Galectin 3 rs4644 gene polymorphism is associated with metabolic traits in Serbian adolescent population

Background Among many genes which have been analyzed to understand obesity and related metabolic traits among children and adolescents, not many studies are conducted on LGALS3 gene, especially in population of children. A positive correlation of circulating galectin 3 serum levels with impaired blood glucose, high blood pressure and higher values of serum lipids and was found in general population. The aim was to investigate possible association of rs4644 with body mass index, glycaemia, and lipid profile in Serbian adolescents. Methods The study included 72 boys and 79 girls, 14-15 years of age. Among boys 51 (67.1%) had normal values of BMI, 11 (14.5%) were overweight, and 14 (18.4%) were obese. Among girls, 53 (63.9%) had normal BMI, 16 (19.3%) were overweight, and 14 (16.9%) were obese. Diabetes type 1 or 2, genetic syndromes, generalized inflammation, cardiovascular and malignant diseases were criteria for exclusion. Genotyping was performed by Real time PCR.


Introduction
Children and adolescent obesity has become a worldwide problem, with high incidence not only in the western world, but also in developing countries.Problem of obesity is not only excessed body weight, but also risk factors for developing a metabolic syndrome (MetS) (1).Metabolic syndrome includes several factors such as glucose intolerance hypertension, insulin resistance, dyslipidemia, and diabetes mellitus type 2 (2).Since for obesity is characterized hypertrophy of white adipose tissue, macrophage together with adipocyte are secreting pro-inflammatory cytokines and chemokines, thus exceeded body weight might also have impact on inflammatory status, which is in correlation with decreased glucose tolerance and insulin sensitivity (3).Among many genes which have been analyzed in order to understand obesity and related metabolic traits among children and adolescent, not many studies are conducted on LGALS3 gene, particularly in population of children.However, Galectin 3 (Gal-3) is a protein encoded by LGALS3 gene located on the chromosome 14 (4).This protein belongs to the family of b-galactosidebinding lectins which counts fourteen more members.Gal-3 is involved in many metabolic processes including basic physiological processes such as cell proliferation, adhesion and programed cell death.Also, in humans, Gal-3 is expressed in various tissues such as epithelial cells, mast cells, immune cells including monocytes, macrophages, dendritic cell, etc (5).Previous researchers have found a positive correlation of circulating galectin 3 serum levels in general population with higher risk of cardiovascular, renal diseases, high blood pressure as well as with higher values of serum lipids and obesity (6).Also, galactines promote preadipocytes maturation into the lipidloaded adipocyte, which might contribute to central obesity (7).Moreover, deletion of LGALS3 gene in mice have shown defectiveness in adipose tissue maturation, which was accompanied by increased body inflammation, higher glucose level and impaired insulin sensitivity (8).Within the LGALS3 gene, common single nucleotide polymorphisms (SNP) have been described as rs4644 C > A, which leads to the substitution of histidine to proline at residue 64 (9).This SNP have been linked with higher serum levels of galactin 3 protein (9,10).The aim of this research was to investigate possible association of rs4644 with BMI (body mass index), glycaemia, and lipid profile in Serbian adolescents.

Research design
Participants enrolled in the study were children 14 to 15 years of age, of both genders who were randomly selected from Yugoslav Study of the Precursors of Atherosclerosis in School Children (YUSAD).Among selected children (n=151), 72 were boys and 79 were girls.Anthropometric data including age, gender, height, weight as well as biochemical parameters such as fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) values were recorded during annual pediatric examinations at 11 primary health care centers from all parts of Serbia.Children's BMI was classified in three different groups, according to the previously published charts for Serbian adolescents (normal weight, overweight above 85 th percentile, and obese above 95 th percentile (11).Ethics Committee of Faculty of Medicine, University of Belgrade, Serbia approved study design and study protocol, (approval number 29/X-9 from 12.10.2017.).For all children included in the study, parents or guardians signed informed consent form.Selected criteria for exclusion from the study were cardiovascular, malignant, genetic diseases as well as general inflammation, both types of diabetes mellitus, cerebral palsy and chronic immobility.

Biochemical and molecular-genetic analysis
Prior to the blood sample collection, all participant fasted for 12 hours.Fasting blood glucose, TC, TG, HDL-C values were measured as described previously, and LDL-C values were calculated by Friedwald's equation (11).Molecular-genetic analysis were performed at the Institute of Human Genetics, Faculty of Medicine, University of Belgrade, Serbia.Prior to genotyping, the total genomic DNA was iso-the girls carriers of CA+AA genotypes, p=0.041, and p=0.045, respectively.The higher frequency of obese was found among group of girls who were carriers of CC genotype, p=0.049.No statistically significant association was observed among other analyzed parameters in neither examined groups.Results: Our research indicates that there is an association between the CC genotype of rs4644 polymorphism with obesity and higher triglycerides level in the group of female adolescents.

Statistical analysis
Quantitative variables were expressed as mean ± standard deviation.Depending on the variable dis-tribution, Student's t-test or the Mann-Whitney test was used to assess the association of BMI, fasting blood glucose and lipid parameters with selected genotype.
For further analysis we grouped CA and AA genotypes.In line with that, statistically significant results were obtained among group of girls carriers CC genotype, where they had higher BMI in comparison to the girls carriers of CA+AA genotypes (p=0.041) .Also, in the same analyzed group, girls carriers of CC genotype had statistically higher mean values of TG compared to the girls carriers of CA+AA genotypes (p=0.045).No other statistically significance was obtained among other analyzed parameters in neither groups, regarding rs4644 genotype, which is presented in Table III.

Discussion
In the recent years many studies are conducted on human and animal models, which have found the positive correlation of higher circulating levels of Gal-3 protein and impaired glucose metabolism, insulin sensitivity, obesity, hypercholesterolemia, and inflammation factors (13).The level of its expression varies through the lifespan, with the highest levels of circulating Gal-3 during embryogenesis and development.Higher serum values of circulating protein were found in obese and diabetic patients in comparison to the nondiabetic patient (14)(15)(16)(17)(18). Also, animal models are suggesting that the elevated levels of galectin-3 found in the altered metabolic processes such as lipid metabolism, obesity, diabetes and metabolic syndrome might be acting at the level of pancreatic islets and adipose tissues (4,13).The study of Liao et al. (19) conducted in a group of patient with coronary artery disease have found the association of rs4644 CC genotype with higher galectin-3 levels, followed by CA and AA genotype.The results of our research regarding BMI and its associations with rs4644 genotype have showed a statistical significance among girls carriers of CC genotype with higher BMI in comparison to the girls who were carriers of CA+AA genotype (p=0.041).The higher frequency of obese was found among group of girls who were carriers of CC genotype (p=0.049).No association was found among the group of boys regarding BMI and rs4644 genotype.Moreover, girl carriers of CC genotype also had statistically higher mean values of TG in comparison to the girls carriers of CA or AA genotype (p=0.045).No association was found among group of boys in neither analyzed parameters.Since, studies have found two common SNPs (rs4644 and rs4652) are affecting the protein properties of the LGALS3 gene product, leading to the higher expression of Gal-3 protein (5-7), research of Florido et al. (20) have shown that higher BMI was in direct correlation with elevated galectin-3 levels.Also, patients with severe obesity and elevated galectin-3 had >4-fold higher risk of developing heart failure.One of the studies conducted by Zhen et al (7), reported elevated expression of Galectin-3BP and positive correlation with obesity and MetS in 932 Chinese adolescents.On the other side Besma et al. (21) obtained no statistically significant results regarding BMI among patients with type 2 diabetes mellitus regarding rs4644 genotype.One of the possible explanation of these results are differences in gonadal hormonal status.Namely, in the puberty increased secretion of progesterone, estrogen and androgen hormones in female compared to adolescent boys, which might lead to increased BMI and visceral adiposity (22).Also, in the period of adolescence, boys tend to have more physical activities than girls, which might be one of the reason for difference in BMI (23).On the other side impact of increased secretion of testosterone in adolescent boys, leads to the higher lipase activity in the liver, which causes lowering of the lipid parameters as well as lower BMI (24).However, many of these studies measured the circulating level of protein in particular populations, but association of rs4644 genotype was not addressed.Also, available data indicated that these studies were conducted in the population of adult patients, but not many data are available in the population of children and adolescents.However, the importance of association studies in children and adolescents refereeing to rs4644 polymorphism is reflecting on the comorbidities accompanying elevated BMI and lipid parameters primarily with cardiovascular and metabolic diseases since many studies showed association of this polymorphic variant with insulin resistance, diabetes mellitus type 2 and cardiovascular events (9,25,26).Thus, the importance of monitoring these biochemical parameters in adolescents might contribute to the lowering incidence of metabolic syndrome and other diseases associated with obesity-related parameters.

Conclusion
In this research we have found a statistically significant correlation between CC genotype of rs4644 polymorphism with higher BMI and mean values of triglyceride levels.However, one of the limitations of the study was relatively small sample size, and lack of non-genetic information such as eating habits, or level of physical activity.For further analysis and better understanding of children and adolescent obesity and other metabolic parameters, non-genetic factors and other polymeric variants among LGLAS3 gene, as well as other genetic variants should be taken into consideration.

Table I
Mean values and range of analyzed parameters depending on gender.