- Email: [email protected]
Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study
Clinical Nutrition xxx (xxxx) xxx
Contents lists available at ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Randomized Control Trials
Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study Mohsen Mazidi a, Dimitri P. Mikhailidis b, Maciej Banach c, d, e, *, Abbas Dehghan f, ** a
Department of Biology and Biological Engineering, Food and Nutrition Science, Chalmers University of Technology, Gothenburg, Sweden Department of Clinical Biochemistry, Royal Free Campus, University College London Medical School, University College London (UCL), London, UK c Department of Hypertension, Chair of Nephrology and Hypertension, Medical University of Lodz, Poland d Polish Mother's Memorial Hospital Research Institute (PMMHRI), Lodz, Poland e Cardiovascular Research Centre, University of Zielona Gora, Zielona Gora, Poland f MRC-PHE Centre for Environment and Health, School of Public Health, Imperial College London, London, United Kingdom b
a r t i c l e i n f o
s u m m a r y
Article history: Received 24 April 2019 Accepted 3 December 2019
Background: Conventional observational studies have suggested that 25-hydroxyvitamin D (25(OH)D) is inversely associated with telomere shortening. We aimed to apply two-sample Mendelian randomization (MR) to assess the causal association between serum 25(OH) D and telomere length (TL). Methods: MR was implemented by using summary-level data from the largest genome-wide association studies (GWAS) on vitamin D (n ¼ 73 699) and TL (n ¼ 37 684). Inverse variance weighted method (IVW) was used to estimate the causal estimates. Weighted median (WM)-based method, and MR-Egger, leaveone-out were applied as sensitivity analysis. Results: The results of MR demonstrated no effect of 25(OH)D on TL (IVW ¼ b:-0.104, p ¼ 0.219, WM ¼ b:-0.109, p ¼ 0.188; MR Egger ¼ b:-0.127, p ¼ 0.506). None of the 25(OH)D-related single nucleotide polymorphisms (SNPs) were significantly associated with TL. Heterogeneity tests did not detect heterogeneity. Furthermore, MR pleiotropy residual sum and outlier (MR-PRESSO) did not highlight any outliers (p ¼ 0.424). Results of leave-one-out method demonstrated that the links are not driven because of the single SNPs. Conclusions: Our study does not support any causal effect of 25(OH) D on TL. © 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Telomere length Mendelian randomization 25-Hydroxyvitamin D 25(OH)D Aging
1. Introduction Telomeres are a repetitive DNA sequences that protect chromosomes from deterioration and fusion during mitosis [1]. They are sensitive to oxidative stress and inflammation [1]. Telomere length (TL) is considered as an indicator of chronological age and observational studies proposed it is related to non-communicable diseases and pre-mature death [2]. Studies have shown that low vitamin D levels might affect mortality [3]. Moreover, several biological effects such as inhibition of the inflammatory response and roles in non-skeletal tissue and cells have been attributed to 25-hydroxyvitamin D (25(OH)D)
[1,4e7]. The Mendelian Randomization (MR) approach is able to circumvent residual bias, confounding factors and reverse causation by using genetic variants that are associated with an exposure as instruments (25(OH)D) to test for associations with an outcome (¼TL) [8]. Thus, if vitamin D status is causally related to mortality risk, we have speculated that it may be exerting its effects via prevention of telomere attrition. The link between 25(OH)D and TL is controversial following results from observational studies [9e17]. Therefore, we explored whether higher 25(OH)D status limits the rate of telomere attrition in a causal manner. 25(OH)D concentrations could represent a modifiable treatment target for the maintenance of TL.
2. Methods * Corresponding author. Department of Hypertension, WAM University Hospital in Lodz, Medical University of Lodz, Zeromskiego 113; 90-549 Lodz, Poland. Fax: þ48 42 639 37 71. ** Corresponding author. E-mail addresses: [email protected] (M. Banach), a.dehghan@ imperial.ac.uk (A. Dehghan).
2.1. Study design A two-sample MR study design was used. In a 2-sample MR, the summary statistics were provided from various studies for the
https://doi.org/10.1016/j.clnu.2019.12.008 0261-5614/© 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
2
M. Mazidi et al. / Clinical Nutrition xxx (xxxx) xxx
association of the genetic instruments with the exposure and outcome. In our study, we obtained the summary statistics from the largest genome wide association studies (GWAS) on vitamin D (25(OH)D) (exposure [18]) and TL (outcome [19]). We applied methods to estimate the unbiased effect of vitamin D (25(OH)D) on TL. 2.2. Genetic predictors of exposures We used six SNPs identified to be associated with circulating 25(OH)D concentration by the SUNLIGHT meta-GWAS, which are samples of European ancestry (79 366 discovery samples and 42 757 replication samples) (Table 1). The genetic association of the instruments with 25(OH)D was obtained from a meta-analysis of 31 studies. The study does not provide detailed information on the demographics of the participants or their vitamin D (25(OH)D) levels. GWAS were performed within each cohort according to a uniform analysis plan. Additive genetic models using linear regression on natural-log-transformed 25(OH)D were fitted and a fixed-effects inverse variance weighted (IVW) meta-analysis across the contributing cohorts was performed [18]. 2.3. Association of genetic instruments with outcome We retrieved the association of the six genetic instruments with TL using data from the largest available published GWAS on TL [19]. All individuals were of European descent [19]. 2.4. Mendelian randomisation analysis We combined the effect of six instruments using inverse variance weighted (IVW) method as implemented in TwoSampleMR package running under R. We assessed the heterogeneity using Q value for IVW. To address potential effect of pleiotropic variants on the final effect estimate, we conducted sensitivity analysis including weighted median (WM) and MR-Egger. Sensitivity analysis was conducted using the leave-one-out method. The weighted median (WM) estimate, as the weighted median of the SNP-specific estimates, provides correct estimates as long as SNPs accounting for 50% of the weight are valid instruments. WM MR allows some variants to be invalid instruments provided at least half are valid instruments. It uses inverse variance weights and bootstrapping to estimate confidence intervals (CIs) [20]. MR-Egger has an ability to make estimates by assumption of all SNPs are invalid instruments as long as the assumption of instrument strength independent of direct effect (InSIDE) is satisfied [20]. MR-Egger allows free estimation of the intercept, although further assumptions, such as the independence between instrument strength and direct effects, cannot be easily verified. Average directional pleiotropy across genetic variants was assessed from the p-value of the intercept
Table 1 Summary results of the 6 genetic loci of serum vitamin D. SNP
Nearest gene
GX
GX SE
EA
OA
EAF
p-value
rs3755967 rs10741657 rs12785878 rs10745742 rs8018720 rs17216707
GC CYP2R1 NADSYN1/DHCR7 AMDHD1 SEC23A CYP24A1
0.089 0.031 0.036 0.019 0.019 0.026
0.0023 0.0022 0.0022 0.002 0.0027 0.0027
T A T T C T
C G G C G C
0.28 0.4 0.75 0.4 0.82 0.79
4.74E-343 2.05E-46 3.80E-62 2.10E-20 1.11E-11 8.14E-23
All of the serum vitamin D markers were associated at genome-wide significance (p < 5 108). EA: effect allele; OA: other allele, EAF: effect allele frequency; GX: the per-allele effect on standard deviation units of the telomere length; GX SE: standard error of GX.
term from MR-Egger [20]. Causal estimates in MR Egger are less precise than those obtained by using IVW MR [21]. Analysis using MR-Egger has a lower false positive rate but a higher false negative rate than IVW [22]. Further, to assess heterogeneity between individual genetic variant estimates, we used the Q0 heterogeneity statistic [23] and the MR pleiotropy residual sum and outlier (MR-PRESSO) test [23]. The Q0 statistic uses modified 2nd order weights that are a derivation of a Taylor series expansion and take into account uncertainty in both numerator and denominator of the instrumental variable ratio (this eases the no-measurement-error [NOME] assumption) [23]. The MR-PRESSO framework relies on the regression of variant-outcome associations on variant-exposure associations and implements a global heterogeneity test by comparing the observed distance (residual sums of squares) of all variants to the regression line with the distance expected under the null hypothesis of no pleiotropy [24]. In case of evidence of horizontal pleiotropy, the test compares individual variants’ expected and observed distributions to identify outlier variants. Further we applied on MR-Robust Adjusted Profile Score (RAPS) this method is able to correct for pleiotropy using robust adjusted profile scores. We consider as results, causal estimates that agreed in direction and magnitude across MR methods, pass nominal significance in IVW MR, and did not show evidence of bias from horizontal pleiotropy using heterogeneity tests. We used R version 3.4.2 (R Core Development Team 2017). The MR studies assume that the SNPs (instrumental variables) are associated with the outcome only via the exposure [25], so we performed sensitivity analysis excluding SNPs with potentially pleiotropic effects. To assess the instrumental variable analysis “exclusion-restriction” assumption we used Ensembl release (http://useast.ensembl.org/index.html). Ensembl contains a base of SNP phenotypes.
2.5. Ethics This investigation uses published or publicly available summary data with no involvement of participants in the study. No original data were collected for this manuscript. Ethical approval for each of the studies included in the investigation can be found in the original publications (including informed consent from each subject). The study conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
3. Results The list of all instruments associations for 25(OH)D is shown in Table 1. The results of MR, as beta-coefficient for interested outcomes per increase in 25(OH)D, demonstrate no effect on TL (MR Egger ¼ b:-0.127, SE: 0.174, p ¼ 0.506; WM ¼ b:-0.109, SE: 0.082, p ¼ 0.188; IVW ¼ b:-0.104, SE: 0.085, p ¼ 0.219; and RAPS ¼ b:0.105, SE:0.081, p ¼ 0.196, respectively). Results of the forest and scatter plots for each outcome are shown in Fig. 1A and B. None of the SNPs that were associated with 25(OH)D concentration were significantly associated with TL (Fig. 1). Heterogeneity tests highlighted no trace of heterogeneity (IVW Q ¼ 6.137, p-value ¼ 0.293; MR-Egger intercept ¼ 0.00121, p-value ¼ 0.886). MR-PRESSO did not highlight any outliers (p ¼ 0.424). Further the result of the MRRAPS (b: 0.105, p ¼ 0.196) was identical with the IVW (b: 0.104, p ¼ 0.219) prediction, which again indicated no chance of pleiotropy. Results of leave-one-out method demonstrated that the links are not driven because of the single SNPs.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
M. Mazidi et al. / Clinical Nutrition xxx (xxxx) xxx
3
significant determinant of the offspring's TL [15]. Results from the Nurses' Health Study (n ¼ 1424) also stated that higher plasma levels of 25(OH)D may be associated with longer TL [11]. Moreover, in the group of 2160 women at mean age 49.4 years it was suggested that higher vitamin D concentrations were associated with longer TL (r ¼ 0.07, p ¼ 0.001), and this relation persisted after adjustment for age (r ¼ 0.09, p < 0.0001) and other covariates (age, season of vitamin D measurement, menopausal status, use of hormone replacement therapy, and physical activity; p for trend across tertiles ¼ 0.003) [16]. Unmeasured or residual confounding is mentioned as the reasons for this controversy across observational studies (27). Our study, however, benefits from Mendelian Randomization approach, which is known for circumventing the confounding effect and reverse causation. Our analysis has certain strengths and limitations. Our approach, MR, is a powerful tool for detection of the causal impact, which makes our study superior to all former observational studies. Moreover, we benefited from largest GWAS on vitamin D and TL. However, MR analysis is known to have limited statistical power. Therefore, lack of finding in our analysis might be due to small causal effects that were not detectable in our study. Our study used genetic instruments and estimation of genetic associations using GWAS that were mainly based on sample of European ancestry. It is known that vitamin D concentrations and telomere dynamics vary by ethnicity. This might affect the generalizability of our findings and therefore the results of our study should be interpreted with caution for other ethnicities. In conclusions, our results found no evidence to support a causal association between 25(OH)D and TL, a marker of cellular senescence. Funding
Fig. 1. The forest (Fig. 1A) and scatter plots (Fig. 1B) of genetic associations with plasma 25-hydroxyvitamin D (25(OH)D) level against genetic associations with telomere length. The slopes of each line represent causal associations for each method.
This manuscript was written independently; no company or institution supported it financially. No professional writer was involved in the preparation of this analysis. Declaration of interest
4. Discussion This study was set out to investigate the causal association between 25(OH)D and telomere length, a biological index of aging. Given the lack of 25(OH)D measures in large scale TL studies, a twosample MR approach [26], that allowed genetic variants to be used as unconfounded measures of 25(OH)D, was used. Our results from MR analyses did not support any causal effect of 25(OH)D concentration on TL. Based on our best knowledge, there is no study available that have evaluated the link between 25(OH)D and TL in casual fashion, while available observational studies highlighted controversial results on the association between 25(OH)D and TL [9e17]. In line with our findings, a cross-sectional analysis of 25(OH)D and TL among 2483 men who were selected from Health Professionals Follow-up Study [10] reported that 25(OH)D was not associated with TL (multivariable-adjusted p-trend ¼ 0.69) [10]. In another cross-sectional analysis of associations of 25(OH)D and TL using data gathered on 5096 participants from Northern Finland Birth Cohort, Williams et al. stated that vitamin D status is unlikely to be an important determinant of TL [9]. In contrast, a cross sectional study between 4347 participants reported a possible positive association between 25(OH)D levels and TL [13]. Interestingly, crosssectional study that included 106 healthy pregnant women without adverse obstetric outcomes and their offspring, reported that maternal vitamin D concentration during pregnancy might be a
All authors have no conflict of interest to declare. Acknowledgments The material presented in this manuscript is original and has not been submitted for publication elsewhere. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.clnu.2019.12.008. References [1] Blackburn EH, Epel ES, Lin J. Human telomere biology: a contributory and interactive factor in aging, disease risks, and protection. Science (New York, NY) 2015;350(6265):1193e8. [2] Herrmann M, Pusceddu I, Marz W, Herrmann W. Telomere biology and agerelated diseases. Clin Chem Lab Med 2018;56(8):1210e22. [3] Afzal S, Brondum-Jacobsen P, Bojesen SE, Nordestgaard BG. Genetically low vitamin D concentrations and increased mortality: Mendelian randomisation analysis in three large cohorts. Br Med J 2014;349:g6330. [4] Ilincic B, Stokic E, Stosic Z, Kojic NE, Katsiki N, Mikhailidis DP, et al. Vitamin D status and circulating biomarkers of endothelial dysfunction and inflammation in non-diabetic obese individuals: a pilot study. Arch Med Sci : AMS 2017;13(1):53e60. [5] Faridi KF, Lupton JR, Martin SS, Banach M, Quispe R, Kulkarni K, et al. Vitamin D deficiency and non-lipid biomarkers of cardiovascular risk. Arch Med Sci : AMS 2017;13(4):732e7.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
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[6] Mazidi M, Karimi E, Rezaie P, Vatanparast H. The impact of vitamin D supplement intake on vascular endothelial function; a systematic review and meta-analysis of randomized controlled trials. Food Nutr Res 2017;61(1): 1273574. [7] Calton EK, Keane KN, Newsholme P, Zhao Y, Soares MJ. The impact of cholecalciferol supplementation on the systemic inflammatory profile: a systematic review and meta-analysis of high-quality randomized controlled trials. Eur J Clin Nutr 2017;71(8):931e43. [8] Smith GD, Ebrahim S. Mendelian randomization': can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol 2003;32(1):1e22. [9] Williams DM, Palaniswamy S, Sebert S, Buxton JL, Blakemore AI, Hypponen E, et al. 25-Hydroxyvitamin D concentration and leukocyte telomere length in young adults: findings from the Northern Finland Birth cohort 1966. Am J Epidemiol 2016;183(3):191e8. [10] Julin B, Shui IM, Prescott J, Giovannucci EL, De Vivo I. Plasma vitamin D biomarkers and leukocyte telomere length in men. Eur J Nutr 2017;56(2):501e8. [11] Liu JJ, Prescott J, Giovannucci E, Hankinson SE, Rosner B, Han J, et al. Plasma vitamin D biomarkers and leukocyte telomere length. Am J Epidemiol 2013;177(12):1411e7. [12] Schottker B, Hagen L, Zhang Y, Gao X, Holleczek B, Gao X, et al. Serum 25hydroxyvitamin D levels as an aging marker: strong associations with age and all-cause mortality independent from telomere length, epigenetic age acceleration, and 8-isoprostane levels. J Gerontol Ser A Biol Med Sci 2019;74(1):121e8. [13] Mazidi M, Michos ED, Banach M. The association of telomere length and serum 25-hydroxyvitamin D levels in US adults: the National Health and Nutrition Examination Survey. Arch Med Sci 2017;13(1):61e5. [14] Beilfuss J, Camargo Jr CA, Kamycheva E. Serum 25-hydroxyvitamin D has a modest positive association with leukocyte telomere length in middle-aged US adults. J Nutr 2017;147(4):514e20. [15] Kim JH, Kim GJ, Lee D, Ko JH, Lim I, Bang H, et al. Higher maternal vitamin D concentrations are associated with longer leukocyte telomeres in newborns. Matern Child Nutr 2018;14(1).
[16] Richards JB, Valdes AM, Gardner JP, Paximadas D, Kimura M, Nessa A, et al. Higher serum vitamin D concentrations are associated with longer leukocyte telomere length in women. Am J Clin Nutr 2007;86(5):1420e5. [17] Liu JJ, Cahoon EK, Linet MS, Little MP, Dagnall CL, Higson H, et al. Relationship between plasma 25-hydroxymitamin D and leucocyte telomere length by sex and race in a US study. Br J Nutr 2016;116(6):953e60. [18] Jiang X, O'Reilly PF, Aschard H, Hsu YH, Richards JB, Dupuis J, et al. Genomewide association study in 79,366 European-ancestry individuals informs the genetic architecture of 25-hydroxyvitamin D levels. Nat Commun 2018;9(1): 260. [19] Codd V, Nelson CP, Albrecht E, Mangino M, Deelen J, Buxton JL, et al. Identification of seven loci affecting mean telomere length and their association with disease. Nat Genet 2013;45(4):422e7. 7e1-2. [20] Bowden J, Davey Smith G, Haycock PC, Burgess S. Consistent estimation in mendelian randomization with some invalid instruments using a weighted median estimator. Genet Epidemiol 2016;40(4):304e14. [21] Bowden J, Davey Smith G, Burgess S. Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int J Epidemiol 2015;44(2):512e25. [22] Burgess S, Bowden J, Fall T, Ingelsson E, Thompson SG. Sensitivity analyses for robust causal inference from mendelian randomization analyses with multiple genetic variants. Epidemiology 2017;28(1):30e42. [23] Bowden J, Del Greco MF, Minelli C, Davey Smith G, Sheehan N, Thompson J. A framework for the investigation of pleiotropy in two-sample summary data Mendelian randomization. Stat Med 2017;36(11):1783e802. [24] Verbanck M, Chen CY, Neale B, Do R. Detection of widespread horizontal pleiotropy in causal relationships inferred from Mendelian randomization between complex traits and diseases. Nat Genet 2018;50(5):693e8. [25] Lawlor DA, Harbord RM, Sterne JA, Timpson N, Davey Smith G. Mendelian randomization: using genes as instruments for making causal inferences in epidemiology. Stat Med 2008;27(8):1133e63. [26] Burgess S, Scott RA, Timpson NJ, Davey Smith G, Thompson SG. Using published data in Mendelian randomization: a blueprint for efficient identification of causal risk factors. Eur J Epidemiol 2015;30(7):543e52.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
Contents lists available at ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Randomized Control Trials
Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study Mohsen Mazidi a, Dimitri P. Mikhailidis b, Maciej Banach c, d, e, *, Abbas Dehghan f, ** a
Department of Biology and Biological Engineering, Food and Nutrition Science, Chalmers University of Technology, Gothenburg, Sweden Department of Clinical Biochemistry, Royal Free Campus, University College London Medical School, University College London (UCL), London, UK c Department of Hypertension, Chair of Nephrology and Hypertension, Medical University of Lodz, Poland d Polish Mother's Memorial Hospital Research Institute (PMMHRI), Lodz, Poland e Cardiovascular Research Centre, University of Zielona Gora, Zielona Gora, Poland f MRC-PHE Centre for Environment and Health, School of Public Health, Imperial College London, London, United Kingdom b
a r t i c l e i n f o
s u m m a r y
Article history: Received 24 April 2019 Accepted 3 December 2019
Background: Conventional observational studies have suggested that 25-hydroxyvitamin D (25(OH)D) is inversely associated with telomere shortening. We aimed to apply two-sample Mendelian randomization (MR) to assess the causal association between serum 25(OH) D and telomere length (TL). Methods: MR was implemented by using summary-level data from the largest genome-wide association studies (GWAS) on vitamin D (n ¼ 73 699) and TL (n ¼ 37 684). Inverse variance weighted method (IVW) was used to estimate the causal estimates. Weighted median (WM)-based method, and MR-Egger, leaveone-out were applied as sensitivity analysis. Results: The results of MR demonstrated no effect of 25(OH)D on TL (IVW ¼ b:-0.104, p ¼ 0.219, WM ¼ b:-0.109, p ¼ 0.188; MR Egger ¼ b:-0.127, p ¼ 0.506). None of the 25(OH)D-related single nucleotide polymorphisms (SNPs) were significantly associated with TL. Heterogeneity tests did not detect heterogeneity. Furthermore, MR pleiotropy residual sum and outlier (MR-PRESSO) did not highlight any outliers (p ¼ 0.424). Results of leave-one-out method demonstrated that the links are not driven because of the single SNPs. Conclusions: Our study does not support any causal effect of 25(OH) D on TL. © 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Telomere length Mendelian randomization 25-Hydroxyvitamin D 25(OH)D Aging
1. Introduction Telomeres are a repetitive DNA sequences that protect chromosomes from deterioration and fusion during mitosis [1]. They are sensitive to oxidative stress and inflammation [1]. Telomere length (TL) is considered as an indicator of chronological age and observational studies proposed it is related to non-communicable diseases and pre-mature death [2]. Studies have shown that low vitamin D levels might affect mortality [3]. Moreover, several biological effects such as inhibition of the inflammatory response and roles in non-skeletal tissue and cells have been attributed to 25-hydroxyvitamin D (25(OH)D)
[1,4e7]. The Mendelian Randomization (MR) approach is able to circumvent residual bias, confounding factors and reverse causation by using genetic variants that are associated with an exposure as instruments (25(OH)D) to test for associations with an outcome (¼TL) [8]. Thus, if vitamin D status is causally related to mortality risk, we have speculated that it may be exerting its effects via prevention of telomere attrition. The link between 25(OH)D and TL is controversial following results from observational studies [9e17]. Therefore, we explored whether higher 25(OH)D status limits the rate of telomere attrition in a causal manner. 25(OH)D concentrations could represent a modifiable treatment target for the maintenance of TL.
2. Methods * Corresponding author. Department of Hypertension, WAM University Hospital in Lodz, Medical University of Lodz, Zeromskiego 113; 90-549 Lodz, Poland. Fax: þ48 42 639 37 71. ** Corresponding author. E-mail addresses: [email protected] (M. Banach), a.dehghan@ imperial.ac.uk (A. Dehghan).
2.1. Study design A two-sample MR study design was used. In a 2-sample MR, the summary statistics were provided from various studies for the
https://doi.org/10.1016/j.clnu.2019.12.008 0261-5614/© 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
2
M. Mazidi et al. / Clinical Nutrition xxx (xxxx) xxx
association of the genetic instruments with the exposure and outcome. In our study, we obtained the summary statistics from the largest genome wide association studies (GWAS) on vitamin D (25(OH)D) (exposure [18]) and TL (outcome [19]). We applied methods to estimate the unbiased effect of vitamin D (25(OH)D) on TL. 2.2. Genetic predictors of exposures We used six SNPs identified to be associated with circulating 25(OH)D concentration by the SUNLIGHT meta-GWAS, which are samples of European ancestry (79 366 discovery samples and 42 757 replication samples) (Table 1). The genetic association of the instruments with 25(OH)D was obtained from a meta-analysis of 31 studies. The study does not provide detailed information on the demographics of the participants or their vitamin D (25(OH)D) levels. GWAS were performed within each cohort according to a uniform analysis plan. Additive genetic models using linear regression on natural-log-transformed 25(OH)D were fitted and a fixed-effects inverse variance weighted (IVW) meta-analysis across the contributing cohorts was performed [18]. 2.3. Association of genetic instruments with outcome We retrieved the association of the six genetic instruments with TL using data from the largest available published GWAS on TL [19]. All individuals were of European descent [19]. 2.4. Mendelian randomisation analysis We combined the effect of six instruments using inverse variance weighted (IVW) method as implemented in TwoSampleMR package running under R. We assessed the heterogeneity using Q value for IVW. To address potential effect of pleiotropic variants on the final effect estimate, we conducted sensitivity analysis including weighted median (WM) and MR-Egger. Sensitivity analysis was conducted using the leave-one-out method. The weighted median (WM) estimate, as the weighted median of the SNP-specific estimates, provides correct estimates as long as SNPs accounting for 50% of the weight are valid instruments. WM MR allows some variants to be invalid instruments provided at least half are valid instruments. It uses inverse variance weights and bootstrapping to estimate confidence intervals (CIs) [20]. MR-Egger has an ability to make estimates by assumption of all SNPs are invalid instruments as long as the assumption of instrument strength independent of direct effect (InSIDE) is satisfied [20]. MR-Egger allows free estimation of the intercept, although further assumptions, such as the independence between instrument strength and direct effects, cannot be easily verified. Average directional pleiotropy across genetic variants was assessed from the p-value of the intercept
Table 1 Summary results of the 6 genetic loci of serum vitamin D. SNP
Nearest gene
GX
GX SE
EA
OA
EAF
p-value
rs3755967 rs10741657 rs12785878 rs10745742 rs8018720 rs17216707
GC CYP2R1 NADSYN1/DHCR7 AMDHD1 SEC23A CYP24A1
0.089 0.031 0.036 0.019 0.019 0.026
0.0023 0.0022 0.0022 0.002 0.0027 0.0027
T A T T C T
C G G C G C
0.28 0.4 0.75 0.4 0.82 0.79
4.74E-343 2.05E-46 3.80E-62 2.10E-20 1.11E-11 8.14E-23
All of the serum vitamin D markers were associated at genome-wide significance (p < 5 108). EA: effect allele; OA: other allele, EAF: effect allele frequency; GX: the per-allele effect on standard deviation units of the telomere length; GX SE: standard error of GX.
term from MR-Egger [20]. Causal estimates in MR Egger are less precise than those obtained by using IVW MR [21]. Analysis using MR-Egger has a lower false positive rate but a higher false negative rate than IVW [22]. Further, to assess heterogeneity between individual genetic variant estimates, we used the Q0 heterogeneity statistic [23] and the MR pleiotropy residual sum and outlier (MR-PRESSO) test [23]. The Q0 statistic uses modified 2nd order weights that are a derivation of a Taylor series expansion and take into account uncertainty in both numerator and denominator of the instrumental variable ratio (this eases the no-measurement-error [NOME] assumption) [23]. The MR-PRESSO framework relies on the regression of variant-outcome associations on variant-exposure associations and implements a global heterogeneity test by comparing the observed distance (residual sums of squares) of all variants to the regression line with the distance expected under the null hypothesis of no pleiotropy [24]. In case of evidence of horizontal pleiotropy, the test compares individual variants’ expected and observed distributions to identify outlier variants. Further we applied on MR-Robust Adjusted Profile Score (RAPS) this method is able to correct for pleiotropy using robust adjusted profile scores. We consider as results, causal estimates that agreed in direction and magnitude across MR methods, pass nominal significance in IVW MR, and did not show evidence of bias from horizontal pleiotropy using heterogeneity tests. We used R version 3.4.2 (R Core Development Team 2017). The MR studies assume that the SNPs (instrumental variables) are associated with the outcome only via the exposure [25], so we performed sensitivity analysis excluding SNPs with potentially pleiotropic effects. To assess the instrumental variable analysis “exclusion-restriction” assumption we used Ensembl release (http://useast.ensembl.org/index.html). Ensembl contains a base of SNP phenotypes.
2.5. Ethics This investigation uses published or publicly available summary data with no involvement of participants in the study. No original data were collected for this manuscript. Ethical approval for each of the studies included in the investigation can be found in the original publications (including informed consent from each subject). The study conforms to the ethical guidelines of the 1975 Declaration of Helsinki.
3. Results The list of all instruments associations for 25(OH)D is shown in Table 1. The results of MR, as beta-coefficient for interested outcomes per increase in 25(OH)D, demonstrate no effect on TL (MR Egger ¼ b:-0.127, SE: 0.174, p ¼ 0.506; WM ¼ b:-0.109, SE: 0.082, p ¼ 0.188; IVW ¼ b:-0.104, SE: 0.085, p ¼ 0.219; and RAPS ¼ b:0.105, SE:0.081, p ¼ 0.196, respectively). Results of the forest and scatter plots for each outcome are shown in Fig. 1A and B. None of the SNPs that were associated with 25(OH)D concentration were significantly associated with TL (Fig. 1). Heterogeneity tests highlighted no trace of heterogeneity (IVW Q ¼ 6.137, p-value ¼ 0.293; MR-Egger intercept ¼ 0.00121, p-value ¼ 0.886). MR-PRESSO did not highlight any outliers (p ¼ 0.424). Further the result of the MRRAPS (b: 0.105, p ¼ 0.196) was identical with the IVW (b: 0.104, p ¼ 0.219) prediction, which again indicated no chance of pleiotropy. Results of leave-one-out method demonstrated that the links are not driven because of the single SNPs.
Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008
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significant determinant of the offspring's TL [15]. Results from the Nurses' Health Study (n ¼ 1424) also stated that higher plasma levels of 25(OH)D may be associated with longer TL [11]. Moreover, in the group of 2160 women at mean age 49.4 years it was suggested that higher vitamin D concentrations were associated with longer TL (r ¼ 0.07, p ¼ 0.001), and this relation persisted after adjustment for age (r ¼ 0.09, p < 0.0001) and other covariates (age, season of vitamin D measurement, menopausal status, use of hormone replacement therapy, and physical activity; p for trend across tertiles ¼ 0.003) [16]. Unmeasured or residual confounding is mentioned as the reasons for this controversy across observational studies (27). Our study, however, benefits from Mendelian Randomization approach, which is known for circumventing the confounding effect and reverse causation. Our analysis has certain strengths and limitations. Our approach, MR, is a powerful tool for detection of the causal impact, which makes our study superior to all former observational studies. Moreover, we benefited from largest GWAS on vitamin D and TL. However, MR analysis is known to have limited statistical power. Therefore, lack of finding in our analysis might be due to small causal effects that were not detectable in our study. Our study used genetic instruments and estimation of genetic associations using GWAS that were mainly based on sample of European ancestry. It is known that vitamin D concentrations and telomere dynamics vary by ethnicity. This might affect the generalizability of our findings and therefore the results of our study should be interpreted with caution for other ethnicities. In conclusions, our results found no evidence to support a causal association between 25(OH)D and TL, a marker of cellular senescence. Funding
Fig. 1. The forest (Fig. 1A) and scatter plots (Fig. 1B) of genetic associations with plasma 25-hydroxyvitamin D (25(OH)D) level against genetic associations with telomere length. The slopes of each line represent causal associations for each method.
This manuscript was written independently; no company or institution supported it financially. No professional writer was involved in the preparation of this analysis. Declaration of interest
4. Discussion This study was set out to investigate the causal association between 25(OH)D and telomere length, a biological index of aging. Given the lack of 25(OH)D measures in large scale TL studies, a twosample MR approach [26], that allowed genetic variants to be used as unconfounded measures of 25(OH)D, was used. Our results from MR analyses did not support any causal effect of 25(OH)D concentration on TL. Based on our best knowledge, there is no study available that have evaluated the link between 25(OH)D and TL in casual fashion, while available observational studies highlighted controversial results on the association between 25(OH)D and TL [9e17]. In line with our findings, a cross-sectional analysis of 25(OH)D and TL among 2483 men who were selected from Health Professionals Follow-up Study [10] reported that 25(OH)D was not associated with TL (multivariable-adjusted p-trend ¼ 0.69) [10]. In another cross-sectional analysis of associations of 25(OH)D and TL using data gathered on 5096 participants from Northern Finland Birth Cohort, Williams et al. stated that vitamin D status is unlikely to be an important determinant of TL [9]. In contrast, a cross sectional study between 4347 participants reported a possible positive association between 25(OH)D levels and TL [13]. Interestingly, crosssectional study that included 106 healthy pregnant women without adverse obstetric outcomes and their offspring, reported that maternal vitamin D concentration during pregnancy might be a
All authors have no conflict of interest to declare. Acknowledgments The material presented in this manuscript is original and has not been submitted for publication elsewhere. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.clnu.2019.12.008. References [1] Blackburn EH, Epel ES, Lin J. Human telomere biology: a contributory and interactive factor in aging, disease risks, and protection. Science (New York, NY) 2015;350(6265):1193e8. [2] Herrmann M, Pusceddu I, Marz W, Herrmann W. Telomere biology and agerelated diseases. Clin Chem Lab Med 2018;56(8):1210e22. [3] Afzal S, Brondum-Jacobsen P, Bojesen SE, Nordestgaard BG. Genetically low vitamin D concentrations and increased mortality: Mendelian randomisation analysis in three large cohorts. Br Med J 2014;349:g6330. [4] Ilincic B, Stokic E, Stosic Z, Kojic NE, Katsiki N, Mikhailidis DP, et al. Vitamin D status and circulating biomarkers of endothelial dysfunction and inflammation in non-diabetic obese individuals: a pilot study. Arch Med Sci : AMS 2017;13(1):53e60. [5] Faridi KF, Lupton JR, Martin SS, Banach M, Quispe R, Kulkarni K, et al. Vitamin D deficiency and non-lipid biomarkers of cardiovascular risk. Arch Med Sci : AMS 2017;13(4):732e7.
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Please cite this article as: Mazidi M et al., Impact of serum 25-hydroxyvitamin D 25(OH) on telomere attrition: A Mendelian Randomization study, Clinical Nutrition, https://doi.org/10.1016/j.clnu.2019.12.008