Abstract
BACKGROUND:
Obesity, as a medical condition, results from interactions between environmental and genetic factors. The rs17782313 polymorphism, located 188kb downstream of the Melanocortin 4 Receptor (MC4R) gene, is one of the essential candidate genetic markers that has shown the highest association with obesity in different populations.
OBJECTIVE:
This study aimed to investigate the possible associations of rs17782313 polymorphism near the MC4R gene with obesity/overweight, body mass index (BMI), and hedonic hunger among women from the Iranian Azeri ethnic group.
METHODS:
Five hundred sixty-three women, composed of 396 patients with obesity/overweight and 167 unrelated healthy controls, were genotyped for the rs17782313 polymorphism by applying the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method.
RESULTS:
This population was in Hardy-Weinberg equilibrium (P = 0.878). The study confirmed a significant association of rs17782313 with obesity, where subjects carrying the C/C genotype had higher odds of obesity (OR = 2.681, P = 0.005, 95%CI:1.340–5.365). Also, C allele carriers have statistically significantly higher BMI scores than those carrying the T allele (P = 0.029). However, no significant associations were found among PFS scores and genotypic/allelic groups of rs17782313 polymorphism (P = 0.368).
CONCLUSIONS:
Our findings suggest that rs17782313 polymorphism is strongly associated with obesity and BMI but not with hedonic hunger among Northwest Iran women. Moreover, the sequencing data analysis in several homozygous and heterozygous carriers of the C allele led to identifying a novel frameshift variant with TCT deletion (rs534212081) in the 166 upstream of rs17782313, which has not been reported so far.
Introduction
Obesity is defined as a systemic disease that shows the excessive and abnormal accumulation of body fat, leading to adverse health effects [1]. To highlight the threats of obesity to public health, the World Health Organization (WHO) declared obesity a global epidemic [2]. During the past decades, the prevalence of obesity and overweight has been increasing at a rapid pace in both western societies and the developing world [3]. In 2016, more than 1.9 billion adults, 18 years and older, were overweight, and of these, over 650 million were obese [4]. Notably, if this trend persists, the global obesity prevalence is predicted to rise to 18%in men and surpass 21%in women by 2025 [5]. It is noteworthy that the Middle East, including the Arabian Peninsula, Eastern Mediterranean, Turkey and Iran, and North Africa, are no exceptions to the global increase in obesity [6]. According to the WHO, in 2016, the prevalence of obesity among adults in Iran was 25.8%. Women with 32.2%had a higher proportion than men with 19.3%, and referring to its outbreak in 1975 (6.5%), it is a statement of a rising trend in the prevalence of obesity in Iran [4].
The pathogenesis of obesity is obviously complex, involving multiple interactions among behavioral, environmental, and genetic factors [7]. It is estimated that genetic factors contribute to the development of obesity at 40–70%. Some of these factors are obesity or leptogenic genes that may influence obese people’s response to weight loss or weight management [8]. Genome-wide association studies (GWAS) have identified variants in several genes that may play roles in weight gain, body fat distribution, energy intake, and expenditure; however, genes are the major cause of obesity in a few cases [9, 10]. The Leptin-Melanocortin pathway has been suggested clearly as a major pathway for energy expenditure and food regulation. The melanocortin-4 receptor (MC4R), the key member of the Leptin-Melanocortin pathway, is a 332-amino-acid protein. It belongs to the family of seven trans-membrane G-protein-coupled receptors (GPCR) and is expressed in brain regions involved in the regulation of food intake [11, 12]. It is documented that the transmission of the MC4R signal is accomplished through the integration of a satiety signal provided by its agonist α-melanocyte-stimulating hormone (α-MSH), as well as an orexigenic signal provided by its antagonist agouti-related protein (AGRP) [13]. These ligands are expressed in distinct neuronal populations of the hypothalamus arcuate nucleus and are regulated by the adipocyte-secreted hormone, leptin, to control food intake and maintain long-term energy homeostasis [14]. In addition, MC4R mRNA is also found in distinct brain areas, contributing to the reward circuit and regulation of feeding behavior, such as the dopamine (DA) mesolimbic systems and nucleus accumbens (NAc). Such observations suggest that the melanocortin system plays a role in the hemostatic control of feeding and its hedonic aspects [15–17]. Therefore, these findings consider the MC4R as an important genetic marker for obesity [18].
Approximately 25 genetic variants have been found in the MC4R gene coding region, associated with common and severe forms of obesity in humans [19]. Furthermore, there exist numerous polymorphisms outside of the coding region of the MC4R gene associated with the predisposition of polygenic obesity. Precise molecular mechanisms for these polymorphisms have not been determined so far, but these variants are likely to have substantial effects on the expression of the MC4R gene [20]. A meta-analysis of GWAS carried out in the Caucasian population revealed that the rs17782313 polymorphism mapped 188 kb downstream of the MC4R gene is closely correlated with the elevated body mass index (BMI) [18, 21]. Studies in different populations of different breeds, including children, adolescents, and adults, confirm this variant’s association with all types of obesity [21, 22]. Gender also affects the relationship between polymorphism and obesity markers and is more effective in women than men [23].
In view of the importance of studies focusing on obesity in the women population, we performed an association study to evaluate the role of rs17782313 polymorphism with susceptibility to obesity/overweight, BMI, and hedonic hunger among women from the Turkish Azeri ethnic group. This would be the first study of rs17782313 polymorphism of the MC4R gene in the Iranian Azeri population to the best of our knowledge. Moreover, limited studies worldwide have evaluated the association of rs17782313 with hedonic hunger.
Materials and Methods
Study population and sample collection
This study was conducted from December 2016 to September 2018 in Tabriz, the capital of the East Azerbaijan province in northwestern Iran. The study procedures were reviewed and approved by the Tabriz University of Medical Science Ethics Committee and the IR.TBZMED.REC.1397.347 registry number already obtained. Participants in this study were invited through announcements and flyer distribution in the city’s public areas and were informed about the study. The participants were randomly selected among those who were minded to participate in the study. After applying the inclusion and exclusion criteria, participants who met the criteria were asked to come to the clinic within 2–3 hours after breakfast. Written consents were received, and the demographic and the Persian version of the power of food scale (PFS-P) questionnaires were performed on them. A total of unrelated 563 women aged 17 to 59 years (mean±SD age, 35.85±9.18) met the inclusion criteria and were selected for the study, dividing into three groups; Obese (300 subjects), overweight (96 subjects), and control (167 subjects). The criteria for entering the study were 17 to 59 years and living in Tabriz for at least five consecutive years. On the other hand, according to the exclusion criteria, the subjects with a BMI of less than 18.5 kg/m2, pregnant or lactating, those with recent weight loss or participating in weight loss programs during the last six months, and those with the diagnosis of any psychotic disorders, substance abuse, alcoholism, or a serious medical illness such as cardiovascular and endocrine diseases were deprived of the current study.
Anthropometric parameters
All participants were measured for body weight and height, wearing light clothes but without shoes after a 12 hours fasting period. A calibrated electronic scale (SECA Birmingham, UK) with an accuracy of 0.1 kg was the primary tool for measuring the weight; meanwhile, a fixed stadiometer was used to measure height to the nearest 0.1 cm. Additional anthropometric measures were also used to enhance the study’s accuracy, including body fat percentage, lean body mass, body water, waist circumference, and hip circumference. BMI was also calculated by dividing the participant’s weight in kilograms by the square of height in meters (kg/m2). According to the BMI definitions, published by the World Health Organization (WHO) in 2000, subjects were classified as healthy weight (BMI = 18.5–24.9), overweight (BMI = 25.0–29.9), or obese (BMI≥30.0) subjects [24].
Hedonic hunger
Participants’ hedonic motivation to food was evaluated using the Power of Food Scale (PFS) questionnaire, which was confirmed in reliability and validity by Aliasghari et al. [25]. PFS is a way of measuring the level of appeal for highly palatable dishes when an individual is in physiological satiety (hedonic hunger) [26]. The questionnaire includes five items answered on a 5-point Likert-type scale ranging from 1 (do not agree at all) to 5 (strongly agree). The PFS involves three factors of Food Available (PFS-FA), Food Present (PFS-FP), and Food Tasted (PFS-FT). Each factor’s score was calculated by averaging the scores of the factor’s all related items, and the total score was also the mean of three factors. A score of 1 to 5 is possible for each participant indicating the participant’s responsiveness to the food environment, and higher scores represent greater responsiveness to the food environment [25, 27].
Blood sampling, DNA extraction, and genotyping
In the next step, after collecting about 4 ml of venous blood samples from all participants in EDTA anticoagulant polypropylene tubes, genomic DNA was prepared from peripheral blood leukocytes using the simple salting out procedure. The rs17782313 was genotyped by a polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method. Briefly, the 330-bp region encompassing the rs17782313 polymorphism site in 188 kb downstream of the MC4R gene was amplified by PCR with previously reported primer pairs (Macrogen, Seoul, South Korea) and protocols [28]. The PCR products were then digested overnight at 37° C with three units of AvaI restriction enzyme (Thermofisher Scientific, Massachusetts, US). The genotype for RFLP products was assessed by electrophoresis on a 3%agarose gel and visualized under ultraviolet light. Also, a number of PCR products were purified and sent to the company for sequencing (Macrogen, Seoul, South Korea) and then analyzed with Chromas Pro 2.1.3. (Technelysium Pty Ltd, South Brisbane, AU).
Statistical analysis
Statistical analysis was performed using Statistical Analysis System (SAS®, version 9, NC, USA) and IBM Statistical Package for the Social Science (IBM SPSS Statistics®, version 25, Chicago, IL, USA). GLMPower PROC of SAS performed the power analysis, and the power for the BMI was 0.69. The Kolmogorov–Smirnov test assessed the distribution normality of the age, BMI, and PFS score variables. According to the results, the Mann-Whitney test was used to compare age among study groups. Descriptive data were compared by the means, and the rs17782313 polymorphism was evaluated using chi-square for Hardy-Weinberg equilibrium. Logistic regression was used to compare genotypic and allelic frequencies between patients (obese or overweight participants or both of them) and healthy controls. The associations between rs17782313 polymorphism and BMI, as well as the PFS scores and the interaction effect of rs17782313 and overweight/obesity on hedonic hunger, were evaluated using the analysis of Covariance (ANCOVA). To investigate the impact of the rs17782313 on BMI, we also implemented a linear regression analysis. It should be noted that all of the analysis except allelic frequencies was adjusted for the age effect. For interpretation, probability values of 0.05 or less were regarded as statistically significant.
Results
Demographic and anthropometric characteristics of participants
Baseline demographic and anthropometric characteristics of participants are presented in Table 1. Three hundred and ninety-six (70.34%) of the participants were overweight/obese. The mean age and BMI were 35.85±9.18 years and 30.26±6.72 kg/m2, respectively. The mean of participants’ PFS scores was 2.98±0.86. In a succession, 216 (38.40%), 272 (48.30%), 75 (13.30%) of the participants had T/T, T/C, and C/C genotypes. The distribution of the rs17782313 genotype within all study groups, including normal (P = 0.753), overweight (P = 0.629), obese (P = 0.978), and total population (P = 0.878), indicated the Hardy-Weinberg equilibrium.
Demographic and anthropometric characteristics of normal, overweight, and obese participants
Demographic and anthropometric characteristics of normal, overweight, and obese participants
*Standard Deviation.
In order to examine the hypothesis that the MC4R non-coding variant increases the risk of obesity, logistic regression models were conducted to estimate the possibility of being overweight and/or obese, considering the rs17782313 genotypes. First, participants were divided into two groups of control (BMI < 25) and patient (BMI≥25). There was no significant difference in the genotypic groups even after applying the age effect (P = 0.058). Alleles were also analyzed within two groups, and the C allele showed a significant difference (P = 0.041) (Table 2).
Distribution of genotype and allele frequencies in patients (obese and/or overweight) and controls
Distribution of genotype and allele frequencies in patients (obese and/or overweight) and controls
*OR: Odds Ratio. **AOR: Adjusted Odds Ratio, consider the effect of age. ***Based on logistic regression.
Three different analyses were also performed on genotype and allele frequencies in subgroups, including controls and overweights (25≤BMI < 30), controls and obese individuals (BMI≥30), and overweight and obese individuals. Concerning the control/overweight and overweight/obese groups, any significant differences were not observed for genotypes and alleles even after adjusting for age (Table 2). However, significant differences were observed in the comparison of alleles in control and obese groups (P = 0.020), which was also a valid condition for genotypes with and without age effect (P = 0.044, P = 0.030) (Table 2).
In a further analysis, genotypic and allelic frequencies were compared simultaneously in normal, overweight, and obesity study groups considering the age effect. Although the model showed a significant difference in the distribution of genotypes between the three groups (P = 0.037), the genotypic frequencies in the overweight group did not show a significant difference from the normal group. However, the frequency of C/C genotype among the obese group was higher than in controls (P = 0.005) (Table 3). The difference in allelic distribution among groups was on the threshold of significance (P = 0.052), and the frequency of the C allele in the obese group was significantly higher than the normal group (P = 0.020). In contrast, there was no significant difference with the allelic frequency of overweight individuals (P = 0.562) (Table 3).
Comparison of genotypic and allelic frequencies in control, overweight, and obese participants
*OR: Adjusted Odds Ratio, consider the effect of age. **OR: Odds Ratio. ***Based on logistic regression.
For a quantitative study, BMI means were compared in genotypic and allelic groups based on the additive, dominant, and recessive models. ANCOVA results demonstrated significant differences among all three models (Table 4). A linear regression analysis between rs17782313 and BMI adjusted for age was also performed, and the association remained the same for dominant (P = 0.029) and recessive (P = 0.020) models. However, the results showed that the association between rs17782313 and BMI in the additive model is increased significantly (P = 0.006) (Table 4).
Relationship between rs17782313 and BMI using additive, dominant, and recessive models
Relationship between rs17782313 and BMI using additive, dominant, and recessive models
*Based on ANCOVA adjusted for age. **Based on linear regression adjusted for age.
In terms of hedonic hunger, the PFS scores’ means were also compared among carrier groups as an additive, dominant, and recessive models to assess the possible association with rs17782313. ANCOVA showed no significant differences in the mean PFS scores among the three genotypic groups. Similarly, dominant and recessive models were not shown any significant differences in PFS scores (Table 5). The interaction effect of rs17782313 with overweight/obesity was also examined, and any significant differences in PFS scores were not observed in additive, dominant, and recessive models (Table 5).
PFS Score comparison by allele carrier status and interaction results of rs17782313 and overweight/obesity on hedonic hunger
PFS Score comparison by allele carrier status and interaction results of rs17782313 and overweight/obesity on hedonic hunger
*Based on ANCOVA adjusted for age and BMI. **Based on ANCOVA adjusted for age, the interaction effect of rs17782313 and overweight on hedonic hunger. ***Based on ANCOVA adjusted for age, the interaction effect of rs17782313 and obesity on hedonic hunger.
Furthermore, analysis of sequencing data (Figure 1) in a number of heterozygous and homozygous samples for rs17782313 polymorphism demonstrated a novel frameshift variant with deletion of TCT nucleotides (rs534212081) in the 166 upstream of rs17782313 (Figure 2).
Sequencing result of a sample with T/C genotype indicating the TCT frameshift polymorphism (rs534212081).
BLAST result of a sample with C/C genotype indicating the TCT frameshift polymorphism (rs534212081) in 166bp upstream of rs17782313.
In the current study, the association of rs17782313 T > C polymorphism in 188 bp downstream of the MC4R gene with overweight/obesity, BMI, and hedonic hunger was evaluated in 563 women from the Iranian Azeri Turkish ethnic group. This is the first study of rs17782313 association with obesity in the Iranian Azeri Turkish population to the best of our knowledge. Also, limited studies all around the world were evaluated the association of rs17782313 with hedonic hunger. The overall results showed significant relationships between rs17782313 polymorphism and obesity (P = 0.005) as well as the higher levels of BMI (P = 0.006). However, there was no significant difference in the genotypic frequencies between the control and patient groups (obese and overweight), which caught the significance threshold after adjusting the age effect (P = 0.058). In terms of alleles, minor allele (C) had significant differences with both obesity (P = 0.020) and obesity/overweight conditions (P = 0.041). By contrast, any significant associations were not observed in the comparison of both genotype and allele frequencies in overweight/control groups as well as in overweight/obese individuals. Regarding hedonic hunger, there were no significant differences in PFS scores among genotype groups (P = 0.666) and dominant (P = 0.809) and recessive (P = 0.368) carrier status. Also, the interaction analysis of rs17782313 and overweight/obesity did not show any significant differences in PFS scores in the study models.
Obesity is a condition with an increasing prevalence worldwide and becoming a threat to global health by promoting the risk of cardiovascular diseases, type 2 diabetes, osteoarthritis, depression, and certain types of cancer [29–31]. Like many other medical conditions, obesity has also resulted from an interaction between genetic and environmental factors, among which genetic factors are particularly important [32]. Studies of severe and common forms of obesity have suggested that the MC4R gene is an essential genetic regulator of energy homeostasis and fat accumulation in the body [33]. The MC4R protein belongs to a family of transmembrane receptors coupled to the G protein (GPCR), widely expressed in the hypothalamus [34]. Huszar et al. in 1997 showed that mice that do not have both of mc4r alleles (mc4r –/–mice) develop a maturity-onset hyperphagic obesity syndrome by ten weeks of age, while the heterozygous mice for the deletion of mc4r (mc4r +/– mice) indicate a moderate obesity phenotype [35]. In 2008, genome-wide association data from 16,876 individuals of European descent were analyzed to identify common variables affecting the body mass index (BMI), and after the variants in FTO, the strongest association signal was for rs17782313 in 188 kb downstream of MC4R. In the same year, a study on 5724 women in the United States indicated the association of rs17782313 polymorphism with food intake, weight change, and diabetes [23]. In another study, thirteen candidate genetic loci for obesity were evaluated in China, and rs17782313 was recognized as one of the most influential factors [36]. Many other studies have been conducted in different populations of Asia, Europe, America, and Africa, all of which showed the role of rs17782313 in the incidence of obesity and increased BMI.
Our findings are broadly consistent with several studies reported from all around the world. Studies of Sull et al. in the adult population of Korea, Srivastava et al. in the Northern India population, and Cyrus et al. in the Saudi Arabians illustrated associations between rs17782313 and obesity and/or the higher means of BMI [37–39]. In addition, reports from Iranian Persian (Mozafarizadeh et al.), Turkish (Kirac et al.), and Russian Tatar (Kochetova et al.) populations, which are ethnically and geographically similar to the Azeri population, indicated statistically significant associations of rs17782313 with obesity and BMI [40–42].
Genetics are not solely correlated to BMI phenotypic variation in individuals but also impact human feeding actions [43]. High levels of the MC4R expression in dopamine-rich regions and the hypothalamic regions indicate non-homeostatic (hedonic) effects along with hemostatic pathways [44, 45]. Complementary studies of Valladares et al., Ho-Urriola et al., Vega et al., and Obregón et al. in the Chilean population have demonstrated the association of rs17782313 polymorphism with different feeding behaviors such as food enjoyment, food responsiveness, satiety responsiveness, and uncontrolled eating [46–49]. Moreover, rs17782313 was significantly associated with emotional eating in European ancestry (Yilmaz et al.) and binge eating in the Brazilian population (Magno et al.) [45, 50]. In this study, to investigate a possible mechanism of rs17782313 in obesity, hedonic hunger as an influential factor has been studied. Our observations were in harmony with the results of similar studies of Yilmaz et al. and Obregón et al., and no significant associations were observed among PFS scores and genotypic and allele carrier groups [45, 49].
Several strengths can be considered for this study. We assessed the association of rs17782313 polymorphism with obesity/overweight and BMI in the Turkish Azeri ethnic group for the first time. In addition, there are very few studies globally investigating the relationship of the rs17782313 polymorphism with hedonic hunger. We also detected a novel upstream polymorphism in all sequenced C allele carriers, indicating a possible linkage between this polymorphism and the rs17782313. Undoubtedly, there are also some limitations to this study. Due to the lack of information on hedonic hunger among Iranian Azeri men, our current study is restricted only to female participants. Thus, more comprehensive studies, including men and women from the Iranian Azeri ethnic group, are recommended to determine whether this association has a real gender difference. Besides, the greater sizes of population studies can bring more profound results to help in expanding results.
In conclusion, our observations confirm the association of the rs17782313 polymorphism mapped in 188kb downstream of the MC4R gene with obesity and BMI among women in Northwest Iran. However, no association was found between hedonic hunger and rs17782313. Findings from the sequencing data of several samples with one and/or two C alleles also demonstrated the existence of a novel variant (rs534212081) in 166bp upstream of the rs17782313. Further studies will clarify the relationship of this variant with rs17782313 and its possible role in obesity. Finally, this study may contain important information about people’s genetic susceptibility to obesity and is considered a prognostic tool to recognize people with higher obesity risks, which can help manage personalized lifestyles.
Footnotes
Acknowledgments
We gratefully thank all participants included in the study and their families. This project was financially supported by the Center of Excellence for Biodiversity (University of Tabriz). The results of this paper are from Mahan Narjabadifam’s M.Sc. thesis.
Funding
The authors report no funding.
Conflicts of interest
The authors have no conflict of interest to report.