¶ Writing group.
The authors have declared that no competing interests exist.
Conceived and designed the experiments: CMOS HWu QY KK WHLK HWa OB CSF MBoc. Performed the experiments: CMOS HWu QY KK IG AMZ AK CSt ZK TH GE LJL VG DEA LF DSS BMP OHF AHo AGU JCMW IJD JMS MP EMB PV SBe CH VV SBa IR OP JW HC JSK JCC AHa CSc MN RB UV PBM MJB JMT JMG TDS WH APM LL WM BOB BRW CG CM WHLK HWa OB CSF MBoc. Analyzed the data: CMOS HWu QY KK IG AMZ AK AT ZK YL TH JD KL AVS GE LJL VG TT GL AD FR LML LP FM JH AMac SBe CH VV JFW WZ AHo AGU FR KE SP MM FDE AMah WH APM MEK ACB CG WHLK HWa OB CSF MBoc. Contributed reagents/materials/analysis tools: IG YL VG EB DH AS DSS BMP AGU FR IJD JMS MP PV GD VV CH JSK JCC PBM MM SYS APM MEK TM WHLK HWa CSF MBoc. Wrote the paper: CMOS IG AK HWa OB CSF MBoc. Revising and reviewing the manuscript for important intellectual content: CMOS HWu QY KK IG AMZ AK CSto AT ZK MM AD WZ GE GL TT LP LML CH KL KM SP DF RS SU ACB MEK AMah FDE VG LJL AMac EB DEA CT YN MJB JMG JMT DSS BMP SBe PV VV AFW TZ MBob IK PN EMB KE JD TBH SBa DH ABS GG DR APA AR TM CM GD JMS JCC BOB BRW JH FM SHW HC APM OHF AHo AGU FR UV AHa RB WH SYS PL HH CSc PBM PG NP MC CG WM LL TDS AVS IR JFW OP IJD MP LF YL BK JSK JCMW MN WHLK HWa OB CSF MBoc.
Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from
Calcium is vital to many biological processes and its serum concentration is tightly regulated. Family studies have shown that serum calcium is under strong genetic control. Apart from
Normal calcium homeostasis is regulated by three major hormones acting on their corresponding receptors in gut, kidney, and bone: parathyroid hormone (PTH) release governed by the calcium-sensing receptor (CASR), calcitonin, and the active metabolite of vitamin D, 1,25(OH)2-D. Despite heritability estimates of 33–78%, the genetic determinants of serum calcium are poorly understood
The discovery analysis consisted of 39,400 individuals from 17 population-based cohorts of European descent (
Discovery analysis | Replication analysis | Meta-analysis | ||||||||||||||||||
Markers |
chr | Position | Nearby Genes | A1 | A2 | N | Freq A1 | Effect A1 | SE | N | Freq A1 | Effect A1 | SE | N | Freq A1 | Effect A1 | SE | |||
rs1801725 | 3 | 123486447 | t | g | 39400 | 0.15 | 0.069 | 0.004 | 6.5E-59 | 21654 | 0.15 | 0.076 | 0.007 | 3.6E-30 | 61054 | 0.15 | 0.071 | 0.004 | 8.9E-86 | |
rs1550532 | 2 | 233929587 | c | g | 39400 | 0.31 | 0.018 | 0.003 | 4.6E-08 | 21598 | 0.31 | 0.019 | 0.005 | 0.0002 | 60998 | 0.31 | 0.018 | 0.003 | 8.2E-11 | |
rs780094 | 2 | 27594741 | t | c | 39400 | 0.41 | 0.020 | 0.003 | 3.7E-11 | 21558 | 0.42 | 0.008 | 0.005 | 0.049 | 60958 | 0.42 | 0.017 | 0.003 | 1.3E-10 | |
rs10491003 | 10 | 9368657 | t | c | 38361 | 0.09 | 0.027 | 0.006 | 1.6E-06 | 21679 | 0.10 | 0.028 | 0.008 | 0.0003 | 60040 | 0.09 | 0.027 | 0.005 | 4.8E-09 | |
rs7481584 | 11 | 2985665 | a | g | 39400 | 0.29 | −0.021 | 0.003 | 9.2E-10 | 21611 | 0.30 | −0.013 | 0.005 | 0.008 | 61011 | 0.30 | −0.018 | 0.003 | 1.2E-10 | |
rs7336933 | 13 | 41457076 | a | g | 39400 | 0.15 | −0.023 | 0.004 | 1.6E-07 | 21528 | 0.14 | −0.022 | 0.007 | 0.0009 | 60928 | 0.15 | −0.022 | 0.004 | 9.1E-10 | |
rs1570669 | 20 | 52207834 | a | g | 39400 | 0.66 | −0.018 | 0.003 | 4.0 E-08 | 21566 | 0.66 | −0.020 | 0.005 | 4.5E-05 | 60966 | 0.66 | −0.018 | 0.003 | 9.1E-12 |
P values are corrected for inflation using genomic control. Replication criteria: overall genome-wide significance (
Chr, chromosome. Effect A1 = beta regression coefficient for allele A1; SE, standard error.
one-sided P values.
The
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 (
Fourteen SNPs from Stage 1 were sent for Stage 2 validation in ≤21,679 additional Europeans: the twelve independent (≥1 Mb apart) SNPs with lowest P values (6.5E-59 to 8.1E-06) in Europeans and two additional genome-wide significant loci (rs9447004 and rs10491003) from a combined sample including 8318 Indian-Asians (
We found no significant association of the 7 replicated SNPs known to provide reliable tags for copy number variations (CNVs) in people of European-descent from the Hypergene dataset. For all the SNPs, the calculated correlation was below 0.002. We also explored a list of SNPs tagging CNVs from the GIANT consortium. Out the 7 SNPs tested, only the rs1570669 was in slight linkage disequilibrium (r2 = 0.54) with one SNP of the WTCCC2 list (rs927651). The corresponding SNP tags the CNVR7875.1 CNV located 455b from the SNP of interest.
For each of the 7 replicated SNPs, we identified all proxy SNPs with r2>0.8 in HapMap CEU (releases 21, 22, and HapMap 3 version 2) using the online SNAP database (
Proposed functions of the genes mapping into the associated intervals (±250 kb) are in
We here summarize the information on genes located within ±250 kb from the top SNP at each locus. Because it is a gene dense region, details of genes located in the
rs10491003, located within a long non-coding RNA with
This region is located in the imprinted gene domain of 11p15.5, an important tumor suppressor gene region
In Indian-Asians, all 7 replicated SNPs had beta-coefficients that were direction-consistent with the primary analysis and 3 were statistically significant (
We conducted analyses of related bone mineral and endocrine phenotypic traits for the 7 replicated loci (
Lumbar bone density | Femoral bone density | Serum phosphorus | 25OH Vitamin D | Parathyroid hormone | |||||||||||||||||
Markers | Gene | A1 | N | Effect A1 | SE | N | Effect A1 | SE | N | Effect A1 | SE | N | zscore | N | Effect A1 | SE | |||||
T | 32948 | −0.011 | 0.012 | 0.4 | 16190 | 22537 | −0.426 | 0.7 | 4181 | ||||||||||||
C | 16190 | 20371 | −0.888 | 0.4 | 4181 | ||||||||||||||||
T | 32946 | −0.009 | 0.008 | 0.3 | 16190 | 22520 | −0.699 | 0.5 | 4181 | 0.0002 | 0.010 | 1.0 | |||||||||
T | 31797 | 0.007 | 0.016 | 0.6 | 32740 | 0.015 | 0.015 | 0.3 | 16190 | −0.001 | 0.010 | 0.9 | 22543 | −1.328 | 0.2 | 4181 | 0.018 | 0.018 | 0.3 | ||
A | 31667 | 0.013 | 0.009 | 0.2 | 32948 | 0.006 | 0.009 | 0.5 | 16190 | 0.011 | 0.006 | 0.08 | 20366 | −1.630 | 0.1 | 4181 | −0.006 | 0.011 | 0.6 | ||
A | 30992 | −0.006 | 0.013 | 0.7 | 32152 | 0.000 | 0.012 | 1.0 | 16190 | 0.0115 | 0.008 | 0.1 | 20437 | 0.648 | 0.5 | 4181 | −0.010 | 0.013 | 0.4 | ||
A | 31739 | −0.004 | 0.009 | 0.7 | 32900 | 16190 | 0.0040 | 0.006 | 0.5 | 20385 | 0.144 | 0.9 | 4181 |
NA, not available. P values<0.05 were considered as statistically significant. A1, effect allele. β, regression coefficient for allele A1, SE, standard error. P, two-sided P value. Zscore, z score.
We selected biologically plausible gene(s) at each locus for
The expression (based on delta CT [cycle threshold] normalized to actin) of the selected genes is compared to the expression of the
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
In order to determine regulation of gene expression by calcium intake, we measured gene expression levels in mice fed low and high calcium diets (0.17% vs. 1.69% calcium) for one week, with normal diet as control (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. *:
We have identified and replicated one known and six new loci for serum calcium near genes linked to bone metabolism and endocrine control of calcium. Of these, 4 loci (
The vast majority of total body calcium is bound in the skeleton as hydroxyapatite and other calcium-phosphate complexes
The A allele of rs1570669 (
We observed specific expression patterns of several genes in the mouse nephron:
Several of the newly identified loci harbor genes linked to the hormonal control of serum calcium. First, the association of
Strengths of this study are the large sample size and consistent mouse studies to support the statistical associations and advance our knowledge of the biology at these loci. Human and mice largely share physiological processes linked to calcium metabolism, including tissue-specific gene expression. Limitations include the lack of a direct marker of bone remodeling and the potential for bias in gene selection for experimental follow-up. Mice may display subtle differences in the regulation of the genes tested compared to humans.
We have identified and replicated one known and six new loci for serum calcium near genes linked to bone metabolism and endocrine control of serum calcium. Supporting experimental mouse studies suggest a role for dietary calcium in bone-specific gene expression. Further work is needed to identify the causal variants and to understand how they influence calcium homeostasis.
In each human study, the local institutional review board approved the study and participants signed written informed consent, including for DNA analyses. The experimental protocol in mice was approved by the local veterinarian authorities and fulfilled Swiss federal regulations for experiences with animals.
A list of all discovery and replication studies, their sample size, mean serum calcium levels, age and serum albumin as well as proportion of women can be found in
Detailed information on the genotyping plateforms and data cleaning procedures for each discovery and replication cohort can be found in
In each discovery study, genotyping was performed using a genome-wide chip and nearly 2.5 million SNPs were genotyped or imputed using the HapMap CEU panels release 22 or 21 as the reference. Each study applied quality control before imputation. Detailed imputation information is provided in
We conducted look-ups for femoral and lumbar bone density in the GEnetic Factors of OSteoporosis (GEFOS) dataset
The Hypergene dataset (a 4206 samples case-control study concerning hypertension genotyped using the Illumina 1M chip) has been used to call CNVs and to check their correlation with the SNPs of interest. The CNVs calls have been done using pennCNV software
We queried the AmiGo 1.8 gene ontology database for each gene located within ±250 kb of the seven replicated SNPs, including rs1801725 (
For each of the 7 replicated SNPs, we identified all proxy SNPs with r2>0.8 in HapMap CEU (releases 21, 22, and HapMap 3 vers. 2) using the online SNAP database (
The rs1801725 SNP encodes a missense variant in exon 7 of the CASR gene leading to an alanine to serine substitution (A986S). Given the key physiological role of CASR in calcium homeostasis (monogenic disorders of calcium balance), this gene was the logical candidate for analysis in mouse at this previously identified locus.
For the 6 newly identified loci, the precise rationale for gene selection varied from one locus to the other, but the main criteria was to focus on the most biologically relevant gene. Rs1550532 on chromosome 2 is an intronic SNP of
Rs780094, on chromosome 2, is located in intro 16 of
Rs10491003 on chromosome 10 is located within a long non-coding RNA. For this locus, we selected
Rs7481584 is located within
For rs7336933, we selected the two only genes (
Finally, rs1570669 is an intronic SNP of
As animal experiments started while the replication process was underway, we had also initially selected the following genes for analysis in mouse: RSG14 and
Five C57bl/6 mice (Janvier) per group were fed, for one week, three different diets in which the percentage of calcium were 0.17% (low calcium diet), 0.82% (normal calcium diet) and 1.69% (high calcium diet) and had free access to water. 12∶12 hours light/dark alternance was imposed. At the end of the week of the specific diet, spot urine were collected and mice were anesthetized. Blood was collected by retro-orbital puncture. Organs were immediately harvested and snap frozen. RNA was extracted using Trizol (Invitrogen) and reversed transcribed with PrimeScriptTM RT reagent Kit (Takara Bio Inc). Calcium, sodium, phosphate and creatinine in plasma and urine were analyzed at the central lab of the Lausanne University hospital using a Cobas-Mira analyzer (Roche).
A separate set of three mice was kept under normal calcium diet. Proximal Tubule (Prox), thick ascending limb of the loop of Henle (TAL), distal convoluted tubule and connecting tubule (DCT-CNT) and cortical collecting duct (CCD) were isolated by microdissection of the left kidney after the mice were perfused with Liberase TM (Roche Diagnostics)
Comparison of groups was performed using unpaired Student's t-test.
QQ-plot of uncorrected serum calcium GWAS meta-analysis. Quantile-quantile plot showing observed p-values of the uncorrected serum calcium meta-analysis vs. expected p values by chance. The second genomic control step was applied to correct for the post meta-analysis of λ = 1.03.
(PDF)
Regional association plot for the CASR locus. Regional association plot showing −log10 p-values for the association of all SNPs ordered by their chromosomal position with uncorrected serum calcium at the
(PDF)
Regional association plot for the newly identified loci. Regional association plot showing −log10 p-values for the association of all SNPs ordered by their chromosomal position with uncorrected serum calcium within the replicated loci. The −log10 P value for each SNP is colored according to the correlation of the corresponding SNP with the SNP showing the lowest p-value (index SNP) within the locus using different colors for selected levels of linkage disequilibrium (r2). Correlation structures correspond to HapMap 2 CEU.
(PDF)
Manhattan plot of corrected serum calcium. Manhattan plot showing −log10 (P values) for all SNPs analyzed, ordered by their chromosomal position. The values correspond to the association of albumin-corrected serum calcium, including age and sex as covariates in the model as well as study-specific covariates if needed.
(PDF)
QQ-plot of corrected serum calcium. Quantile-quantile plot showing observed p-values of the corrected serum calcium meta-analysis vs. expected P values by chance in Europeans at discovery. The second genomic control step was applied to correct for the post meta-analysis of λ = 1.03.
(PDF)
Relative expression of genes in non-replicated loci in kidney, duodenum and tibia. The expression (based on delta CT normalized to actin) of the selected genes is compared to the expression of the
(PDF)
Relative expression in segments of kidney tubules of genes located in non-replication loci. 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) of the selected genes is compared to the expression of the CASR gene in the PROX. Data are means of values obtained from 3 mice fed a normal diet.
(PDF)
Relative expression of genes in non-replicated loci under various calcium diets. Data are means± SEM of values obtained from 5 mice fed a low (0.17%) and high (1.69%) calcium diet compared to mice fed a normal calcium diet (0.82%). Expression levels were normalized to actin. Statistical difference was calculated using unpaired t-test. *: P value≤0.05 (low compared to high); §: P value≤0.05 (low compared to normal); #: P value≤0.05 (high compared to normal).
(PDF)
Characteristics of study participants in discovery and replication cohorts. Data are mean (SD) unless otherwise specified for each discovery and replication studies.
(DOCX)
SNPs brought forward for replication that did not replicate. Chr, chromosome. A1, effect allele. A2, non-effect allele. Effect A1, regression coefficient for the A1 allele. SE, standard error. Freq A1,frequency of allele A1.
(DOCX)
SNPs with P value<5*E-05 for uncorrected calcium in Europeans (discovery). Chr, chromosome. Position, position on build 36. A1, allele 1 (effect allele). A2, allele 2. Freq A1, frequency of allele 1. InRefGen, gene symbol if SNP is located within a specific gene.
(DOCX)
Comparison of association with uncorrected versus corrected serum calcium. Chr, chromosome. Freq A1, frequency of allele A1. Beta, regression coefficient for the A1 allele. SE, standard error. A1, allele 1 (effect allele). Only replicated loci are included in this table.
(DOCX)
Genome-wide significant loci for corrected calcium in Europeans (discovery). Chr, chromosome. Position, position on build 36. A1, allele 1 (effect allele). A2, allele 2. Freq A1, frequency of allele 1. InRefGen, gene symbol if SNP is located within a specific gene.
(DOCX)
eQTL analysis for the seven genome-wide replicated loci for serum calcium. We used the online eQTL database of the University of Chicago (
(DOCX)
Details on genes located in the GCKR genomic region.
(DOCX)
Gene Ontology classification (AmiGo). Data are GO numbers, ontology and mechanism/location from the AmiGo 1.8 gene ontology database for each gene located within ±250 kb of the seven replicated SNPs, including rs1801725 (CASR).
(DOCX)
OMIM disorders associated with the genes located within the replicated loci. This table includes all Mendelian disorders or other types of genetic disorders included in the OMIM database described for each gene located within ±250 kb of any of the six new loci and for
(DOCX)
Association of replicated serum calcium loci in other ethnic groups. Chr, chromosome. Position, position on build 36. A1, allele 1 (effect allele). A2, allele 2. Freq A1, frequency of allele 1. Effect A1, regression coefficient for the A1 allele. SE, standard error. NA, not available.
(DOCX)
Plasma and Urine electrolytes values by calcium diet in mice. Data are means ± SEM of values obtained from 3 to 5 mice. *: P value≤0.05 compared to normal or high calcium diet.
(DOCX)
Study information.
(DOCX)
Genotyping information for each cohort (discovery, replication and look-ups).
(DOCX)
Study specific acknowledgements.
(DOCX)
The full list of acknowledgments for each study is provided in the Supporting Information files (