- Open Access
The association between variant rs9939609 in the FTO gene with free leptin index and the risk of obesity in the Indonesian children population
Egyptian Journal of Medical Human Genetics volume 23, Article number: 108 (2022)
Several studies have reported that fat mass and obesity-associated gene (FTO), especially the rs9939609 polymorphism, are associated with obesity and high leptin levels. The free leptin index (FLI) is known to be accurate in determining the function of leptin. The aim of this study was to determine the association between the rs9939609 variant in the FTO gene and FLI and the risk of obesity among children living in Medan, Indonesia.
This case–control study included 212 children aged 6–12 years who were genotyped for variants of the rs9939609 FTO gene. The case group consisted of children with obesity who were < 6 years old, and the control group had a normal body mass index and came from the Bataknese or Chinese ethnicities. Anthropometric measurements were performed. Serum leptin and soluble leptin receptor (sOB-R) levels were measured. Odds ratio and 95% confidence intervals were calculated to determine the risk of having obesity associated with the risk allele.
In this study, there was no significant association between leptin, soluble leptin receptor, or free leptin index and the rs9939609 FTO gene; however, the risk allele (A) of FTO rs9939609 had a significant association with obesity in Chinese ethnicity subjects (p < 0.05). The AA/AT genotype had an increased risk of FLI 5.889 times compared to the TT genotype. Multivariate analysis showed that the rs9939609 polymorphism in the FTO gene played a role in obesity through the FLI.
It was concluded that the rs9939609 polymorphism played a significant role in obesity through FLI. Further studies are still needed.
Childhood obesity has been alarmingly growing, and it is the most common nutritional problem among children in developed and developing countries. It is related to substantial morbidity and mortality, which includes cardiovascular, respiratory, gastrointestinal, endocrine, and psychosocial morbidities.  Along with the consistent increase in the prevalence, the morbidity and mortality resulting from the condition should be considered. Among the many factors that contribute to the increasing prevalence of childhood obesity are environmental and genetic factors. 
Some genes, such as the fat-mass and obesity-associated gene (FTO), are strongly related to obesity and overweight. FTO acts as a gene that has been recently studied and identified to be associated with obesity. Some of these gene polymorphisms are associated with obesity and metabolic disorders. [3, 4] The FTO gene role to date has not been fully understood, nor do the variants of this gene act as the most critical genetic determinants for body weight and obesity.  The FTO gene was found to be a highly polymorphic gene with more than 1500 single-nucleotide polymorphisms (SNPs). rs9939609 is known to be the most frequent polymorphism associated with the obesity phenotype. 
Leptin regulates energy balance and food intake through different pathways in the central and nervous systems.  Soluble leptin receptor (SOB-R) levels give an idea of free leptin levels and free leptin index (FLI), which is the ratio between leptin and sOB-R. This free leptin index is known to be accurate in determining the function of leptin. 
This study aimed to determine the association between the rs9939609 polymorphism of the FTO gene and the free leptin index and the risks of obesity in children. This study conducted the first FTO research focusing on children with obesity in Indonesia.
This is an observational analytic study using a case–control design. The minimum sample size for this study was estimated using the formula of two group proportion, with an α at 0.05, power of 80%, p1 0.42, and p2 0.58. The final minimum sample size was 80 cases for each group. The case subjects of this study need to meet the inclusion criteria, including children aged 6–12 years old with body mass index (BMI) > 95th percentile according to the Center for Disease (CDC) 2000 BMI curve, who were obese since < 6 years old and came from Bataknese or Chinese ethnicities. Meanwhile, the control subjects’ inclusion criteria consisted of children with BMI 3–85th percentile according to the CDC 2000 BMI curve and came from a similar population to the case subjects. The research was conducted at ten elementary schools in Medan with dominant ethnicities of Bataknese and Chinese.
During the study period, parents or caregivers completed a structured questionnaire to obtain information about age at onset of obesity, family history of obesity, and sedentary lifestyle. All participants provided informed consent before the data were collected.
Measurements of body weight, height, and waist circumference were also performed. Weighing scales used the SECA brand scale (sensitivity 0.1 kg) to measure the child's weight (kg); height was measured by the SECA brand standing height meter (sensitivity 0.1 cm), while an elastic measuring tape (cm) was used to measure waist circumference. The CDC 2000 BMI curve, ages 2–20 years, was used to determine the child's nutritional status. Waist circumference was measured in a standing position by attaching an anthropometric tape to the waist, passing through a point on the side of the body, between the 12th costae and the upper end of pelvis, and one centimeter below the navel.
Blood samples were collected by Prodia laboratory personnel under the provision of researchers. Leptin and sOB-R levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit based on the manufacturer’s instructions and standard guidelines. The free leptin index is the ratio between leptin and sOB-R levels.
For the genetic studies, a whole blood sample was collected from each child and mixed with ethylenediaminetetraacetic acid (EDTA). A TaqMan assay for the rs9939609 polymorphism (Assay ID C_25638153_10; Applied Biosystems, Foster City, CA) was used to perform genotyping. Real-time PCR was performed in 25-µl reactions, which consisted of 12.5 µl of TaqMan Genotyping Master Mix, 1.25 µl of TaqMan genotyping assay mix (20X) that includes the primer and probe, 11.25 µl of DNase-free, RNase-free water, and approximately 20 ng of template deoxyribonucleic acid (DNA) in 5 µl. Thermal cycling was performed on a Biorad CFX96 (Biorad, Munchen, Germany). This method was performed at a temperature of 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 90 s. CFX96TM Real-Time PCR software (Bio-Rad, Munich, Germany) was used to conduct the allelic discrimination method for SNP genotyping. Three selected genotypes, AA, AT, and TT, were regenotyped using oligonucleotides 5’-AAGAGATGATCTCAAATCTACTTTATFAFFATA-3’ (forward) and 5’-AGGATAGTTTCGATCTATTGACCTC-3’ (reverse) (IDT DNA, Singapore) through sequencing by Sanger’s dideoxy chain termination reaction using Big-Dye Terminator v.1.1 Cycle Sequencing Kit and the ABI 3500 sequencer (Applied Biosystems, Foster City, USA).
Univariate analysis was performed to explain the characteristics of each studied variable. Normally distributed data were analyzed using the chi-square test and are presented as the mean ± SD. Data are presented in tables and diagrams. The association between the rs9939609 polymorphism in the FTO gene and leptin levels, soluble leptin receptor, and free leptin index was assessed using one-way ANOVA. The odds ratio (OR) between the rs9939609 polymorphism in the FTO gene and the free leptin index was determined using multiple logistic regression tests. Multivariate analysis was conducted to evaluate and confirm the hypotheses and control for confounding variables. The backward LR method of multiple logistic regression analysis was used. Statistical significance in this study was obtained when the p-value was less than 0.05.
A total of 212 children were included in this study, divided into 105 subjects in the case group and 107 subjects in the control group. Table 1 shows that more than 50% of Bataknese subjects in the case and control groups had the TT allele, as did Chinese subjects. The TT allele was found in the majority of male and female subjects in both groups, with 64.1% in the case group and 36.4% in the control group for males, and 63.2% in the case group and 51.2% in the control group for females. In the case group, 66.7% of the TT allele group had a family history of obesity, followed by 31.9% in the AT allele group and 1.4% in the AA allele group. Finding in the control group was also similar, with 53.9% in the TT allele group, 34.6% in AT allele group, and 11.5% in AT allele group. There was a significant difference in age between AA, AT, and TT in the control group, while family history of obesity exhibited significance in the case group with a p-value of < 0.05. Children in the Chinese ethnic group had a significant difference between AA, AT, and TT both in the case group and control group, with a p-value < 0.05.
Table 2 reports no significant association between leptin, soluble leptin receptor, and free leptin index with the rs9939609 FTO gene in all alleles because the p values were 0.559, 0.091 and 0.464, respectively.
We generated a receiver operating characteristic (ROC) curve to determine the value of the leptin, sOB-R, and FLI cutoff points. The results were 8.63, 30.75, and 0.28 for leptin, soluble leptin receptor, and free leptin index, respectively. The odds ratio between genetic variation and leptin, sOB-R, and FLI in the ethnic groups is shown in Table 3. Subjects in the control group who had the AT/AA genotype had a 3.69 times risk of rising leptin, a 2.29 times increased risk of having high sOB-R, and a 5.889 times increased risk of having a high FLI. It has been shown that the case group had a lower risk than the control group in all analyses.
Multivariate analysis was performed to determine and control the confounding factors in this study, as shown in Table 4. It is observed that the 15–20 h and > 20 h sedentary lifestyle had a significant effect on the occurrence of obesity with p-value < 0.05 and had a risk of 17.17 and 18.11 times for the occurrence of obesity. This multivariate analysis also showed that the rs9939609 polymorphism in the FTO gene played a significant role in obesity by influencing FLI.
The FTO gene has been identified and associated with obesity by genome-wide association studies (GWAS), and several polymorphisms of this gene were associated with obesity or obesity phenotypes, such as high BMI. Subsequent studies also found that children who had the FTO risk allele had an increase in energy intake. This gene was relatively frequent (it was estimated that 16% of the population had one allele variant). The presence of this gene was associated with an average increase of 1.5 kg of body weight. 
The FTO gene’s most critical genetic polymorphism is rs9939609, and it is known that this polymorphism is associated with BMI and predisposition to obesity in childhood and adulthood.  Children and adults who held at least one risk allele of rs9939609 in the FTO gene (homozygous = AA and heterozygous = AT) had a more significant energy intake than the wild-type allele (TT). 
In this study, we found a significant difference between Chinese ethnicity and the rs9939609 FTO gene. Along with our study, a study in Malaysia also reported a significant association between Chinese ethnicity and the rs9939609 FTO gene in children with obesity.  A study conducted in Singapore found an association between the FTO gene variant and obesity in Chinese and Malay ethnicities living in Singapore.  A study in Beijing reported an association between the rs9939609 FTO gene and BMI and the risk of obesity in children and adolescents.  Another study conducted on Han children aged 7–18 years also showed that the genetic variation rs9939609 was associated with obesity and dietary choices in children and adolescents in China.  We were only able to compare the Chinese ethnicities in this study with Chinese in Malaysia or in China, and we found that the frequency of polymorphism rs9939609 of the FTO gene in our research was almost similar to that in the Chinese ethnic groups in these countries.
We obtained different results in Bataknese patients, and there was no significant difference between the Bataknese and rs9939609 FTO genes. The possible explanation for this different result is that obesity is a condition with multifactorial and heterogeneous causes that can occur due to the interrelated interactions between gene factors and the environment such as diet, physical activity patterns, the interaction between gene factors and genes, and the interaction between genetic factors and the environment. These interactions make it difficult to explain the specific relationship between genetic variants and obesity. In addition, the FTO gene was involved in the pathophysiology of obesity and played a role in controlling food intake and energy output.  The FTO gene is also known to be part of the neuroregulation (leptin) pathway of energy metabolism in adipose tissue, and barriers to leptin signaling can inhibit downstream changes in adipose tissue that stimulate FTO expression; thus, it was thought that there was a relationship between the leptin signaling pathway and FTO 
Our study demonstrated that children in the control group who had the AA/AT genotype had an increased risk of high leptin of 3.69 times and high FLI 5.889 times compared to children with the TT genotype. However, we found no significant association between the FTO gene and leptin, sOB-R or FLI. Several studies have identified an association between the rs9939609 FTO gene polymorphism and leptin and leptin receptors. Studies from Austria and Spain reported a similar result to our study, which found no significant association between the rs9939609 FTO gene and leptin in children and adults with obesity and normal weight. [18, 19] A study in Poland showed an association between waist circumference and higher FTO expression. 
Meanwhile, another study identified an association between the rs9939609 FTO gene and a high level of leptin, and this single-nucleotide polymorphism role was thought to trigger leptin resistance and induce dysregulation of food intake and energy output, resulting in obesity.  A study in Denmark also reported an association between the rs9939609 polymorphism in the FTO gene and an increase in serum leptin levels, which was thought to occur due to an increase in adipocytes.  A study conducted in Pakistan reported that the rs9939609 FTO gene polymorphism was associated with leptin and leptin receptors in adult women (aged > 18 years) but not in children.  A study in Iran showed that the homozygous risk allele (A) for FTO rs0039609 had higher serum leptin (p = 0,005; F: 5,131), and there were no significant differences between the TT and AT genotypes. 
Our multivariate analysis found a significant association between the rs9939609 polymorphism in the FTO gene and obesity through FLI. Unfortunately, we were unable to compare our study results with those of previous studies because no study reported an association between the rs9939609 polymorphism in the FTO gene and the FLI.
One study demonstrated that sedentary behavior of > 22 h a week acted as a vital risk factor for overweight and obesity.  In this study, we also looked for an association between sedentary lifestyle, such as the number of times children spent sitting in activities, excluding time spent in school, and the incidence of obesity in children. This study obtained results that were similar to those in previous studies, i.e., we found that the majority of children with obesity had a sedentary lifestyle of > 20 h per week compared to the control group. Additionally, FLI > 0.28 was significantly associated with the weekly duration of sedentary habits in subjects. It is essential to prevent obesity in children by increasing physical activity and limiting their sedentary activities.
The strength of this study is that there has not been a similar previously conducted study in Indonesia. This study is the first to evaluate the association between the rs9939609 polymorphism in the FTO gene and the free leptin index in children in Indonesia. This study obtained a significant difference between the rs9939609 polymorphism and the occurrence of obesity in the Chinese ethnic group in Medan, Sumatera Utara, Indonesia.
This study has limitations. We only examined one polymorphism of the FTO gene, rs9939609, because based on the literature, rs9939609 was the most frequent polymorphism associated with the obesity phenotype; therefore, in this study, we chose the rs9939609 polymorphism to determine whether it has an association with obesity in children in Medan. From the literature, it is known that several other FTO gene variants have been reported to be associated with obesity, namely, rs1421085, rs8050136, rs17817449, and rs1121980; thus, further research is recommended to determine the role of other FTO gene polymorphisms in the incidence of obesity in children in Indonesia.
Our study provided evidence that the rs9939609 polymorphism played a significant role in obesity indirectly through FLI. Nevertheless, further studies and analyses are needed. The results of this study are expected to improve public health services in preventing obesity and providing education to children and parents to change their lifestyles, especially children who have a risk allele for the tendency to have obesity in the future.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Body mass index
Center for disease control
Ethylene diamine tetraacetic acid
Free leptin index
Fat mass and obesity associated gene
Genome wide association study
Receiver operating characteristic
Single nucleotide polymorphism
Soluble leptin receptor
Sidik SM, Ahmad R (2004) Childhood obesity: contributing factors, consequences and intervention. Malays J Nutr 10:13–22
Freemark M. Childhood obesity. In: Brook’s clinical pediatric endocrinology. 6th Ed. West Sussex: wiley-Blackwell. 2009. p. 528–52
Karasawa S, Daimon M, Sasaki S et al (2010) Association of the common fat mass and obesity associated (FTO) gene polymorphism with obesity in a Japanese population. Endocr J 57:293–301
Larder R, Cheung MK, Tung YC et al (2011) Where to go with FTO? Trends Endocrinol Metab 22:53–59
Hubáček JA, Pikhart H, Peasey A et al (2011) FTO variant, energy intake, physical activity and basal metabolic rate in Caucasians. The HAPIEE study Physiol Res 60:175–183
Jacobson JA, Klovins J, Kapa I, Danielsson P, Svensson V, Ridderstråle M, Gyllensten U, Marcus C, Fredriksson R, Schiöth HB (2008) Novel genetic variant in FTO influences insulin levels and insulin resistance in severely obese children and adolescents. Int J Obes 32:1730–1735
Huang KC, Lin RC, Kormas N, Lee LT, Chen CY, Gill TP, Caterson ID (2004) Plasma leptin is associated with insulin resistance independent of age, body mass index, fat mass, lipids, and pubertal development in non diabetic adolescents. Int J Obes Relat Metab Disord 28:470–475
Misra M, Millier KK, Almazan C, Ramaswamy K, Aggarwal A, Herzog DB, Neubauer BJ, Klibanski A (2004) Hormonal and body composition predictors of soluble leptin receptor, leptin, and free leptin index in adolescent girls with anorexia nervosa and controls and relation to insulin sensitivity. J Clin Endocrinol Metab. 89:3486–95
Wilding JPH (2010) Pathophysiology and aetiology of obesity. Medicine 39:6–10
Gu HF, Alvarsson A, Brismar K (2010) The common FTO Genetic polymorphism rs9939609 is associated with increased bmi in type 1 diabetes but not with diabetic nephropathy. Biomark Insights 5:29–32
Tanofsky KM, Han JC, Anandalingam K et al (2009) The FTO gene rs9939609 obesity-risk allele and loss of control over eating. Am J Clin Nutr 90:1483–1488
Chey WW, Fan SH, Say YH (2013) Association of fat mass and obesity associated (FTO) gene rs9939609 variant with obesity among multi-ethnic Malaysians in Kampar. Perak Sains Malaysiana 42:365–371
Tan JT, Dorajoo R, Seielstad M et al (2008) FTO variants are associated with obesity in the Chinese and Malay populations in Singapore. Diabetes 57:2851–2857
Xi B, Shen Y, Zhang M et al (2010) The common rs9939609 variant of the fat mass and obesity-associated gene is associated with obesity risk in children and adolescents of Beijing. China BMC Med Genet 11:107
Yang M, Xu Y, Liang L et al (2014) The effects of genetic variation in FTO rs9939609 on obesity and dietary preferences in Chinese Han children and adolescents. PLoS ONE 9:1–9
Wang L, Yu Q, Xiong Y et al (2013) Variant rs1421085 in the FTO gene contribute childhood obesity in Chinese children aged 3–6 years. Obes Res Clin Pract 7:14–22
Qi L, Kang K, Zhang C et al (2008) Fat mass-and obesity-associated (FTO) gene variant is associated with obesity: longitudinal analyses in two cohort studies and functional test. Diabetes 57:3145–3151
Mangge H, Renner W, Almer G, Weghuber D, Möller R, Horejsi R (2011) Rs9939609 variant of the fat mass and obesity-associated gene and trunk obesity in adolescents. J Obes 36:186368
Olza J, Ruperez AI, Gil-Campos M, Leis R, Fernandez-Orth D, Tojo R, Cañete R, Gil A, Aguilera CM (2013) Influence of FTO variants on obesity, inflammation, and cardiovascular disease risk biomarkers in Spanish children: a case-control multicentre study. BMC Med Genet 14:1–11
Czogala W, Czogala M, Strojny W, Ator GW, Wolkow P, Wojcik M, Multanowski MB, Tomasik P, Edrychowicz AW et al (2021) Methylation and expression of FTO and plag1 genes in childhood obesity: insight into anthropometric parameters and glucose-lipid metabolism. Nutrients 13:1683
Labayen I, Ruiz JR, Ortega FB, Dallongeville J, Jiménez-Pavon D, Castillo MJ, Henauw S, González-Gross M, Bueno G, Molnar D, Kafatos A, Diaz LE, Meirhaeghe A, Moreno LA (2011) Association between the FTO rs9939609 polymorphism and leptin in European adolescents: a possible link with energy balance control. HELENA Study Int J Obes 35:66–71
Andreasen CH, Stender-Petersen KL, Mogensen MS et al (2008) Low physical activity accentuates the effect of the FTO rs9939609 polymorphism on body fat accumulation. Diabetes 57:95–101
Shaheed A, Rana S, Saeed S et al (2013) Common variant of FTO gene, rs9939609, and obesity in Pakistani females. Biomed Res Int 324093:1–7
Mehrdada M, Doaei S, Gholamaliza M, Fardaeid M, Fararouei M, Eftekharif MH (2020) Association of FTO rs9939609 polymorphism with serum leptin, insulin, adiponectin and lipid profile in overweight adults. Adipocyte 9:51–59
Thibault H, Contrand B, Saubusse E, Baine M, Maurice-Tison S (2009) Risk factors for overweight and obesity in French adolescents: Physical activity, sedentary behavior and parental characteristics. Nutrients 26:192–200
We are thankful to Prof. Harun Alrasyid Damanik for his assistance throughout our study .Nadhira Anindita Ralena, M.D. and Megan Quinka Dwidara Toding, M.D. in helping us in drafting and submitting our manuscript. We are also grateful to the children who participated in our study and all field workers, data, and laboratory personnel.
All funding for this research was conducted from personal expenses and no grant was received.
Ethics approval and consent to participate
This study was approved by The Ethics Committee of The Medical School, Universitas Sumatera Utara, Medan, Indonesia, and informed consent was received from participants and legal guardians at the time of participation.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Lubis, S.M., Fattah, M. & Batubara, J.R.L. The association between variant rs9939609 in the FTO gene with free leptin index and the risk of obesity in the Indonesian children population. Egypt J Med Hum Genet 23, 108 (2022). https://doi.org/10.1186/s43042-022-00321-w