Association of Estrogen Receptor-α Gene & Metallothionein-1 Gene Polymorphisms in Type 2 Diabetic Women of Andhra Pradesh

Abstract

Type 2 diabetes mellitus (DM) is a multifactorial disease where both genetic and environmental factors contribute to its pathogenesis. Estrogen plays an important role in type 2 DM pathogenesis. A number of polymorphisms have been reported in the estrogen receptor (ESR1), including the XbaI and PvuII restriction enzyme polymorphisms of ESR1,which may be involved in disease pathogenesis. Metallothioneins (MT) act as potent antioxidants against various oxidative damages. Very few studies have indicated the association between Estrogen Receptor-α, MT1 gene polymorphisms with type2 DM. A total of 100 type 2 diabetic women and 100 age, sex matched controls were recruited. Using the PCR based RFLP method, the PvuII and XbaI polymorphisms of ESR1 and in MT1A (rs8052394 and rs11076161) gene polymorphisms were analysed. The genotype distribution and frequency of mutated allele showed no significant differences between diabetic and non-diabetic groups in PvuII (χ2 = 2.443; P = 0.1181) or XbaI (χ2 = 1.789; P = 0.1812) and rs8052394 (χ2 = 1.154; P = 0.2840) or rs11076161 (χ2 = 0.4141; P = 0.5199), polymorphisms. This is the first Indian study to conclude that ESR1 and MT1 gene polymorphisms are not associated with increased susceptibility to type 2 diabetes in Indian women.

Introduction

India leads the world with largest number of diabetic subjects earning the dubious distinction of being termed the “diabetes capital of the world”. According to the Diabetes Atlas 2006 published by the International Diabetes Federation, the number of people with diabetes in India, currently around 40.9 million is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken [1, 2]. Type 2 diabetes mellitus (DM) is a multifactorial disease where both genetic and environmental factors contribute to its pathogenesis. Therefore, it is helpful to identify the population with genetic predisposition and to protect them from exposure to environmental risks.

Estrogens exert its physiological effects mainly through the estrogen receptors (ERs), ERα and ERβ, and might prevent menopause syndrome, osteoporosis, arteriosclerosis, and diabetes, etc. ER is found in many tissues, including the pancreatic islets1 and skeletal muscle, adipose tissue, and cardiovascular system, which all participate in the pathogenesis of type 2 diabetes. Epidemiological findings have indicated that the prevalence of type 2 DM is significantly lower in patients on estrogen replacement therapy, and estrogen seems useful for the treatment of diabetes [10, 17]. Type 2 DM is a heterogeneous disorder caused by a combination of genetic and acquired abnormalities that affect insulin sensitivity and insulin secretion.

Estrogen has been implicated in adipose tissue biology [18], stimulation of liver fatty acid metabolism, suppression of hepatic glucose production, 9 reduction of both hyperglycemia and plasma insulin levels. It also helps in protection of pancreatic β-cell function/survival and insulin secretion in conditions of oxidative stress, and the blunting of neuroendocrine and metabolic responses to hypoglycemia. Estrogen modulates insulin secretion, regulates K-ATP channel activity, and regulates calcium signals via plasma membrane receptors [3, 4]. Thus, the gene encoding ERα gene (ESR1, gene map locus 6q25.1) is a potential candidate gene for susceptibility to type 2 diabetes. A number of polymorphisms in ESR1 have been reported. The PvuII and XbaI restriction fragment length polymorphisms (RFLPs) are two commonly employed markers for genetic analyses of the ESR1;both are located in intron 1 of the ESR1. Association between ESR1 and disease has been demonstrated in several cases. An association has been found between the T allele (an allele with a T/C transition) and the absent expressed ESR1 gene in breast.

Cancer cell and breast tumor tissue. PvuII polymorphism may affect the splicing of ER mRNA, resulting in the alteration of protein expression. XbaI and PvuII RFLPs of ESR1 may play a role in the pathogenesis of such diseases as breast and prostate cancer, osteoporosis, Alzheimer’s disease, cardiovascular diseases, and stroke.

MTs are a family of proteins with low molecular mass and high affinity to certain metal ions. MTs exist in multiple organs with several molecular forms, including isoforms of MT1, MT2, MT3, and MT4 [19, 20]. A multigene family with at least 14 closely related and pseudo genes encodes MT proteins, and the most MT genes, including the functional MT genes (MT1A, MT1B, MT2A), lie on human chromosome 16 [7]. MTs can function on the regulation of zinc and copper homeostasis and also act as potent antioxidants against various oxidative damages. Major cause of diabetes and diabetic complications is related to the induction of oxidative stress, we and others have demonstrated protection by increased MT expression in the pancreas [21, 22] and in the heart against initiation of diabetes and diabetic cardiomyopathy, respectively. Furthermore, MT was found also to play a critical role in energy metabolism [23, 24].

Genomic scanning has suggested chromosomal regions possibly containing T2DM susceptibility genes. The chromosomal regions in ESR1 is 6q 25.1 [3–5] and MT1 is located on chromosome 16q22 [6–8]. Therefore, any changes in ESR1 or MT1 genes may be associated with T2DM predisposition. We investigated whether type 2 DM is associated with ESR1 or MT1gene polymorphisms in Andhra Pradesh women.

The prevalence of type 2 DM and associated traits such as obesity, dyslipidemias, and hypertension in the overall population has become a worldwide challenge for health care systems. Estrogen has beneficial effects on lipids and lipoproteins metabolism [9–11]. In order to find if the ESR1 variant relates to serum lipid level, which may contribute to the mechanism causing type 2 diabetes, the relationship between the gene polymorphism and serum lipid level was also investigated.

Materials and Methods

Study Design

The participants were selected who were Hyderabad residents, aged 30–70 years and had no significant liver damage or renal dysfunction. Patients and control subjects had no history of a sex-hormone-dependent disease and never received hormone replacement therapy. Informed written consent was obtained from each individual.

A total of 100 T2DM female patients and 100 age and sex matched controls of Indian population were recruited for the genetic analysis at the Institute of Genetics and Hospital for Genetic Diseases (Hyderabad, Andhra Pradesh, India). They were diagnosed as T2DM based on the World Health Organization 1999 guideline that was adopted from the American Diabetes Association 1997 standard.

Biochemical Analyses

Blood samples were collected from the patients after a 12-h fasting. Total cholesterol, HDL cholesterol, and triglyceride levels were determined by standard methods using commercial kits from ERBA (Transasia Biomedical LTD, India).

Genotyping

PCR–RFLP assays were used to determine the ESR1 and MT1 genotypes. The oligonucleotide primers used to determine the PvuII and XbaI polymorphisms within the ESR1 gene were the same as those used by Yaich et al (forward, 5′-TGCCACCCTATCTGTATCTTTTCCTATTCTCC-3′; reverse, 5′-TCTTTCTCTGCCACCCTGGCGTC-GATTATCTGA-3′). Genomic DNA used for PCR amplification was extracted from the whole blood sample using a DNA extraction kit (Hi media, India). The PCR product of approximately 1.3 kb was completely digested with restriction endonuclease PvuII or XbaI for 8 h, and electrophoresed in a 1.5% agarose gel. The PvuII polymorphism, which is caused by a T/C transition in intron 1, is located approximately 0.4 kb upstream of exon 2. The XbaI polymorphism, which is caused by an A/G transition in intron 1, is approximately 50 bp away from the PvuII polymorphism site [12].

The oligonucleotide primers used to determine the rs8052394and rs11076161 polymorphisms within the MT1 gene were rs8052394 (forward: 5-ACTAAGTGTCCTCTGGGGCTG, reverse: 5-AATGGGTCACGGTTGTATGG 3) and rs11076161 (Forward: 5-TTCGGGATTAAGGACATAAAGC-3, reverse: 5-GAAATGGATCATTGGCCTACTC-3). The PCR amplification was performed in a 25-l reaction mixture with each primer, for which the conditions included an initial denaturation at 94°C for 5 min, followed by 35–40 cycles of 94°C for 45 s, 55–60°C for 1 min and 72°C for 1 min, and a final elongation at 72°C for 10 min. A 15-l aliquot of the PCR products was completely digested with 7–10 units of restriction enzymes (Table 1).

Table 1.

The primer sequence for PCR–RFLP in a Indian population

Gene	RFLP	Primer sequence
ESR1	PvuII, XbaI	Forward, 5′-CTG CCACCC TAT CTG TAT CTT TTC CTA TTC TCC-3 Reverse, 5′-TCT TTC TCT GCC ACC CTGGCG TCG ATT ATC TGA-3′
MT1 A	rs8052394	Forward: 5-ACTAAGTGTCCTCTGGGGCTG 3 Reverse: 5-AATGGGTCACGGTTGTATGG 3
MT1 B	rs11076161	Forward: 5-TTCGGGATTAAGGACATAAAGC-3 Reverse: 5-GAAATGGATCATTGGCCTACTC-3

Statistical Analysis

All quantitative variables were presented as mean ± standard deviation while categorical variables were summarized by absolute frequencies and percentages. Allele frequencies were calculated for each genotype by allele counting. Continuous variables were compared using independent two-sample t test, and categorical variables were compared using Chi-square test.

Results

A total of one hundred patients with diabetes and 100 controls without diabetes were genotyped for the four common polymorphisms PvuII, XbaI of ESRI gene and rs8052394, rs11076161 of MT1 gene. All participants were aged 35–86 years. The mean age of the patients with diabetes and controls were 44.6 ± 9.89 years and 40.4 ± 6.19 years (P = 0.0798), respectively.

Genotyping Analysis for the Association with T2DM

All four polymorphisms were detected in the study. The frequencies of polymorphisms in patients with diabetes compared with non-diabetic group is shown in Table 2. The blood LDL, HDL, total cholesterol, and triglyceride levels of the participants were measured to determine their relations with type 2 diabetes. Lipid profile of the participants are summarized in Table 3.

Table 2.

Genotype distribution and allele frequencies of PvuII, XbaI and rs8052394, rs11076161 polymorphisms in type 2 diabetic patients (n = 100) and control subjects (100)

ESR1
PvuII
Subjects	TT (%)	CT (%)	CC (%)	T	C	OR (95% CI)
Patients	39	43	18	121 (63.6%)	79 (41.5%)	1.387
Controls	52	32	16	136 (68%)	64 (32%)
For PvuII χ2 = 2.443; P = 0.1181, heterozygous T/C, homozygous mutant C/C, normal T/T

XBaI
Subjects	GG (%)	AG (%)	AA (%)	G	A	OR (95% CI)
Patients	75	20	5	170 (85%)	30 (15%)	1.492
Controls	86	10	4	186 (89.4%)	22 (10.5%)
For XbaI χ2 = 1.789; P = 0.1812, heterozygous G/A, homozygous mutant A/A, normal G/G

MT1A
rs8052394
Subjects (MT1A)	GG (%)	GA (%)	AA (%)	A	G	OR (95% CI)
Patients	86	8	6	20 (10%)	180 (90%)	1.476
Controls	90	6	4	14 (7%)	186 (93%)
For MT1A (Pst1) χ2 = 1.154; P = 0.2840, heterozygous G/A, homozygous, mutant A/A, normal G/G

rs11076161
Subjects (MT1B)	AA (%)	AG (%)	GG (%)	G	A	OR (95% CI)
Patients	90	7	3	13 (6.5%)	187 (93.5%)	1.321
Controls	92	6	2	10 (5%)	190 (95%)
For MT1B (Tsp5091) χ2 = 0.4141; P = 0.5199, heterozygous A/G, homozygous, mutant G/G, normal A/A

Table 3.

Lipid profile of the study subjects

Characteristics	Women with diabetes (n = 100)		Women without diabetes (n = 100)		P value
	Mean ± SD	Median	Mean ± SD	Median
Age	44.6 ± 9.89	41	40.4 ± 6.19	40	0.0798
GLU	127.7 ± 61.74	113.65	110.85 ± 71.83	82	0.5178
CH	165 ± 29.42	162.9	174 ± 20.21	171	0.6824
TG	259.7 ± 2 82.6	185.22	186.5 ± 108.73	150	0.7211
HDL	37.35 ± 20.47	31.92	30 ± 1 2.21	25	0.7164
LDL	82.79 ± 33.5	96.8	106 ± 25.65	113	0.8825
VLDL	59.95 ± 56.55	37	37.05 ± 21.72	30	0.5318

TC total cholesterol; TG triglycerides; LDL-c low-density lipoprotein cholesterol; HDL-c high-density lipoprotein cholesterol

Discussion

Type 2 DM is thought to be a multifactorial disease and both genetic and acquired factors contribute to its pathogenesis. Identification of the susceptibility genes for type 2 DM thus may lead to primary prevention of the disease. In most patients, type 2 DM results from genetic changes, each has a partial and additive effect. The inheritance pattern is thus complex, and environmental factors play an important role in favoring or delaying the expression of the disease.

Sex steroids clearly have an impact on insulin resistance risk. Recent data have revealed a surprising role of estradiol in regulating energy metabolism, which opened new insights into the role of the two estrogen receptors. 11 Therefore, estrogen receptors seem to play a role in the prevention or in the occurrence of diabetes type 2 [25]. ERα polymorphisms have attracted great interest in the last few years and the PvuII and XbaI are the most extensively investigated issues. The abnormal expression of MT may cause uncontrolled oxidative stress and inflammatory responses and may accelerate the development of diabetic complications.

We report for the first time no significant difference in the ESR1 and MT1 gene polymorphisms in Indian population. There are few studies showing the significant association of ESR1 gene (Pvu)II) polymorphisms in the Chinese [13], African–Americans and European–Americans [14]. We did not observe any association between PvuII polymorphism and diabetes or significant relation between XbaI polymorphism and diabetes. Our findings correlated with the Iranian study [15].

Study from Chinese people of Han (397 T2DM and 454 controls) supports that there was a significant Association of MT1A gene (rs8052394) polymorphism with T2DM patients [16] but no association with MT1B gene rs11076161 [15]. In our study no significant association of the MT1A (rs8052394) gene and MTIB (rs11076161) gene polymorphisms was observed in Indian women of type 2 DM.

We also demonstrated that ESR1 and MT1 gene polymorphisms are not significantly associated with the lipid levels, since the patients were on treatment. The present findings are limited in the way that they were obtained from a relatively small study population and our non-significant findings may be caused by the poor statistical power. The results of our study should be considered exploratory and confirmed by additional studies, which include larger sample size and other polymorphisms in estrogen receptor and MT genes. This will help to identify the population with genetic predisposition and to protect them from exposure to environmental risks.

This is the first Indian study to show that ESR1 and MT1 gene polymorphisms are not associated with increased susceptibility of Indian women to type 2 DM.