Polymorphisms of the Vitamin D Receptor Gene Predict the Onset of Surgical Menopause in Caucasian Females
By Dvornyk, Volodymyr; Long, Ji-Rong; Liu, Peng-Yuan; Shen, Hui; Et al
Abstract
We tested association of four single nucleotide polymorphisms (SNPs) of the vitamin D receptor gene (VDR) with age at surgical and natural menopause in a sample of Caucasians composed of 153 women with surgical and 260 with natural menopause. A significant association was observed between age at surgical menopause and two SNPs, rs1544410 (BsmI) and rs731236 (TaqI) (p < 0.05). For rs1544410, homozygotes of the minor allele, AA, had about two-fold higher risk of surgical menopause than homozygotes of the major allele, GG (95% confidence ratio (CI) 1.09-3.82). For rs731236, the CC subjects had a greater chance of surgical menopause than the TT subjects (odds ratio = 2.01, 95% CI 1.07-3.78). Since rs1544410 and rs731236 are in strong linkage disequilibrium, the haplotypes based on these two loci were also tested. The haplotype AC was highly significantly associated with age at surgical menopause (p = 0.008). Women with this haplotype had surgical menopause on average 2.8 years earlier than non-carriers. These results reveal the potential effect of the VDR gene on ovaries and uterus, and suggest that its SNPs can be used as predictors of genetic susceptibility for early surgical menopause and respective causal health problems.
Keywords: Association, surgical menopause, vitamin D receptor gene
Introduction
Early onset of menopause results in premature exposure to low estrogen levels and is associated with a number of postmenopausal health problems as well as higher risk of mortality. Early menopause has been shown to increase risks for various cancers [1-3], cardiovascular diseases [4], osteoporosis [5] and lowtrauma fractures [6]. Several studies reported a number of genetic factors underlying premature or early menopause [7,8].
Surgical menopause is a common cause of the early menopause. It usually results from bilateral oophorectomy or hysterectomy. Although surgical menopause itself is not a disease, it is usually performed after the diagnosis of a number of diseases, including adenomyosis, various cancers, endometriosis, fibroid tumors and others. Hysterectomy shows considerable heritability (h^sup 2^ = 59%), as do its two main indications: fibroids (h^sup 2^ = 69%) and menorrhagia (h^sup 2^ = 55%) [9]. Like surgical menopause, natural menopause is largely determined genetically [10,11].
The gene (VDR) encoding the vitamin D receptor (VDR) may be one of the potential candidate genes underlying the onset of menopause. VDR not only mediates the action of 1,25-dihydroxyvitamin D^sub 3^ in calcium/phosphate translocating tissues, primarily intestine, but also elicits a myriad of apparent bioactivities in other major cell systems, including immune, neural, epithelial and endocrine [12,13]. In particular, it is ubiquitously expressed in the reproductive organs and tissues [14].
In the present study, we tested effect of four single nucleotide polymorphisms (SNPs) in VDR on the onset of menopause in a population-based sample of Caucasian women.
Materials and methods
A random sample of 413 Caucasian postmenopausal females recruited in the Omaha metropolitan area (Nebraska, USA) was used in the present study. They came from our previous genetic epidemiological studies [15,16], which were approved by the Creighton University Institutional Review Board. Among these females, 153 suffered surgical menopause (surgical menopause females, SMF) and 260 had natural menopause (natural menopause females, NMF). All study subjects signed informed-consent documents before entering the project. Information about the age at menopause, smoking habits, age at menarche, number of pregnancies, and use of oral contraceptives before menopause was recorded for each subject by nurse- administered questionnaires. SMF had hysterectomy, or hysterectomy plus oophorectomy. Because surgical menopause is usually performed for disorders which are complex (e.g. endometriosis [17]) and may have common etiology (e.g. adenomyosis and endometrial hyperplasia [18]), it was considered in the present study as a surrogate phenotype without specifying a cause for which the operation was assigned. Such an approach is widely used in population association studies of other complex disorders. For example, bone mineral density (BMD) is commonly considered a surrogate phenotype when studying osteoporosis, although a wide variety of factors may contribute to BMD [19]. The age of surgical menopause was denned as the age at the date of operation. The age of natural menopause was defined as the date of last menses followed by 12 months of no menses.
Genomic DNA was extracted from peripheral blood leukocytes using a commercial isolation kit following the procedure recommended by the manufacturer (Centra Systems, Minneapolis, MN, USA). Four SNPs within the VDR gene were genotyped: rs2238136, rs2228570 (FokI), rs 1544410 (BsmI) and rs731236 (TaqI). The genotyping procedure for all SNP markers was similar, involving polymerase chain reaction and invader assay reaction (Third Wave Technology, Madison, WI, USA), and is detailed elsewhere [15]. The error rate for this technique is <0.8% [20].
The χ^sup 2^ test was employed to examine Hardy-Weinberg equilibrium (HWE) of the four studied markers. Multivariate regression analysis was performed to test the effects of the studied polymorphisms and other potential factors such as smoking, number of pregnancies, age of menarche and use of oral contraceptives on the age at surgical and natural menopause. The number of minor allele homozygotes for rs2238136 was small (only eight) and thus they were combined with the respective heterozygotes into one group (Table II). Therefore, the subjects were divided into two groups, with or without the minor allele. Pairwise linkage disequilibrium (LD) between SNPs within the gene was calculated by the measure D’ [21]. Using D’ = 0.7 as an arbitrary limit for useful LD in association studies [22], the rs 1544410 and rs731236 pair fell below this threshold (D’ = 0.926) [16]. Therefore, the haplotypes reconstructed based on these two markers were tested for association with age at menopause. For those SNPs or confounding factors that were found to be associated with age at surgical menopause, the odds ratio (OR) with 95% confidence interval (CI) were calculated. All statistical analyses were conducted using SPSS version 10 (SPSS, Inc., Chicago, IL, USA) and results are reported as mean + standard error.
Results
Age, height, weight and age at menarche were similar between subjects of the two groups (SMF and NMF). In the SMF group, more women were smoking and took oral contraceptives before menopause than in the NMF group. SMF had fewer pregnancies (3.5 0.2) than NMF (4.1 0.2). Age at menopause differed significantly between SMF and NMF: 40.4 0.6 and 48.9 0.3 years, respectively (Table I).
The χ^sup 2^ test revealed deviations from HWE at rs2228570 in SMF, rs 1544410 and rs731236 in NMF (Table II). There was a clear prevalence of heterozygotes over homozygotes at these polymorphisms. The deviations may be caused by population stratification/admixture at these loci.
Table I. Characteristics of women involved in the study.
Table II. Association analyses for the four single nucleotide polymorphisms (SNPs) with age at menopause (mean standard error) in Caucasian females.
Significant associations were observed for age at surgical menopause at two markers, rs 1544410 (BsmI) and rs731236 (TaqI), with p values of 0.045 and 0.030, respectively. For the marker rs1544410, SMF carrying a minor allele had menopause on average 2.4 years earlier than non-carriers: 39.8 0.7 vs. 42.2 1.1 years, respectively. The AA subjects had about two-fold higher risk of surgical menopause than the GG individuals (95% CI 1.09-3.82). For rs731236, subjects with the minor allele had surgical menopause on average 3 years earlier than those without this allele (39.5 0.7 vs. 42.5 1.1 years, respectively). The CC subjects had increased risk of surgical menopause compared with TT women (OR = 2.01, 95% CI 1.07-3.78). Suggestive association was obtained between rs2228570 and age at surgical menopause (p = 0.055). Homozygotes at minor allele appeared to have a later onset of surgical menopause. In the NMF group, no significant association was found between any of the studied markers and age at menopause (Table II).
Haplotype AC (reconstructed from rs 1544410 and rs731236) was significantly associated with age at surgical menopause (p = 0.008). Women with this haplotype had surgical menopause on average 2.8 years earlier than non-carriers (39.4 0.8 vs. 42.2 0.8 yrs, respectively).
Discussion
In this population-based association study, we have shown for the first time that allelic variants of the VDR gene are associated with surgical menopause, which is usually assigned for disorders of the ovaries and uterus. Homozygotes at the minor alleles of markers rs 1544410 and rs731236 had higher risk of early surgical menopause. These results could be expected based on the following data. VDR is a member of the ligand-dependent receptor superfamily mediating the signal of steroid hormones [23]. It is found ubiquitously in many tissues and organs including ovary, oviduct and uterus [14]. VDRknockout mice had uterine hypoplasia with impaired folliculogenesis [2\4]. It is possible that VDR expressed in the reproductive organs influences their functions and thus alters the onset of menopause. In addition, the VDR gene was previously reported as a predictor of advanced onset of menarche in Japanese girls [25], which also suggests a relationship between VDR and the reproductive system.
Several studies have provided indirect evidence of the association between the VDR gene and the reproductive system. Vitamin D and its metabolites are related to endometriosis and medical menopause [26,27]. Vitamin D-dependent calcium-binding protein, calbindin, was expressed in uterine (luminal) epithelium of pregnant rats and was not observed in non-pregnant rats [28]. If one supposes that calbindin and enhanced calcium metabolism have a protective effect on uterine epithelium, this may explain why the number of pregnancies is a significant factor associated with surgical menopause. Given that the VDR gene is ubiquitously expressed in the reproductive organs and tissues [14], its contribution to the various diseases associated with surgical menopause seems probable. Likewise, polymorphisms of the VDR gene were reported to have effects on the risk of various disorders associated with menopause and estrogen depletion, such as osteoporosis [29], cardiovascular disease [30], breast cancer [31] and others.
It is not entirely clear how the three SNPs of the VDR gene may affect age at surgical menopause. For the rs2228570 polymorphism, which results in an amino acid replacement Met/Thr in exon 2, we can suppose that the allelic variants may have different activity. The other two polymorphisms, rs2228570 and rs 1544410, are anonymous and may be in LD with a truly functional mutation elsewhere in the VDR gene or in the nearby genomic region. These two intronic markers may also act as a regulatory element in mediating transcription and/or stability of mRNA, or function as a protein-binding site. For the rs2238136 marker, no association was observed with age at menopause. The potential reason is that it may be not in LD with the functional polymorphisms.
VDR is one but not the only gene contributing to the risk for early abnormal menopause. Krauss and colleagues [7] reported premature ovarian failure and premature menopause at the age of 24- 37 years due to an interstitial deletion of the long arm of the X chromosome. The genetic deficiency of the blood enzyme galactose-1- phosphate uridyl transferase (reduced activity) might be associated with infertility and early menopause [32]. Recently, factor V Leiden mutation was reported to accelerate the onset of natural menopause [8].
Overall, the present study is one of the very few association studies of age at menopause and the first one to provide evidence for an association of the VDR gene with age at surgical menopause. The results obtained suggest the potential effect of this gene on the uterus and ovaries, and may implicate use of VDR gene polymorphisms in clinical practice for risk estimation of genetic susceptibility to early surgical menopause.
Acknowledgements
The investigators were partially supported by grants from Health Future Foundation, the National Institutes of Health, the State of Nebraska (LB595 and LB692), US Department of Energy, Chinese National Science Foundation, the Ministry of Education of the People’s Republic of China, the Huo Ying Dong Education Foundation, and Hunan Normal University. The study also benefited from 211 State Key Research Fund to Xi’an Jiaotong University.
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VOLODYMYR DVORNYK1,2*, JI-RONG LONG1,3*, PENG-YUAN LIU1,4, HUI SHEN1, ROBERT R. RECKER1, & HONG-WEN DENG1,5,6
1 Osteoporosis Research Center and Department of Biomedical Sciences, Creighton University, Omaha, Nebraska, USA,
2 Dep\artment of Biological Sciences, Kent State University, Kent, OH, USA, 3 Vanderbilt Center for Health Services Research,
Nashville, Tennessee, USA, 4 Department of Surgery, Washington University in St Louis, St Louis, Missouri, USA, 5 The Key
Laboratory of Biomedical Information Engineering of Ministry of Education and Institute of Molecular Genetics, Xi’an Jiaotong
University, Xi’an, People’s Republic of China, and 6 Laboratory of Molecular and Statistical Genetics, College of Life Sciences,
Hunan Normal University, Changsha, Hunan, People’s Republic of China
(Received 20 April 2005; revised 16 September 2005; accepted 23 September 2005)
Correspondence: H.-W. Deng, Departments of Orthopedic Surgery and Basic Medical Sciences, University of Missouri-Kansas City, 2411 Holmes Street, Room M3-CO3, Kansas City, Missouri, 64108-2792, USA. Tel: 1 816 235 5354. Fax: 1 816 235 6517. E-mail: dengh@umkc.edu
* Contributed equally to this work.
Copyright Taylor & Francis Ltd. Oct 2006
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