Adiponectin Gene Polymorphisms and Possible Susceptibility to Metabolic Syndrome among the Sudanese Population: A Case-Control Study

Results There is a significant difference in genotypic frequencies of the rs266729, rs2241766, and rs1501299 SNPs and allele frequencies (P  < 0.05) between the MetS patients and non-MetS group. MetS patients had a significantly higher serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) in the GG genotype of rs2241766 (P  < 0.05). Additionally; the TT genotype of rs1501299 had higher SBP, serum TG, TC, and LDL-C (P  < 0.05). Multivariate logistic regression analysis showed hypertension, hyperglycemia, BMI, WC, serum TG, ADIPOQ rs2241766 (TG allele), and ADIPOQ rs1501299 (GT allele) had independently predicted the incidence of metabolic syndrome in the Sudanese population. The three investigated SNPs of ADIPOQ were in a moderate linkage disequilibrium (LD) connection according to the LD measures (D' = 0.54, 0.62, and 0.69, respectively). The CTT, CGG, and GTG haplotypes, which consist of three alleles of −11377C > G, +45T > G, and +276G > T, were shown to report 1.788-, 1.622,- and 1.641-fold risks toward MetS susceptibility in Sudanese's population, respectively. Conclusion Along with clinical and biochemical signs, the ADIPOQ gene's genetic variants (rs266729, rs2241766, and rs1501299), CTT, CGG, and GTG haplotypes are connected to the MetS risk among the Sudanese population.


Introduction
Te most recent pandemic of disorders that has afected humans is metabolic syndrome (MetS). Since obesity and type 2 diabetes (T2D) are included in the MetS category, it has ensnared millions worldwide [1,2]. MetS, as determined by the Adult Treatment Panel III criteria of the National Cholesterol Education Program (NCEP ATP III), was identifed when at least three of the following factors were present: abdominal obesity (WC ≥ 102 cm, ≥88 cm in men and women, respectively), elevated TG (≥150 mg/dL), reduced HDL-C (<40 mg/dL, <50 mg/dL in men and women), increased blood pressure (≥130/85 mmHg), and elevated fasting plasma glucose (≥110 mg/dL) [3,4]. MetS was present in 16.6% of the Sudanese population [4]. Because of the increased frequency of MetS and its impact on public health, early detection and management of MetS are crucial to preventing the onset of T2D, cardiovascular disease, and other consequences.
Te ADIPOQ gene, situated on chromosome 3q27 [5,6] and only expressed in human adipose tissue, is responsible for producing the plasma protein adiponectin [7,8]. T2D, obesity, and metabolic syndrome have all been linked to the 3q27 gene [9,10]. Numerous researchers have looked into the adiponectin gene area in various populations to fnd genetic variations that substantially infuence the pathophysiology of obesity, metabolic syndrome (MetS), diabetes, and related complications [11].
Tree exons make up the ADIPOQ obesity-associated variants, and the rs2241766, rs266729, and rs1501299 were frequently studied [8]. Te minor allele G of the SNP rs266729, which is situated in the gene's promoter region, changes the sequence of the binding site (SP1), resulting in a loss of the SP1 binding efect and perhaps reducing the transcription activity of the adiponectin promoter [10]. Te SNP rs2241766, found in exon 2 of the gene, may have polymorphic efects on the stability or shearing of precursor mRNA or the level of proteins. Te ADIPOQ gene's second intron has the SNP rs1501299 and studies suggest that this polymorphism may impact how well the exon next to it functions [9]. Te polymorphism of these two locations infuences the body's insulin sensitivity and stimulates the development of T2D [9]. Terefore, this study aimed to ascertain the genotypes, haplotypes, and allelic distribution of the ADIPQ gene polymorphisms (rs266729, rs2241766, and rs1501299) and its relationship to MetS in Sudanese population. Additionally, ADIPQ gene SNPs based on BMI, HbA1c, and the lipid profle in the Sudanese people are investigated.

Methodology
Four hundred and twenty middle-aged adult Sudanese participants (210 had MetS and 210 non-MetS) were recruited for this community-based case-control study between July 2019 and July 2021. After getting ethical approval from the Research Committee of the Sudan University of Science and Technology (DSR-IEC-04-08), all participants received written informed consent forms after being told of the study's purpose. Patients with liver illness, renal failure, autoimmune disorders, and corticosteroid medication and patients with incompletely documented data were excluded from this study based on their medical records. Participants were divided into two groups (MetS and non-MetS), matched according to age and gender and based on whether they had MetS, as determined by the NCEP ATP III criteria.

Data Collection and Analysis.
Patients' weights were measured using a weight balance, their heights were measured using a meter, and their waists were measured using stretch-resistant tape between the bottom edge of the lower rib and the iliac crest. A mercury sphygmomanometer was used to check the patient's blood pressure (SBP and DBP). Five milliliters of fasting venous blood was drawn in the morning and divided into two containers: a whole blood container (EDTA) for HbA1c measurement and DNA extraction and a plain container for serum TG, TC, LDL-C, and HDL-C measurements. Automated spectrophotometers (Mindray-BA-88A) and ichroma (Boditech-I-CHROMA Reader) were used to measure serum TG, TC, LDL-C, HDL-C, and HbA1c, respectively. BMI was calculated by dividing body weight/kg by height/m 2 [12].

DNA Extraction.
Genomic DNA was extracted from whole blood using a guanidine extraction protocol [13]. Te quality of DNA was assessed using 2.0% gel electrophoresis.

Screening of ADIPOQ SNPs.
PCR-RFLP was used to amplify and genotype ADIPOQ variants −11377C/G (rs266729), +45T/G (rs2241766), and +276G/T (rs1501299). Te amount of the PCR reaction mixture was 20 μL, and the annealing temperatures varied depending on the primers (Table 1). A 2.0% agarose gel stained with ethidium bromide was used to test the amplifed products for electrophoresis. Table 1 shows the specifcation of the restriction enzymes used in this study, obtained from Fermentas, Termo Fisher Scientifc Inc., USA. Following the manufacturer's recommendations, 1U of the relevant restriction enzyme was used to digest 5 μL of the amplifed products in a total reaction volume of 10 μL. Te 100 base pair DNA ladder and the digestion products were resolved on 2.0% agarose gels stained with ethidium bromide and seen under a UV transilluminator (SYNGENE-InGeniusL). Gel photos of the ADIPOQ gene are shown in Figure 1

Statistical Analysis
Statistical Package for Social Sciences (SPSS; Chicago, USA, version 20.0) was used for all statistical analyses. Te onesample Kolmogorov-Smirnov test was used to detect the normal distribution of data for baseline parameters. Te Hardy-Weinberg equilibrium (HWE) was evaluated for all the SNPs in patients and controls by comparing the observed and expected frequencies of the genotypes using the chisquared test. An independent t.test was used to analyze baseline parameters. Te distribution of the genotypes and allele frequencies of ADIPOQ SNPs for patients and control were compared using the chi-squared test with two × two contingency tables using GraphPad Prism 6 software. Te association of SNPs with other parameters was performed using the one-way ANOVA test. Te association of the ADIPOQ SNPs with the risk of MetS was assessed by the odds ratio (OR) with 95% confdence intervals (CI) and logistic regression to predict risk factors. Linkage disequilibrium analysis of the three adiponectin gene SNP loci haplotypes was performed using SHEsis online software (http://analysis.bio-x.cn).  Table 2 summarizes the clinical and biochemical characteristics of the participants. MetS patients showed substantially greater BMI, WC, SBP, and DBP when compared to non-MetS individuals (P � 0.001). In addition, compared to non-MetS individuals, MetS patients had considerably higher HbA1c, serum TG, TC, and LDL-C levels while having signifcantly lower HDL-C levels. Genetic analysis showed that the ADIPOQ gene polymorphisms −11377C/G, +45T/G, and +276G/T were detected in all the participants. PCR products of C/G SNP (rs266729), T/G SNP (rs2241766), and G/T SNP (rs1501299) were 250 bp, 305 bp, and 413 bp, respectively ( Figure 2). Restriction digestion was carried out following amplifcation. For the rs266729 genotype, the wild genotype (CC) produced a single band of 250 bp; the heterozygous genotype (CG) had three fragments of 250 bp, 137 bp, and 113 bp; and the homozygous mutant genotype (GG) produced two bands of 137 bp and 113 bp. For rs2241766, the wild genotype (TT) had a single band 305 bp; the heterozygous genotype (TG) produced three fragments 305 bp, 202 bp, and 103 bp; and the homozygous mutant genotype (GG) produced two bands 202 bp and 103 bp. For rs1501299, the wild genotype (GG) was digested and made two bands, 270 bp and 143 bp; the heterozygous genotype (GT) had three rounds, 413 bp, 270 bp, and 143 bp; and the homozygous mutant genotype produced a single band, 413 bp ( Figure 1). Table 3 summarizes the genotypes and allele frequencies of ADIPOQ SNPs (rs266729, rs2241766, and rs1501299).

Discussion
Metabolic syndrome (MetS) is a recognized risk factor for the onset of cardiovascular disease and other degenerative illnesses, which are the major causes of morbidity and mortality [2,10,11]. Te adiponectin gene has been identifed as a human adiposity marker after signifcant research into the relationships between genetic diferences in the adiponectin gene and several obesity measurements and metabolic syndrome in numerous ethnic cultures [11]. Tis study was carried out to determine the relationship between the risk of MetS in the included studies and the polymorphisms in the ADIPOQ gene (SNP-11377C > G, SNP +45T > G, and SNP +276G > T). LD analysis revealed that   [14]. Additionally, this study demonstrated a statistically signifcant diference in the genotype distribution of +45T > G (rs2241766) between MetS cases and non-MetS controls (P � 0.03). Tere were signifcantly more carriers of the TG and GG genotypes in the MetS group (27.6% and 4.8%) than in the non-MetS group (16.2% and 1.0%). It is also discovered that the rs2241766G allele increases the risk of developing metabolic syndrome in the study participants by 2.293 folds (P � 0.001). Our results are supported by research by Zahary et al. that indicates the genetic polymorphisms at the ADIPOQ +45T > G (rs2241766) loci enhanced the risk factor for the onset of MetS [15]. When looking at the relationship between the +276G > T (rs1501299) variation and MetS, it was shown that the Tminor allele contributed to a 2.228 fold (P � 0.001) increased risk of susceptibility to metabolic syndrome (MetS) in the current population. Our results are in line with research by Zahary et al., which suggests the genetic variants at the ADIPOQ +276G > T (rs1501299) loci enhanced the risk factor for developing MetS [15]. Tese signifcant associations persisted after the adjustments to hypertension,   [11]. Te results of the current investigation showed that the mutant genotypes −11377C > G (rs266729), +45T > G (rs2241766), and +276 G> T (rs1501299) were not signifcantly associated with any of the obesity indices (BMI and WC). Our fndings are in line with research by Ogundele et al. that found no correlation between +276G > T (rs1501299) and obesity indicators in the Nigerian population [8]. Our results, however, are the exact reverse of those obtained by Cui et al., who found a connection between central obesity and the ADIPOQ gene [7].
From another point of view, it is critical to assess if the polymorphisms afect the alteration of MetS components and other obesity-related disorders following a change in lifestyle, such as dieting. De Luis et al. demonstrated that in this situation the CC genotype of the ADIPQ-11377C > G polymorphism is connected to increase in adiponectin levels and decrease in total cholesterol, LDL-C, glucose, and insulin levels as well as the homeostasis model assessment for insulin resistance after weight loss [16]. All parameters showed the same improvement after a diet high in polyunsaturated fat [17]. One interventional study of 60 severely obese patients evaluated 32 months after bariatric surgery revealed that C-allele homozygotes decreased BMI and the lipid profle in obese people [18].
Our haplotype analysis revealed that the MetS participants had signifcantly higher frequencies of the CTT, CGG, GTG, CGT, and GTT haplotypes. Te results of the association analysis revealed that the CTT, CGG, GTG, CGT, and GTT haplotypes increased the risk of developing the metabolic syndrome in the research participants by 1.788 (P 0.001), 1.622 (P 0.033), 1.641 (P 0.027), 5.863 (P 0.001), and 5.621 (P 0.011) times, respectively.
Te current case-control study has both strengths and limitations. To the best of our knowledge, this is the frst study in Sudan to provide information on the genetic relationship between ADIPOQ variants and susceptibility to the metabolic syndrome risk. Te study gathered enough information about lifestyle choices that might be  International Journal of Endocrinology  International Journal of Endocrinology 9 confounding factors in the onset of disease. Tese variables may also play a role in fguring out how genes and the environment interact.
One limitation of the current study was that no SNPs with proven relationships published in the literature were genotyped due to cost-efectiveness concerns. Based on information from the literature search and their higher allele frequency in the SNP database, the SNPs for the current study were selected.
In conclusion, the results of this study give strong support for the hypothesis that the metabolic syndrome risk in the Sudanese population is strongly infuenced by the ADIPOQ-11377C > G (rs266729), +45T > G (rs2241766), and +276G > T (rs1501299) polymorphisms and their haplotype combinations. Given the signifcance of gene-gene interactions, more studies are required to determine the substantial contributions of ADIPOQ gene variants to the emergence of obesity and other characteristics of metabolic diseases to clarify the pathological processes.

Data Availability
Te datasets used and/or analyzed during the current study can be found at the following link: https://fgshare.com/ articles/dataset/ADIPOQ_gene_data/21687407.

Ethical Approval
Ethical approval was obtained before the start of the study from the Ethics Committee of the Sudan University of Science and Technology (no. DSR-IEC-04-08). All the procedures used in this study met the current guidelines of the Helsinki Declaration.

Conflicts of Interest
Te authors declare that they have no conficts of interest.