ADRB3 polymorphism rs4994 (Trp64Arg) associates significantly with bodyweight elevation and dyslipidaemias in Saudis but not rs1801253 (Arg389Gly) polymorphism in ARDB1

Background In some populations, obesity and body weight related disorders show a correlation with polymorphisms in three subtypes of beta-adrenoceptor (β1, β2, and β3) [ADRB1, ADRB2 and ADRB3] genes. We scanned for the polymorphism of Arg389Gly (rs1801253) in ADRB1 and Trp64Arg (rs4994) in ADRB3 genes in Saudi population to determine association, if any, of these polymorphisms with obesity and related disorders. Methods We studied 329 non-related adults (33.1% men and 66.9% women), aged 18–36 years. Anthropometric measurements were recorded, and Body mass index (BMI) and waist/hip ratio were calculated; leptin, insulin, lipidogram, and glucose concentrations were determined. ADRB1 and ADRB3 polymorphisms (Arg389Gly and Trp64Arg, respectively) were screened by DNA sequencing. The subjects were divided into three groups according to BMI: normal weight (BMI < 25 kg/m2), overweight (BMI ≥25.1–29.9 kg/m2) subjects, and obese (≥30 kg/m2). Results In the age-matched groups of the normal weight, overweight and obese male and female subjects, all anthropometric parameters were found to be significantly higher, and in the obese group, all biochemical parameters were significantly elevated compared to the normal weight controls. The allelic frequency of Gly389 ADRB1 did not differ amongst the three groups, whereas the frequency of Arg64 of ADRB3 gene was significantly higher in the overweight and obese subjects, compared with the normal weight subjects. In addition, subjects carrying Arg64 allele regardless of their BMI had a greater waist and hip circumference, W/H ratio, plasma cholesterol, triglyceride, LDL, leptin, insulin, and glucose level compared to those with the wild-type Trp allele. Conclusion The results of this study have shown a significant association between the Trp64Arg polymorphism in ADRB3 gene and the development of overweight and obesity in Saudi populations. It also has an influence on the levels of lipid, insulin, leptin, and glucose, whereas, Arg389Gly polymorphism in ADRB1 is not associated with overweight, obesity or dyslipidaemias in Saudis.


Background
Obesity and diabetes mellitus Type 2 (T2DM) are rapidly growing public health problems in the Saudi population [1,2]. A high risk of T2DM and cardiovascular complication is also associated with increased obesity [2,3]. Several epidemiological and clinical studies have shown that human obesity is a multifactorial disorder, with both genetic predisposition and environmental factors contributing to its etiology [4,5]. Extensive studies have linked different genetic loci to obesity, and strong linkage has between reported between beta (β) adrenoceptor polymorphisms (ADRB) and obesity or weight gain [6][7][8][9][10]. There are three subtypes of ARDB: β-1, β-2, and β-3, and these are involved in development, behavior, smooth muscle tone, heart function, and energy metabolism [11]. The three β -subtypes (β-1, β-2, and β-3) coexist in both white adipose tissue (WAT) and brown adipose tissue (BAT). In WAT, ADRB signaling is thought to stimulate lipolysis in response to fasting, whereas in BAT, it mediates heat production in response to cold exposure or overfeeding via activation of the uncoupling protein-1(UCP1) [12][13][14]. Catecholamines are regulators of lipolysis, and act via β1-, β2-, β3-(stimulatory), and α2-(inhibitory) adrenoceptor subtypes in adipose tissue. Most of the sympathetic nervous system-mediated energy expenditure in skeletal muscles takes place via the coupling of catecholamines with β2adrenoceptors [15,16] and plays important roles in energy expenditure and control of body weight [9][10][11][12][13][14][15][16][17]. Recently, it has been shown that ARDBs are polymorphic with single nucleotide polymorphism (SNPs) exerting functional consequences affecting receptor activity and regulation, and hence may contribute to the pathophysiology of obesity and related disorders [7][8][9]. Several studies have shown an association between ADRB polymorphism and BMI, T2DM, hypertension [HT] and dyslipidaemias, while other studies have failed to show such an association .
In our previous report, we have shown a link between obesity-related disorders in Saudi population and Gln27Glu polymorphism in ARDB2 gene [9]. In the present study, we have investigated the relationship between polymorphism in ARDB1 (rs1801253) and ARDB3 (rs4994) genes and obesity and related disorders in the Saudi population.

Study group
The study was approved by the local ethics committee at the Umm Al Qura University, Makkah Al Mukaramah, Saudi Arabia (IRB No. 235). It included randomly chosen 329 unrelated Saudi subjects [men = 109 (33.1%) and women = 220 (66.9%)], from a nationwide population, with ages ranging from 18 to 36 years. Informed written consent was obtained from all study subjects before their participation.

Anthropometric and biochemical measurements
Anthropometric measurements included the recording of height, weight, waist and hip circumference by standard methods. Body mass index (BMI; kg/m 2 ) and waist-to-hip ratio (WHR) were calculated [54]. For biochemical studies, 20 ml of blood was drawn by venipuncture from an antecubital vein without compression, following an overnight fast, from each subject under study. 10 ml blood was placed in ethylenediaminetetraacetic acid (EDTA) coated tubes, 2 ml in tubes containing fluoride and the rest in plain tubes. All samples were immediately centrifuged at room temperature to collect the plasma/serum. Plasma glucose was determined in duplicate by a glucoseoxidase method adapted to an autoanalyzer (Hitachi 704, Boehringer Mannheim, Germany). Enzymatic methods using commercial kits (Boehringer Mannheim) were used to estimate total serum cholesterol, triglycerides, low-density lipoprotein [LDL] and high-density lipoprotein [HDL]. Plasma insulin and leptin concentrations were estimated by radioimmunoassay (RIA) using Human Insulin Specific RIA kit and Human Leptin RIA Kit, respectively (Linco Research, St Louis, MO).

Genotyping of ADRB1 polymorphism (rs1801253)
The genomic DNAs of all the subjects were extracted from peripheral blood leukocytes using Gentra Systems Kit (Minneapolis, MN, cat # D5500). The DNA fragment containing codon 389 of the ARDB1 gene was amplified by polymerase chain reaction (PCR) using a sense primer (5'-CTCTTCGTCTTCTTCAACTGGCT-3′) and an antisense primer (5'-CAACAAGGAACATCAGCAAGC-3′). The PCR conditions consisted of an initial denaturation step at 95°C for 15 min, followed by 34 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final extension of 10 min at 72°C. With these primers, a PCR product was verified on 2% agarose gel electrophoresis. Nucleotide Genotyping of ADRB3 polymorphism (rs4994) The DNA fragment containing codon 64 of the ADRB3 gene was amplified by PCR using a sense primer (5′-CCAGTGGGCTGCCAGGGG-3′) and an antisense primer (5′-GCCAGTGGCGCCCAACGG -3′). The PCR conditions were also the same as mentioned above for ADRB1 studies. Nucleotide sequencing was carried out by the ABI Big Dye Terminator protocol using ABI 3100 Avant Genetic Analyzer.

Statistical analysis
Stat View program for Windows (version 8.0; SAS) was used to conduct all data analyses. Based on the value of BMI, the total population was grouped as normal weight (BMI < 25 kg/m 2 ), overweight (BMI ≥25.1-29.9 kg/m 2 ), and obese (≥30 kg/m 2 ). The data obtained was analyzed separately for each group and is presented as mean ± SEM. The comparisons of anthropometric parameters and biochemical and hormonal variables between overweight, obese and normal weight control subjects were carried out using the independent student's t-test and ANOVA.
The data were further grouped according to the three genotypes of ARDB1 and ARDB3 genes, and the anthropometric measurements, age, BMI, fasting serum insulin, leptin, glucose level and lipid profile, were compared by the Mann-Whitney U-test. To study the influence of the ARDB1 and ARDB3 genotypes on BMI, multivariable logistic regression was used. The different parameters were correlated using Pearson's correlation coefficient (r), and the p-value was obtained. Frequency distribution analysis was performed using the chi-square test, and frequencies of the different alleles in different groups (normal, overweight and obese) were compared. Relative risk was estimated by the odds ratios (ORs) and their 95% confidence intervals (CIs), and a p-value ≤0.05 was considered statistically significant.

Obesity-related anthropometric and biochemical characteristics
Using a BMI cut-off point of BMI < 25 kg/m 2 , BMI ≥ 25.1-29.9 kg/m 2 ) and BMI > 30 kg/m 2 for normal weight, overweight and obese, as recommended by the World Health Organization [55] there were 115, 68, 146 individuals in the normal weight, overweight and obese groups, respectively.  The anthropometric characteristics of the study subjects are presented in Table 1. All the three groups matched in their age and no statistically significant differences were observed amongst the groups. All other parameters except HDL, were significantly higher in overweight and obese subjects as compared to the normal weight (control) group (p < 0.0001) as shown in Table 1. The results of biochemical and hormonal parameters are presented in Table 2 for the three groups. The value for each parameter was elevated significantly, in the overweight and obese subjects, except HDL, which was significantly lower when compared to the normal weight group (Table 2).

Polymorphism in ADRB1 Arg389Gly genotype
The genotype and allele frequencies of the ADRB1 Arg389Gly (rs1801253) polymorphism were calculated in each group and results were compared. There was no significant difference in the genotype and allelic frequency of ADRB1 Arg389Gly between control and obese or overweight subjects, as shown in Table 3. The ADRB1 genotypes (CC, CG, GG) were grouped separately, and the phenotypic characteristics, biochemical and hormonal parameters were calculated separately for each genotype. No significant differences were observed in the results obtained for the three genotypes (Table 4).

Polymorphism in ADRB3 Trp64Arg genotype
The frequencies of Trp64Arg (rs4994) alleles and genotypes were calculated. The overweight and obese subjects had a significantly higher genotype and allele frequency of Arg64 compared with normal weight subjects. The genotype and allele frequencies in the total group, are presented in Table 5 with the OR, CI, χ 2 and p-value. Furthermore, the study groups were separated according to their Trp64Arg genotypes (TT, TC, CC), and the phenotypic characteristics were obtained. The value of the different parameters in the different genotypes is   (Table 6).

Discussion
This is the first study to report the relationship between ADRB1 and ADRB3 gene polymorphism and obesityrelated phenotypes in the Saudi population. Since in our previous report, we had observed an association between Gln27Glu polymorphism of ADRB2 gene, obesity, and other related disorders in Saudi population [9] our interest was to explore if any association existed between ADRB1 (rs1801253) and ADRB3 (rs4994) and these abnormalities. Hence, during this study, a large cohort of 329 subjects was genotyped for Gly389Arg in ADRB1 (rs1801253) and Trp64Arg in ADRB3 (rs4994) genes and the association of the variant allele with metabolic parameters were analyzed. The results of our investigation have clearly highlighted the strong association with ADRB3 polymorphism, where the mutant Arg allele in ADRB3, significantly associates with a greater body weight and higher BMI, elevated leptin and dyslipidaemias both in heterozygous and homozygous. It also results in elevated blood glucose level and hyperinsulinaemia. However, the ADRB1 polymorphism Arg389Gly, shows no association either with the BMI or related disorders. The ADRB1 is a candidate gene for obesity due to its role in catecholamine mediated energy homeostasis. In 2008, Ohshiro and coworkers had shown a strong association between mutations in the ADRB1 and massive obesity in Japanese [56]. In obese individuals, the degree of weight loss during a very low calorie diet has been shown to correlate with changes in ADRB1 protein concentration in adipose tissue [57,58]. An investigation involving a population cohort of 761 women indicated that women carrying the Gly49 genotype had greater elevation in BMI over 15 years compared to those with the Ser49 genotype [59]. Dionne et al., [7] reported that Gly389Arg exhibited a strong relationship with obesity in Caucasian women. In contrast, some studies have reported the Arg389Gly polymorphism had no significant relationship to obesity in Danish [21], Swedish [60] and Japanese [56] subjects, suggesting that it has no role in human obesity. Masuo and Lambert [10] have reviewed various studies about the relationship of polymorphism in either Gly49-Gly389 or Ser49Gly-Arg389Gly in ADRB1 with obesity or obesity related disorders and have reported contradicting findings among different populations which means that the relationship of ADRB1 polymorphism to obesity could vary between different populations. In fact, our findings for Arg389 Gly polymorphism in ADRB1 amongst Saudi population are in line with earlier published reports [7,21,61,62] and suggest that ADRB1 Arg389Gly polymorphisms do not contribute to obesity and related disorders in the Saudi population. The ADRB3 gene is expressed in adipose tissues and stimulates the mobilization of lipids from the WAT and increases thermo-genesis in BAT [10]. Mutation of ADRB3 in WAT could slow lipolysis and thereby cause the retention of lipids in adipocytes and may contribute to visceral obesity in humans. Interestingly, extensive studies have investigated the role of Trp64Arg (rs4994) in the development of obesity, T2DM, hypertension (HT), cardiovascular disease CAD, dyslipidaemias. Table 7 summarizes some of the studies reported from different populations and shows that there are extensive contradictions between populations, between genders and within different female age groups. Some studies suggest a strong relationship between obesity and Trp64Arg polymorphism in some populations [20, 26-28, 31-33, 35, 37-40, 45, 48, 51, 61-65]. Whereas, other studies on same or different populations have failed to show any association [21,36,41,46,49,50,52]. Even within the same population there are contradictory reports (Table 7). This makes the study of Trp64Arg polymorphism necessary in every population. The present study has found a strong relationship between obesity and Trp64Arg polymorphism in the Saudi population. This is a similar association that we have shown earlier to exist between Gln27Glu polymorphism in ADRB2 gene and obesity related disorders such as hypertriglyceridemia, hyperinsulinemia, and hyperleptinemia in Saudis [9].
The ADRB2 and ARDB3 gene polymorphisms may act as predictive markers for obesity and obesity related disorders in Saudis.

Conclusion
Our present results shows for the first time that obesity and related disorders in Saudis are linked to polymorphism of Trp64Arg (rs4994) in ADRB3 gene but not to Arg389Gly (rs1801253) in ARDB1 gene.