Trp Homozygotes at Codon 64 of ADRB3 Gene Are Protected Against the Risk of Type 2 Diabetes in the Kashmiri Population

Abstract

The prevalence of type 2 diabetes mellitus has reached epidemic proportions worldwide. Type 2 diabetes is a consequence of complex interactions among multiple genetic variants and environmental risk factors. Polymorphisms in various candidate genes confer susceptibility to diabetes. This study was undertaken to analyse a single nucleotide polymorphism Trp64Arg (C↔T) in the ADRB3 gene and elucidate its effects on type 2 diabetes and its associated risk factors. The study included 200 type 2 diabetes patients and 300 age and gender matched healthy controls belonging to the ethnic Kashmiri population. Polymerase chain reaction-restriction fragment length polymorphism technique was used for genotyping and the results were validated by direct sequencing assay. Genotypes for Trp64Arg polymorphism were in Hardy-Weinberg equilibrium (χ²=0.48, p=NS). Frequency of the Arg64 allele was 40% and 10.2% in cases and controls, respectively (p<0.05; odds ratio 5.89; 95% CI; 3.69-9.39). The Arg64 allele was directly related to higher body mass index, waist-to-hip ratio, dyslipidemia and uncontrolled disease status. The study signifies that the Arg64 allele of the ADRB3 gene is a genotypic risk factor and confers susceptibility to type 2 diabetes, whereas the homozygous Trp64 genotype exerted a protective effect in our population.

Introduction

Type 2 diabetes mellitus (T2D) is a heterogeneous disorder characterized by two interrelated metabolic defects: insulin resistance coupled with impaired insulin secretion by β-cells in the pancreas (Doria et al., 2008). The number of people developing T2D has increased rapidly in the last 30 years, at first in America and Europe, and more recently in Asia where it is said to be reaching epidemic proportions (Chan et al., 2009). The etiology involves both genetic and environmental factors. Age, physical activity patterns, dietary habits, lifestyle, and obesity play a crucial role in the modulation and occurrence of T2D (Christopher et al., 2011). In the past few years, major advances relating to the genetic bais of T2D have been made and different susceptibility genes have been identified that interact with environmental factors, during gestation, early childhood, and later in life (Dabelea et al., 2000; Voight et al., 2010; Herder and Roden, 2011; Hochberg et al., 2011). This has cumulated in the discovery and confirmation of more than 40 diabetes-associated loci (Zeggini et al., 2008; Dupuis et al., 2010; Saxena et al., 2010; Voight et al., 2010; Christopher et al., 2011).

The β₃-adrenergic receptor (ADRB3) is a G protein-coupled receptor mainly expressed in adipose tissue, and contributes to variations in energy expenditure and body fat distribution (Clement et al., 1995; Shuldiner and Sabra, 2001; Lowell and Bachman, 2003). Molecular abnormalities in the β₃-adrenergic receptor are related to the development of obesity and T2D. Experimental studies on rodent models show decreased expression of β₃-adrenergic receptor (Muzzin et al., 1991; Collins et al., 1994), whereas β₃-specific agonists have compelling effects on obesity and diabetes in both animals and humans (Mitchell et al., 1992). A base change (T→C) in the first cytoplasmic region of the ADRB3 gene causes the substitution of the coding sequences from tryptophan into arginine in 64th position, and this is able to influence the receptor's affinity to nor-epinephrine and its interaction with G proteins in adipocytes (Walston et al., 1995). The Trp64Arg polymorphism (rs 4994) is reported to be associated with early development of T2D, lower resting metabolic rate, abdominal obesity and insulin resistance in the homozygous variant (Arg64Arg) compared to the homozygous wild (Trp64Trp) and heterozygous (Trp64Arg) genotypes (Clement et al., 1995; Kadowaki et al., 1995; Widen et al., 1995; Walston et al., 2000; Corella et al., 2001; Hao et al., 2004; Cruz et al., 2010). A cellular transfection study also showed an association with decreased glucose dependent insulin secretion (Perfetti et al., 2001). Conversely, some studies did not find any association between this polymorphism and T2D (Susulic et al., 1995; Awata and Katayama, 1996; Pietri-Rouxel et al., 1997; Rissanen et al., 1997; Büettner et al., 1998; Oeveren van-Dybicz et al., 2001). This discrepancy among various studies can in part be explained by age, ethnicity, or population differences. In this study, we examined for the first time, the potential role of Trp64Arg polymorphism in the ADRB3 gene as a contributor to T2D and associated risk factors in the Kashmiri population.

Methods

Study population

A total of 500 subjects belonging to the Kashmir valley and willing to participate voluntarily were selected for the study. 200 T2D subjects and 300 non diabetic controls matched for age and gender were screened for ADRB3 Trp64Arg polymorphism. Written informed consent was obtained from each participant. The study was approved by Institutional Ethics Committee.

Phenotypic and clinical parameters

Anthropometric measurements, including body mass index (BMI) and waist-to-hip ratio (WHR) were calculated for each subject. BMI was expressed as kg/m². BMI was used to reflect the total body fat, while WHR is an indirect measurement of body fat centralization. Hypertension was defined as a systolic pressure >140 mmHg or diastolic pressure >90 mmHg. Blood collection from the cubital vein took place after 12 h of fasting in the morning. Laboratory tests included fasting plasma glucose, lipid panel [total cholesterol, triglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL)], and HbA1c levels. Lipid panel analysis was performed on the “Sinhron CX4-DELTA” biochemical autoanalysator (“Beckman”, USA).

Genotyping

DNA was extracted from peripheral leukocytes by the Phenol-Chloroform method with slight modifications. ADRB3 polymorphism was detected using the polymerase chain reaction-Restriction fragment length polymorphism method. A 255 bp region encompassing the Trp64Arg polymorphism was amplified using forward primer 5′-CCAGTGGGCTGCCAGGGG-3′ and reverse primer 5′-GCCAGTGGCGCCAACGG-3′ in a thermal cycler (“Biorad” USA). The amplification protocol was one cycle of denaturation at 94°C for 3 min, annealing at 65°C and extension at 72°C; 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s and extension at 72°C for 45 s; and one final elongation cycle at 72°C for 10 min. The amplicons were digested overnight at 37°C using MspI restriction enzyme that differentiates the three genotypes: a 255 bp uncut (Trp64Trp) wild homozygous, a 158 and 97 bp variant homozygous (Arg64Arg) and a 255, 158 and 97 bp heterozygous (Trp64Arg) genotype. Digested fragments were resolved on a 3% agarose gel by electrophoresis, stained with ethidium bromide, and photographed on a gel scanning imager. Genotyping results were confirmed by DNA sequencing (BigDye^® Terminator v3.1Cycle Sequencing Kits, with ABI 3130xl Genetic Analyzer; Applied Biosystem, Foster City, CA).

Statistical analyses

The Statistical Package for Social Sciences (SPSS, version 16.0) and Java Stat software were used for statistical analyses. Data were described as mean±SD and percentage. Fischer's exact test and odds ratio analysis was used for genotyping analysis where p value of <0.05 was considered to be significant. Hardy and Weinberg equilibrium was determined by χ² analysis.

Results

The study consisted of 200 T2D patients (101 males and 99 females) and 300 control subjects (150 males and 150 females) belonging to the ethnic population of the Kashmir valley. Mean age of T2D cases and controls was 50.1±11.4 and 49.4±12.2 years, respectively. The most common age group among participants was 50-60 years. 30.5% of T2D subjects had a familial history of disease. The subjects were screened for Trp64Arg (T→C) polymorphism in the ADRB3 gene. In our population the frequency of Trp64Trp, Trp64Arg and Arg64Arg genotypes was 58%, 33.8%, and 2.2%, respectively. Genotypes for Trp64Arg polymorphism were in Hardy-Weinberg equilibrium in our population (χ²=0.48, p=NS). Trp64Trp was the most common genotype in controls, whereas Trp64Arg was commonly found in cases. Among T2D patients, 24.5% were wild homozygous (Trp64Trp) where as 71% were heterozygous (Trp64Arg). Among the controls 80.3% and 19% of the subjects exhibited wild homozygous and heterozygous genotypes, respectively. The homozygous variant (Arg64Arg) was rarely present in our population (4.5% in T2D patients and 0.7% in control population). Genotypic analyses and frequency distribution are shown in Table 1.

Table 1.

Genotyping Analysis and Frequency Distribution of Trp64Arg Polymorphism

Genotypes	T2D Patients n=200	Controls n=300	Odds ratio (95% CI)	χ²	p-Value
Trp64Trp^a	49	241
Trp64Arg	142	57	12.25 (7.9-18.9)	147.05	<0.0001
Arg64Arg	9	2	22.1 (4.6-105.6)	147	0.000008
Trp64Arg+Arg64Arg	151	59	12.5 (8.1-19.3)	153.5	<0.001

Fischer's exact test and odds ratio analysis were used for genotyping analysis where p-value of<0.05 at 95% confidence interval (CI) was considered to be significant.

Trp64Trp: Reference allele.

T2D, type 2 diabetes mellitus.

The frequency distribution shows a positive association between the presence of the Trp64Arg allele and T2D, whereas the homozygous Trp64 allele seems to exert a protective effect against T2D in our population (Fig. 1). Intragroup analysis of ADRB3 Trp64Arg genotypes with clinical parameters and diabetic risk factors was suggestive of the deranged status and severity of T2D with respect to the presence of the Arg64 allele (Table 2). The highest association was observed in anthropometric measurements. Presence of the 64Arg allele was directly related to higher BMI and WHR in a dose-dependent manner with most severe association in the subjects carrying the Arg64Arg genotype. Carriers of Trp64Arg were characteristic of higher BP levels. Mean blood glucose levels were also higher in Trp64Arg genotype but the data did not reach statistical significance. However HbA1c levels showed statistically significant association with Arg64 allele. Presence of the Arg64 allele also correlated with TG and LDL levels. Genotypic correlation elucidates a protective effect conferred by Trp64Trp genotype and predisposition to T2D and higher incidence of risk factors in carriers of Trp64Arg and Arg64Arg genotypes.

FIG. 1.

Frequency distribution of Trp64Arg genotype in T2D cases and controls. T2D, type 2 diabetes mellitus.

Table 2.

Intragroup Analysis of Clinical Parameters Across Genotypes

Parameter	Trp64Trp (n=49)	Trp64Arg (n=142)	Arg64Arg (n=9)	p-Value	p^a-Value
Age (years)	50.3±12.2	50.5±10.9	49.6±9.3	0.915	0.847
BMI (kg/m²)	22.9±3.7	25.9±5.3	30.5±3.3	<0.005	<0.0005
WHR	0.85±0.09	0.91±0.09	1.01±0.06	<0.005	<0.0005
BP (Systolic)	123.5±17.4	133±21.1	130±23.5	0.003	0.416
BP (Diastolic)	78.2±9.3	83.2±10.7	78.9±3.4	0.004	0.836
BG-F (mg/dl)	192.2±60.0	213.4±93.5	196.4±42.5	0.07	0.803
BG-PP (mg/dl)	273.6±95.9	303.4±121.9	232.2±56.9	0.122	0.095
HbA1c (%)	7.8±1.0	10.4±2.5	9.0±1.3	<0.005	0.027
TGs (mg/dl)	156.6±51.9	226.5±131.0	250±99.6	<0.005	0.025
LDL (mg/dl)	94.6±19.6	108.4±26.7	120.1±25.3	0.001	0.018
HDL (mg/dl)	40.2±7.5	38.3±9.8	33.0±6.6	0.218	0.009
Chol (mg/dl)	152.2±32.0	168.6±57.1	200.1±47.6	0.728	0.335

BMI, body mass index; WHR, waist-to-hip ratio, BP, blood pressure, BG-F, blood glucose fasting, BG-PP, blood glucose post prandial; TGs, triglycerides; LDL, low density lipoproteins; HDL, high density lipoproteins; Chol, cholesterol.

p: Trp64Trp versus Trp64Arg, p^a: Trp64Trp versus Arg64Arg.

Discussion

The nonsynonymous Trp64Arg polymorphism (rs4994) located within the ADRB3 gene has been previously associated with T2D, obesity, insulin resistance and hypertension (McFarlane-Anderson et al., 1998; Ringel et al., 2000; Oizumi et al., 2001; Gjesing et al., 2008; Kurokawa et al., 2008). To examine the potential role of inherited defects in this gene as contributors to T2D, we evaluated the Kashmiri population, an ethnic group with very high impaired fasting glucose and impaired glucose tolerance levels (Zargar et al., 2001) and an alarming increase in the prevalence of T2D in the recent past (Zargar et al., 2000; 2008; Ahmad et al., 2011). We observed a significant association between Trp64Arg polymorphism and T2D, indicating a potential role of this polymorphism in key components of the T2D, in accordance to what has been described in previous studies (Fujisawa et al., 1998; Ringel et al., 2000; Oizumi et al., 2001; Xiu et al., 2004; Bracale et al., 2007; Yoshida et al., 2007). The homozygous Arg64 was rarely present in our population. Similar results were observed in the Kyrgyz population (Mirrakhimov et al., 2011).

Our results indicate that Trp64 homozygotes were relatively protected against diabetic risk factors and metabolic abnormalities. Studies examining the relation between Trp64Arg polymorphism and BMI show stronger association in Asian populations as compared to Caucasians (Fujisawa et al., 1998; Kurokawa et al., 2001). We observed a dose-dependent difference in anthropometric parameters across the genotypes. Association between Trp64Arg carrier state and increased blood pressure was also observed in our study which is in consensus with the experimental data indicating that ADRB3 modulates peripheral vascular tone and increase blood pressure (Shen et al., 1994). Clinical studies have shown relationship between Trp64Arg genotype and higher mortality among hypertensive subjects (Tonolo et al., 1999; Iwamoto et al., 2011).

A remarkable observation in our study was that mean blood glucose levels (both fasting and post prandial) did not vary when Trp64Trp was compared with Trp64Arg and Arg64Arg genotypes. However, when HbA1c levels were compared the data was highly significant. The reasons for this observation remain speculative but it has been postulated that plasma glucose and HbA1c levels differ substantially depending on the glycemic control of the studied population (Kilpatrick et al., 2007). Discrepancy in plasma glucose and HbA1c levels can also be attributed to a limited number of plasma glucose measurements recorded over a limited time period, erythrocyte survival and certain genetic elements (Snieder et al., 2001; Virtue et al., 2004). Genetic polymorphisms are major causes of inter-individual differences in drug response and adverse drug reaction (Yang et al., 2009). Association of the Arg64 allele with poor glycemic index provides an insight for assessing the drug efficacy and response to treatment among different genotypes of ADRB3 Trp64Arg polymorphism in the Kashmiri population.

Studies examining the role of Trp64Arg polymorphism with serum lipids have shown inconsistent results (Widen et al., 1995; Gagnon et al., 1996; Urhammer et al., 1996; Corella et al., 2001; Mirrakhimov et al., 2011). We found a positive association between TGs and LDL levels across the genotypes but lower HDL levels reached statistical significance only in the case of Arg64 homozygotes. This observation is contradictory to one study that found an association for HDL levels only (Mirrakhimov et al., 2011).

There are large frequency differences between Asian and European populations for many gene variants screened by candidate gene studies that were not identified in any of the larger genome wide association studies. Although a meta-analysis showed a modest contribution of the Arg64 variant to susceptibility to T2D (Fujisawa et al., 1998), it was not reported as one of the significant genes identified in recent genome-wide association studies carried out in European populations. It is therefore imperative to conduct this type of study in different population groups to understand the population distribution of allele frequencies and confirm the results of this study. In the Kashmir valley, the increase in the prevalence of diabetes coincides with a parallel increase in obesity (Masoodi et al., 2010). Given the importance of obesity in diabetes, these findings suggest that Trp64Arg polymorphism in ADRB3 gene may confer an increased risk of T2D. Further research in our population is necessary to decipher the mechanisms through which this missense polymorphism has an impact on diabetic traits.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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