Published by Oxford University Press on behalf of the International Epidemiological Association ß The Author 2009; all rights reserved. Advance Access publication 7 August 2009
International Journal of Epidemiology 2009;38:1725–1734 doi:10.1093/ije/dyp273
Influences on the reduction of relative telomere length over 10 years in the population-based Bruneck Study: introduction of a well-controlled high-throughput assay Silvia Ehrlenbach,1 Peter Willeit,2 Stefan Kiechl,2 Johann Willeit,2 Markus Reindl,2 ¨ tter1* Kathrin Schanda,2 Florian Kronenberg1 and Anita Brandsta
Accepted
9 July 2009
Background Telomeres play a key role in the maintenance of chromosome integrity. Short telomeres are linked to age-associated diseases and cancer. Our aim was to determine the decrease rate of relative telomere length (RTL) over 10 years and whether this rate was influenced by age, sex and smoking behaviour. Methods
We compared RTL in 510 sample pairs from the longitudinal population-based Bruneck Study, which were collected in 1995 and recollected in 2005, and additionally determined RTL from 159 participants who died during follow-up. RTL were determined by a high-throughput real-time PCR assay and by applying a mathematical model.
Results
The telomeres shortened, on average, by 455 bp over 10 years. The RTL shortening rate was highly correlated with baseline RTL (r ¼ 0.674, P < 0.001). Participants who died within the observed period had considerably shorter telomeres than those who survived (median RTL of 0.98 vs 1.49; P < 0.001). In contrast to previous studies, smoking behaviour had no influence on RTL and on telomere shortening.
Conclusion This is the first comprehensive longitudinal study of individuals who were, on average, 60 at baseline, and who were re-evaluated 10 years later. Our methodology proved to be a reliable tool for a rapid, accurate and cost-efficient determination of RTL with a low amount of DNA. Keywords
Relative telomere length, absolute telomere length, longitudinal study, mortality, telomere attrition rate, smoking, real-time PCR
Introduction Telomeres, the ends of linear chromosomes, consist of large but variable numbers of short tandem repeats with the sequence motif TTAGGG embedded in a 1
2
Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria. Department of Neurology, Innsbruck Medical University, Innsbruck, Austria.
nucleoprotein complex. These ends are involved in the maintenance of cellular stability.1 The DNA shortens with each replication round as described by the
* Corresponding author. Division of Genetic Epidemiology, Department of Medical Genetics, Molecular and Clinical ¨pfstrasse Pharmacology, Innsbruck Medical University, Scho 41, A-6020 Innsbruck, Austria. E-mail:
[email protected]
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‘end-replication problem’.2 This phenomenon is based on the fact that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis, as the process of DNA replication only works in the 50 to 30 direction, and DNA polymerase requires binding of an RNA primer. This results in a progressive loss of a small 50 nucleotide segment with each DNA replication round and telomere shortening. The telomeres of human newborns span 8.3 kb.3 Primary human cells lose about 25–100 bases of TTAGGG repeats per cell division.4 When telomeres shrink to a critical length, they lose capping function, and a DNA-damage response is induced that leads to a permanent cell cycle arrest at the senescence stage.5 The minimal functional telomere length (TL), and whether this length varies among cell types has, however, not been clearly defined. Even in senescent human cells, telomeric double-stranded repeats are readily detectable, suggesting that several kilobases of TTAGGG repeats are required at all times.6 Nevertheless, it has been shown that telomere structure, not TL, is the main determinant of functional telomeres.7 As decreased TL indicates advanced age, the telomere length constitutes a biological marker for the age at the cellular level.1 The field of telomere biology has progressed considerably from the simplistic view that telomeres function only as non-coding buffer zones at the ends of linear chromosomes. Telomeres are now viewed as highly specialized and regulated complexes in which length and structure determine integrity and function.6 Shortened telomeres were shown to be correlated with age and important age-associated pathologies such as cardiovascular disease (CVD), diabetes mellitus, Alzheimer’s disease, obesity, HIV, schizophrenia and osteoporosis.8–12 In addition, recent studies provided evidence that telomere shortening is a common genetic alteration in carcinoma and that telomere length may thus affect risk of cancer.13–15 The assessment of telomere attrition rates and their impact on aging processes requires large-scale longitudinal studies, which could shed some light on the importance of genetic and life style factors on the individual telomere shortening rate.9 Several studies have been published concerning longitudinal approaches for determining the TL.16–23 However, most of these studies included an observation period of only a few months, only a low number of samples, involved only males or included participants from extreme age groups only. Different approaches to determine the TL have been published. As a method for ascertaining absolute TL, the telomere restriction fragment (TRF) method, which degrades genomic DNA to short fragments but leaves intact the telomeres, was described.24 TRF analysis has been the standard method for TL analysis as it works with a wide variety of sample types and it can measure a wide range of TLs if combined with
pulsed-field agarose gel electrophoresis. However, apart from other numerous drawbacks, the TRF technique is time consuming, difficult to quantify and requires large amounts of DNA. TL analysis was considerably improved with the development of fluorescence in situ hybridization (FISH) methods.25 Compared with TRF analysis, FISH methods provide increased sensitivity, specificity and resolution.26 Limitations of the FISH methods are that they require specialized and expensive equipment and are time consuming and labour intensive. In the study at hand, we applied a quantitative polymerase chain reaction (qPCR) assay for the measurement of relative telomere lengths (RTLs) as described by Cawthon,27 which uses far less DNA and requires much less time to perform than the traditional Southern blot method for measuring (TRF) lengths. We aimed to determine telomere attrition rates in the longitudinal prospective Bruneck Study over a time frame of 10 years and to study the influence of age, gender and smoking behaviour on telomere attrition rates.
Materials and methods Study population The Bruneck Study is a prospective population-based survey designed to investigate the epidemiology and pathogenesis of atherosclerosis.28 Briefly, the study population was recruited as a sex- and age-stratified random sample of all inhabitants of Bruneck, Italy (125 women and 125 men in the 5th to 8th decades each, n ¼ 1000). At the 1990 baseline, 93.6% of recruited subjects participated, with data assessment completed in 919 subjects. Follow-up examinations were performed in 1995, 2000 and 2005.28 Smoking behaviour was ascertained by participant self-report and classified in three categories: current or active smokers, past or ex-smokers (abstinence from smoking for 430 days) and non-smokers (
