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received: 23 December 2015 accepted: 22 August 2016 Published: 14 September 2016

Genetically Low Vitamin D Levels, Bone Mineral Density, and Bone Metabolism Markers: a Mendelian Randomisation Study Shan-Shan Li1,2,*, Li-Hong Gao1,2,*, Xiao-Ya Zhang1,2, Jin-We He1,2, Wen-Zhen Fu1,2, Yu-Juan Liu1,2, Yun-Qiu  Hu1,2 & Zhen-Lin Zhang1,2 Low serum 25-hydroxyvitamin D (25OHD) is associated with osteoporosis and osteoporotic fracture, but it remains uncertain whether these associations are causal. We conducted a Mendelian randomization (MR) study of 1,824 postmenopausal Chinese women to examine whether the detected associations between serum 25OHD and bone mineral density (BMD) and bone metabolism markers were causal. In observational analyses, total serum 25OHD was positively associated with BMD at lumbar spine (P = 0.003), femoral neck (P = 0.006) and total hip (P = 0.005), and was inversely associated with intact parathyroid hormone (PTH) (P = 8.18E-09) and procollagen type 1 N-terminal propeptide (P1NP) (P = 0.020). By contract, the associations of bioavailable and free 25OHD with all tested outcomes were negligible (all P > 0.05). The use of four single nucleotide polymorphisms, GC-rs2282679, NADSYN1-rs12785878, CYP2R1-rs10741657 and CYP24A1-rs6013897, as candidate instrumental variables in MR analyses showed that none of the two stage least squares models provided evidence for associations between serum 25OHD and either BMD or bone metabolism markers (all P > 0.05). We suggest that after controlling for unidentified confounding factors in MR analyses, the associations between genetically low serum 25OHD and BMD and bone metabolism markers are unlikely to be causal. Vitamin D insufficiency is an increasingly prevalent public health issue worldwide and is regarded as one of the foremost modifiable risk factors for a several common diseases and conditions, including osteoporosis and osteoporotic fracture1–3. Serum 25-hydroxyvitamin D (25OHD) levels are the established clinical marker of vitamin D status. The findings from most observational studies have suggested associations between low serum 25OHD levels and secondary hyperparathyroidism, elevated levels of bone turnover markers and excessive bone loss1,3. Moreover, bone mineral density (BMD) and bone turnover markers are among the robust predictors of osteoporotic fracture risk4,5. However, meta-analyses of randomized controlled trials have provided little evidence that vitamin D supplements alone provide benefits of BMD improvement or fracture prevention6–8, thus suggesting vitamin D may not have a causal effect on bone health9,10. Notably, these findings require cautious interpretation. Observational investigations are susceptible to potential confounding factors and reverse causality11,12, whereas the dosage of vitamin D supplements, research duration, baseline vitamin D levels of the study population, and genetic factors inevitably affect the intervention response7,8,13. Therefore, whether these associations are causal remains uncertain. An alternative approach to a causality study is a Mendelian randomization (MR) analysis, an increasingly used method that draws a causal inference from the observational data. MR analyses assess genetic variants predicted risk factors to screen their causal effects on the outcomes of interest12,14. Because the genetic variants are randomly assorted at conception, similarly to randomized trials, the MR approach is largely free of both residual confounding factors and reverse causation. Moreover, the MR approach has successfully clarified the causal effect of low 25OHD levels on the increased risk of type 2 diabetes15 and of high urate levels on a reduced rate 1

Metabolic Bone Disease and Genetics Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China. 2Shanghai Key Clinical Center for Metabolic Disease, Shanghai 200233, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Z.-L.Z. (email: [email protected])

Scientific Reports | 6:33202 | DOI: 10.1038/srep33202

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Total sample (n = 1824)

Minimum

Age (years)

65.5 ±​  8.9

43.1

Maximum 94.9

Height (cm)

154.2 ±​  6.0

133.0

175.0

Weight (kg)

55.8 ±​  8.4

29.0

81.0

BMI (kg/m2)

23.5 ±​  3.3

14.2

36.5

Total 25OHD (ng/mL) Vitamin D binding protein (mg/L) Albumin (g/L)

18.3 (13.3–23.8)

4.0

57.4

152.9 (100.7–240.8)

43.7

482.9

46.0 (44.0–48.0)

21.0

55.0

Bioavailable 25OHD (ng/mL)

3.7 (2.4–5.5)

0.4

16.3

Free 25OHD (pg/mL)

8.8 (5.9–13.5)

1.1

38.9

40.7 (32.2–52.1)

17.4

100.3

PTH (pg/mL) P1NP (g/L)

57.0 (43.5–73.7)

16.4

143.0

392.5 (282.0–533.0)

88.0

1030.0

ALP (U/L)

72.9 ±​  16.8

23.0

112.0

Ca (mmol/L)

2.33 ±​  0.10

2.08

2.60

P (mmol/L)

1.16 ±​  0.14

0.80

1.60

Cr (μ​mol/L)

58.9 ±​  10.6

34.0

102.0

Beta-CTX (ng/L)

BUN (mmol/L)

5.1 ±​  1.3

1.9

10.2

Lumbar 1–4 BMD (g/cm2)

0.877 ±​  0.141

0.419

1.695

Femoral neck BMD (g/cm2)

0.722 ±​  0.110

0.362

1.301

Total hip BMD (g/cm2)

0.765 ±​  0.118

0.321

1.260

Table 1.  Baseline characteristics of the study participants. Normally distributed variables are presented as the means ±​ standard deviation, and non-normally distributed variables are presented as medians (interquartile range).

of Parkinson disease progression16, and has confirmed that genetically low urate levels and decreased BMD are not causally related17. Our previous study of 2,897 healthy Chinese subjects has suggested that the GC, CYP2R1 and NADSYN1 polymorphisms within the vitamin D metabolic pathway are genetic determinants of variations in serum 25OHD levels18, and these findings have provided a basis for our present MR analyses in a Chinese population. In this study, we sought to verify whether serum 25OHD levels had a strong causal effect on bone health by using the MR approach. First, we examined the observational associations between the total serum 25OHD and BMD and bone metabolism markers [i.e., intact parathyroid hormone (PTH), Beta-CrossLaps of type I collagen containing cross-linked C-telopeptide (Beta-CTX) and procollagen type 1 N-terminal propeptide (P1NP)] using ordinary least squares (OLS) models. Second, we calculated the free and bioavailable (free +​  albumin bound) 25OHD levels by using Vermeulen formulas based on directly measured values of the total serum 25OHD, vitamin D binding protein (DBP) and albumin levels19, and determined whether the bioavailable and free 25OHD levels were more closely associated with BMD and bone metabolism markers. Then, we determined whether the verified observational associations were causal by using different two stage least squares (TSLS) models based on the assumptions of the MR analyses12,14,20.

Results

General characteristics of the study population.  Of the 2,013 participants, 158 subjects (7.9%) were excluded because they had diseases or took medications that might affect bone or vitamin D metabolism. We excluded an additional 31 subjects (1.5%) with abnormal plasma glucose, serum calcium, or phosphorus levels and those with abnormal renal or liver function. 1,824 participants were included in our study, and their general characteristics are summarized in Table 1. The average age was 65.5 (SD 8.9) years, the average BMI was 23.5 (SD 3.3) kg/m2, and the median (25th and 75th percentiles) total serum 25OHD level was 18.3 (13.3, 23.8) ng/mL. Observational relationships between serum 25OHD and clinical traits.  OLS regression analyses

provided strong evidence of observational associations between the total serum 25OHD levels and BMD at different sites (i.e., lumbar spine at L1-L4, femoral neck, and total hip); these associations remained robust after adjusting for age, season and BMI (Table 2). The absolute changes in BMD at L1-L4, femoral neck, and total hip were 0.047 g/cm2 (P =​ 0.003), 0.031 g/cm2 (P =​ 0.006) and 0.034 g/cm2 (P =​ 0.005) per unit increase in adjusted total serum 25OHD, respectively. Subjects with lower total 25OHD levels were shown to have significantly higher PTH values (Beta =​  −​0.103, P =​ 8.18E-09) and P1NP values (Beta =​  −​0.088, P =​ 0.020) independent of age, season and BMI. However, no significant associations were observed between the serum levels of bioavailable or free 25OHD and BMD, PTH, Beta-CTX or P1NP (all P >​ 0.05) after controlling for age, season, and BMI in the OLS regression analyses (Supplementary Table 1). We conducted ANOVA tests for normal data and Kruskal-Wallis tests for non-normal data in the subsequent analyses to address possible bias resulting from the multicollinearity of the present analyses. However, we still did not identify any significant associations between the serum levels of bioavailable or free 25OHD and the tested outcomes (Supplementary Tables 2 and 3).

Scientific Reports | 6:33202 | DOI: 10.1038/srep33202

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P Valuea

Effect estimatesa

Effect estimatesb

P Valueb

Lumbar 1–4 BMD (g/cm )

0.043

0.008

0.047

0.003

Femoral neck BMD (g/cm2)

0.036

0.004

0.031

0.006

2

0.039

0.004

0.034

0.005

PTH (pg/mL)

−​0.114

2.302E-11

−​0.103

8.181E-09

Beta-CTX (ng/L)

−​0.029

0.350

−​0.023

0.483

P1NP (g/L)

−​0.082

0.029

−​0.088

0.020

Total hip BMD (g/cm2)

Table 2.  Observational associations between the total serum 25OHD levels and the clinical variables. The False Discovery Rate (FDR) method was used to control the family-wise error rate when multiple hypotheses tests were performed. The null hypothesis was tested using alpha =​ 0.05 (two-sided). Significant values are presented in bold. Serum PTH, Beta-CTX, P1NP, and 25OHD levels were log-transformed to approximate normality. aEffect estimates are presented as changes in the clinical variables per unit increase in the logtransformed serum 25OHD levels. bAdjusted for age, season and BMI.

Chromosome

Gene (SNP)

SNP Property

Allele

Functional Change

MAF

HWE test P Value

4

GC (rs4588)

Exon 11

G/T

p.Thr436Lys

0.333

0.755

4

GC (rs7041)

Exon 11

A/C

p.Asp432Glu

0.253

0.805

4

GC (rs2282679)

Intron 12

T/G

NA

0.338

0.747

4

GC (rs1155563)

Intron 1

T/C

NA

0.409

0.683

11

NADSYN1 (rs2276360)

Exon 3

G/C

p.Val74Leu

0.461

0.120

11

NADSYN1(rs12785878)

intron2

G/T

NA

0.462

0.177

11

CYP2R1 (rs2060793)

5′​-flanking

G/A

NA

0.379

1.000

11

CYP2R1(rs10741657)

5′​-flanking

G/A

NA

0.379

0.808

11

CYP2R1 (rs10766197)

5′​-flanking

G/A

NA

0.345

0.573

20

CYP24A1 (rs6013897)

3′​-flanking

T/A

NA

0.162

0.611

Table 3.  25OHD single nucleotide polymorphisms (SNPs) selected for this study. SNP, single nucleotide polymorphism; Allele, major allele/minor allele; NA, not available; MAF, minor allele frequency; HWE, HardyWeinberg Equilibrium.

Associations between SNPs with total serum 25OHD levels, clinical traits and potential confounding factors.  Table 3 presents the basic information pertaining to the SNPs. The minor allele fre-

quencies (MAFs) of the 10 SNPs selected in the present study were similar to the HapMap-CHB reference data, and none of the SNPs failed the quality control checks. Four SNPs (GC-rs2282679, NADSYN1-rs12785878, CYP2R1-rs10741657 and CYP24A1-rs6013897) were considered as candidate instrumental variables (IVs) by default on the basis of their repeatable genome-wide significant associations with the 25OHD levels from genome-wide association study (GWAS) data21. Because high linkage disequilibrium was identified in our study between GC-rs4588, NADSYN1-rs2276360 and CYP2R1-rs2060793 and candidate IVs GC-rs2282679 (r2 =​  0.95), NADSYN1-rs12785878 (r2 =​  0.99) and CYP2R1-rs10741657 (r2 =​ 0.99), respectively, we selected the last 3 SNPs as proxies in the subsequent MR analyses. For the remaining 3 SNPs, the minor alleles of both GC-rs1155563 and CYP2R1-rs10766197 were significantly related to decreased total serum 25OHD levels after adjusting for age, BMI and season of blood draw, but these fell below the significance threshold after False Discovery Rate (FDR) correction (Table 4). Therefore, these 3 SNPs were excluded from the subsequent analyses. We then examined whether the 4 selected IVs were associated with tested outcomes (i.e., BMD, PTH and P1NP) or potential confounding factors (i.e., age, BMI, Ca, P, Cr and BUN), and no significant evidence of associations was identified after FDR correction (Supplementary Tables 4 and 5). Therefore, 4 SNPs, including rs2282679, rs12785878, rs10741657 and rs6013897, were considered to be the genetic IVs in the subsequent MR analyses.

Evaluation of causal associations between 25OHD levels and clinical outcomes.  We used both

single instrument model and multiple instruments model in the present study. According to previous GWAS data21, GC-rs2282679 exhibited the strongest association with the 25OHD levels and was used as the IV in the single instrument model. The multiple instruments models, including unweighted allele scores model and weighted allele scores model, were performed on the basis of the sum of the number of effect alleles of the 4 candidate IVs. The variability in the log-transformed total serum 25OHD levels explained by each IV ranged from 0.7% to 1.1% (Table 5). The lowest relative TSLS/OLS bias ratio was observed in both the single instrument model and the weighted allele scores model. The F-statistic values, which indicated the instrument strength of the MR analyses, are shown in Table 5. As a rule of thumb, all 3 MR analyses models were considered to have strong instruments for the F-statistic values obtained from first stage regression analyses with values more than 10; thus, all 3 MR models were used to assess the causal association.

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SNP

Beta

SE

lower limit

rs7041

0.010

0.009

−​0.007

0.027

0.229

rs1155563

−​0.017

0.008

−​0.031

−​0.002

0.024

rs10766197

−​0.016

0.008

−​0.032

−​0.001

0.039

Table 4.  Effect of the genetic polymorphisms on the total serum 25OHD levels. SNP, single nucleotide polymorphism; Beta, regression coefficient; SE, standard error; CI, confidence interval. The False Discovery Rate (FDR) method was used to control the family-wise error rate when multiple hypotheses tests were performed. The null hypothesis was tested using alpha =​ 0.05 (two-sided). Significant values obtained after FDR correction are presented in bold. The analyses were performed under additive models by adjusting for age, season and BMI. The total serum 25OHD levels were log-transformed to approximate normality. Beta refers to the changes in the log-transformed total serum 25OHD levels per each additional copy of the minor allele.

R2

F-statistic

Relative TSLS/ OLS bias ratio

0.011

20.26

0.050

  Unweighted allele scores

0.007

12.84

0.078

  Weighted allele scores

0.011

20.26

0.050

Models Single instrument model  rs2282679 Multiple instruments model

Table 5.  Instrument strength and relative sample bias of the two stage least squares (TSLS) models on the total serum 25OHD levels. OLS, ordinary least squares. The multiple instruments model was based on the sum of the number of effect alleles of rs2282679, rs12785878, rs10741657, and rs6013897. The serum 25OHD levels were log-transformed to approximate normality.

As presented in Table 6, using the SNP (rs2282679) for the single instrument analyses, we found no significant evidence of a causal effect of the total serum 25OHD levels on BMD, PTH or P1NP (all P >​ 0.05), with SE values ranging from 0.056 to 0.139. The Hausman Test indicated significant differences in the IV estimates compared with the OLS estimates of the effect of the 25OHD levels on the 4 tested outcomes after FDR correction (all P 

Genetically Low Vitamin D Levels, Bone Mineral Density, and Bone Metabolism Markers: a Mendelian Randomisation Study.

Low serum 25-hydroxyvitamin D (25OHD) is associated with osteoporosis and osteoporotic fracture, but it remains uncertain whether these associations a...
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