Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations

Abstract

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR, the genes associated with serum calcium are largely unknown. We conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. We characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression in bone in response to dietary calcium in mice. Our results shed new light on the genetics of calcium homeostasis.

Figure 1. Genome-wide association for serum calcium in discovery analysis in Europeans.

Manhattan plot showing −log₁₀(P values) for all SNPs in the discovery GWAS for uncorrected serum calcium in Europeans (N = 39,400), ordered by chromosomal position. The plot is truncated at −log10 P values of 10 (truncated −log₁₀P values for GCKR and CASR). The values correspond to the association of uncorrected serum calcium, including age and sex as covariates in the model as well as study-specific covariates if needed. The gene closest to the SNP with the lowest P value is listed at each locus. Six loci reached genome-wide significance (P<5E-08) at discovery analysis (GCKR, DGKD, CASR, VKORC1L1 (in grey on chromosome 7), CARS and CYP24A1. The seven loci that reached genome-wide significance at the combined analysis following replication are highlighted in red (GCKR, DGKD, CASR, GATA3, CARS, DGKH-KIAA0564 and CYP24A1).

Figure 2. Relative mRNA expression of replicated genes in three calcium-transporting tissues (kidney, duodenum, tibia).

The expression (based on delta CT [cycle threshold] normalized to actin) of the selected genes is compared to the expression of the CASR gene in the duodenum, thereby providing a relative expression. Cut-off was set at delta CT≤15. Data are means ± standard error of the mean (SEM) of values obtained from 5 mice fed a normal diet. GCKR was not expressed.

Figure 3. Relative mRNA expression of identified genes in kidney tubule segments.

The renal tubular segments analyzed were the proximal tubule (PROX), the thick ascending limb of the loop of Henle (TAL), the distal convoluted tubule and connecting tubule (DCT-CNT), and the cortical collecting duct (CCD). The expression (based on the delta CT [cycle threshold]) of the selected genes is compared to the expression of the CASR gene in the PROX, thereby providing a relative expression. Data are means of values obtained from 3 mice fed a normal diet. GCKR was not expressed.

Figure 4. Relative mRNA expression of identified genes from mice fed a low (0.17%) and high (1.69%) calcium diet compared to mice fed a normal calcium diet (0.82%).

Data are means± SEM of values obtained from 5 mice for each diet group. Expression levels were normalized to actin. Statistical significance of the difference between diets was calculated using unpaired t-test. *: P≤0.05 (low compared to high); §: P≤0.05 (low compared to normal); # P≤0.05 (high compared to normal).