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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 697-700
Copyright © 2003 by The Endocrine Society

Calcium Sensing Receptor Gene A986S Polymorphism and Responsiveness to Calcium Supplementation in Postmenopausal Women

Robert Young, Fiona Wu, Neil Van de Water, Ruth Ames, Greg Gamble and Ian R. Reid

Departments of Medicine and Molecular Medicine, University of Auckland, Auckland, New Zealand

Address all correspondence and requests for reprints to: R. P. Young, M.D., University of Auckland, Faculty of Medicine and Health Science, Private Bag 92019, Auckland, New Zealand. E-mail: rp.young{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Recently several studies in adolescent girls or premenopausal women have implicated the calcium sensing receptor (CASR) gene A986S polymorphism in calcium and bone metabolism. However, the role of this genetic variant in postmenopausal women, specifically the development of osteoporosis, is unknown. This study reports the findings of a randomized, double-blind, placebo-controlled study of healthy postmenopausal women followed for 2 yr while taking placebo or supplementary calcium. Specifically, we examined the relationship between the CASR A986S polymorphism, bone biochemical profile, and bone mineral density at baseline and after 2 yr of treatment. We found no effect of this genetic variant in postmenopausal women at baseline or in response to calcium supplementation. These results are in contrast to those in young or premenopausal women, and they provide no support for an important role for the CASR A986S polymorphism in osteoporosis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GENETIC FACTORS HAVE a strong influence on bone mass, and a number of candidate genes associated with bone mineral density (BMD) and osteoporotic risk have been proposed (1). Serum calcium has a central role in bone metabolism and has been shown to be under tight physiological control mediated by the calcium sensing receptor (CASR; Ref. 2). Distributed on the extracellular surface of parathyroid chief cells and renal tubular epithelium, these receptors control calcium levels by altering PTH secretion and renal calcium reabsorption. The CASR has also been implicated in the regulation of osteoclastic bone resorption (3). In two studies of healthy young women (4, 5), the rarer S allele of the A986S polymorphism was associated with elevated serum calcium. In a study of adolescent girls, the CASR A986S polymorphism was associated with both calcium levels and BMD, although the latter association disappeared after adjustment for body size and physical activity (6). This polymorphism is believed to be of pathophysiological significance because it encodes for an amino acid change (alanine to serine in position 986, located in exon 7 of the CASR gene) in the cytoplasmic tail of the receptor, implicating it in intracellular signaling. On the basis of these studies, it has been proposed that this genetic variant may be a candidate gene for osteoporosis. Given the importance of age-related effects on both serum calcium concentration and BMD decline, we examined the role of the A986S polymorphism on serum calcium, BMD, and BMD response to calcium supplementation in healthy postmenopausal women.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

During a 2-yr randomized double-blind placebo-controlled trial of the effect of calcium supplementation (1000 mg/d) on bone loss in 135 healthy white postmenopausal women (7), a random sample of 102 subjects (mean ± SD age, 58 ± 5 yr) were genotyped for the A986S polymorphism (5). These women had been recruited originally through advertisement, and respondents completed a medical questionnaire. The exclusion criteria were: a history of disorders of calcium metabolism; renal, thyroid or hepatic dysfunction; systemic disease; use of hormone replacement therapy in the last 3 yr; use of glucocorticoids for the last 6 months; or concurrent use of anticonvulsants or thiazide diuretic agents. This study was approved by the local ethics committee, and all participants gave written informed consent. BMD was measured at baseline and every 6 months thereafter by dual energy x-ray absorptiometry (Lunar Corp., Madison, WI). Serum and urinary indices of calcium and bone metabolism were measured at baseline, 3, 12, and 24 months.

Analysis of CASR A986S polymorphism

The CASR A986S polymorphism was genotyped using minor modifications of a recently published method (5). Using an allele-specific mutation-selective amplification technique, both the 986 S and A alleles were detected. Two upstream primers (0.75 µM 5'-GCT-TTG-ATG-AGC-CTC-AGA-AGA-TCG-3' and 0.5 µM 5'-ACG-GTC-ACC-TTC-TCA-CTG-ACG-TTT-GAT-GAG-CCT-CAG-AAG-TAC-T-3') and a common downstream primer (0.5 µM 5'-CTC-TTC-AGG-GTC-CTC-CAC-CTC-T-3') were used. Included in the 20 µl reaction volume were 2 µl of 10x PCR buffer [500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl (pH 8.3)], 50 ng of DNA template, 0.2 mM each of the four deoxynucleotide triphosphates, and 0.5 U of ampliTaq Gold (Amersham Pharmacia Biotech, Arlington Heights, IL). Genomic DNA underwent 30 cycles at 94 C denaturation for 15 sec, reannealing at 57 C for 39 sec, elongation at 72 C for 30 sec, and final elongation at 72 C for 5 min. Allele-specific fragments were separated by gel electrophoresis for 1.5 h at 5V/cm, stained with ethidium bromide, and visualized under ultraviolet transillumination. In each amplification, AA, AS and SS control samples, previously typed by sequence analysis (made available by Dr. Betty Wong, University Health Network, Quebec, Canada), were used to ensure fidelity of the method.

Statistical analysis

Differences between normally distributed variables were compared in subjects with and without the S allele (AA subjects compared with those with either the AS or SS genotype) in a two-way comparison using an unpaired t test. Differences between these variables in each genotype (AA, AS, and SS) were also sought using ANOVA (three-way comparison) with post hoc analyses using the method of Tukey. A 5% significance level was maintained.


   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Table 1Go shows the genotype distribution, baseline biochemical, hormonal, and bone density data. The mean age, years since menopause, height, and weight were similar between the three (AA, AS, SS) genotype groups, although in the two-way comparison the mean age of the S allele group (AS or SS group) was 2 yr younger than the AA group. In the three-way comparison, serum total calcium and alkaline phosphatase levels were significantly lower in the SS group compared with the AA or the AS group. In the two-way comparisons, no differences were found. All other baseline serum and urinary indices and bone density measurements were similar between the groups.


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  		Table 1. Baseline clinical, biochemical, hormonal, and bone density data according to the CASR A986S genotype

 
Ninety-two women who had undergone genotyping completed 2 yr of the calcium study. The mean urinary calcium-to-creatinine ratio and 24-h urinary calcium excretion during the study period were higher in the calcium-treated group, but urinary calcium excretion was similar across all of the genotypes (regardless of grouping) in response to calcium treatment. The other indices of calcium metabolism and bone turnover were not different between the calcium and placebo groups, and no genotype-treatment interactions were seen. Calcium supplementation reduced bone loss as reflected by significantly smaller changes in BMD in those receiving calcium supplementation compared with placebo at the lumbar spine (-0.008 ± 0.031 vs. -0.013 ± 0.036; P < 0.005) and total body (-0.018 ± 0.022 vs. -0.029 ± 0.022; P < 0.05). There was a trend toward reduced bone loss at the hip. Genotype did not influence response to calcium treatment or rate of bone loss in the placebo group (all P > 0.05; Fig. 1Go).



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  		Figure 1. Change in lumbar spine (A) and total body (B) BMD over 2 yr according to treatment and genotype groups (mean ± SE).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The distribution of the A986S polymorphism genotypes in our group of white postmenopausal women was in accordance with Hardy-Weinberg equilibrium, with allele and genotype frequencies comparable to those reported in other studies (4, 5, 6). The latter studies recruited adolescent or premenopausal women and reported that the S allele (SS genotype or combined AS/SS genotypes) of the A986S polymorphism was associated with elevated total serum calcium (4, 5, 6) and reduced BMD (6). However, in contrast to these studies, in our cohort of healthy postmenopausal women, the SS genotype was associated with lower total calcium than those women with the AA and AS genotypes. Of note, there was no difference in total calcium between the AA and AS/SS groups. Because the number of subjects with the SS genotype is small, ranging between 1 and 5% for all studies reported to date, it is possible that sampling error may account for these differences. In this regard, it should be noted that our study was similarly powered to two of the three other studies (4, 6) with an SS genotype frequency of 5% in a cohort of 102 subjects. Even in the larger study that recruited 387 young women, only 6 (2%) had the SS genotype (5). An alternative explanation is that the effect of age may be contributing to our conflicting findings. Several studies have confirmed that serum calcium levels increase with age (8, 9, 10). Although several mechanisms have been proposed to explain this age effect, it is not clear why a genetic effect on serum calcium in adolescent and premenopausal women would be lost in postmenopausal women. Given that the SS genotype is found in only 1–5% of the cohort studies to date, conclusions about genetic effects from such a small group are limited until much larger cohorts are studied, but it can be concluded that the simple presence of the S allele in postmenopausal women is most unlikely to be associated with a biologically significant difference in serum calcium.

The relationship between the CASR A986S polymorphism and BMD requires further discussion. Lorentzon et al. (6) reported that adolescent subjects with the S allele had 3–5% lower BMD at all sites (P values between 0.02 and 0.06), although these associations disappeared after adjustment for body size and physical activity. In a multiple regression analysis, they found physical activity, rather than the CASR A896S polymorphism, predicted BMD. Although the authors concluded that the CASR A896S polymorphism may influence BMD through effects on the amount of physical activity, this association could also be due to chance alone. In our study, calcium supplementation was shown to reduce the decline in BMD over a 2-yr follow-up, but no convincing effect of the CASR A896S polymorphism was seen on baseline BMD or response to treatment. In particular, although there was an apparent trend toward decrease in bone turnover indices and an increase in baseline BMD (for spine and total body) in the SS group compared with AA and AS groups in our study (not significant), this was not apparent when the AS/SS groups were combined. Thus, there was no evidence of a biological (i.e. genetic dose) effect from the S allele when comparing these parameters between AA, AS, and AS/SS groups (Table 1Go). A recently published cross-sectional study of Hungarian postmenopausal women also failed to find any relationship between the A986S genotype (comparing AA with AS/SS) and BMD (lumbar spine and femoral neck) in nonosteoporotic or osteoporotic women (11). The present study has a power of 80% to detect a difference in BMD of 0.6 Z-score between groups at baseline, equating to an absolute BMD difference of about 4% in the total body. Thus, small effects of this polymorphism on BMD cannot be ruled out, but an effect of major biological significance is most unlikely. This conclusion is consistent with the results of the other studies discussed. One other study has implicated the CASR gene in osteoporosis. In a cross-sectional study of elderly (mean age, 72 yr) Japanese women, the association between a CA-repeat polymorphism and BMD was examined (12). The polymorphism has no known function and lies within 110 kb of the CASR gene. The association was with one of the more common repeats (n = 18) and only just reached significance (uncorrected P = 0.03). Future studies will be needed to better understand the relationship between this polymorphism and the A986S polymorphism, but differences in the populations studied alone could account for disparate results between these studies.

In conclusion, although the CASR polymorphism appears a determinant of serum calcium in adolescent and premenopausal women, our results in postmenopausal women failed to show any relationship between this polymorphism and serum calcium, baseline BMD, or BMD decline with and without calcium supplementation. The role of the CASR A986S polymorphism as an important candidate polymorphism for calcium metabolism and osteoporosis must be in some doubt.


   Acknowledgments
 


   Footnotes
 
This work was supported by the Health Research Council of New Zealand.

Abbreviations: BMD, Bone mineral density; CASR, calcium sensing receptor.

Received March 6, 2002.

Accepted October 21, 2002.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Eisman JA 1999 Genetics of osteoporosis. Endocr Rev 20:788–804[Abstract/Free Full Text]
  2. Pearce SHS, Thakker RV 1997 The calcium-sensing receptor: insights into extracellular calcium homeostasis in health and disease. J Endocrinol 154: 371–378
  3. Kameda T, Mano H, Yamada Y, Takai H, Amizuka N, Kobori M 1998 Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem Biophys Res Commun 245:419–422[CrossRef][Medline]
  4. Cole DE, Peltekova VD, Rubin LA, Hawker GA, Vieth R, Liew CC, Hwang DM, Evroski J, Hendy GN 1999 A986S polymorphism of the calcium-sensing receptor and circulating calcium concentration. Lancet 353:112–115[CrossRef][Medline]
  5. Cole DE, Vieth R, Trang HM, Wong BYL, Hendy GN, Rubin LA 2001 Association between total calcium and the serum A986S polymorphism of the calcium-sensing receptor. Mol Genet Metabol 72:168–174[CrossRef][Medline]
  6. Lorentzon M, Lorentzon R, Lerner UH, Nordstrom P 2001 Calcium sensing receptor gene polymorphism, circulating concentrations and bone mineral density in healthy adolescent girls. Eur J Endocrinol 144:257–261[Abstract]
  7. Reid IR, Ames RW, Evans MC, Gamble GD, Sharpe SJ 1993 Effect of calcium supplementation on bone loss in postmenopausal women. N Engl J Med 328:460–464[Abstract/Free Full Text]
  8. Nordin BEC, Need AG, Hartley TF, Philcox JC, Thomas DW 1989 Improved method for calculating calcium fractions in plasma: reference values and effects of menopause. Clin Chem 35:14–17[Abstract/Free Full Text]
  9. Tanasijevic MJ, Ginsburg E, Wybenga DR, Barbieri RL, Winkelman JW 1995 Total serum calcium reference intervals in postmenopausal outpatients. Gynecol Obstet Invest 39:266–270[Medline]
  10. Squier TC, Bigelow DJ 2000 Protein oxidation and age-dependent alterations in calcium homeostasis. Front Biosci 5:D504—D526
  11. Takacs I, Speer G, Bajnok E 2002 Lack of association between calcium sensing receptor gene "A986" polymorphism and bone mineral density in Hungarian postmenopausal women. Bone 30:849–852[Medline]
  12. Tsukamoto K, Orimo H, Hosoi T, Miyao M, Ota N, Nakajima T, Yoshida H, Watanabe S, Suzuki T, Emi M 2000 Association of bone mineral density with polymorphism of the human calcium-sensing receptor. Calcif Tissue Int 66:181–183[CrossRef][Medline]



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