The Leiden Longevity Study was approved by the medical ethics committee of the Leiden University Medical Center. Written informed consent was obtained from all participants.

We performed 3 sensitivity analyses. First, we excluded participants who used supple- ments. Second, we analyzed the data from a ran- dom subpopulation (279 offspring and 275 con- trols) of participants for whom we had information about sun exposure and tanning bed use; we adjusted for these variables in this analysis. Third, we restricted the analysis to par- ticipants included in the study as a couple to fur- ther minimize the possible effect of environ- mental differences.

Serum measurements

The in vitro quantitative determinations of 25(OH) vitamin D3 and intact parathyroid hor- mone levels in plasma (blood anticoagulated in ethylenediaminetetraacetic acid [EDTA]) were performed with electrochemiluminescence immunoassays on a Cobas e411 analyzer from Roche Diagnostics. Creatinine levels, which we used as a crude measure of kidney function, were measured by the kinetic alkaline picrate method, implemented on an Abbott ci8200 according to the manufacturer’s manual.

Using data for the entire study population, we repeated the analyses separately for each of the included SNPs. We also analyzed all 3 SNPs together with the potential confounding factors to test whether a difference in vitamin D level was driven by one of these genotypes.

Other variables

Height and weight were self-reported. Data about habitual vitamin D intake and vitamin D and mul- tivitamin supplement use (use or no use) were obtained via a validated food frequency question- naire.12 Information about disease history was obtained from the participants’ general practition- ers, and medication use was obtained from the participants’ pharmacies. Information on sun exposure and tanning bed use was obtained by a separate questionnaire in a subpopulation.

In all analyses, we corrected for familial rela- tionships among offspring, using residual weight. We considered p values below 0.05 to be statistically significant.

Results

Data on serum vitamin D levels were available for 2344 participants in the Leiden Longevity Study (1620 offspring and 724 controls). How- ever, data on genetics, self-reported weight and height, and data from the food frequency ques- tionnaire were missing for 582 (35.9%) offspring and 263 (36.3%) controls, leaving a total of 1038 offspring (from 380 families) and 461 controls for the analyses.

Genotyping

We determined the genotypes of offspring and controls at 3 single nucleotide polymorphisms (SNPs): rs2282679 (GC, group specific compo- nents, vitamin D binding protein), rs3829251 (NADSYN1, nicotinamide adenine dinucleotide synthetase 1) and rs2060793 (CYP2R1, cyto chrome P450, family 2, subfamily R, polypeptide 1). These SNPs have been shown in genome-wide association studies to be associated with vitamin D levels.11 We obtained genome-wide SNP data for the offspring and controls by use of Illumina Infinium HD Human660W-Quad BeadChips and Illumina OmniExpress BeadChips.13

The characteristics of the study population are shown in Table 1. The offspring were slightly older than the controls, but the groups were simi- lar in the percentage of women and BMI. Intake of vitamin D via food was similar in offspring and controls. The prevalence of type 2 diabetes and hypertension, as well as use of the corre- sponding medications, were lower in the off- spring group than in the control group.

Vitamin D levels

vitamin D levels between groups was also observed among participants who were excluded because of missing values (offspring 64.6 nmol/L v. controls 68.0 nmol/L; p = 0.04).

The level of vitamin D was inversely correlated with that of parathyroid hormone (p < 0.001) and was higher in participants with high sun exposure than in those with low sun exposure (72.5 nmol/L v. 62.9 nmol/L; p < 0.001). Participants who used a tanning bed had higher levels of vitamin D (1–5 times per year: 76.2 nmol/L; 6–10 times per year: 82.1 nmol/L) than those who never used a tan- ning bed (68.8 nmol/L; p < 0.001).

We found no difference in the serum levels of parathyroid hormone between offspring and con- trols (39.5 and 39.4 pg/mL, respectively; p = 0.98). In the matched analysis, offspring had lower serum parathyroid hormone levels (37.4 pg/mL) than controls (39.1 pg/mL; p = 0.049).

Using a model adjusted for age and sex, we found that offspring had lower levels of vitamin D than controls (64.3 v. 68.5 nmol/L; p = 0.002) (Table 2). These results did not change when we included the other possible confounding factors. The statistically significant difference between off- spring and controls persisted when we restricted the analyses to participants who did not use supple- ments (vitamin D or multivitamin), and when we also adjusted for sun exposure and tanning bed use in a random subpopulation. The results were also consistent in our matched analysis (participants who also had a partner included in the study).

Genetic variation associated with 25(OH) vitamin D levels

Vitamin D levels by genotype at SNPs in the CYP2R1, GC and NADSYN1 genes for offspring and controls are shown in Table 3 and Appendix 1 (available at www.cmaj.ca /lookup /suppl /doi: 10 .1503 /cmaj.120233/-/DC1). The common genetic variants of the CYP2R1 and GC genes were significantly associated with serum vitamin D levels in offspring and controls. The difference in vitamin D levels between the offspring and controls persisted when we stratified the analyses by genotype for the 3 SNPs, except for carriers of the homozygous variant genotype for the CYP2R1 and NADSYN1 genes (Table 3).

Vitamin D levels were not associated with disease history (e.g., type 2 diabetes, hyperten- sion and myocardial infarction) (Appendix 1, available at www.cmaj.ca /lookup /suppl /doi: 10 .1503 /cmaj.120233/-/DC1). The difference in