Telomere length as a putative biomarker of health and disease
The telomeres of human chromosomes consist of long tracts of the hexanucleotide repeat sequence T2AG3 which help to maintain the integrity of the genome. Telomeric DNA is gradually lost with cellular proliferation, until the length of any given telomere reaches a critical level that triggers cell senescence and death. Telomere dysfunction leading to shortened telomeres is now implicated in a range of severe inherited premature ageing syndromes with reduced longevity. Average telomere length, usually measured in leukocytes from peripheral blood, decreases as humans age and shows considerable heterogeneity in the wider population. This variability is in part believed to reflect individual lifetime exposure to environmental, genetic and biological stressors that impact on telomere maintenance. Previous research suggests that these stressors increase the rate of telomere shortening with potential impact on age-related disease and mortality later in life. Thus there is interest in the application of telomere length measures as a biomarker of general health or “biological age”, and the possibility of using mean telomere length to gauge individual disease risk, and to promote lifestyle changes to improve health. The principle aim of this thesis was to explore the impact of environmental and genetic factors on telomere length, with the additional aim of developing improved methods for accurately measuring telomere length. Establishment of the monochrome multiplex quantitative polymerase chain reaction (MMQPCR) assay for measuring average telomere length proved problematic on the Roche LightCycler® 480 qPCR platform. Temperature heterogeneity across the Peltier block led to severe position effects resulting in high assay variability which impacted on the T/S ratio. Therefore, this assay was abandoned. A modified form of the original two-plate qPCR assay was determined to be more compatible with this platform, with the choice of hot-start Taq polymerase, intercalating dye, and careful attention to quality control of the assay revealed as critical factors. This assay was validated by comparison with the gold standard, TRF (Terminal restriction fragment) method. The modified telomere length assay was then applied to two longitudinal cohorts, the Christchurch Health and Development Study (CHDS) and the Canterbury Health, Ageing and Life Course (CHALICE) study. Extensive phenotypic data were available to comprehensively assess the effect of life course stressors on telomere length. No associations were found in either cohort between telomere length and life course stress, although for the CHDS cohort, Māori and Pacific Island participants were found to have a significantly higher average leukocyte telomere length than Caucasians. For the CHALICE cohort, the only significant association observed was between telomere length and gender, with females having longer telomere length than men. Whole exome analysis was used to assess the potential impact of rare genetic variation on telomere length in six subjects from the CHALICE cohort who had the shortest telomeres. The top fifteen variants arising from this analysis were genotyped in the entire CHALICE cohort to determine if they were over-represented in individuals with short telomeres. Two variants reached statistical significance but were found to be very rare in this cohort, and would need to be followed up in a larger cohort before any firm conclusion could be drawn about their relevance to telomere length. Finally, attempts were made to develop a novel method of accurately measuring telomere length by directly counting the telomeric repeats from individual chromosomes using the MinIONTM nanopore sequencer. Preliminary data demonstrated that some telomere repeats were detectable, but the method was error prone with few perfect consecutive repeat sequences obtained. Nanopore sensing holds some potential as an alternate approach for measuring telomere length, but further advances in the sequencing chemistry, base calling and bioinformatics analysis of these data are required to achieve this goal.
Advisor: Kennedy, Martin; Hampton, Mark; Pearson, John
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Pathology (UOC)
Publisher: University of Otago
Keywords: Telomere; qPCR; Biomarker; Ageing; MinION
Research Type: Thesis