Association between Total Serum Calcium and the A986S Polymorphism of the Calcium-Sensing Receptor Gene

Molecular Genetics and Metabolism 72, 168 –174 (2001) doi:10.1006/mgme.2000.3126, available online at http://www.idealibrary.com on

Association between Total Serum Calcium and the A986S Polymorphism of the Calcium-Sensing Receptor Gene David E. C. Cole,* ,† ,‡ ,1 Reinhold Vieth,* ,§ Hoang M. Trang,‡ Betty Y.-L. Wong,‡ Geoffrey N. Hendy,㛳 and Laurence A. Rubin† *Department of Laboratory Medicine & Pathobiology, and †Department of Medicine, University of Toronto, ‡The Toronto General Hospital Genetic Repository, University Health Network, and §Mount Sinai Hospital, Toronto, Ontario; and 㛳Departments of Medicine, Physiology, and Human Genetics, McGill University, Montreal, Quebec, Canada Received August 16, 2000; and in revised form October 30, 2000; published online January 24, 2001

In healthy subjects, circulating calcium concentrations show a gaussian distribution and can be considered a quantitative trait, subject to genetic and environmental influence. Twin studies indicate a high degree of heritability. Whitfield and Martin (1) found a heritability coefficient (h 2 ) of 0.61 ⫾ 0.06 for total calcium, but acknowledged that albumin concentrations might account for a significant portion of the genetic effect. Study of a smaller cohort from an Australian twin registry found that more than three-quarters of the variance (78%) in plasma total calcium was attributable to additive genetic effects (2), and a similar analysis of U.S. Veterans Twin Registry (NHLBI cohort) data identified small but significant differences in variances for calcium and albumin concentrations of dizygotic as compared to monozygotic twins (3,4). The ability to maintain extracellular calcium concentrations [Ca 2⫹] 0 in a narrow physiological range is mediated by the calcium-sensing receptor (CASR) which acts to coordinate calcium regulation by the parathyroid gland and the kidney (5,6). At the cell surface, the CASR glycoprotein comprises a large extracellular domain, a membrane-spanning motif common to G protein-coupled receptors, and an intracellular tail. Mutations of the CASR gene can cause either loss or gain of function, but most lead to significant alterations in [Ca 2⫹] 0 (7). Inactivating mutations are found in patients with familial hypocalciuric hypercalcemia (FHH), secondary hyperparathyroidism, and neonatal severe hyperparathyroidism (NSHPT) (5,8,9). In affected patients, higher than normal [Ca 2⫹] 0 is required to suppress PTH release from the

Serum calcium is under tight physiological control, but it is also a quantitative trait with substantial genetic regulation. Mutations of the CASR gene cause familial hypocalciuric hypercalcemia or autosomal dominant hypoparathyroidism, depending on whether they decrease or increase, respectively, ligand binding to the receptor protein. We described an association between ionized calcium and a common polymorphism (A986S) found in the cytoplasmic tail of this G protein-coupled receptor. We report here on an independent study of 387 healthy young women. Genotyping was performed by allele-specific amplification and serum chemistries were measured by automated clinical assay. Frequencies of SS, AS, and AA genotypes were 6, 107, and 274, respectively, yielding a 986S allele frequency of 15.4%. Mean total serum calcium (Ca T) was significantly higher in the SS (9.88 ⴞ 0.29 mg/dL, P ⴝ 0.015) and AS groups (9.45 ⴞ 0.05 mg/dL, P ⴝ 0.002), than in the AA group (9.23 ⴞ 0.04 mg/dL). In multiple regression modeling, the A986S genotype remained an independently significant predictor of CaT (P < 0.0001) when serum albumin, globulin, inorganic phosphate, and creatinine covariates were included. These data are the first to show significant association between a common polymorphism and concentrations of a serum electrolyte. The A986S polymorphism is also a potential predisposing factor in disorders of bone and mineral metabolism. © 2001 Academic Press

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To whom correspondence should be addressed at University of Toronto, Room 415, Banting Building, 100 College Street, Toronto, Ontario M5G 1L5, Canada. Fax: (416) 978-5650. E-mail: [email protected]. 168 1096-7192/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

SERUM CALCIUM AND CASR GENE

parathyroid gland and enhance calcium excretion by the kidney. Conversely, activating mutations result in autosomal dominant hypocalcemia (7,10) and the calcium-sensing mechanisms maintains subnormal [Ca 2⫹] 0 with reduced PTH secretion and increased calcium excretion. Mutant CASRs expressed in vitro (11) show altered intracellular signaling in response to fluctuations in [Ca 2⫹] 0, consonant with the patient phenotype. This correlation between CASR mutations and altered calcium concentrations suggested that CASR might be a genetic determinant of [Ca 2⫹] 0 in the general population. To test this hypothesis, we assayed total calcium, albumin, and other analytes in 163 subjects who had been genotyped for the CASR A986S polymorphism (12). In a multivariate regression model, biochemical and genetic parameters accounted for 74% of the total calcium variation, and the A986S genotype was independently predictive. In a subset of 84 patients prospectively studied, ionized calcium also showed a positive association with the 986S variant. In this report, we describe results of a study whose objective was to determine whether a larger sample size would allow detection of a significant predictive effect of A986S on plasma total calcium—without correction for albumin. We find that CASR A986S is independently predictive of total calcium, an effect that appears dependent on gene dosage. This constitutes the first identification of a novel polymorphic variant contributing substantially to the population distribution of a commonly assayed serum electrolyte. MATERIALS AND METHODS Blood Collection and Sample Preparation Leukocyte DNA was extracted from 389 adults who had been prospectively recruited to a larger study aimed at measuring indices of bone and mineral metabolism in healthy women 18 to 35 years of age (13). Informed consent was obtained for the metabolic studies and the collection of genomic DNA for the analyses presented here. Excluded from the present study were subjects reporting other than caucasian ethnicity and relatives already in the parent study. The women in this sample reside in metropolitan Toronto, a demographically diverse urban region without substantial founder subpopulations or genetic evidence of recent admixture effects. These studies received ethical approval from the

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Women’s College Hospital institutional review board. Genotyping Genotyping was performed by an allele-specific mutagenically separated amplification technique (13,14,15) (Fig. 1). Both CASR alleles were detected using two upstream primers (0.75 ␮M 5⬘-GCTTTGATGAGCCTCAGAAGAtCG, and 0.5 ␮M 5⬘-ACGGTCACCTTCTCACTGAcgTTTGATGAGCCTCAGAAGtACT for the A986 and the 986S alleles, respectively), and a common downstream reverse primer— 0.5 ␮M 5⬘-CTCTTCAGGGTCCTCCACCTCT. In the forward primers, noncomplementary nucleotides (introduced to minimize heteroduplexing) are indicated in lower case, and the allele-specific complementary nucleotide in each primer at the 3⬘ position is in bold (see GenBank Accession No. U20760). Included in the 20-␮l reaction volume were 2 ␮l of 10X PCR-buffer (500 mM KCl, 15 mM MgCl 2, 100 mM Tris-HCl, pH 8.3, and 0.01% gelatin), 50 ng of DNA template, 0.2 mM each of the four dNTPs (Pharmacia, Baie D’Urfe, Quebec), and 0.5 units of ampliTaq Gold (Perkin-Elmer, Branchburg, NJ). Genomic DNA was subjected to 30 cycles of amplification (Perkin-Elmer GeneAmp PCR System 2400, PE Biosystems, Foster City CA) at 94°C denaturation for 15 s, reannealing at 57°C for 30 s, elongation at 72°C for 30 s, and a final elongation at 72°C for 5 min. Allelic-specific fragments were separated by gel electrophoresis for 1.5 h at 5 V/cm in 0.75X TAE buffer (30 mM Tris-base, 15 mM acetate, 0.75 mM EDTA, pH 8.0) and visualized under UV transillumination after staining with 0.5 ␮g/ml (w/v) ethidium bromide. Fidelity of this genotyping method was confirmed by sequence analysis. A portion of exon 7 was amplified from leukocyte DNA by a method modified from Janicic et al. (16), and direct sequencing of a nested 201-bp PCR product was performed by the dideoxy method with unidirectional (5⬘ to 3⬘) fluorescently labeled primers (17). Biochemical Analysis Serum analytes were assayed on coded sample aliquots from 387 subjects using the Cobas Integra700 multifunctional analytical system (Roche Diagnostics, Basel, Switzerland) (18). The total calcium method is based on dye binding with o-cresophthalein complexone reagent. Similarly, creatinine was estimated using a kinetic Jaffe´

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tribution of genotype to prediction of serum total calcium was modeled using multivariate linear regression analysis, with the SPSS 8.0 statistics package (SPSS Inc, Chicago IL). RESULTS CASR Genotypes The proportions of subjects heterozygous and homozygous for the CASR 986S genotype were 107/387 (27.6%) and 6/387 (1.6%), respectively, with no evidence of departure from Hardy-Weinberg equilibrium (P ⫽ 0.31, Fisher exact test). The allele frequency of the 986S allele in this group (15.4%) was not different from that previously reported (16.3%) (12). Moreover, the coancestry coefficient ( ␪ p ⫽ 0.028) for the subsample used the present study and the subsample reported earlier did not differ significantly from zero (95% confidence limits: ⫺0.002 to 0.072), suggesting that the two samples were not genetically different at the CASR A986S locus. FIG. 1. Electrophoretic pattern of CASR amplicons generated by mutagenically separated PCR. Shown in lane 1 is an M3 ladder (Elchrom Scientific, Cham, Switzerland). In lanes 2 to 4, typical patterns for AA, AS, and SS samples are shown, where the A986 product is 217 bp, and the 986S product is 238 bp in length. Constant amplification products of 141 and 201 bp (homozygous G990 and homozygous Q1011, respectively) are also seen in this multiplex reaction that genotypes the two contiguous polymorphic sites (data not shown).

method, total protein by the modified Biuret technique, albumin by bromcresol green dye-binding assay, and inorganic phosphate by a modification of the phosphomolybdate reaction, according to the manufacturer’s instructions. Serum globulin (nonalbumin protein) was calculated as the difference between total protein and albumin concentrations. Statistical Analysis Genotype frequencies and equilibria (19) were analyzed using the GDA Program (P. O. Lewis, and D. Zaykin, 2000, Genetic Data Analysis: Computer Program for the Analysis of Allelic Data, Version 1.0 (d15), a free program distributed over the internet from the GDA Home Page at http://alleyn.eeb. uconn.edu/gda/). Biochemical covariates of serum total calcium were examined by conventional bivariate correlation analysis. Differences in biochemical values relative to genotype were tested using one-way ANOVA and Tukey post hoc comparisons. The con-

Clinical and Genetic Covariates of Total Calcium Bivariate Pearson correlation coefficients for age, height, weight, and serum biochemical data are summarized in matrix fashion (Table 1). The strong association between height and weight is expected, but neither variable was associated with age—also expected for this young adult cohort (18 to 35 years of age). Of the biochemical parameters, only serum creatinine showed a positive correlation with age, a well-documented phenomenon that may reflect agerelated changes in renal function (20). The primary analyte of interest, total calcium, showed the strongest association with albumin but was also correlated with total protein, globulin, inorganic phosphate, and creatinine, reflecting the importance of calcium-binding constituents in the determination of total calcium and the role of renal function in calcium regulation. Analysis of analyte data grouped by A986S genotype (Table 2) showed no differences for age or weight, and the apparent difference (P ⫽ 0.03) for height between A986S heterozygotes and both homozygous groups did not persist when the alpha statistic was adjusted by the Bonferroni method for multiple hypothesis testing. In contrast, univariate ANOVA for total serum calcium by A986S genotype (Fig. 2) was highly significant (F 1,386 ⫽ 16.5, P ⫽ 6.0 ⫻ 10 ⫺6 ). Mean serum calcium was significantly

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TABLE 1 Bivariate Pearson Correlation Matrix for Clinical Measures Clinical variable

Total calcium

Age

Weight

Height

Albumin

Total protein

Globulin

Inorganic phosphate

Age Weight Height Albumin Total protein Globulin Inorganic phosphate Creatinine

⫺0.104 ⫺0.100 ⫺0.060 0.652 0.690 0.344 0.352 0.251

0.066 ⫺0.018 ⫺0.060 ⫺0.038 0.004 ⫺0.007 0.150

0.392 ⴚ0.229 ⴚ0.212 ⫺0.013 ⫺0.102 0.006

⫺0.052 ⫺0.049 ⫺0.013 ⫺0.048 0.069

0.725 0.044 0.284 0.157

0.726 0.195 0.189

⫺0.004 0.124

0.203

Note. All correlation coefficients in italics are significant at P ⬍ 0.01; those in italics and bold are significant at P ⬍ 0.001.

greater (Tukey test) in AS heterozygotes (9.45 ⫾ 0.05 mg/dL, P ⫽ 0.002) or SS homozygotes (9.88 ⫾ 0.29 mg/dL, P ⫽ 0.015) in comparison to those with the AA homozygous genotype (9.23 ⫾ 0.04 mg/dL). The A986S Effect in a Multivariate Regression model of Serum Total Calcium Since total serum calcium shows strong covariation with other serum analytes, we conducted a stepwise linear regression analysis, including the A986S genotype, and the serum covariates—albumin, globulin, inorganic phosphate, and creatinine. In the final model (Table 3), the overall regression was highly significant (P ⬍ 10 ⫺10 ), including the A986S genotype (F 1,382 ⫽ 33.8, P ⫽ 1.7 ⫻ 10 ⫺8 ). In this model, approximately three-quarters (R 2 ⫽ 0.77) of the variance in serum calcium is attributed to the TABLE 2 Clinical and Biochemical Data (Mean ⴞ SE) by A986S Genotype

Age (year) Height (in) a Weight (lb) Albumin (g/dL) Total protein (g/dL) Globulin (g/dL) Serum creatinine (mg/dL) Inorganic phosphate (mg/dL)

AA (n ⫽ 274)

AS (n ⫽ 107)

SS (n ⫽ 6)

26.9 ⫾ 0.3 65.1 ⫾ 0.2 140 ⫾ 2 4.3 ⫾ 0.1 6.6 ⫾ 0.1 2.27 ⫾ 0.02

27.2 ⫾ 0.4 64.3 ⫾ 0.3 138 ⫾ 3 4.3 ⫾ 0.1 6.6 ⫾ 0.1 2.31 ⫾ 0.03

26.7 ⫾ 1.7 65.0 ⫾ 1.3 132 ⫾ 7 4.5 ⫾ 0.2 6.8 ⫾ 0.2 2.25 ⫾ 0.11

0.71 ⫾ 0.01

0.71 ⫾ 0.01

0.79 ⫾ 0.04

3.44 ⫾ 0.03

3.47 ⫾ 0.04

3.29 ⫾ 0.14

a Height of AS heterozygotes significantly different from AA heterozygotes by ANOVA and TUKEY test, P ⫽ 0.03 uncorrected for multiple testing.

five independent variables. Serum albumin (R 2 ⫽ 0.64) accounts for more than three-quarters (84%) of the explained variability, and the A986S genotype (R 2 ⫽ 0.04) accounts for nearly a third (32%) of the rest. DISCUSSION The biochemical and endocrine regulation of serum calcium has been the subject of intense scrutiny for many years, and there is no doubt that PTH is of paramount importance as the principal moment-tomoment modulator of extracellular calcium concentrations. Brown and colleagues (21) demonstrated that a key factor in the ability of the parathyroid gland to respond to hypocalcemia is the presence of a calcium-sensing receptor that transduces reduced extracellular calcium binding into intracellular events that stimulate PTH release. Less attention has been paid to constitutive or genetic factors in the determination of [Ca 2⫹] 0, although twin studies (1–3) indicate substantial heritability of total serum calcium. It has been suggested that the correlation between the presence of activating and inactivating muta-

FIG. 2. Serum total calcium (mean ⫾ SE) by A986S genotype.

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TABLE 3 Multivariate Regression Analysis of A986S Effect on Total Calcium a Variable

Coefficient ( ␤ˆ i )

SE

R2

t

P

Serum albumin Serum globulin A986S genotype Serum inorganic phosphate Serum creatinine

0.026 0.013 0.058 0.16 0.0015

0.002 0.001 0.01 0.032 0.001

0.431 0.1 0.04 0.032 0.006

16.5 9.10 5.91 5.04 2.42

⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 0.016

a Shown in the table are the estimate for the regression coefficient ( ␤ˆ i ) with standard error (SE), standardized variance (R 2 ), t statistic, and probability (P) by stepwise analysis, with a constant ( ␤ˆ 0 ) of 0.641 ⫾ 0.075. In the final model with all five variables, the overall regression (F 1,384 ⫽ 270.0) was highly significant (P ⬍⬍ 10 ⫺10 ).

tions of the CASR gene and altered concentrations of free and total calcium points to CASR as an attractive candidate locus for genetic determinants of extracellular calcium (6). Although the polymorphisms in exon 7 described by Heath et al. (A986S, R990G, and Q1011E) (15) do not involve the domains thought to be associated with altered binding of cation, two of the three encode amino acid changes at positions conserved in mammalian CASR orthologs. The fact that we were previously unable to identify any significant association between CASR polymorphisms and total calcium was related to the strong covariation with serum albumin, which binds about half of the serum calcium. Biochemical studies have confirmed that total calcium concentrations are also dependent on binding to other proteins and small molecules, particularly inorganic phosphate (22). Efforts to model the degree of binding in normal and pathologic states have met with variable success (22–24). In our initial study (12), we found that calcium concentrations were positively associated with the presence of the 986S allele, whether blood-ionized calcium was measured or total calcium was corrected for albumin and other cofactors (12). We had a large population of adults from which to sample (13), allowing us to test the hypothesis that a significant association with total calcium would emerge, if a larger independent subset of this population were analyzed. Neither the genotype distribution nor the biochemical data in this second subset reported here were different from the first, but the association between the 986S allele and increased total calcium was indeed highly significant (P ⬍ 10 ⫺5 ). When the effects of important biochemical covariates (creatinine, inorganic phosphate, globulin, albumin) were taken into account by multivariate regression modeling, the significance was substantially increased.

The clinical significance of this finding depends on three issues. The first is whether the association we have identified is confounded, either by population stratification or by ascertainment bias. As for the latter, we selected sequential samples from a group of healthy young adult women (18 –35 years of age) recruited for a study of genetic and clinical determinants of bone mineral density and excluded anyone whose current illness(es) or medication(s) might modify bone and mineral metabolism. We also analyzed only the subset of women who self-reported Caucasian ethnicity. While we cannot exclude population stratification, we have no evidence that this is so. Tests for Hardy-Weinberg disequilibrium were negative for the A986S marker as well as the R990G and Q1011E polymorphisms nearby (data not shown). The second issue is whether the association is specific to our population or can be generalized to others. Kanazawa et al. (25) found only one A986S heterozygote in a small sample of 31 Japanese high school students, and concluded that the CASR986 is unlikely to be significant in their population(s). They also reported a much higher frequency of the R990G polymorphism (57% of 88 chromosomes)—similar, in fact, to that in our sample of Asian Canadians (55% of 82 chromosomes) (7). Kanazawa et al. (26) also failed to find a significant association between circulating calcium and the R990G polymorphism in another study, but their sample size was small (n ⫽ 44) and their population (high school students) may have included adolescents who had not finished growing and therefore still had actively mineralizing skeletons. We too have observed a low frequency of the 986S allele in Afro-Americans (5% of 74 chromosomes) and the Q1011E polymorphism appears to have the highest prevalence (24% of 74 chromosomes) (7). Whether either the R990G or Q1011E

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polymorphism shows association with calcium remains to be seen. Is the increased prevalence of the 986S allele in Caucasians happenstance? We have previously suggested that the persistence of private, inactivating CASR mutations (genetically lethal in homozygotes) may have offered some heterozygote advantage in the past (9). This would also be so of the 986S polymorphism, if it conferred some resistance to the hypocalcemic effects of hypovitaminosis D caused by reduced sunlight exposure at higher latitudes among early Europeans, the predominant ancestors of our Caucasian population. If vitamin D and dietary calcium were limiting in the past, as historical records indicate, then there could have been positive selection for the “hypercalcemic” 986S allele. Tests of this hypothesis would require considerably more data for different Caucasian populations and detailed study of disequilibria among polymorphic loci closely linked to the CASR locus. The final issue is whether the effect is large enough to influence clinical status. At first glance, the fact that the A986S genotype explains only 4% of the total variance in serum calcium suggests not. Indeed, this study emphasizes the important role that the genetics of biochemical covariates may play. More than likely, genes regulating serum albumin (including the albumin gene itself) will prove to have the largest impact on total calcium. In this context, though, it is reassuring that the association of the CASR genotype, predicted to act on the ionized calcium fraction only, is significant in the genetic analysis of total calcium. In the physiological model relating PTH secretion to [Ca 2⫹] 0 (27), CASR mutations may affect either the calcium setpoint (ED 50), altering resting ionized calcium, or the degree of parathyroid gland responsiveness (slope), or both (27). It is therefore predictable that the effect of a CASR polymorphism may manifest as disease susceptibility, in part or completely related to parathyroid dysfunction, rather than in differences of fasting serum calcium concentrations. Some evidence for a predictive effect of CASR has been reported for urinary calcium in renal stone disease (28), bone mineral density of postmenopausal Japanese women (29), and primary hyperparathyroidism (30), but these reports await confirmation. In summary, the present study reaffirms the A986S polymorphism as a genetic predictor of [Ca 2⫹] 0. Clinical significance lies in its potential role as a predictor in diseases of bone and mineral metabolism. More rigorous tests of association should

therefore focus on well-defined populations and families with prevalent disorders of calcium regulation, including idiopathic hypercalciuria, hyperparathyroidism, and osteoporosis. ACKNOWLEDGMENTS Supported in part by grants from NSERC/DFC (CRD 22678599) and MRC Canada.

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