The Associations Of Plasma Metals And TERT-CLPTM1L Variants With Peripheral Leukocyte Telomere Length

Leukocyte telomere length?TL?is widely considered as an index of cellular aging,which was previously reported to be associated with the development of many chronic diseases,such as cardiovascular diseases,diabetes,and malignancies.As we all know,TL would shorten with the cell division,and the rate of shortening could be influenced by environment exposure and genetic inheritance.Generally,human is simultaneously exposed to multiple metals in the living and working environment.On one hand,some essential metals could protect human body by taking part in the constitution of key protein and enzyme.For example,zinc and selenium are necessary part of copper-zinc superoxide dismutase and glutathione antioxidant enzyme,respectively,and they were identified to relieve chromosome damage and decrease risk of cancer incidence.On the other hand,some heavy metals like chromium and lead,were reported to have adverse effect by producing reactive oxygen species?ROS?.However,few studies have illustrated the associations between multiple metals exposure and TL.Moreover,genome-wide association studies have reported several telomere-related chromosome regions,like 5p15.33,but seldom results revealed the interactions of genetic and environment factors on TL.In this study,we included a total of 842 coke-oven workers,determined their plasma concentrations of 23 metals and detected the relative TL in peripheral leukocytes.The genetic background of this population was also genotyped by using Illumina genotyping chip.We aimed to investigate the associations between plasma levels of these metals and TL in peripheral leukocyte,testify the relationships between TERT-CLPTM1 L variants and leukocyte TL,and explore the combined effects of metals and genetic variants.There are two parts in this study as shown in the following.Part ?.Associations between plasma levels of metals and peripheral leukocyte telomere length among coke-oven workersObjective: We aimed to study the associations between plasma levels of metals and leukocyte TL.Methods: We enrolled 842 coke-oven-workers in this study.All participants were interviewed face-to-face to collect information on their demographic characteristics and lifestyles by using a standardized occupational questionnaire.Fasting blood and urine samples were also collected from each individual.Plasma concentrations of 23 metals were detected by using inductively coupled plasma-mass spectrometry?ICP-MS?,and the relative TL in peripheral leukocyte was measured by quantitative real-time PCR.We conducted generalized linear models to investigate the association between each single metal exposure and leukocyte TL,with adjustment for the covariates including age,gender,body mass index?BMI?,smoking status,alcohol drinking status,urinary levels of the sum of 10 polycyclic aromatic hydrocarbon?PAH?metabolites and plasma levels of anti-7,8-dihydrodiol-9,10-epoxide benzo[a]pyrene?BPDE?-albumin adducts.The Bonferroni-correction,backward regression?P<0.1?,and LASSO regression model were then used to select the positive metal that had the consistently significant association with TL.In addition,we investigated relationship between the positive metal and leukocyte TL among different subgroups,classifying our population by age??40 and >40 years?,gender?males and females?,BMI??25 and >25 kg/m²?,smoking status?ever smoking and never smoking?,and alcohol drinking status?ever drinking and never drinking?,respectively.The interaction effects of positive metal with age,gender,BMI,smoking and alcohol drinking status were assessed by introducing a multiplicative interaction term?plasma levels of Mn * characteristics' subgroups?into the generalized linear models.Results: In the single-metal models,we found that the levels of Aluminum?Al?,Chromium?Cr?,Iron?Fe?,Manganese?Mn?,Nickel?Ni?,Antimony?Sb?,Titanium?Ti?,and Vanadium?V?showed positively associations with leukocyte TL?all P<0.05?,however,only the effects of Al and Mn were still significant after Bonferroni-correction?P=0.001 and P<0.001,respectively,all below 0.05/22?0.002?.In the multiple-metal models,all metals were simultaneously included as covariates in the regression models and we used the backward selection method with P<0.01 to identify the effective metals.The results indicated that only the association of plasma Mn with TL remained significant in the analyses.The LASSO regression model was also used to pick up the positive metals and only the plasma levels of Mn showed consistently significant association.After adjusting the covariates,we found one percentage increase in plasma concentration of Mn was associated with 0.153% increase in leukocyte TL [? and 95% confidence interval?95%CI?=0.153?0.075,0.230?,P<0.001].The restricted cubic spline analysis indicated a linear dose-response relationship between plasma level of Mn and leukocyte TL?P<0.001?.Then,the study population were also classified into four subgroups?Q1-Q4?by quartiles of plasma concentration of Mn.When compared with subjects with the lowest level of plasma Mn??1.97 ?g/L,Q1 subgroup?,subjects with the highest level of plasma Mn?>4.21 ?g/L,Q4 subgroup?had significantly elevated leukocyte TL [??95%CI?=0.230?0.078,0.383?,P=0.003].There was a significantly increased trend in elevated TL with increasing plasma level of Mn(Q1 to Q4 subgroup,P_trend=0.003).The stratification analysis indicated that the association of plasma Mn with leukocyte TL showed consistently significant in each subgroup of age,BMI,and smoking status.The significant association of plasma Mn with leukocyte TL was also shown among males [??95%CI?=0.167?0.077,0.257?,P<0.001] and never alcoholdrinkers [??95%CI?=0.168?0.075,0.262?,P<0.001].However,the marginal significant association of plasma Mn with leukocyte TL was shown among females [??95%CI?=0.126?-0.020,0.271?,P=0.091] and ever alcohol-drinkers [??95%CI?=0.136?-0.005,0.277?,P=0.059].There were no significant interactions between age,gender,BMI,smoking and alcohol drinking status and plasma Mn on leukocyte TL(all P_interaction>0.05).Conclusions: At the normal exposure level,we found the plasma level of Mn showed linear positive association with leukocyte TL,which indicated that the elevated leukocyte TL was observed with the increased level of plasma Mn.However,the underlying mechanisms were unclear.Additionally,considering the important influence of genetic factors on leukocyte TL,the following part of this study was conducted to further investigate the combined effects of plasma Mn with TERT-CLPTM1 L variants on leukocyte TL.Part ?.TERT-CLPTM1 L variants and their combined effects with plasma Mn on peripheral leukocyte telomere lengthObjective: We aimed to testify the associations between TERT-CLPTM1 L variants and leukocyte TL,and further investigate the combined effects between plasma Mn and these variants on leukocyte TL.Methods: Among the study population,we determined the genotypes by Illumina genotyping chip and increased the desity of single nucleotide polymorphism?SNP?markers by imputation.We further selected the SNPs within TERT-CLPTM1 L region?GRCh37: Chromosome 5,1253282¹345180?by the criteria of minor allele frequency>0.05 and imputation quality score>0.3.Then,we used the dominant,recessive,and additive models to investigate the associations between TERT-CLPTM1 L variants and leukocyte TL,under the generalized linear regression models,with adjustment for age and gender.For SNPs with positive associations,we calculated their linkage disequilibrium?LD?profiles,and conducted the expression Quantitative Trait Loci?e QTL?analysis by online tool GTEx?http://www.gtexportal.org/home/?.Considering the results from above analyses and previous publications,the tagging SNPs?tag SNPs?of TERT-CLPTM1 L region were selected,and their combined effects with plasma Mn(classifying into high and low Mn exposure subgroups by the 75^th percentile of plasma Mn concentration)on leukocyte TL were further testified.Results: A total of 164 SNPs within TERT-CLPTM1 L region were selected in this study.Based on the results from three genetic models,we identified 34 SNPs that had significantly associations with leukocyte TL?all P<0.05?.Among these variants,LD analysis found that 31 SNPs were included in two blocks,and e QTL analysis indicated 22 SNPs could significantly influence the both expression levels of TERT and CLPTM1 L.Considering the above results and previous publications,five tag SNPs were selected?rs33954691,rs6554759,rs465498,rs2455393,and rs31489?for the combined effects analysis.The significant association between plasma Mn and leukocyte TL was mainly observed in rs33954691 GG carriers [??95%CI?=0.273?0.108,0.438?,P=0.001],rs6554759 AA carriers [??95%CI?=0.205?0.066,0.344?,P=0.004],rs465498 AA carriers [??95%CI?=0.200?0.040,0.360?,P=0.014],rs2455393 GT+TT carriers [??95%CI?=0.296?0.108,0.485?,P=0.002],and rs31489 CC carriers [??95%CI?=0.210?0.050,0.369?,P=0.010].No interaction effects were found between these 5 tag SNPs and plasma Mn on leukocyte TL(all P_interaction>0.05).Compared to subjects with rs33954691 GA+AA genotype and low plasma Mn,those with rs33954691 GG genotype and high Mn had significantly elevated leukocyte TL [??95%CI?=0.370?0.188,0.551?,P<0.001].Compared with rs6554759 AA carriers with low Mn,the leukocyte TL for subjects carrying rs6554759 AA or AG+GG carriers and had high plasma Mn had significantly lengthened TL [??95%CI?=0.206?0.068,0.344?,P=0.004,and ??95%CI?=0.491?0.122,0.861?,P=0.009,respectively].Similarly,compared to subjects with rs465498 AA genotype and low plasma Mn,the rs465498 AA or AG+GG carriers with high plasma Mn had significantly increased TL [??95%CI?=0.206?0.050,0.361?,P=0.010,and ??95%CI?=0.376?0.150,0.601?,P=0.001,respectively].Moreover,compared to the rs2455393 GG carriers with low Mn,the rs2455393 GT+TT carriers with high Mn had higher levels of leukocyte TL [??95%CI?=0.400?0.211,0.588?,P<0.001].For rs31489,compared with CC carriers with low Mn,the high Mn exposure either rs31489 CC or CA+AA carriers had both elevated TL [??95%CI?=0.215?0.059,0.370?,P=0.007,and ??95%CI?=0.362?0.135,0.589?,P=0.002,respectively].The combined analysis indicated that rs33954691 GG,rs6554759 AG+GG,rs2455393 GT+TT,rs465498 AG+GG,rs2455393 GT+TT,and rs31489 CA+AA carriers with high plasma Mn had lengthened leukocyte TL.Conclusions: Our study found the significant associations between 34 TERT-CLPTM1 L variants and leukocyte TL,and identified five tag SNPs?rs33954691,rs6554759,rs465498,rs2455393,and rs31489?could combined with plasma level of Mn on affecting the leukocyte TL.These results provided epidemiological proof for the combined effect of environmental and inherited factors on TL,and hinted scientific clues for the early prevention of telomere-related diseases.