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Журнал «Здоровье ребенка» Том 18, №4, 2023

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Асоціації варіантів гена GHRL із розвитком ожиріння та метаболічних порушень у дітей

Асоціації варіантів гена GHRL із розвитком ожиріння та метаболічних порушень у дітей

Авторы: A. Abaturov, A. Nikulina
Dnipro State Medical University, Dnipro, Ukraine

Рубрики: Педиатрия/Неонатология

Разделы: Клинические исследования

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Резюме

Актуальність. Однонуклеотидні варіанти (single nucleotide variant — SNV) гена греліну (GHRL) супроводжуються продукцією дефектного протеїну препрогреліну, що може призводити до розвитку ожиріння та метаболічних порушень. Мета: вивчити асоціації гена SNV GHRL із розвитком різних фенотипів ожиріння в дітей. Матеріали та методи. Обстежено 252 пацієнтів з ожирінням віком 6–18 років. Основну групу (n = 152) становили діти з метаболічно нездоровим ожирінням (МНО). Контрольну групу (n = 100) представили діти з метаболічно здоровим ожирінням (MЗO). У 31 дитини основної та 21 дитини контрольної групи проведено повногеномне секвенування (CeGat, Німеччина). Рівень інтерлейкіну (IL) 1β у сироватці крові визначали методом імунохемілюмінесцентного аналізу, IL-6 — методом імуноферментного аналізу (Synevo, Україна). Результати. Асоціація з розвитком MНO була вищою для T-алеля SNV rs696217 гена GHRL у здорових осіб (t = 2,31; p < 0.05) та пацієнтів з ожирінням (t = 2,06; p < 0,05). Генотип GT SNV rs696217 був пов’язаний з інсулінорезистентністю (r = 0,40; p < 0,05) у групі MНO і зворотно корелював з умістом холестерину (r = –0,45) та холестерину ліпопротеїнів низької щільності (r = –0,39). Генотип TA SNV rs4684677 корелював із рівнем IL-6 (r = 0,74) у групі MЗO та з IL-1β (r = 0,35) у групі MНO, p < 0,05. Профілактика трансформації MЗO в MНO визначається T-алелем SNV rs34911341 (t = 2,29, p < 0,05). Висновки. Міссенс-варіанти rs696217, rs4684677 гена GHRL є SNV, високо асоційованими з ожирінням та розвитком метаболічних порушень.

Background. Single nucleotide variants (SNVs) of the ghrelin (GHRL) gene are accompanied by the production of a defective preproghrelin protein, which can lead to the development of obesity and metabolic disorders. The purpose was to study the associations of SNVs of the GHRL gene in children with the development of various obesity phenotypes. Materials and methods. Two hundred and fifty-two obese children aged 6–18 years were examined. The main group (n = 152) was represented by patients with metabolically unhealthy obesity (MUO). The control group (n = 100) included children with metabolically healthy obesity (MHO). Whole genome sequencing (CeGat, Germany) was performed in 31 children of the main group and 21 controls. Serum levels of interleukin-1β were measured using a chemiluminescent immunoassay, interleukin-6 — by enzyme-linked immunosorbent assay (Synevo, Ukraine). Results. The association with the development of MUO was higher for the T allele of SNV rs696217 in healthy individuals (t = 2.31; p < 0.05) and obese patients (t = 2.06; p < 0.05). The GT genotype SNV rs696217 was associated with insulin resistance (r = 0.40; p < 0.05) in the MUO group and inversely correlated with levels of cholesterol (r = –0.45) and low-density lipoprotein cholesterol (r = –0.39) in children with MHO. The TA SNV rs4684677 genotype correlated with IL-6 levels (r = 0.74) in the MHO group and with IL-1β (r = 0.35) in children with MUO, p < 0.05. Prevention of the transformation of MHO into MUO is determined by the T allele SNV rs34911341 (t = 2.29, p < 0.05). Conclusions. The missense variants rs696217 and rs4684677 of the GHRL gene are SNVs highly associated with obesity and the development of metabolic disorders.


Ключевые слова

грелін; аналіз однонуклеотидних варіантів генів; діти; метаболічно нездорове ожиріння; метаболічно здорове ожиріння

ghrelin; analysis of single nucleotide gene variants; children; metabolically unhealthy obesity; metabolically healthy obesity

doi: http://dx.doi.org/10.22141/2224-0551.18.4.2023.1596

Introduction

Over the past decades, due to the pandemic nature of the spread of obesity, the problem of its treatment and prevention of metabolic disorders in children has been in the focus of medical community [22]. Evidence has now been obtained that a sedentary lifestyle, excessive consumption of high-ca–lorie foods, sleep disorders and genetic predisposition cause a high risk of developing polygenic obesity [2]. Mutations of genes involved in the regulation of appetite, of the activity of hedonic pathways, the formation of food preferences, carbohydrate and fat metabolism, as well as the development of adipocytes, the distribution of adipose tissue in the body may predetermine excessive accumulation of fat [1, 4, 16, 32].
According to the results of the Human Obesity Gene Map project, about 500 genes are associated with the development of human obesity, among which the ghrelin-obestatin preproprotein gene (growth hormone secretagogue receptor ligand, GHRL) is noted [8, 10, 34–39, 46].
The GHRL gene is located on the short arm of chromosome 3 (3q25-26). Structurally, the human GHRL gene consists of five exons, four introns and encodes the 117 amino acid sequence of preproghrelin [24, 39].
As a result of post-translational modifications, preproghrelin is processed to form two biologically active molecules: ghrelin, consisting of 28 amino acid residues (sequence from 24 to 51 amino acid residues of the preproghrelin molecule), and obestatin, formed by 23 amino acid residues (sequence from 76 to 98 amino acid residues of the preproghrelin molecule) [45, 50].
The orexigenic hormone ghrelin was identified in 1999 as a peptide secreted by A-like cells in the gastric mucosa. The acylated form of ghrelin has the ability to bind with the N-terminal region of its molecule to receptors 1a that stimulate the secretion of growth hormone (growth hormone secretagogue receptor 1a — GHSR1a), which are located on the membranes of neurons of the hypothalamus, endotheliocytes, intestinal epithelial cells, cardiomyocytes, adipocytes, β- and α-cells of the pancreas, cells of the adrenal glomerulus, osteoblasts [9, 17, 24, 45]. A rhodopsin-like high-affinity G-protein-coupled receptor, class A GHSR1a, which has seven transmembrane α-helical domains, is activated by the acylated form of ghrelin and is blocked by liver-expressed antimicrobial peptide 2. Ghrelin-mediated induction of GHSR1a receptors, which are located on NPY/AgRP neurons, causes an increase in appetite and changes in eating behavior, while excitation of GHSR1a, which are located on other cells, preferentially activates mechanisms that promote hyperglycemia. Ghrelin inhibits insulin secretion by β-cells and stimulates the production of glucagon secretion by pancreatic α-cells, regardless of food intake, stimulates lipogenesis, inhibits thermogenesis, has a cardioprotective and antiatrophic effect on muscle tissue [28, 29, 35, 36, 53], while obestatin, interacting with receptors such as G-protein coupled receptor 3, glucagon-like peptide-1 receptor and GHSR, causes effects opposite to ghrelin. Ghrelin stimulates appetite and growth hormone secretion, and obestatin blocks these effects. Obestatin suppresses food intake and promotes weight loss [50, 51].
Non-synonymous single nucleotide variant (SNV) of the GHRL gene is accompanied by the production of a defective preproghrelin protein, which can lead to the deve–lopment of obesity and metabolic disorders [10, 30].
However, the associations of single nucleotide variants of the GHRL gene with obesity-related metabolic disorders remain practically unexplored.
The purpose was to study the associations of SNV of the GHRL gene in children with the development of various obesity phenotypes.

Materials and methods

Ethical approval
Participants provided written informed consent, and research protocols and procedures were approved according to the ethical standards of the Helsinki Declaration 2013 and by the Human Research Ethics Committee of Dnipro State Medical University (ethical approval DSMU/EC/19/1107). Time of data collection: January 2020 — February 2023.
Informed consent: informed consent was obtained from all individual participants included in the study.
Study design: observational, analytical, longitudinal, cohort study [44].
Inclusion criteria: polygenic obesity (BMI ≥ 97th percentile), age of 6–18 years.
Exclusion criteria: monogenic and secondary forms of obesity; hereditary syndromes accompanied by obesity; di–seases whose treatment requires the use of medications that affect carbohydrate and lipid metabolism; pregnancy.
Setting. At the Children’s Endocrinology Department of the Communal Non-profit Enterprise “Dnipro City Clinical Hospital 9” of the Dnipro City Council, 252 children aged 6–18 years with a diagnosis of obesity were examined. To verify the diagnosis, the classification of obesity recommended in clinical practice was used: Order of the Ministry of Health of Ukraine No. 254 dated 27.04.2006 “Protocol for the provision of medical care to obese children” and Order of the Ministry of Health of Ukraine No. 1732 dated 24.09.2022 “About the approval of Standards of medical care “Obesity in children”.
The main group (n = 152) was represented by children with metabolically unhealthy obesity (MUO), the control group (n = 100) was formed from patients with metabolically healthy obesity (MHO).
Criteria for inclusion in the main group: the presence of abdominal obesity [3] and two of the following criteria: hyperglycemia and/or hyperinsulinemia; dyslipidemia; systo–lic blood pressure and diastolic blood pressure above the 90th percentile for a given age, gender and height [15].
Immunochemical examination
The studies were carried out in a certified Synevo laboratory (Dnipro, Ukraine). The material for the study was venous blood.
To study carbohydrate metabolism disorders, the level of basal glycemia and insulinemia was determined by immunochemical testing with electrochemiluminescence immunoassay. Obese children were included in the main group with a glycemic level equal to or greater than 5.6 mmol/L and/or they had an increase in insulinemia above 90th percentile according to the percentile curves recommended by the IDEFICS consortium for the European population depending on the age and gender of a child [13, 33].
To study lipid metabolism disorders, the level of high-density lipoproteins (HDL-C), low-density lipoprotein cholesterol and triglycerides was determined by the enzymatic colorimetric method using Roche Diagnostics kits (Switzerland) on the Cobas 6000 analyzer. Obese children were included in the main group with HDL-C ≤ 1.03 mmol/L or less than 10th percentile of the age norm or an increase ≥ 1.7 mmol/L or more than the 90th percentile of the age norm [14].
Molecular and immunological examination
To study the role of pro-inflammatory markers in the development of meta-inflammation in childhood obesity, serum levels of IL-1β, IL-6 were determined in the certified Synevo laboratory (Dnipro, Ukraine). Interleukin-1β was investigated by immunochemical method with chemiluminescence immunoassay. Analyzer and test system was Immulite (Siemens AG, Germany). The reference IL-1β value was 0–5 pg/ml. Interleukin-6 was determined by enzyme-linked immunosorbent assay using a Cobas 6000/Cobas 8000 kit provided by Roche Diagnostics (Switzerland). The reference IL-6 value was 1.5–7.0 pg/ml.
Molecular genetic testing
To study the contribution of GRLN SNV variants to the formation of MUO, a molecular genetic examination was carried out using the method of next generation sequencing according to the recommendations of The American College of Medical Genetics and Genomics [12] in 52 patients (31 children from the main group and 21 controls) with venous blood sampling in a certified CeGat laboratory (Tubingen, Germany) using the Illumina СSPro® Certified Service Provider Program.
Average amount of DNA (μg) in samples was 0.875. Library Preparation: quantity used 50 ng. Library Preparation Kit: Twist Human Core Exome plus Kit (Twist Bioscience). Sequencing parameters: NovaSeq 6000; 2 × 100 bp. QC va–lues of sequencing, Q30 value: 96.07 %.
Bioinformatic analysis
Bioinformatic analysis — demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (version 2.20). Adapters were trimmed with Skewer, version 0.2.2 [20]. DNA-Seq: trimmed raw reads were aligned to the human reference genome (hg19-cegat) using the Burrows-Wheeler Aligner, BWA-mem version 0.7.17-cegat [26]. ABRA version 2.18 and Genotype Harmonizer v. 1.4.20 were used for local restructuring of readings in target regions to improve more accurate detection of indels in the genome during mutagenesis [11, 31].
Reference sequence was obtained from the National Center for Biotechnology Information RefSeq database [39].
Statistical analysis
Statistical analysis of the obtained results was carried out using a package of application programs Statistica 6.1 (No. AGAR909E415822FA) with the help of a personal computer based on an Intel processor Pentium 4. Depen–ding on the test result, parametric and nonparametric statistical methods were used. Correlation analysis was used to analyze 100 indicators of clinical, laboratory-instrumental and molecular genetic examinations in 252 children. To assess the relationship between quantitative traits, correlation analysis was used according to the Pearson method, and between qualitative traits, a non-parametric ranking method was used according to Spearman’s analysis (r). Only essential connections were taken into account (p < 0.05).

Results

Whole genome sequencing of obese children identified four SNV of the GHRL gene: rs696217, rs4684677, rs34911341, and rs139684563. The distribution of genotype frequencies was in Hardy-Weinberg equilibrium in both groups of obese children.
Molecular genetic characteristics of the identified SNV of the GHRL gene are presented in Table 1.
The most highly pathogenic among the identified SNVs of the GHRL gene are three nonsynonymous variants rs696217, rs4684677, rs34911341 (СADD = 22.6, 24.3, 25.5, respectively).
Associations of SNV GHRL gene with obesity phenotypes in children
The frequency of occurrence of SNV of the GHRL gene in children with different obesity phenotypes is presented in Table 2.
In children with the MUO phenotype, the frequency of the mutant T allele for SNV rs696217 of the GHRL gene was significantly higher than the frequency of this polymorphism among healthy Europeans of non-Finnish origin (t = 2.31; p < 0.05) and children with the MHO (t = 2.06; p < 0.05).
According to the analysis data, the frequency of the T allele SNV rs34911341 of the GHRL gene in children with the MUO phenotype was significantly lower than in patients with the MHO (t = 2.29, p < 0.05).
Associations of SNV GHRL gene 
with inflammatory activity
Correlation analysis revealed that production of pro-inflammatory cytokines in obese children depended on the SNV rs4684677 genotype of the GHRL gene. Thus, the AT genotype SNV rs4684677 in children with MHO was highly associated with the level of IL-6 (r = 0.74), and in patients with MUO — with IL-1β concentration (r = 0.35) in the blood serum. Carriers of the A allele compared with non-carriers had a higher level of pro-inflammatory interleukins.
Associations of SNV GHRL gene with disorders of carbohydrate metabolism
It was found that of all SNV of the GHRL gene identified in patients with obesity, only rs696217 genetic variant was associated with carbohydrate metabolism disorders. This association was noted exclusively in children with MUO. SNV rs696217 of the GHRL gene appeared to be moderately associated with the HOMA index (r = 0.40). Children with the MUO and the GT genotype SNV rs696217 of the GHRL gene had a higher HOMA index than those with the MHO phenotype and the wild GG genotype of SNV rs696217 GHRL gene.
Associations of SNV GHRL gene with lipid metabolism disorders
It was found that SNV rs696217 of the GHRL gene in children with the MHO phenotype is inversely related to the serum level of cholesterol and low-density lipoprotein cholesterol: r = –0.45; r = –0.39. The remaining identified SNV of the GHRL gene were not associated with the blood lipids of obese children.

Discussion

According to the results of whole genome sequencing, SNVs rs696217, rs4684677, rs34911341, and rs139684563 of the GHRL gene are found in obese children. Three non-synonymous variants rs696217, rs4684677, rs34911341 have a high level of pathogenicity (СADD = 22.6, 24.3, 25.5, respectively). It should be noted that the presence of SNV rs139684563 of the GHRL gene in obesity was revealed by us for the first time. The missense mutation rs139684563 (C>A, T), which is accompanied by the replacement of a glycine re–sidue with an arginine residue at position 18 (Gly18Arg) of the preproghrelin molecule, is very rare in the European population (AF = 0.08 %) [21]. In children with the MUO phenotype, the frequency of the T allele SNV rs696217 of the GHRL gene was significantly more common than in healthy Europeans of non-Finnish origin and children with the MHO.
We found that SNVs rs696217, rs4684677 of the GHRL gene are associated with pro-inflammatory status and metabolic markers in obese children. Also, E. Becer and M.C. Ergo–ren believe that SNVs of the GHRL gene are associated with the development of obesity and metabolic syndrome in adults [5]. However, according to the results of C. Bing et al. [6], the SNV rs696217 of the GHRL gene has no effect on the development of metabolic disorders in obese adults.
It was shown that the missense variant of SNV rs696217 (G>T), located in the second exon of the GHRL gene, leads to the replacement of a leucine residue with a methionine residue at position 72 (Leu72Met) of the preproghrelin mole–cule [30]. The SNV rs696217 of the GHRL gene is believed to be highly associated with the development of obesity. According to E. Becer and M.C. Ergoren [5], the T allele of SNV rs696217 of the GHRL gene is significantly associated with waist and hip circumferences. In the Tur–kish Cypriot population, the frequency of occurrence of the minor T allele SNV rs696217 in obese individuals is significantly higher than in people with physiological body weight. Individuals with the GT or TT genotype are at higher risk of develo–ping obesity compared to those with the GG genotype SNV rs696217 [52]. A low-calorie diet as a treatment for obesity in female individuals with SNV rs696217 of the GHRL gene does not result in weight loss [42]. We found that the GT SNV rs696217 genotype of the GHRL gene is associated with markers of metabolic disorders in obese children. In persons with metabolic syndrome, the frequency of the T allele of the currently most studied SNV rs696217 of the GHRL gene is 8.6 % [6]. According to our data, this level of occurrence of the T allele SNV rs696217 of the GHRL gene is typical for children with the MHO phenotype, and in children with the MUO, it reaches 19 %. In the MUO, the GT SNV rs696217 genotype contributes to the development of insulin resistance, and in the MHO, this genotype prevents the development of dyslipidemia. The results of stu–dies on the relationship between SNV rs696217 of the GHRL gene and the risk of developing carbohydrate metabolism disorders are controversial. According to the research by E.A. Rivera-León et al. [41], the G allele of SNV rs696217 of the GHRL gene was more common in type 2 diabetes mellitus (T2DM). At the same time, F.E. Joatar et al. [23], J. Liu et al. [27] emphasize the absence of association between SNV rs696217 of the GHRL gene and the risk of developing T2DM. Buraczynska M. et al. [7] also did not find a significant association between SNV rs696217 of the GHRL gene and the risk of developing T2DM, but showed that the presence of the T allele of SNV rs696217 is associated with a higher risk of hypertension (OR = 2.50, 95% CI 1.68–3.73, p < 0.001). Regarding the association of SNV rs696217 of the GHRL gene and the blood lipids, M. Su et al. [47] demonstrated that after a high-carbohydrate diet, carriers of the SNV rs696217 T allele of the GHRL gene had a significantly lower serum triglyceride/HDL ratio than those with the wild genotype. Also, the T allele (Met72) compared to the G allele (Leu72) is associated with a lower risk of developing metabolic-associated liver disease [48].
According to our data, SNV rs4684677 of the GHRL gene was not associated with either the development of obesity or the differentiation of the obesity phenotype in children. At the same time, M. Gueorguiev et al. [18] showed the association of SNV rs4684677 of the GHRL gene with the risk of developing obesity. It is believed that obestatin Q90L does not sufficiently block ghrelin-induced appetite activity, and therefore the SNV rs4684677 A allele of the GHRL gene contributes to the development of polyphagia [19]. We have demonstrated for the first time that the missense variant of SNV rs4684677 (T>A) located in the third exon of the GHRL gene, which leads to the replacement of a glutamine residue with a leucine residue at position 90 (Gln90Leu) of the preproghrelin molecule, is positively associated with the pro-inflammatory status of obese children.
We did not reveal any relationship between the nonsynonymous SNV rs34911341 (C>T) GHRL gene and the markers of metabolic disorders. We showed that the frequency of mutant T alleles of SNV rs34911341 of the GHRL gene was significantly lower in individuals with MUO than the allelic frequency of these polymorphisms among individuals with MHO.
It is known that this genetic variant leads to the replacement of arginine with a glutamine residue at position 51 –(Arg51Gln) of the preproghrelin molecule, which prevents the proteolytic cleavage of preproghrelin, and as a result, causes a decrease in the serum level of ghrelin [49]. There is scientific evidence that the T allele of SNV rs34911341 (C>T) of the GHRL gene is a protective factor that prevents the development of T2DM [54].
Thus, in obese children, SNVs rs696217, rs4684677 of the GHRL gene are associated with the level of pro-inflammatory activity and laboratory markers of metabolic disorders (Fig. 1).
The GT genotype SNV rs696217 in obese children is associated with the risk of developing the MUO.

Conclusions

1. Missense variants rs696217, rs4684677 of the GHRL gene in children are associated with the development of obesity and metabolic disorders induced by obesity. The development of the MUO phenotype in children is determined by the T allele of SNV rs696217.
2. SNV rs34911341 of the GHRL gene is associated with the MHO phenotype and prevents the formation of metabolic disorders in children.
3. The TA genotype SNV rs4684677 of the GHRL gene in obese children is associated with a pro-inflammatory status.
4. Variants rs696217 of the GHRL gene are associated with certain features of carbohydrate and lipid metabolism in obese children. Children with the CT genotype SNV rs696217 and the MUO have a higher level of basal hyperinsulinemia and insulin resistance, and those with the MHO have a low level of atherogenicity.
5. Determination of the SNV genotype of the GHRL gene will make it possible to predict the likelihood of obesity and to personalize the development trajectory for various metabolic disorders associated with obesity in children.
 
Received 22.04.2023
Revised 19.05.2023
Accepted 21.05.2023

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