Longer leukocyte telomere length increases cardiovascular mortality in type 2 diabetes patients

Abstract Aims Leukocyte telomere length (LTL), as a biomarker of biological aging, is associated with the prevalence and complications of diabetes. This study aims to investigate the associations between LTL and all‐cause and cause‐specific mortality in patients with type 2 diabetes. Methods All participants with baseline LTL records were included from the National Health and Nutrition Examination Survey 1999–2002. Death status and its causes were ascertained for National Death Index based on International Classification of Diseases, Tenth Revision code. Cox proportional hazards regression models were established to estimate the hazard ratios (HRs) of LTL associating with all‐cause and cause‐specific mortality. Results The study enrolled 804 diabetic patients with the mean follow‐up of 14.9 ± 2.59 years. There were 367 (45.6%) all‐cause deaths, 80 (10.0%) cardiovascular deaths, and 42 (5.2%) cancer‐related deaths. Longer LTL was associated with reduced all‐cause mortality, whereas this association disappeared after adjusting for other variables. Compared with the lowest tertiles of LTL, the multivariable‐adjusted hazard ratio of cardiovascular mortality was 2.11 (95% confidence interval [CI] 1.31–3.39; p < .05) in the highest tertiles. In terms of cancer mortality, the highest tertile was negatively correlated with the risk of cancer mortality (HR 0.58 [95% CI 0.37, 0.91], p < .05). Conclusion In conclusion, LTL was independently associated with the risk of cardiovascular mortality in patients with type 2 diabetes and was negatively correlated with the risk of cancer mortality. Telomere length may be a predictor of cardiovascular mortality in diabetes.


| INTRODUCTION
Diabetes is a leading cause of morbidity and mortality worldwide. 1,2 Patients with diabetes have 2 4 times risk to develop cardiovascular diseases (CVDs). 3 An international registry found that the risk of cardiovascular death was greater in patients with diabetes compared with nondiabetes (16.5% versus 13.1%; adjusted hazard ratio [HR] 1.27). 4 In addition, diabetes was associated with a greater risk of heart failure, nonfatal myocardial infarction, and stroke. 5 Besides, the diabetes status was involved in aging, oxidative stress, and chronic inflammation, promoting shortening of telomeres.
Telomeres are the TTAGGG repetitive DNA sequences at the ends of eukaryotic chromosomes and are responsible for maintaining the genome integrity during cell division. 6 Excess oxidative stress and proinflammatory mediators like tumor necrosis factor-alpha, interferon-gamma, interleukin-6 and interleukin-10 were two mechanisms contributing to age-dependent leukocyte telomere length (LTL) shortening. 7,8 Highly shortened telomeres can cause cellular senescence and subsequent arrest in the cell cycle. 9 Epidemiological studies have shown that shorter LTL was associated with a number of age-related disorders such as CVDs, neurodegenerative diseases, and cancers. 10-12 A community-based cohort study found that shorter LTL was associated with increased overall cardiovascular, respiratory, digestive, and musculoskeletal mortality but not cancerrelated mortality. 13 The telomere system played an important role in the pathogenesis and progression of diabetes mellitus as well as in its vascular complications. 14 However, the prognostic effect of telomere length on cardiovascular mortality or cancer mortality remains unclear in diabetes patients.
Hence, this study was designed to evaluate the relationship between LTL and all-cause and cause-specific mortality in type 2 diabetes by using data from the National Health and Nutrition Examination Survey (NHANES).

| Study population
Our study included individuals from the NHANES. A total of 1162 patients with type 2 diabetes were included from NHANES cycle of 1999-2002. After excluding those with missing LTL records (n = 245) and with cancer or pregnancy (n = 113), a total of 804 participants were enrolled in our study. Figure 1 depicted the selection process. The protocol was approved by the Ethics Review Board of National Center for Health Statistics (Protocol # 98-12).

| Leukocyte telomere length measurement
Genomic DNA was extracted from the buffy coat in the peripheral blood sample using QIAamp 96 DNA Blood Kits (Qiagen, Valencia, CA) according to the manufacturer's protocol. Relative telomere length was measured by comparing the ratio of telomere repeat number (T) to a single copy gene number for albumin (S) in the experimental sample to a standardized reference sample value using multiplexed quantitative polymerase chain reaction method. All experimental samples were assayed in duplicate, and the average value of the two replicates was used for the final analysis for each subject. The mean of the T/S ratio values was calculated, and the largest and the smallest T/S ratio values were marked as potential outliers. If the absolute value of the log of the ratio between the recalculated mean (excluding the potential outliers) to the F I G U R E 1 The flow chart of participants selection. DM, diabetes mellitus; LTL, leukocyte telomere length; NHANES, National Health and Nutrition Examination Survey. value of the potential outlier was >0.4, then the value was marked as an outlier. The interassay coefficient of variability for LTL was 4%.

| Covariates collection
Information on sex, age, race, education level, smoking status, alcohol habit, prior comorbid illness, and medication use were obtained from standardized questionnaires. Body mass index (BMI, kg/m2) was calculated as weight divided by height squared. Race was classified as non-Hispanic white, non-Hispanic black, Mexican American, or other. Education levels were categorized into less than high school, high school or equivalent, and college or above. Family income-topoverty ratio was classified as 0-1.0, 1.0-3.0, or >3.0, indicating a low, middle, and high income status, respectively. Smoking habits were defined as current, past and never. Previous history of diseases (hypertension and CVD) and medications (aspirin, hypoglycemic drugs, and lipid-lowering drugs) were determined from self-reported questionnaires.

| End points
The primary outcome was all-cause mortality and secondary outcomes included death from CVD, diabetes, or malignant neoplasms. Mortality status was obtained by linkage to the National Death Index by 31 December 2015. Causes of death included malignant tumors, CVDs, respiratory diseases, Alzheimer's disease, diabetes, nephropathy-related diseases, accidental death, and other causes. CVDs were defined as International Classification of Diseases, Tenth Revision (ICD-10) codes I00-I09, I11, I13, or I20-I51. Malignant neoplasm was defined as ICD-10 codes C00-C97 (Supplementary  Table S1).

| Statistical analysis
Descriptive statistics were presented according to the levels of LTL (T1: <0.83, T2: 0.83-1.02, T3: >1.02; Figure 2) and group differences were explored by one-way analysis of variance and chi-square tests. First, we explored the association of LTL tertiles with overall survival using Kaplan-Meier analysis. Associations between levels of LTL and the risk of all-cause mortality was estimated by classical Cox regression models. We fitted Cox model by specifying the event of interest, and by censoring for competing events. Multiple imputation based on predictive mean matching was performed for covariates with missing values using package mice form R (Supplementary Table S2). All analysis were performed using R version 3.6.

| RESULTS
This study included 804 participants with 14.9 ± 2.59 year of follow-up. The baseline characteristics of the study population according to LTL tertiles (T1: <0.83, T2: 0.83-1.02, T3: >1.02) were shown in the Table 1. Compared with the lowest group, participants in the highest group were more likely to be younger, non-Hispanic black, and have higher BMI.

| DISCUSSION
In our study, we found that longer leukocyte telomere length increased the risk of cardiovascular mortality in patients with type 2 diabetes. However, leukocyte telomere length was not associated with all-cause mortality in diabetic patients. Telomere length may be a predictor of cardiovascular mortality in diabetes.
Previous study reported that shorter LTL was associated with a higher risk of mortality in the general population, which could be explained by increased systemic inflammation and oxidative stress. 15 It was understandable that our univariate survival analysis also suggested that lower LTL was associated with T A B L E 1 Characteristics of the study population according to the leukocyte telomere length. increased all-cause mortality. However, after adjusting many covariates, the association disappeared in our study, which suggested that other factors could mediate their association. In the general population, LTL was negatively 16 or U-shaped 17 associated with the risk of all-cause mortality. On contrary to the previous studies, we did not' observe a significant association between LTL and all-cause mortality in diabetic patients. We speculated that telomeres have already shortened substantially among diabetes subjects, 18 and given the presence of other major risk factors for death, the independent effect of telomere length on mortality may be reversed. Besides, Chen et al found that shortened relative leukocyte telomere length was associated with an increased risk of all-cause mortality in type 2 diabetes. 19 The difference may be due to (a) different population (US population in our study and Asian population in the previous study); (b) a lower level of LTL in our study (0.96) compared with the previous study (4.6), which could be attributed to the different methods used for the measurement of LTL; and (c) a high all-cause mortality in our study (45.6%), although it was not implausible, given the starting ages of the subjects and the long follow-up time.
T A B L E 2 Association of leukocyte telomere length with all-cause and cause-specific mortality. Surprisingly, we found that longer LTL increased the risk of cardiovascular mortality in patients with diabetes. Although a report found shortening of telomeres would protect against cardiovascular mortality in elderly men, 20 our results contrasted with other previous reports that associated shorter telomeres with cardiovascular mortality. 21,22 Chen et al also found that shortened leukocyte telomere length was associated with prevalent and incident cardiovascular complications in type 2 diabetes. 23 There may be a competing risk against cardiovascular mortality in diabetic patients and it was stronger in shorter LTL subgroups. The relation should be interpreted with some caution until more studies are designed to explore this association. In consistent with previous reports, we found an inverse correlation between LTL and cancer mortality.
Our study has some limitations. First, some covariates were self-reported. Second, we used mean LTL measured in leukocyte cells, which are a heterogeneous mix of cells with varying telomere length. It is possible that the association may vary depending on cell type used to measure telomere length. Third, data on LTL was collected only once at baseline, and it was unclear whether longitudinal changes over time could affect the association with mortality. Finally, heart diseases or other variables may not be present at baseline.

| CONCLUSION
We found that leukocyte telomere length was independently associated with the risk of cardiovascular mortality, not all-cause mortality, in patients with type 2 diabetes. Telomere length may be a predictor of cardiovascular mortality in diabetes.

AUTHOR CONTRIBUTIONS
Xiao Mingbing and Wu Xiaohui designed the study; Chen Ziwei, Shen Yao, and He Jing wrote the main manuscript; Shen Yao and Zhu Weida prepared the figures and tables.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.