Genes and Immunity (2014) 15, 578–583 © 2014 Macmillan Publishers Limited All rights reserved 1466-4879/14 www.nature.com/gene SHORT COMMUNICATION Genome-wide association study of vitamin D levels in children: replication in the Western Australian Pregnancy Cohort (Raine) study D Anderson1, BJ Holt1, CE Pennell2, PG Holt1, PH Hart1 and JM Blackwell1 This genome-wide association study (GWAS) utilises data from the Western Australian Pregnancy Cohort (Raine) Study for 25- hydroxyvitamin D (25(OH)D) levels measured in blood collected at age 6 years (n = 673) and at age 14 years (n = 1140). Replication of significantly associated genes from previous GWASs was found for both ages. Genome-wide significant associations were found both at age 6 and 14 with single nucleotide polymorphisms (SNPs) on chromosome 11p15 in PDE3B/CYP2R1 (age 6: rs1007392, P = 3.9 × 10 − 8; age14: rs11023332, P = 2.2 × 10 − 10) and on chromosome 4q13 in GC (age 6: rs17467825, P = 4.2 × 10 − 9; age14: rs1155563; P = 3.9 × 10 − 9). In addition, a novel association was observed at age 6 with SNPs on chromosome 7p15 near NPY (age 6: rs156299, P = 1.3 × 10 − 6) that could be of functional interest in highlighting alternative pathways for vitamin D metabolism in this age group and merits further analysis in other cohort studies. Genes and Immunity (2014) 15, 578–583; doi:10.1038/gene.2014.52; published online 11 September 2014 INTRODUCTION RESULTS AND DISCUSSION In humans, vitamin D is obtained primarily through exposure to Descriptive statistics of the study participants are shown in ultraviolet B radiation from sunlight and secondarily through a Table 1. Differences were seen between estimated 25(OH)D levels diet including vitamin D-rich foods or supplements.1 A sunny for the 12 serum samples measured with both enzyme climate does not ensure immunity to vitamin D deficiency. For immunoassay (EIA) and isotope-dilution liquid chromatography- example, a recent Australian population-based health survey tandem mass spectrometry at the age 6 years follow-up estimated prevalence of vitamin D deficiency (serum 25- (Supplementary Figure S1A) but good agreement is seen between hydroxyvitamin D (25(OH)D)o50 nmol l − 1) amongst adults to the two assays at the age 14 years follow-up (Supplementary be 23%.2 This is within the range of deficiency prevalence found in Figure S1B). In light of this, a larger sample of 50 sera from the age other national population-based studies carried out in the United 6 years follow-up were remeasured with liquid chromatography States, Canada, United Kingdom and New Zealand where separation coupled tandem mass spectrometry and it can be seen estimates of deficiency ranged from 18 to 36%.3 Detrimental that the EIA appears to overestimate 25(OH)D levels at higher effects of prolonged vitamin D deficiency on musculoskeletal concentrations (Supplementary Figure S1C), which was also found health include development of rickets in children and osteoma- to be the case in a previous study of 100 serum samples.13 The lacia in adults.4,5 Associations have also been found between 25 main results presented here for both the age 6 years and age 14 (OH)D levels and many other diseases6 including cancer, years follow-ups are for the GWAS performed on standardised 25 cardiovascular disease, influenza type A, rheumatoid arthritis, (OH)D levels (described in Methods; standardisation equations and both type 1 and type 2 diabetes mellitus. shown in Supplementary Figure S2) with results for the GWAS Previous twin and family-based studies provide evidence performed on 25(OH)D levels determined by EIA presented in the that 25(OH)D levels are determined by a significant genetic supplementary information. component, with estimates of heritability varying widely from 23 Manhattan plots of the GWAS analyses are shown in Figure 1 to 80%.7 A number of genome-wide association studies (GWASs) and Supplementary Figure S3. Quantile-quantile plots of the have identified genetic variants associated with 25(OH)D observed versus expected − log10 P-values did not reveal levels,8–11 with only one of these focussing on children.10 This evidence for systematic bias in either the age 6 year follow-up study presents results from a GWAS utilising data from The (inflation factor λ = 1.01; Supplementary Figure S4A) or age 14 year Western Australian Pregnancy Cohort (Raine) Study12 (hereinafter follow-up (inflation factor λ = 1.01; Supplementary Figure S4B) referred to as the Raine Study) for 25(OH)D levels measured results. Supplementary Tables S1 and S2 list the top 50 associated in blood collected at both the age 6 years and age 14 years single nucleotide polymorphisms (SNPs) for the age 6 years follow-ups. Replication of significantly associated genetic variants follow-up and the age 14 years follow-up, respectively. Table 2 with 25(OH)D levels from previously published GWASs was also summarises results for the top SNP in each of the genes of assessed. functional interest identified by these top 50 SNPs. Regional plots 1Telethon Kids Institute, The University of Western Australia, Subiaco, Western Australia, Australia and 2School of Women's and Infants' Health, The University of Western Australia, Perth, Western Australia, Australia. Correspondence: Professor JM Blackwell, The University of Western Australia, Telethon Kids Institute, 100 Roberts Road, Subiaco, 6008, Western Australia, Australia. E-mail: [email protected] Received 15 May 2014; revised 15 July 2014; accepted 17 July 2014; published online 11 September 2014 Novel pathways for vitamin D metabolism in children D Anderson et al 579 of association are shown in Figure 2 for both the age 6 years Table 1. Characteristics of the Raine Study participants at the age 6 follow-up and the age 14 years follow-up. years and age 14 years follow-upsa Genome-wide significant associations were found for SNPs fi Age 6 years Age 14 years located within intronic regions of the group-speci c component follow-up follow-up (vitamin D binding protein) (GC) gene on chromosome 4q13 for − (n = 673) (n = 1140) both the age 6 years (lowest P = 4.2 × 10 9, rs17467825, Figure 2a) and the age 14 years follow-ups (lowest P = 3.9 × 10 − 9, rs1155563, Male (n (%)) 364 (54) 592 (52) Figure 2c). The GC gene encodes a vitamin D binding protein that Age, years (mean (s.d.)) 5.9 (0.19) 14.1 (0.21) transports vitamin D to various tissues of the body.14 Significant BMI (median (IQR)) 16 (14.8–16.5) 20 (18.5–23.0) 25(OH)D, nmol l − 1 101 (84–123) 81 (68–98) associations were also found for SNPs located on chromosome (median (IQR)) 11p15 within either the phosphodiesterase 3B, cGMP-inhibited (PDE3B) or cytochrome P450, family 2, subfamily R, polypeptide 1 − Caucasian mother and father (n (%)) (CYP2R1) genes for both the age 6 years (lowest P = 3.9 × 10 8, Yes 645 (96) 1073 (94) rs1007392, Figure 2b) and the age 14 years follow-ups (lowest No 28 (4) 67 (6) P = 2.2 × 10 − 10, rs11023332, Figure 2d). The top associated SNPs b on chromosome 11p15 are located in an intron of PDE3B but the Season of blood collection (n (%)) true functional association is believed to be with CYP2R1 as these Summer 126 (19) 189 (17) Autumn 165 (25) 320 (28) SNPs are in strong linkage disequilibrium with the top associated Winter 205 (30) 308 (27) SNPs in CYP2R1 (Figure 2b and d). The CYP2R1 gene encodes an 15 Spring 177 (26) 323 (28) enzyme that converts vitamin D to 25(OH)D. Nevertheless, it is also plausible that the association could be with PDE3B because Abbreviations: BMI, body mass index; IQR, interquartile range; 25(OH)D, there is evidence that vitamin D and phosphodiesterase 3B are 25-hydroxyvitamin D. BMI is defined as weight(kg)/height(m2). aDescriptive both involved in a common pathway controlling lipid statistics are shown for those participants with genome-wide SNP 16,17 genotyping data. bThe four seasons are defined as Summer (December metabolism. Specifically, it is thought that parathyroid to February), Autumn (March to May), Winter (June to August) and Spring hormone promotes conversion of 25(OH)D to 1α,25-dihydroxyvi- (September to November). tamin D3 (1,25(OH)2D) which then stimulates calcium influx in adipocytes. This influx of calcium inhibits lipolysis through Figure 1. Manhattan plots showing the association between standardised 25(OH)D levels and SNP allele dosages for the age 6 years follow-up (a) and for the age 14 years follow-up (b). The top SNPs meeting genome-wide significance (P ⩽ 5×10− 8) are shown in red and SNPs within 400 kilobases of these top SNPs are highlighted in red. © 2014 Macmillan Publishers Limited Genes and Immunity (2014) 578 – 583 Novel pathways for vitamin D metabolism in children D Anderson et al 580 Table 2. Top associated SNPs in/near genes of functional interest for both the age 6 and age 14 years follow-ups ̂ SNP Chr Locationa Major/Minor allele MAFb Risk allele P-valuec eβ (95% CI)d Neighbouring gene(s)e (SNP functional class)f Age 6 years follow-up rs17467825 4 72605517 A/G 0.30 A 4.2 × 10 − 9 1.07 (1.05–1.09) GC rs10766192 11 14811686 A/G 0.41 A 4.0 × 10 − 8 1.06 (1.04–1.08) CYP2R1 Age 14 years follow-up rs1155563 4 72643488 T/C 0.28 C 3.9 × 10 − 9 0.94 (0.92–0.96) GC (intron variant) rs10500804 11 14910273 T/G 0.42 G 3.5 × 10 − 10 0.94 (0.92–0.96) CYP2R1 (intron variant) Abbreviations: SNP, single nucleotide polymorphism.