Association of Estrogen Receptor Alpha Gene Polymorphisms and Risk of Fracture

Abstract

The association between estrogen receptor alpha (ESR1) gene polymorphisms and risk of fracture is still controversial and ambiguous. The objective of this study was to evaluate the effect of PvuII polymorphisms of the ESR1 gene on fracture risk in Chinese patients. A population-based control study of elderly subjects was conducted in 120 fracture patients and 120 controls. The PvuII pp genotype of the ESR1 gene was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay. There was no relationship between ESR1 gene PvuII polymorphism and fracture risk. When stratifying by fracture type, it was found that vertebral fracture cases had a significantly higher frequency of the PvuII pp genotype (odds ratio=2.00, 95% confidence interval=1.03, 3.88; p=0.04) than controls. This study suggested that there was a modest but statistically significant association between the PvuII pp genotype of the ESR1 gene and vertebral fracture in Chinese patients. The molecular mechanism underlying this association needs further study.

Introduction

Osteoporosis is a common skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue with increased susceptibility to fracture (Recker, 2005a; 2005b). Osteoporosis is considered to be a multifactorial syndrome with environmental and genetic factors interacting. Genetic factors play an important role in the pathogenesis of osteoporosis (Recker and Deng, 2002; Recker, 2004). From a genetic perspective, osteoporosis is a multifactorial disease resulting from multiple independent gene effects and gene-gene and gene-environment interactions (Rivadeneira et al., 2006). Association analysis of candidate genes is an efficient way to identify the modest but real genetic effects of individual polymorphisms (Lohmueller et al., 2003; Dresner-Pollak et al., 2009; Darabi et al., 2011).

Estrogen receptor alpha (ESR1; 6q25.1) polymorphisms have been strongly associated with bone mineral density (BMD) in Chinese women (Wang et al., 2007). Fracture is a relevant clinical outcome to assess in genetic studies of osteoporosis. A meta-analysis of 13 studies including 1279 cases and 6069 controls suggests a modest but statistically significant association between the ESR1 PvuII pp genotype and vertebral fracture (Lei et al., 2010). However, another meta-analysis by Ioannidis et al. (2002) suggested that the PvuII polymorphisms of the ESR1 gene were not associated with either BMD or fracture risk. Therefore, we decided to perform a population-based control study of elderly subjects to study whether a relationship exists between the PvuII polymorphisms of the ESR1 gene and risk of fracture in Chinese patients.

Materials and Methods

Study subjects

A population-based control study of elderly subjects was conducted in 120 fracture patients and 120 controls between 2007 and 2010 from the second affiliated hospital of Harbin Medical University, China. The Chinese participants were collected from the same geographic region. We recorded the demographic and clinical data. Only participants who lived independently participated in this study. Detailed medical histories were obtained from their general practitioner and by an extensive interview. Participants with known bone disease—on current or previous treatment with drugs that affect bone metabolism, including hormonal replacement therapy and calcium supplements—or with a previous history of cancer were excluded. General practitioners were asked to report on previous and current illness and drug therapy. A full fracture history was evaluated by interview of the patient and by information from the general practitioner. Only radiographically documented fractures were considered. Fractures resulting from car accidents were excluded. For vertebral fractures, only symptomatic fractures were considered, that is, vertebral deformations reported by a radiologist on an X-ray that was performed on clinical grounds, such as back pain. One hundred and twenty patients had suffered from a fragility fracture (60 had a history of vertebral fracture and 60 of hip fracture). Signed informed consent forms were obtained according to the Declaration of Helsinki from all study subjects who participated in this study. The protocol of this study was approved by the Ethical Committee of the second affiliated hospital of Harbin Medical University.

Genotyping

Genomic DNA was prepared from 10 mL of EDTA-preserved whole blood by a simply salting out procedure. The PvuII genotype of the ESR1 gene was determined by using a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. Based on the GenBank reference sequence, the PCR primers were as follows: forward, 5′-CTGCCACCCTATCTGTATCTTTTCCTATTCACC-3′ and reverse, 5′-TCTTTCTCTGCCACCCTGGCGTCGATTATCTGA-3′. PCR condition was as follows: 94°C for 5 min, and then 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 30 cycles, and a final extension at 72°C for 7 min. The amplified DNA was digested with PvuII restriction endonuclease (Fermentas) and electrophoresed in 2.0% agarose gel.

Statistical analysis

Statistical analyses were carried out with a commercially available software package SPSS 11.0 for Windows statistical package (SPSS, Inc., Chicago, IL). Categorical variables were analyzed with the χ² test. Continuous data were analyzed with the Wilcoxon rank sum test. Genotype distribution and allele frequencies were compared to the Hardy-Weinberg equilibrium model using the Pearson χ² test. The value of p<0.05 was considered statistically significant. All statistical tests were two-sided.

Results

Characteristics of the study population

General characteristics of fracture patients and controls were listed in Table 1. No differences were seen in body height, weight, age, gender, smoking status, or calcium intake. The mean age was 65.8 (±6.8) years for the fracture patients and 66.3 (±7.0) years for the controls. Calcium intake was on average 554 (±137) mg/day for the fracture patients and 562 (±141) mg/day for the controls. The genotype distribution was found to be in Hardy-Weinberg equilibrium.

Table 1.

General Characteristics of Fracture Patients and Controls

Characteristics	Fracture	Controls	p-Value
Subjects (n)	120	120	-
Age (years), mean±SD	65.8±6.8	66.3±7.0	0.58
Sex: women/men	79/41	81/39	0.78
Height (cm)	160.1±0.9	159.9±0.8	0.07
Weight (kg)	61.9±1.1	62.1±1.2	0.18
Smoking status: smokers/nonsmokers (n)	21/99	19/101	0.73
Calcium intake (mg/day)	554±137	562±141	0.66
Fracture type: vertebral/hip (n)	60/60	-	-

Association between the genotype and fractures

There was no relationship between ESR1 gene PvuII polymorphism and fracture risk. When stratifying by fracture type, it was found that vertebral fracture cases had a significantly higher frequency of the PvuII pp genotype (odds ratio=2.00, 95% confidence interval=1.03, 3.88; p=0.04) than controls (Table 2). There were no significant differences in genotype frequencies of ESR1 gene PvuII polymorphism between hip fracture patients and controls (Table 2).

Table 2.

Genotype Frequencies of ESR1 Gene PvuII Polymorphism Between Fracture Patients and Controls

		Fracture			Vertebral fracture			Hip fracture
Genotype	Controls n (%)	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p	n (%)	OR (95% CI)	p
PP	28 (23.3)	27 (22.5)	0.95 (0.52, 1.74)	0.88	11 (18.3)	0.74 (0.34, 1.61)	0.44	16 (26.7)	1.20 (0.59, 2.43)	0.62
Pp	62 (51.7)	55 (45.8)	0.79 (0.48, 1.31)	0.37	25 (41.7)	0.67 (0.36, 1.25)	0.21	30 (50.0)	0.94 (0.50, 1.74)	0.83
pp	30 (25.0)	38 (31.7)	1.39 (0.79, 2.45)	0.25	24 (40.0)	2.00 (1.03, 3.88)	0.04	14 (23.3)	0.91 (0.44, 1.89)	0.81
P allele frequency	118 (49.2)	109 (45.4)	0.86 (0.60, 1.23)	0.41	47 (39.2)	0.67 (0.43, 1.04)	0.07	62 (51.7)	1.11 (0.71, 1.71)	0.66
p allele frequency	122 (50.8)	131 (54.6)	1.16 (0.81, 1.66)	0.41	73 (60.8)	1.50 (0.96, 2.35)	0.07	58 (48.3)	0.91 (0.58, 1.40)	0.66

OR, odds ratio; CI, confidence interval.

Discussion

We found that vertebral fracture cases had a significantly higher frequency of the PvuII pp genotype than controls. Our finding is consistent with that of meta-analysis by Lei et al. (2010).

A large number of studies have examined the association between ESR1 gene polymorphisms and BMD (Mesmar and Kanan, 2009). In 2000, Ho et al. studied the association of PvuII polymorphisms of the ESR1 gene and BMD in healthy southern Chinese women. A study of women from northeast Scotland in the United Kingdom by Albagha et al. (2001) found that PvuII polymorphisms of the ESR1 gene are significantly related to BMD. Yamada et al. (2002) found the association of polymorphisms of the ESR1 gene with BMD of the femoral neck in elderly Japanese women. The data by Zhang et al. (2003) suggested that the ESR1 genes maybe associated with the BMD variation in Chinese women. The study by Boot et al. (2004) showed that PvuII polymorphisms of the ESR1 gene were associated with BMD during childhood. But study by Koh et al. (2002) found that there were no significant differences in BMD among PvuII and XbaI genotypes of ESR1. Wang et al. (2007) conducted a meta-analysis to assess their pooled effects. Sixteen eligible studies involving 4,297 Chinese women were identified. They found that the PvuII polymorphism had a very weak association with femoral neck BMD whereas XbaI polymorphism was unlikely to be a predictor of femoral neck or spine BMD in Chinese women (Wang et al., 2007).

The association between ESR1 gene polymorphisms and risk of fracture is still controversial and ambiguous. In 2042 individuals of the Rotterdam Study, a prospective population-based cohort study of elderly subjects, van Meurs et al. (2003) found that ESR1 polymorphism in the 5′ (promoter) region was associated with vertebral fracture risk, lumbar spine BMD, and vertebral bone area in postmenopausal women, but not in men. The results of Salmen et al. (2000) suggested that the pp genotype was a relatively hormone-insensitive genotype, and it appeared that women with the P allele might benefit more from the protective effect of hormone replacement therapy on fracture risk than women with the pp genotype. But a study by Vandevyver et al. (1999) found that in elderly Caucasian women the ESR1 polymorphism was not associated with osteoporosis or fracture history. There were two meta-analyses to study whether a relationship exists between the PvuII polymorphisms of the ESR1 gene and risk of fracture. The meta-analysis by Ioannidis et al. (2002) found that the PvuII polymorphism was not associated with either BMD or fracture risk. But the meta-analysis by Lei et al. (2010) suggested a modest but statistically significant association between the ESR1 PvuII pp genotype and vertebral fracture.

This study has several limitations. First, only participants who were not treated for bone disease and without a history of cancer were studied, so the results are not representative for the whole population. Second, only symptomatic vertebral fractures were considered. The incidence of asymptomatic fractures of spine can therefore be underestimated. Third, this study was limited to participants without any treatment for osteoporosis. Fourth, the sample size used is relatively small, so the statistical power of our study remains very limited.

In conclusion, this study suggested that there was a modest but statistically significant association between the PvuII pp genotype of the ESR1 gene and vertebral fracture in Chinese patients. Our findings highlight the necessity of large studies to achieve sufficient statistical power to further elucidate the complex, multigenic character of osteoporosis. The molecular mechanism underlying this association needs further study.

Footnotes

Acknowledgment

This study was supported by the Scientific Research Staring Foundation for Youth of The 2nd Affiliated Hospital of Harbin Medical University (Grant No. QN2010-16).

Disclosure Statement

No competing financial interests exist.

References

Albagha

, McGuigan

, Reid

et al. 2001. Estrogen receptor alpha gene polymorphisms and bone mineral density: haplotype analysis in women from the United Kingdom. J Bone Miner Res, 16:128-134.

Boot

, van der Sluis

, de Muinck Keizer-Schrama

et al. 2004. Estrogen receptor alpha gene polymorphisms and bone mineral density in healthy children and young adults. Calcif Tissue Int, 74:495-500.

Darabi

, Ani

, Panjehpour

et al. 2011. Effect of estrogen receptor beta A1730G polymorphism on ABCA1 gene expression response to postmenopausal hormone replacement therapy. Genet Test Mol Biomarkers, 15:11-15.

Dresner-Pollak

, Kinnar

, Friedlander

et al. 2009. Estrogen receptor beta gene variant is associated with vascular dementia in elderly women. Genet Test Mol Biomarkers, 13:339-342.

, Yeung

, Kung

. 2000. PvuII polymorphisms of the estrogen receptor alpha and bone mineral density in healthy southern Chinese women. Calcif Tissue Int, 66:405-408.

Ioannidis

, Stavrou

, Trikalinos

et al. 2002. Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density and fracture risk in women: a meta-analysis. J Bone Miner Res, 17:2048-2060.

Koh

, Kim

, Hong

et al. 2002. Estrogen receptor alpha gene polymorphisms Pvu II and Xba I influence association between leptin receptor gene polymorphism (Gln223Arg) and bone mineral density in young men. Eur J Endocrinol, 147:777-783.

Lei

, Yang

, Tu

et al. 2010. Oestrogen receptor-alpha polymorphism and risk of fracture: a meta-analysis of 13 studies including 1279 cases and 6069 controls. J Int Med Res, 38:1575-1583.

Lohmueller

, Pearce

, Pike

et al. 2003. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat Genet, 33:177-182.

10.

Mesmar

, Kanan

. 2009. Possible association of combined vitamin D receptor and estrogen receptor genotypes and low bone mineral density in Jordanian postmenopausal women. Genet Test Mol Biomarkers, 13:603-609.

11.

Recker

. 2004. Genetic research in osteoporosis: where are we? Where should we go next? J Musculoskelet Neuronal Interact, 4:86-90.

12.

Recker

. 2005a. Current trends in osteoporosis. Manag Care, 14:3-8discussion 21-23.

13.

Recker

. 2005b. Summary—Novel therapies for osteoporosis. J Musculoskelet Neuronal Interact, 5:358-359.

14.

Recker

, Deng

. 2002. Role of genetics in osteoporosis. Endocrine, 17:55-66.

15.

Rivadeneira

, van Meurs

, Kant

et al. 2006. Estrogen receptor beta (ESR2) polymorphisms in interaction with estrogen receptor alpha (ESR1) and insulin-like growth factor I (IGF1) variants influence the risk of fracture in postmenopausal women. J Bone Miner Res, 21:1443-1456.

16.

Salmen

, Heikkinen

, Mahonen

et al. 2000. The protective effect of hormone-replacement therapy on fracture risk is modulated by estrogen receptor alpha genotype in early postmenopausal women. J Bone Miner Res, 15:2479-2486.

17.

van Meurs

, Schuit

, Weel

et al. 2003. Association of 5′ estrogen receptor alpha gene polymorphisms with bone mineral density, vertebral bone area and fracture risk. Hum Mol Genet, 12:1745-1754.

18.

Vandevyver

, Vanhoof

, Declerck

et al. 1999. Lack of association between estrogen receptor genotypes and bone mineral density, fracture history, or muscle strength in elderly women. J Bone Miner Res, 14:1576-1582.

19.

Wang

, Tang

, Chen

et al. 2007. Association of estrogen receptor alpha gene polymorphisms with bone mineral density in Chinese women: a meta-analysis. Osteoporos Int, 18:295-305.

20.

Yamada

, Ando

, Niino

et al. 2002. Association of polymorphisms of the estrogen receptor alpha gene with bone mineral density of the femoral neck in elderly Japanese women. J Mol Med (Berl), 80:452-460.

21.

Zhang

, Long

, Liu

et al. 2003. Estrogen receptor alpha and vitamin D receptor gene polymorphisms and bone mineral density: association study of healthy pre- and postmenopausal Chinese women. Biochem Biophys Res Commun, 308:777-783.