Relative Telomere Length Research Articles

Vitamin D as predictor of telomere length in the transition from child to adolescent

Introduction Telomere length is seen as marker for healthy ageing, as accelerated shortening is involved in the development of age-related diseases, including cardiovascular disease. Vitamin D might influence telomere length due to anti-inflammatory activities, because inflammation could decrease telomere length. Evidence supporting this theory is mainly from studies in adults, whether vitamin D in children is a predictor of telomere length is unknown. Therefore, this study investigated the role of vitamin D on telomere length in the transition from childhood to (early) adolescence. Methods Healthy Belgian children (n = 142) were followed-up between 2010–2015 (aged 4.7–10.7 years old at baseline, 50.4% boys). Serum vitamin D (25-hydroxy vitamin D) concentrations were measured at baseline and in 2015 using standardized protocols. Average relative telomere lengths were measured in blood samples using a quantitative real-time PCR method. Cross-sectional and longitudinal age and sex adjusted mixed linear regression models addressed the association between vitamin D and telomere length. Results Telomere length was shorter with increasing age (P = 0.01). Vitamin D was not associated with telomere length in cross-sectional analysis combining both sexes, but in boys higher vitamin D was related to longer telomeres. An interquartile increase in vitamin D (9.4 ng/mL) was associated with a 2.5% (95% confidence interval: 0.1 to 4.9%; P = 0.04) increase in telomere length. Longitudinal analysis showed similar results for boys (P = 0.02). Conclusion This study over a 5-year period suggests that higher vitamin D concentrations are associated with longer telomere length in boys. These findings highlight the potential beneficial effects of vitamin D on ageing, especially since vitamin D concentrations might be easily adjustable through nutritional supplements.

Abstract 1407: Telomere length and melanoma survival

Abstract Telomeres at chromosomal ends are comprised of multiple short repeat sequences. In humans TTAGGG repeats account for 10-15 kb of double-stranded telomeres that end in single stranded G-rich tails of about 150-200 nucleotides. Telomeres in somatic cells undergo gradual shortening due to inherent limitations of DNA replication and limited levels of specialized enzyme telomerase. We previously reported an association between increased telomere length and risk of melanoma. In this study based on 1019 stage I and II cutaneous melanoma patients, we determined the effect of telomere length on patient survival. The telomere length was measured in DNA from peripheral blood cells using monochrome multiplex quantitative real-time PCR where telomere repeat copy number (T) was compared to a single-copy gene, albumin (S) and results presented as relative telomere length based on T/S ratio. A univariate Cox regression showed increased patient survival with increased telomere length (HR 0.65, 95% CI 0.42-1.00, P 0.05). With every decrease in 1 unit of telomere length the estimated odds of survival decreased by an approximate factor of 1.54 (95%CI 1.00-2.38). A model based on median distribution showed short rather than long telomere associated with poor survival with hazard ratio (HR) of 2.05 (95% confidence interval (CI) 1.33-3.16; log rank P 0.001); the model after adjustment with confounding factors including age at diagnosis showed a HR of 1.50 (95%CI 0.96-2.34). Stratification of patients based on median age showed statistically significant effect of decreased telomere length on poor survival in patients below the median age (HR 2.27 95%CI 1.09-4.27) but not in patients above the median age (HR 1.42 95%CI 0.82-2.46). Distribution of the patients into quantiles based on age showed the largest effect of decreased telomere length in patients younger than 30 years (HR 3.90 95%CI 1.28-11.93); the effect decreased with increasing age. Three single nucleotide polymorphisms, rs131178082, rs7726159 and rs6060627, previously shown to be associated with telomere length, were used to construct a weighted genetic score and paradoxically, the patients carrying the alleles associated with increased telomere length showed poor survival with shorter telomeres (HR 2.94 95%CI 1.54-5.62). Multivariate analysis that included, age, sex, outdoor life style, presence of actinic keratosis, number of nevi, tumor location, tumor stage, Breslow thickness and tumor ulceration showed a statistically significant association between composite genetic score and poor survival (HR 2.68 95%CI 1.24-5.80). Thus, the results from the study show that in contrast to association between melanoma risk and increased telomere length, the decreased telomere length associates with poor patient survival. The adverse effect on survival due to decreased telomere length was more pronounced in young rather than old patients and on the patients carrying genotypes for longer telomeres. Citation Format: Sivaramakrishna Rachakonda, Nalini Srinivas, Zaida Garcia-Casado, Celia Requena, Eduardo Nagore, Rajiv Kumar. Telomere length and melanoma survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1407.

Possible association of the TERT promoter polymorphisms rs2735940, rs7712562 and rs2853669 with diabetes mellitus in obese elderly Polish population: results from the national PolSenior study

One of the markers of aging is lymphocyte telomere length (LTL), which is affected by genetic constitution of the organism and environmental conditions, such as development and diseases, including diabetes. The relationship of the later seems to be bilateral. The enzyme responsible for the maintenance of telomere length is a subunit of telomerase—telomerase reverse transcriptase (TERT). The aims of the present study were to (1) determine the influence of the TERT promoter sequence SNP variants on relative telomere length (RTL) in an elderly Polish population and (2) explore the potential associations of the SNPs with the type 2 diabetes mellitus (T2DM) in the obese individuals. Two highly homogenous subgroups of PolSenior participants were investigated, the first constituted 70 relatively healthy respondents and the second 70 individuals with T2DM. Telomere length ratio (T/S value) was measured; 1.5 kb part upstream of the transcription start site of the TERT promoter was sequenced, and the frequencies of polymorphisms were calculated and compared against analysed data. Low-frequency SNPs were evaluated but excluded from further comparative analyses to RTL and glucose metabolism markers. No significant difference in telomere length was found between the two studied subgroups. Univariate statistical analyses showed only a weak association of environmental or genetic factors altering this marker of aging. Approximate frequency of four SNPs in TERT promoter sequence was assessed in Polish population aged 65–95 years, but three of them (rs2735940, rs7712562 and rs2853669) were selected for further analyses. The SNP selection was based on their minor allele frequencies in general population and on published data. The univariate analysis has revealed that carriers of CC SNP (rs2853669) have had the shortest RTL in the T2DM group. Multivariate analysis has also revealed that the genetic effect of TERT promoter CC SNP was strengthened by the incidence of T2DM. The additional variation in RTL in paired groups indicates that in addition to T2DM and genetics, there are other factors contributing to development of the age-related diseases.

TERT promoter hypermethylation is associated with poor prognosis in adrenocortical carcinoma.

Telomere maintenance, most commonly achieved by telomerase activation through induction of the telomerase reverse transcriptase (TERT) gene, is required for cell immortalization, a hallmark of cancer. Adrenocortical carcinoma (ACC) is an endocrine tumor for which TERT promoter mutations and telomerase activation have been reported. The present study assessed alterations of the TERT gene locus and telomere length in relation to clinical characteristics in ACC. In total, 38 cases of ACC with known TERT promoter mutational status were included. TERT promoter methylation densities were assessed by pyrosequencing, and TERT copy numbers and telomere length were determined by quantitative polymerase chain reaction analysis, followed by comparison of the mRNA expression of TERT and clinical parameters. The ACC tissue samples showed increased TERT copy numbers, compared with normal adrenal tissue (NAT) samples (P=0.001). Mutually exclusive TERT copy number gains or promoter mutation were present in 70% of the ACC samples. The ACC tissues exhibited higher levels of CpG promoter methylation of all eight CpG sites investigated within the ‑578 to ‑541 bp (Region A), compared with the NATs (P=0.001). High methylation density at this region was associated with metastatic disease and/or relapse, poor survival rates and higher European Network for the Study of Adrenal Tumor stage (P<0.05). The mRNA expression of TERT was inversely correlated with methylation density at ‑162 to ‑100 bp (Region B). Correlation was observed between relative telomere length and the gene expression of TERT. It was concluded that epigenetic alterations of the TERT promoter are frequent and associated with advanced disease and poorer clinical outcome in ACC.

Telomere shortening associated with increased levels of oxidative stress in sulfur mustard-exposed Iranian veterans

Sulfur Mustard (SM) is the most widely used chemical weapon. It was used in World War 1 and in the more recent Iran-Iraq conflict. Genetic toxicity and DNA alkylation effects of SM in molecular and animal experiments are well documented. In this study, lymphocytic telomere lengths and serum levels of isoprostane F2α were measured using q-PCR and enzyme immunoassay-based methods in 40 Iranian veterans who had been exposed to SM between 1983-88 and 40 non-exposed healthy volunteers. The relative telomere length in SM-exposed individuals was found to be significantly shorter than the non-exposed individuals. In addition, the level of 8-isoprostane F2α was significantly higher in the SM-exposed group compared to controls. Oxidative stress can be caused by defective antioxidant responses following gene mutations or altered activities of antioxidant enzymes. Chronic respiratory diseases and infections may also increaseoxidative stress. The novel finding of this study was a the identification of ‘premature ageing phenotype’. More specifically, telomere shortening which occurs naturally with aging is accelerated in SM-exposed individuals. Oxidative stress, mutations in DNA repair genes and epimutaions may be among the major mechanisms of telomere attrition. These findings may help for a novel therapeutic strategy by telomere elongation or for validation of an exposure biomarker for SM toxicity.

PO-228 Comprehensive molecular characterisation of TNBCs expressing HORMAD1, a driver of homologous recombination deficiency

IntroductionGenomic instability (GIS) is a major tumorigenesis driving factor. Both - amplification of ERBB2 in breast cancer, and acute or cycling tumour hypoxia have been shown to result in increased GIS. Data concerning effects of chronic and mild hypoxia on GIS in cancer cells are conflicting and scarce. Therefore the aim of this study was to explore effects of chronic mild hypoxia (cmH) on GIS in HER2-positive breast cancer cell line SK-BR-3.Material and methodsState of GIS was characterised by proportion of micronuclei containing cells (immunostaining with anti-tubulin and DAPI), chromosomal breakpoints, copy number alterations (Illumina CytoSNP12 v2-1 Bead Chip), expression of genome stability related genes (qPCR); relative telomere length (RTL) (qPCR) after prolonged cultivation of SK-BR-3 (three passages) in hypoxia (2% O2) or normoxia (control).Results and discussionsProlonged exposure to cmH resulted in significant 3.3-fold increase of micronuclei containing cells (hypoxia vs normoxia: 25.38 and 5.86 micronuclei per 1000 cells). Initial adaptation to cmH manifested as contraction of genome and decrease of chromosomal breakpoints (normoxia vs hypoxia: DNA index 2.06 and 1.90; breakpoint count: 4573 and 2678). Further cultivation in cmH resulted in additional reduction of DNA index (1.89) and increase in number of breakpoints (3037). CmH increased expression of ATM dependent DDR genes, significantly decreased expression of dsDNA-break reparation genes (H2AFX, BRCA1, FANCD2) and had no significant effect on aneuploidy–related gene expression. Initial exposure to cmH resulted in major increase of RTL (from 1.1 to 7.8), but further culture in hypoxia showed gradual decrease of RTL (down to 1.3). Low expression levels of telomerase (TERT) through all passages did not change significantly.ConclusionInitial adaptation to hypoxia was characterised by increased RTL, contraction of genome and decrease of genomic heterogeneity. Initial selection of hypoxia-fit SK-BR-3 subpopulations was followed by increased formation of chromosomal rearrangements. CmH in SK-BR-3 activated ATM-CHEK2 branch of DDR and decreased expression of genes involved in reparation of dsDNA breaks. Low expression levels of TERT through all passages did not change significantly and did not correlate with the RTL.

Dyskeratosis congenita with a novel genetic variant in the DKC1 gene: a case report

BackgroundDyskeratosis congenita (DC) is a rare genetic disorder of bone marrow failure inherited in an X-linked, autosomal dominant or autosomal recessive pattern. It has a wide array of clinical features and patients may be cared for by many medical sub specialties. The typical clinical features consist of lacy reticular skin pigmentation, nail dystrophy and oral leukoplakia. As the disease advances, patients may develop progressive bone marrow failure, pulmonary fibrosis, oesophageal stenosis, urethral stenosis, liver cirrhosis as well as haematological and solid malignancies. Several genes have been implicated in the pathogenesis of dyskeratosis congenita, with the dyskerin pseudouridine synthase 1 (DKC1) gene mutations being the X-linked recessive gene.Case presentationHerein, we report a 31-year-old male with history of recurrent febrile episodes who was found to have reticulate skin pigmentation interspersed with hypopigmented macules involving the face, neck and extremities, hyperkeratosis of palms and soles, nail dystrophy, leukoplakia of the tongue, premature graying of hair, watery eyes and dental caries. Several of his male relatives, including two maternal uncles and three maternal cousins were affected with a similar type of disease condition. Pedigree analysis suggested a possible X-linked pattern of inheritance. Genetic testing in the proband showed a novel hemizygous, non-synonymous likely pathogenic variant [NM_001363.4: c.1054A > G: p.Thr352Ala] in the PUA domain of the DKC1 gene. Quantitative polymerase chain reaction for relative telomere length measurements performed in the proband showed that he had very short telomeres [0.38, compared to a control median of 0.71 (range 0.44–1.19)], which is consistent with the DC diagnosis. Co-segregation analysis of the novel mutation and telomere length measurements in the extended family members could not be performed as they were unwilling to provide consent for testing.ConclusionsThe novel variant detected in the DKC1 gene adds further to the existing scientific literature on the genotype-phenotype correlation of DC, and has important implications for the clinical and molecular characterization of the disease.

The aging lung: tissue telomere shortening in health and disease

BackgroundTelomere shortening has been associated with several lung diseases. However, telomere length is generally measured in peripheral blood leucocytes rather than in lung tissue, where disease occurs. Consequently, telomere dynamics have not been established for the normal human lung nor for diseased lung tissue. We hypothesized an age- and disease-dependent shortening of lung tissue telomeres.MethodsAt time of (re-)transplantation or autopsy, 70 explant lungs were collected: from unused donors (normal, n = 13) and patients with cystic fibrosis (CF, n = 12), chronic obstructive pulmonary disease (COPD, n = 11), chronic hypersensitivity pneumonitis (cHP, n = 9), bronchiolitis obliterans syndrome (BOS) after prior transplantation (n = 11) and restrictive allograft syndrome (RAS) after prior transplantation (n = 14). Lungs were inflated, frozen and then scanned using CT. Four tissue cores from distinct lung regions were sampled for analysis. Disease severity was evaluated using CT and micro CT imaging. DNA was extracted from the samples and average relative telomere length (RTL) was determined using real-time qPCR.ResultsThe normal lungs showed a decrease in RTL with age (p < 0.0001). Of the diseased lungs, only BOS and RAS showed significant RTL decrease with increasing lung age (p = 0.0220 and p = 0.0272 respectively). Furthermore, we found that RTL showed considerable variability between samples within both normal and diseased lungs. cHP, BOS and RAS lungs had significant shorter RTL in comparison with normal lungs, after adjustment for lung age, sex and BMI (p < 0.0001, p = 0.0051 and p = 0.0301 respectively). When investigating the relation between RTL and regional disease severity in CF, cHP and RAS, no association was found.ConclusionThese results show a progressive decline in telomere length with age in normal, BOS and RAS lungs. cHP, BOS and RAS lungs demonstrated shorter RTL compared to normal lungs. Lung tissue RTL does not associate with regional disease severity within the lung. Therefore, tissue RTL does not seem to fully reflect peripheral blood telomere length.

Impact of extended working periods on genomic and telomeric DNA and on inflammatory markers: Results of an intervention study with office workers and carpenters

Aim of this study was to clarify if extension of the work phase has an impact on DNA- stability, telomere lengths and inflammatory markers. We conducted an intervention trial with office workers (n = 24) and carpenters (n = 10), who changed their working schedule from 8 to 12 h per day over a period of 3 months. The work of both groups involved only moderate physical activity. We found no evidence for induction of double strand breaks (measured in γH2AX assays) and relative telomere lengths (relTL_36B4 and ALB) in lymphocytes in the two study groups. Furthermore, no overall changes of the levels of C-reactive protein (CRP), interleukin-6 (IL-6) and thiobarbituric acid reactive substances (TBARS) in plasma were detected. However, we found in agreement with earlier investigations a moderate (not significant) increase of the CRP levels with age. Furthermore, significant higher CRP concentrations (P = 0.03) were detected in young individuals (21–30 years) as a consequence of the extended working period. Taken together our findings indicate that prolongation of the working hours has no pronounced impact on DNA stability, telomere shortening and inflammatory markers; but the increase of the CRP concentrations in young workers may be indicative for adverse health effects in this subgroup.

An Association Study between Longitudinal Changes of Leukocyte Telomere and the Risk of Azoospermia in a Population of Iranian Infertile Men

Background:Telomeres are evolutionary, specialized terminal structures at the ends of eukaryotic chromosomes containing TTAGGG repeats in human. Several human diseases have been known to be associated with dramatic changes in telomere length. The aim of the present study was to assess the correlation between the relative leukocyte telomere length (LTL) and infertility in a group of Iranian azoospermic males.Methods:In this case-control pilot study, relative telomere length (RTL) of peripheral blood leukocytes from a total of 30 idiopathic non-obstructive azoospermic males and 30 healthy fertile males was evaluated using real-time PCR. RTL was calculated as T (telomere)/S (single copy gene) ratio and compared between infertile and fertile groups.Results:Patients with azoospermia showed significantly shorter RTL than fertile males (0.54 vs. 0.84, p < 0.05). The area under the receiver operating characteristic (ROC) curve was estimated to be 99.8%, suggesting LTL as a potential marker for the diagnosis of azoospermia.Conclusion:Our findings demonstrated a probable association between telomere shortening and azoospermia in a population of Iranian infertile men affected by idiopathic azoospermia.

A novel heme oxygenase-1 splice variant, 14kDa HO-1, promotes cell proliferation and increases relative telomere length

Alternative splicing is a routine phenomenon which greatly increases the diversity of proteins in eukaryotic cells. In humans, most multi-exonic genes are alternatively spliced and their splice variants confer distinct functions. Heme oxygenase-1 (HO-1, 32 kDa) is an inducible stress responsive protein, which possesses multiple functions in many cellular processes. In the current study, we identified a novel alternative splice isoform of 14 kDa HO-1 generated through exclusion of exon 3, and it is highly expressed in immortalized cells. In contrast to nuclear accumulation of the full-length 32 kDa HO-1, the novel 14 kDa HO-1 isoform is retained in the cytoplasm under ultraviolet (UV) irradiation. Interestingly, the 14 kDa HO-1 is shown to promote cell proliferation and an increase in relative telomere lengths in vivo and in vitro. Thus, we are pioneer to report and confirm the presence of a novel splice form of HO-1 and its distinct role in modulating telomere length and tumor growth.

BPA exposure is associated with non-monotonic alteration in ESR1 promoter methylation in peripheral blood of men and shorter relative telomere length in peripheral blood of women.

The aim of this study was to evaluate the potential association of urinary Bisphenol A (BPA) levels with estrogen receptor alpha (ESR1) promoter % methylation and relative telomere length in a sample of 482 participants. Urinary BPA concentration was measured using organic phase extraction followed by high performance liquid chromatography mass spectroscopy. Peripheral blood ESR1 promoter % methylation and relative telomere length were measured using direct bisulfite sequencing and real-time polymerase chain reaction, respectively. The mean ± SD urinary BPA concentration adjusted for urinary creatinine was 2.90 ± 4.81 (μg/g creatinine) with a median of 1.86 μg/g creatinine (min-max: <LOD -69.85). There was a potentially non-monotonic relationship between adjusted urinary BPA concentrations and ESR1 promoter % methylation in men. As a matter of fact, for the lowest tertile of ESR1 promoter % methylation, the OR and 95% CI of the middle and highest tertiles of urinary adjusted BPA were 2.54 (1.01-6.39) and 1.64 (0.55-4.86) when compared to the lowest BPA tertile, respectively. After adjustment for potential confounders, similar results remained in men and appeared in the whole cohort. As for relative telomere length, there was a significant trend whereby higher adjusted urinary BPA concentrations were significantly associated with shorter relative telomere length in females. For instance, for the shortest relative telomere length tertile, the OR and 95% CI of the middle and highest tertiles of urinary adjusted BPA were 2.91 (1.38-6.16) and 3.19 (1.57-6.49) when compared to the lowest BPA tertile, respectively. This trend remained significant after adjustment for potential confounders.

Occupational exposure to asphalt mixture during road paving is related to increased mitochondria DNA copy number: a cross-sectional study

BackgroundAsphalt workers are exposed to polyaromatic hydrocarbons (PAHs) from hot mix asphalt via both inhalation and dermal absorption. The use of crumb rubber modified (CRM) asphalt may result in higher exposure to PAHs and more adverse effects. Our aim is to assess occupational exposure to PAHs from conventional and CRM asphalt paving by measuring PAH metabolites in urine, and to investigate the effects on mitochondrial DNA copy number (mtDNAcn) and telomere length.MethodsWe recruited 116 workers paving conventional asphalt, 51 workers paving CRM asphalt and 100 controls in Sweden, all males. A repeated-measures analysis included 31 workers paving both types of asphalt. Urine and blood samples were collected pre-working on Monday morning and post-working on Thursday afternoon after 4 days working. PAH metabolites: 1-hydroxypyrene (1-OH-PYR) and 2-hydroxyphenanthrene (2-OH-PH) were measured in urine by LC-MS/MS. Relative mtDNAcn and telomere length were measured by quantitative PCR.ResultsConventional and CRM asphalt workers showed higher 1-OH-PYR and 2-OH-PH than controls (p < 0.001 for all). Relative mtDNAcn were 0.21 units (p < 0.001) higher in conventional asphalt workers and 0.13 units (p = 0.010) higher in CRM asphalt workers compared to controls. Relative telomere length did not differ across occupational groups, but it was positively associated with increment of 2-OH-PH (β = 0.075, p = 0.037) in asphalt workers. The repeated-measures analysis showed no difference in either increment of 1-OH-PYP, or changes in effect biomarkers (mtDNAcn or telomere length) between paving with conventional and CRM asphalt. Increment of 2-OH-PH was smaller after paving with CRM asphalt.ConclusionsRoad asphalt paving in open areas resulted in PAHs exposure, as shown by elevation of PAH metabolites in urine. Asphalt workers may experience oxidative stress, evidenced by alternation in mtDNAcn; however the effects could not be fully explained by exposure to PAHs from the asphalt mixture.

Telomere length was similar in school-age children with bronchopulmonary dysplasia and allergic asthma.

Inflammation is a major factor in the pathophysiology of bronchopulmonary dysplasia (BPD), and it contributes to accelerated telomere shortening and cellular ageing. This study aimed to determine its effect on telomere length and lung function in school-aged children born preterm with BPD. We examined 29 children with BPD, born preterm in Stockholm county 1998-99, along with 28 children with allergic asthma born at term matched for age and gender. At 10years of age, we measured relative telomere length (RTL) in blood by quantitative polymerase chain reaction, lung function by spirometry and inflammation by fractional exhaled nitric oxide and blood cytokines. RTL was not different in preterm born with BPD compared to term born children with asthma. The gender effect was strong in both groups; girls had significantly longer median RTL than boys (1.8 versus 1.5, p<0.01). Short RTL was associated with low forced expiratory flow, also after adjusting for gender, but was not affected by severity of BPD or ongoing inflammation. Telomere length was similar in 10-year-old children born preterm with a history of BPD and term born children with allergic asthma. However, impaired lung function and male gender were associated with short telomeres.

Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer

Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.

Abstract P014: Associations of Depression, and Perceived Stress With Telomere Length in African Americans: The Minority Health Genomics and Translational Research Bio-Repository Database Study

Background: African Americans (AA) experience higher levels of psychological distress. Adverse psychological conditions and mental stress could be associated with accelerated cellular aging, evidenced by shortened leukocyte telomere length (TL), which could underlie this association. Hypothesis: In this study, we hypothesized that depressive symptoms and higher stress level would be associated with shorter telomere length in AA. We also hypothesized that the associations would be attenuated after adjusting for individual-level sociodemographic variable, various disease conditions, and lifestyle factors. Methods: The analysis included 225 women and 142 men aged 30-55 years from the Minority Health Genomics and Translational Research Bio-Repository Database Study. Relative TL (T/S ratio) was measured using quantitative polymerase chain reaction. The 20-item Center for Epidemiologic Studies Depression Scale and 14-item Perceived Stress Scale were used to assess the presence of depressive symptoms and perceived stress respectively. Summation of the scores and the Principal Component Analysis were performed for both the scales. Multivariable linear regression models were used to estimate mean differences in log T/S associated with depressive symptoms and perceived stress after adjusting for sociodemographic variable, various disease conditions, and lifestyle factors. Results: No evidence of association between stress and TL was observed. We found effect modification by hypertension affecting the association between depressive symptoms and log T/S. Depressive symptom, especially higher psychosomatic symptoms, was associated with increased TL in normotensive participants (β= 0.11 for mid-level psychosomatic symptoms and β= 0.12 for high-level psychosomatic symptoms; p value &lt;0.05 for both). No association was observed in hypertensive participants. Conclusions: We found a positive association between psychosomatic depressive symptoms and TL, which do not support the hypothesis that depression is associated with shorter TL and aging in AA. Longitudinal studies with larger samples are needed to better understand and confirm this relationship.

A novel pooled-sample multiplex luminex assay for high-throughput measurement of relative telomere length.

Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness. We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision. The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r = 0.79, P = 1.44 × 10-15 ). In an independent set of samples (n = 550), the new assay showed a negative correlation of RTL with age (r = -0.41), a result providing external validation for the method. We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies.

Association of chronic fatigue syndrome with premature telomere attrition

BackgroundChronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis (ME), is a severely debilitating condition of unknown etiology. The symptoms and risk factors of ME/CFS share features of accelerated aging implicated in several diseases. Using telomere length as a marker, this study was performed to test the hypothesis that ME/CFS is associated with accelerated aging.MethodsParticipant (n = 639) data came from the follow-up time point of the Georgia CFS surveillance study. Using the 1994 CFS Research Case Definition with questionnaire-based subscale thresholds for fatigue, function, and symptoms, participants were classified into four illness groups: CFS if all criteria were met (n = 64), CFS-X if CFS with exclusionary conditions (n = 77), ISF (insufficient symptoms/fatigue) if only some criteria were met regardless of exclusionary conditions (n = 302), and NF (non-fatigued) if no criteria and no exclusionary conditions (n = 196). Relative telomere length (T/S ratio) was measured using DNA from whole blood and real-time PCR. General linear models were used to estimate the association of illness groups or T/S ratio with demographics, biological measures and covariates with significance set at p < 0.05.ResultsThe mean T/S ratio differed significantly by illness group (p = 0.0017); the T/S ratios in CFS (0.90 ± 0.03) and ISF (0.94 ± 0.02) were each significantly lower than in NF (1.06 ± 0.04). Differences in T/S ratio by illness groups remained significant after adjustment for covariates of age, sex, body mass index, waist–hip ratio, post-exertional malaise and education attainment. Telomere length was shorter by 635, 254 and 424 base pairs in CFS, CFS-X and ISF, respectively, compared to NF. This shorter telomere length translates to roughly 10.1–20.5, 4.0–8.2 and 6.6–13.7 years of additional aging in CFS, CFS-X and ISF compared to NF respectively. Further, stratified analyses based on age and sex demonstrated that the association of ME/CFS with short telomeres is largely moderated by female subjects < 45 years old.ConclusionsThis study found a significant association of ME/CFS with premature telomere attrition that is largely moderated by female subjects < 45 years old. Our results indicate that ME/CFS could be included in the list of conditions associated with accelerated aging. Further work is needed to evaluate the functional significance of accelerated aging in ME/CFS.

Prospective association of depression and phobic anxiety with changes in telomere lengths over 11 years.

Although depression and anxiety have been associated with shorter telomeres in cross-sectional studies, the data regarding the prospective relations of depression and anxiety to accelerated telomere length shortening are limited and findings are mixed. We prospectively examined relations of baseline depression and phobic anxiety to subsequent 11-year change in relative leukocyte telomere lengths (LTLs). We selected 1,250 women from a subcohort of the Nurses' Health Study who provided blood specimens at both blood collections (1989-1990 and 2000-2001). Depression was defined by self-reported regular antidepressant use or presence of severe depressive symptoms; anxiety symptoms were assessed using the Crown-Crisp Experiential Index. Using quantitative real-time polymerase chain reaction assay, LTLs were measured as the copy number ratio of telomere repeat to a single control gene. Changes in LTLs were defined in three ways: absolute change, symmetrized percent change, and decile shift. Overall, there were no statistically significant associations of depression or phobic anxiety to subsequent 11-year LTL shortening, despite a point estimates in the direction of greater telomere shortening among participants with versus without depression, across all three metrics of telomere change. The strongest predictor of LTL change was baseline telomere length, and regression-to-the-mean was observed. Baseline depression and phobic anxiety were not significantly associated with 11-year attrition in LTLs among 1,250 mid-life and older women. However, a suggestion of depression and greater subsequent LTL attrition, while not statistically significant, may warrant further inquiry, particularly in prospective studies with larger sample sizes and broader windows of the lifespan.

Elevated Vasodilatory Cyclases and Shorter Telomere Length Contribute to High-Altitude Pulmonary Edema.

High-altitude (HA) genetics is complex with respect to health and disease (HA pulmonary edema i.e., HAPE). Based on the widely recognized fact that oxidative stress is a major trigger of several physiological processes, this study was designed to establish the significance of vasodilatory cyclases and telomere length in HA physiology. The study was performed in three groups, namely HAPE-free sojourners (HAPE-f, n = 150), HAPE patients (HAPE-p, n = 150), and healthy highland natives or highlanders (HLs, n = 150). Variations in soluble guanylyl cyclase β1-subunit (GUCY1B3) and adenylyl cyclase type 6 (ADCY6) were genotyped by the SNaPshot method and/or Fluidigm SNP type genotyping. Plasma GUCY1B3 and ADCY6 levels were estimated using ELISA, and relative telomere length was estimated by qRT-PCR. The rs7638AA genotype was over-represented in HLs compared with HAPE-f and HAPE-p (p = 0.035 and p = 0.012, respectively). Similarly, the rs7638A allele was prevalent in HLs compared with both groups, but significance was attained against HAPE-p (p = 0.012). Significantly elevated plasma levels of GUCY1B3 and ADCY6 were obtained in HAPE-p compared with HAPE-f (p = 0.001 and p = 0.006, respectively) and HLs (p = 3.31E-05 and p = 0.05, respectively). Shorter telomere length was observed in HAPE-p compared with HAPE-f (p > 0.05) and HLs (p = 0.017). Elevated cyclases and shorter telomere length associate with HAPE pathophysiology.