We performed a meta-analysis for genome-wide associations of serum calcium, determined by subtracting the estimated amount of calcium bound to albumin from the total serum calcium, to infer the amount of ionized calcium (see Materials and Methods
). Our study included four cohorts: (i) 5404 European individuals from the Cohorte Lausanne (CoLaus) 
, (ii) 5548 European and Indian Asian individuals from the London Life Sciences Population (LOLIPOP) Study from West London UK 
, (iii) 1196 European individuals from the InCHIANTI Study (Tuscany, Italy) 
, and (iv) 717 individuals of European descent from the Baltimore Longitudinal Study of Aging (BLSA) study based in the Baltimore-Washington DC area 
, totaling 12,865 participants (see for more detailed characteristics of each cohort).
Characteristics of participants, by study.
Genome-wide association scans were performed first independently for each cohort using linear regression and then the effect sizes from each cohort were meta-analyzed (see Materials and Methods
). Due to the possibility of population substructure obscuring effects of genetic variants, meta-analysis was performed separately for (i) combined European and Indian Asian cohorts (N
12,865) and restricted to cohorts of (ii) European (N
8,919), and (iii) Indian Asian descent (N
3,947). The meta-analyses yielded 100 SNPs from the combined cohorts, 70 SNPs when restricting to European cohorts and 22 SNPs restricting to Indian Asian cohorts that exceeded the genome-wide significance threshold of 5×10-8
() (the full list is provided in Table S2A, S2B, S2C
). All SNPs reaching statistical significance clustered around the CASR
locus at 3q13. The most significant SNP in the (i) combined and (ii) European meta-analyses was rs1801725 (p
, respectively) and in the (iii) Indian Asian meta-analysis was rs17251221 (p
). These two SNPs are less than 11 kb apart and are in high linkage disequilibrium with each other (r2
0.946, 0.494, 1.0, 1.0 in HapMap CEU, CHB, JPT, YRI, respectively), and therefore most likely derive from the same association signal. We find that rs1801725 explains 1.26% of the variance in serum calcium, with the effect sizes and standard errors of the serum calcium increasing T
allele in individual cohorts shown in and Table S3
. According to our additive model, each rs1801725 T
allele increases log10
serum calcium (in units mmol/L) by 3.61×10-3
, equivalent to a multiplicative effect of 1.008 on serum calcium (see also Table S2
). At an average serum calcium level of 2.25 mmol/L, each rs1801725 T
allele yields an increase of 0.01874 mmol/L, or 21% of one standard deviation of serum calcium levels in a normal population. The regional pattern of association of SNPs around the CASR
locus, and their linkage disequilibrium with rs1801725, are shown in . Of note, rs1042636, which has been associated with decreased serum calcium 
, also achieved genome-wide significance with the G minor allele associated with decreased serum calcuim (p
). However, conditional on the rs1801725 locus, located 12 bps upstream, the rs1042636 p-value became 3.32×10−4
, indicating that the two loci share contributions to serum calcium levels.
Genome-wide association results.
Comparison of rs1801725 significance across cohorts.
Regional association plot of the CASR locus.
To confirm the rs1801725 signal, we analyzed the association pattern with serum calcium in a separate cohort. We used a subset of 4,126 Icelandic individuals from the deCODE study 
with serum calcium measurements. We found the rs1801725 T
allele to be strongly associated with increased serum calcium (p
), replicating the key meta-analysis result.
While only the CASR
locus reached nominal genome-wide significance for association with serum calcium, the top regions with p<10-5
are shown in . These SNPs cover 12 regions, the significance of which is displayed across cohorts in Figure S3
. There were no SNPs in other candidate genes (which have previously been shown to be involved in disorders associated with disturbed serum calcium levels) that were associated with serum calcium at genome-wide significance. The most significant SNPs within 500 kb of the gene transcripts are shown in . Considering the set of 18,611 distinct SNPs mapping to the set of serum calcium candidate genes excluding CASR
, we find no significant association (at significance level 0.05 and applying the Bonferroni correction for multiple testing, giving a cut-off p-value of 2.69×10-6
, see also Figure S4
). Indeed, fixing the sample size and genome-wide significance threshold our study is well-powered (≥0.80) to detect SNPs explaining at least 0.31% of the variance. Therefore the common SNPs within the candidate genes (excluding CASR) likely play at best a small role in serum calcium regulation.
We analyzed the association of the top SNP with several calcium-related outcomes (coronary heart disease, myocardial infarction, hypertension, stroke, osteoarthritis, osteoporosis and kidney stones). The number of cases and controls for each outcome and each cohort is given in Table S4
. Logistic regression including age and pseudosex (see Materials and Methods
) as covariates did not find any significant association between rs1801725 and the calcium-related outcomes, after correcting for multiple testing (effect sizes and standard errors for the T
allele are listed in Table S5
). Power calculations show that given the sample sizes for the clinical traits above, our study has good power (≥0.80) to detect odds ratios of 1.20, 1.13, 1.77, 1.27, 1.27, 1.24 and 1.75, respectively. As the smallest p-values from calcium-related traits were for osteoarthritis and osteoporosis (bonferroni-corrected p
0.21, 0.44, respectively), we further investigated bone density traits. None of deCODE hip bone mineral density or spine bone mineral density (N
6657 and 6838, respectively) nor InCHIANTI total bone density, trabecular bone density, cortical bone density, cortical bone thickness or cortical bone area (N
1196) bonferroni-adjusted p-values for eight traits were significant.