role of genetic variables in the emergence of prevalent multifactorial diseases and the discovery of novel therapeutic targets were two topical directions of biomedical research. In this instance, single nucleotide polymorphism analysis [4, 5] is thought to be a helpful technique for determining a condition hereditary susceptibility.
Neurological problems may result from autoimmune thyroid disorders that affect the transcriptional activity of the genes that control neurogenesis and neurotrophins. Understanding how genetic variables contribute to the emergence of common multifactorial diseases is crucial, and this problem continues to dominate in biomedical research today.
The aim of the study was to investigate the combined impact of the BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) gene polymorphisms on cognitive impairment in patients with autoimmune thyroiditis and hypothyroidism in the population of Western regions of Ukraine, and to predict the onset of cognitive disorders in these patients.

Materials and methods

Our study included 153 patients with different types of autoimmune thyroiditis and hypothyroidism. Patients were distributed into three groups. Group 1 (n = 16) included patients experiencing postoperative hypothyroidism; group 2 (n = 65) included patients with hypothyroidism resulting from autoimmune thyroiditis (AIT), and group 3 (n = 72) included patients with both AIT and increased serum antibodies (Ab) anti-thyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO). Twenty-five healthy subjects were recruited as a control group, randomly and without age and gender matching. The clinical and biochemical features of the subjects are displayed in Table 1.
Ethical approval. The study fully ensured standards described in the 1975 Helsinki Declaration of Human Rights (amended in 2008). The participants completed and signed a written informed consent before enrolling voluntarily in the research.
To diagnose hypothyroidism, we were guided by recommendations required by the American Association of Clinical Endocrinologists [6]. The corresponding clinical features were considered when verifying AIT, namely the results of a sonogram of the thyroid gland (reduced echogenicity) and circulating antibodies to thyroid antigens, anti-TPO, and anti-Tg were detected.
Blood samples from patients and controls were taken in the morning after a night fast. Using Stat Fax 303/Plus analyzer (Awareness Technology Inc., USA), we determined levels of free thyroxine (normal range 6.0–13.0 pmol/L for males and 7.0–13.5 pmol/L for females), TSH (normal range 0.3–4.0 mIU/mL), anti-TPO Ab (normal range 0–30 IU/mL) and anti-Tg Ab (normal range 0–65 IU/mL) in each individual who participated in the study.
Exclusion criteria were the following: less than 18 years of age, malignancy, inflammation resulting from rheumatic diseases or acute/chronic infection, diabetes mellitus, vascular, chronic diseases of liver and kidneys, and pregnancy. Individuals administering drugs that could influence thyroid function were also ruled out from the study.
We detect a decline in cognitive function using the Mini-Mental State Examination, which has been the most used screening instrument throughout decades [7].
Genotyping of the VDR (rs2228570), BDNF (rs6265), and NMDA (rs4880213) gene polymorphism
When collecting venous blood, we used a sterile vacutainer and stabilized it with K2EDTA. To isolate total DNA from peripheral blood, we applied PREP-RAPID-GENETICS DNA Extraction Kit (DNA-TECHNOLOGY, Catalog No P-021/4), adhering to the manufacturer’s instructions.
DNA amplification and genotyping
The samples were genotyped by TaqMan probes and TaqMan Genotyping Master Mix (4371355) on CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., USA). Polymerase chain reaction (PCR) for TaqMan genotyping was conducted due to the kit instructions (Applied Biosystems, USA). TaqMan Genotyping Master Mix contains DNA polymerase AmpliTaq Gold®, dNTPs, reference dye ROX™, and buffer ingredients. TaqMan probes are target-specific oligonucleotides with reporter dyes attached to the 5’ end of each probe: (VIC® dye on the 5’ end of the Allele 1 probe and 6FAM™ dye on the 5’ end of the Allele 2 probe), and a non-fluorescent quencher the 3’ end of the probe. Genomic DNA was intensified in a 10 μL reaction mix comprising genomic DNA, forward and reverse primers, fluorescent probes, and TaqMan Genotyping Master Mix. Genotyping of the samples conducted on the CFX-Manager™ software using allelic discrimination assays based on the magnitude of relative fluorescence units.
Statistical analysis
We used the Student’s t-test, Pearson’s χ2 test, odds ratio (OR), relative risk (RR), and equality 0 correlation test to determine the difference between groups. The odds ratio and 95% confidence interval (CI) were computed by binary logistic regression where P < 0.05 were regarded as a statistically significant difference between the two groups (StatSoft, Statistica v.12.0).

Results

Our paper studied the combined effect of gene polymorphisms in the studied population. The distribution of the combination of genotypes of polymorphic variants of the BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) genes showed no statistically disparities between the patient group and the control group that are significant (Table 2).
Most of the examined patients were owners of CC/AG/CT combinations of genotypes (16.99 %). CC/AA/CC (10.46 %) and CC/AA/CT combinations of genotypes (10.46 %) also prevailed among patients. The remaining genotype combinations of the three BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) genes were found in individual cases (Table 2). The studied population did not detect carriers of TT/AA/TT, TT/AG/TT, and TT/GG/TT combinations of genotypes.
Analysis of a combination of BDNF (rs6265), VDR (rs2228570), and NMDA (rs4880213) gene genotypes depending on the type of thyroid pathology (Tables 3–5) showed no statistically significant differences in the frequency of genotype combinations between patients with PO, AIT with hypothyroidism, and AIT.
Epidemiological analysis showed that the combinations of genotypes of the analyzed genes are not risk factors for PO, AIT with hypothyroidism, and AIT (Tables 3–5).
According to our studies, carrying a combination of CC/AG/CC genotypes significantly reduces the risk of developing cognitive impairment (OR = 0.1410; 95% CI 0.0181–1.0965; p = 0.0416) (Table 6). At the same time, carriers of a combination of CT/AG/CT genotypes increase the risk of cognitive impairment by more than 5 times (OR = 5.1915; 95% CI 1.2471–21.6107; p = 0.0214) and by 10 times in carriers of a combination of CT/AG/TT genotypes (OR = 10.1224; 95% CI 1.1037–92.8401; p = 0.0281), respectively. Carriers of the CT/AA/CT genotype combination have a 6.4-fold increased risk of cognitive impairment (OR = 6.4062; 95% CI 1.2019–34.1471; p = 0.0253)

Discussion

Neurologic problems can occur in endocrine disorders patients [8]. Hyperthyroidism, or hypothyroidism have been recognized as risk factors for developing progressive cognitive impairment [9, 10]. It should be made abundantly clear that despite decades of extensive research, the connection between thyroid function and cognition has yet to be fully understood. Numerous studies have demonstrated that thyroid conditions can affect cognitive function.
To prevent cognitive repercussions, it is crucial to evaluate thyroid function. Therefore, additional research is required to clarify the relevant relationships and the mechanism behind this interaction.
According to earlier studies, hypothyroidism has been linked to impairments in general cognition, memory, attention/concentration, perceptual function, language, psychomotor speed, and executive function. The most frequently mentioned impairment is impaired verbal memory [11, 12].
Another study discovered that elevated thyroid hormone levels, even within the normal range, are linked to alterations in cognition, behavior, and the structural integrity of the brain [13].
The Val66Met polymorphism (rs6265) in the BDNF gene causes the amino acid at codon 66 to change from valine to methionine, changing how BDNF is transported through cells and secreted [14]. Several studies have found a link between Val66Met polymorphism and reduced hippocampal and prefrontal gray matter volumes, leading to abnormal hippocampal activity, impaired cognitive function, and decreased memory and executive function [15].
Codon 66, found inside the BDNF pro-domain, changes from valine to a methionine amino acid residue due to the C to T substitution in BDNF. A sortilin-binding site is impacted, which causes intracellular trafficking to be disturbed. Affected BDNF proteins are not guided to secretory vesicles in the absence of prober binding to sortilin, which lowers their activity-dependent secretion [16].
According to studies, healthy German subjects with the rs6265 T allele have lower blood serum BDNF concentrations [17]. Subjects with the TT GRIN1 genotype (SNP rs4880213) exhibited significantly fewer instances of disruptive behavior and depression than individuals in the other two groups. The TT SNP GRIN1 rs4880213 genotype, according to F. Mori et al. [16], was associated with reduced intracortical inhibition, elevated glutamatergic excitement, and improved NMDAR glutamate function.
Previously, we showed that the C allele is protective and significantly reduces the chances of lowering the level of BDNF in the blood serum of patients with thyroid pathology in the studied population. On the other hand, the carrier of the T-allele increases the risk of low BDNF in thyroid pathology by almost ten times [18]. In addition, we found a significant decrease in BDNF levels in the experimental group in carriers of the AA and AG genotypes of the VDR gene (rs2228570) by 1.58 (p = 0.038) and 2.39 (p = 0.002) times, respectively, compared to carriers of the AA and AG genotypes of the control group [19].
Considering that the effect of thyroid hormones on the brain is indisputable, and their lack leads to various neurological complications, early diagnosis of nervous system damage in thyroid diseases is crucial for preventing and treating neurological complications.

Conclusions

Carriers of the CC/AG/CC genotype combination of the BDNF (rs6265), VDR (rs2228570) and NMDA (rs4880213) genes have a reduced risk of developing cognitive disorders, while carriers of the CT/AG/CT, CT/AG/TT and CT/AA/CT have an increased risk of cognitive impairment in patients with thyroid pathology.
Ethical approval. Our study was conducted according to the Declaration of Helsinki adopted in 1975 and revised in 2008, and the ethical principles were entirely respected.
Consent to participate. Written informed consent was obtained from the participants.
Data availability. The data of this study is available by request.
 
Received 21.12.2022
Revised 19.01.2023
Accepted 01.02.2023

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