DNA Telomere Length Research Articles

Association between Genetic Variants in DNA and Histone Methylation and Telomere Length

Telomere length, a biomarker of aging and age-related diseases, exhibits wide variation between individuals. Common genetic variation may explain some of the individual differences in telomere length. To date, however, only a few genetic variants have been identified in the previous genome-wide association studies. As emerging data suggest epigenetic regulation of telomere length, we investigated 72 single nucleotide polymorphisms (SNPs) in 46 genes that involve DNA and histone methylation as well as telomerase and telomere-binding proteins and DNA damage response. Genotyping and quantification of telomere length were performed in blood samples from 989 non-Hispanic white participants of the Sister Study, a prospective cohort of women aged 35–74 years. The association of each SNP with logarithmically-transformed relative telomere length was estimated using multivariate linear regression. Six SNPs were associated with relative telomere length in blood cells with p-values<0.05 (uncorrected for multiple comparisons). The minor alleles of BHMT rs3733890 G>A (p = 0.041), MTRR rs2966952 C>T (p = 0.002) and EHMT2 rs558702 G>A (p = 0.008) were associated with shorter telomeres, while minor alleles of ATM rs1801516 G>A (p = 0.031), MTR rs1805087 A>G (p = 0.038) and PRMT8 rs12299470 G>A (p = 0.019) were associated with longer telomeres. Five of these SNPs are located in genes coding for proteins involved in DNA and histone methylation. Our results are consistent with recent findings that chromatin structure is epigenetically regulated and may influence the genomic integrity of telomeric region and telomere length maintenance. Larger studies with greater coverage of the genes implicated in DNA methylation and histone modifications are warranted to replicate these findings.

Relative telomere length: a novel non-invasive biomarker for the risk of non-cirrhotic hepatocellular carcinoma in patients with chronic hepatitis B infection

Background and aimsTelomere length has emerged as a promising risk predictor of various cancers including hepatocellular carcinoma (HCC). However, the majority of studies in this area measured telomere length in hepatocytes and one in lymphocytes with conflicting results. Moreover, no studies have been reported on using circulating DNA telomere length as a non-invasive HCC biomarker. MethodsWe conducted a nested case-control study to determine the relative telomere length (RTL) in serum DNA from 140 hepatitis B virus (HBV)-related HCC cases and 280 frequency-matched cancer-free HBV controls. ResultsCases had a significantly longer RTL (median, 0.31; range, 0.02–2.31) than controls (median, 0.20; range, 0.01–1.60) (P=0.003). Consistently, longer RTLs conferred a significantly increased HCC risk compared to short RTLs in a univariate logistic regression analysis (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.02–2.33, P=0.038). This association attenuated after multivariate adjustment (OR=1.40, 95% CI=0.90–2.19, P=0.132). In a quartile analysis, a significant dose-response relationship was noted in univariate analysis (Ptrend=0.017) which was again attenuated in multivariate analysis (Ptrend=0.079). Further analyses revealed that the significant association between serum RTL and HCC risk was evident in non-cirrhotic (OR=3.54, 95% CI 1.58–7.93 P=0.002), but not cirrhotic (OR=0.95, 95% CI 0.55–1.64, P=0.860) HBV patients. Moreover, the significantly increased HCC risk conferred by cirrhosis was modulated by RTL with a significant interaction effect (Pinteraction=0.013). ConclusionsRTL in circulating cell-free serum DNA could potentially be used as a novel non-invasive biomarker for non-cirrhotic HCC. Prospective cohort studies are warranted to validate this finding and assess its clinical significance in HCC prevention.

Repetitive hyperthermia attenuates progression of left ventricular hypertrophy and increases telomerase activity in hypertensive rats

We investigated the hypothesis that repetitive hyperthermia (RHT) attenuates the progression of cardiac hypertrophy and delays the transition from hypertensive cardiomyopathy to heart failure in Dahl salt-sensitive (DS) hypertensive rats. Six-week-old DS rats were divided into the following five groups: a normal-salt diet (0.4% NaCl) (NS group), a normal-salt diet plus RHT by daily immersion for 10 min in 40°C water (NS+RHT group), a high-salt diet (8% NaCl) (HS group), a high-salt diet (8% NaCl) plus RHT (HS+RHT group), and high-salt diet (8% NaCl) plus RHT with 17-DMAG (HSP90 inhibitor) administration (HS+RHT+17-DMAG group). All rats were killed at 10 wk. Cardiac hypertrophy and fibrosis were noted in the HS group, whereas RHT attenuated salt-induced cardiac hypertrophy, myocardial and perivascular fibrosis, and blood pressure elevation. The phosphorylated endothelial nitric oxide synthase (eNOS) and Akt were decreased in the HS group compared with the NS group, but these changes were not observed in the HS+RHT group. The levels of HSP60, 70, and 90 were elevated by RHT. Moreover, the increased levels of iNOS, nitrotyrosine, Toll-like receptor-4, BNP, PTX3, and TBARS in the HS group were inhibited by RHT. Telomeric DNA length, telomerase activity, and telomere reverse transcriptase (TERT) were reduced in the HS group; however, these changes were partially prevented by hyperthermia. In conclusion, RHT attenuates the development of cardiac hypertrophy and fibrosis and preserves telomerase, TERT activity and the length of telomere DNA in salt-induced hypertensive rats through activation of eNOS and induction of HSPs.

Association of genetic polymorphisms in the telomerase reverse transcriptase gene with prostate cancer aggressiveness.

Telomerase reverse transcriptase (TERT), encoded by the TERT gene, is an essential component of telomerase, essential for the maintenance of telomere DNA length, chromosomal stability and cellular immortality. The aim of the present study was to evaluate the association between common genetic variations across the TERT gene region and prostate cancer (PCa) aggressiveness in a Chinese population. A total of 12 TERT tagging single-nucleotide polymorphisms (SNPs) were genotyped on the Sequenom Mass-ARRAY iPLEX® platform in a case-case study with 1,210 Chinese patients with PCa. Unconditional logistic regression was used to investigate the association of genotypes with PCa aggressiveness, Gleason grade and risk of developing early-onset PCa. It was observed that the C allele of the TERT intron 2 SNP (rs2736100) was significantly associated with reduced risk of PCa aggressiveness [odds ratio (OR)=0.81; 95% confidence interval (CI): 0.66-0.99; P=0.037]. This allele was also significantly correlated with a reduced risk of developing a tumor with a high Gleason score (>7; OR=0.83; 95% CI: 0.70-0.99; P=0.039). The T allele of the intron 4 SNP (rs10069690) was found to be significantly associated with a decreased risk for an aggressive form of PCa (OR=0.76; 95% CI: 0.59-0.97; P=0.030). In addition, the A allele of rs10078761 located at the 3' end of the TERT gene exhibited a statistically significant association with the reduced risk of developing a higher grade disease (OR=0.48; 95% CI: 0.28-0.81; P=0.006). However, no association between TERT polymorphisms and age at diagnosis was observed in the present study. The present findings demonstrated for the first time, to the best of our knowledge, that genetic variations across the TERT gene are associated with PCa aggressiveness in a Chinese Han population.

Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA

In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.

Telomere Length Is An Independent Predictor of Outcome After Therapy in CLL: Results From the UKCLL4 Trial,

Abstract 3884▪▪This icon denotes a clinically relevant abstractTelomere length measurement has been reported to predict time to first treatment (TTFT) and overall survival from diagnosis in chronic lymphocytic leukaemia (CLL). Of note, the study by Rossi et al (Leukemia, 2009), of presentation samples from over 400 CLL patients, showed short telomere length predicted reduced TTFT, independent of established prognostic biomarkers such as disease stage, immunoglobulin heavy chain variable region (IGHV) mutation status and unfavourable genomic aberrations. However, to date no studies have been reported on the value of telomere length as a biomarker in CLL at time of first therapy.We have used the monochrome multiplex quantitative PCR (MMQPCR) method of Cawthon (Nucleic Acids Research, 2009), to determine relative average telomere length in peripheral blood mononuclear cell DNA from treatment naive CLL patients at time of enrolment into the phase III UKCLL4 trial. MMQPCR reports a telomere length value (T/S) which has been shown to correlate linearly with mean telomere terminal restriction fragment length as determined by southern blotting. Telomere length was measured for 375/777 patients selected from the trial, of the 375 cases 157, 85 and 133 were randomised to the Chlorambucil, Fludarabine or Fludarabine plus Cyclophosphamide (FC) arms respectively. Other than an increased proportion of cases from FC arm, the selected cohort did not differ from the whole trial cohort.The median T/S for all cases was 2.60 (range, 0.61–19.05). In a multiple linear regression model, only age at trial entry and IGHV homology to germline had significant predictive effect for T/S (p=0.023 and <0.0001 respectively). Whereas, Beta-2 microglobulin levels (B2M), WBC, time from diagnosis to study entry, percentage ZAP70 positive tumour cells and percentage CD38 positive tumour cells did not. T/S was significantly lower in cases with unmutated compared with mutated IGHV genes, with del 17p compared with no del 17p and with del 11q compared with no del 11q (all p<0.001) but did not significantly differ with disease stage, gender, or between those cases with or without trisomy 12.Splitting cases into two groups based on those with T/S above or below the median, short telomeres were associated with TP53 loss or mutation, del 11q and unmutated IGHV (all p<0.001) but not with gender or disease stage. Cases with short telomeres also showed significantly higher expression of ZAP70 and CD38 (both p<0.001) and of B2M (p=0.03).In univariate survival analysis, cases with short telomeres showed significantly reduced progression free survival after therapy (PFS) (median; 21.1 months v 33.3 months, p<0.001) and overall survival after therapy (OS) (median; 57.5 months v 108.7 months, p<0.001). Stratifying by IGHV mutational status; short telomeres remained associated with significantly reduced PFS and OS in both IGHV mutated cases (PFS, median; 22.1 months v 42.3 months, p=0.002 and OS, median; 75.9 months v not reached, p<0.001) and IGHV unmutated cases (PFS, median; 21.7 months v 25.7 months, p=0.049 and OS, median; 54.8 months v 81.9 months, p=0.005).Multivariate survival analysis, selected short telomere length as an independent predictor of reduced PFS (Hazard ratio (HR) 1.5; 95% CI 1.1–2.0; p=0.011) and OS (HR 1.9; 95% CI 1.3–2.8; p=0.002) in a model containing treatment arm, IGHV mutational status, age, gender, disease stage, del17p, del11q, ZAP70 expression and CD38 expression (Table 1).Table 1 –Multivariate Analysis for PFS and OS in a 236 Patient Cohort from UKCLL4 Trial.OutcomeCovariateHazard Ratio (HR)95% CI for HRSignificance (p)PFSShort telomeres1.501.10–2.050.011Treatment with FC0.360.26–0.49<0.001IGHV unmutated1.731.28–2.35<0.001Deletion 17p6.513.56–11.92<0.001Deletion 11q1.521.08–2.150.017OSShort telomeres1.881.27–2.780.002IGHV unmutated2.441.58–3.77<0.001Age1.051.03–1.07<0.001Deletion 17p6.843.63–12.89<0.001Only covariates selected as significant are listed above.These results demonstrate that telomere length is a powerful independent predictor of outcome after alkylating agent/purine analogue therapy in CLL. The simplicity and low cost of the MMQPCR assay for telomere length measurement warrants validation of these findings and comparison with other assays for telomere length in other large clinical trials. If confirmed, this prognostic biomarker has the potential for more widespread use in CLL. Disclosures:Gonzalez de Castro:The Institute of Cancer Research: Employment.

The relationship between DNA methylation and telomere length in dyskeratosis congenita

The regulation of telomere length (TL) is a complex process, requiring the telomerase enzyme complex and numerous regulatory proteins. Epigenetic regulation may also be important in telomere maintenance. Specifically, methylation at subtelomeres is associated with changes in TL in vitro and in mouse models. Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by exceedingly short telomeres and mutations in telomere biology genes. To understand the interaction between methylation and TL in humans, we measured LINE-1, pericentromeric (NBL2), and subtelomeric (D4Z4) methylation in peripheral blood DNA derived from 40 patients with DC and 51 mutation-negative relatives. Pearson's correlation coefficient and linear regression models were used to evaluate the relationship between age-standardized lymphocyte TL measured by flow FISH and % DNA methylation. No differences in % subtelomeric, LINE-1, or pericentromeric methylation between patients with DC and relatives were noted except for an increase in % subtelomeric methylation in DC patients with a telomerase-complex mutation (TERC, TERT, DKC1, or TCAB1) (63.0% in DC vs. 61.8% in relatives, P = 0.03). Positive correlations between TL and DNA methylation at LINE-1 (r = 0.39, P = 0.01) and subtelomeric (r = 0.32, P = 0.05) sites were present in patients with DC. The positive correlation between TL and % LINE-1 methylation was restricted to TINF2 mutations. In contrast, statistically nonsignificant inverse correlations between TL and % LINE-1 (r = -0.17), subtelomeric (r = -0.20) were present in unaffected relatives. This study suggests an interaction between TL and both subtelomeric and LINE-1 methylation, which may be altered based on mutation status of telomere biology genes.

Risk of renal cell carcinoma in relation to blood telomere length in a population-based case–control study

Background:There are few known risk factors for renal cell carcinoma (RCC). Two small hospital-based case–control studies suggested an association between short blood telomere length (TL) and increased RCC risk.Methods:We conducted a large population-based case–control study in two metropolitan regions of the United States comparing relative TL in DNA derived from peripheral blood samples from 891 RCC cases and 894 controls. Odds ratios and 95% confidence intervals were estimated using unconditional logistic regression in both unadjusted and adjusted models.Results:Median TL was 0.85 for both cases and controls (P=0.40), and no differences in RCC risk by quartiles of TL were observed. Results of analyses stratified by age, sex, race, tumour stage, and time from RCC diagnosis to blood collection were similarly null. In multivariate analyses among controls, increasing age and history of hypertension were associated with shorter TL (P<0.001 and P=0.07, respectively), and African Americans had longer TL than Caucasians (P<0.001).Conclusion:These data do not support the hypothesis that blood TL is associated with RCC. This population-based case–control study is, to our knowledge, the largest investigation to date of TL and RCC.

Abstract A82: Can telomere length be modified in healthy people?

Abstract Short constitutional telomere length has been associated with aging and cancer, but how telomere length is affected by other epidemiologic or environmental variables in health has not been thoroughly studied. Understanding these associations with telomere length in health is important for studies to evaluate the relationship of telomere length and diseases such as cancer. We measured peripheral blood leukocyte DNA telomere length with quantitative PCR in 1977 non-related healthy controls collected through the Colon Cancer Family Registry (an NCI cooperative group) and through the Mayo Biobank and Biobank for Gastrointestinal Health Research, two IRB approved prospective biospecimen collections based at Mayo Clinic, Rochester, MN. The controls were a median age of 50.6 years old, and 49.6% of the controls were male. Ninety-six percent of the controls were white; 1.8% were Asian, and &amp;lt;1% were either black, Pacific Islander, or Native American. Less than 1% of controls were Hispanic. On univariate analysis, as age increased, telomere length decreased (Pearson correlation coefficient = −0.07, 95% CI: −0.11, −0.03). Telomeres were longer in people who consumed more fruits (p&amp;lt;0.01) and vegetables (p&amp;lt;0.01); took aspirin on a regular basis (p&amp;lt;0.01); or had a family history of cancers of any type (p=0.05). Women had longer telomeres than men (p=0.03). Telomeres were shorter in people who consumed alcohol (p&amp;lt;0.01), took folate on a regular basis (p=0.01), or who consumed red meat (p&amp;lt;0.01). Statistical significance was not found in the following variables: tobacco use, diagnosis of diabetes, intake of calcium, use of hormone replacement therapy in women, or body mass index. On multivariate analysis, age, vegetable consumption, family history of colorectal cancer or any type of cancer, and folate intake were identified as being significantly associated with telomere length. Telomere length may be associated with multiple environmental and demographic variables in healthy individuals, and adjusting for these may be important for studies that test for an association between telomere length and diseases such as cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A82.

The Catalytic Subunit of Human Telomerase Is a Unique Caspase-6 and Caspase-7 Substrate

Telomerase is a ribonucleoprotein complex that is essential for persistent cellular proliferation. The catalytic subunit of human telomerase, hTERT, functions as a reverse transcriptase and promotes vitality by maintaining telomeric DNA length. hTERT is tightly regulated with complex but poorly understood positive and negative regulation at several levels including transcription, protein-protein interactions, and post-translation modifications. Because evidence implicates hTERT as an apoptosis inhibitor and because telomerase activity tends to decrease during apoptosis, we hypothesized that hTERT is a caspase substrate leading to down regulation during apoptosis. Caspases are proteases that initiate and execute apoptosis by cleaving target proteins. Indeed, we found that caspases-6 and -7 cleave hTERT during apoptosis in cultured cells. Caspase-6 cleaves at residues D129 and D637, and caspase-7 cleaves at E286 and D628. Three of the caspase cleavage sites are unique motifs. All four caspase motifs appear conserved in TERTs from Old World monkeys and apes, and the caspase-6 sites appear conserved in all primates. The caspase site that cleaves at D129 appears conserved in amniotes. hTERT fragments generated by cleavage were remarkably persistent, lasting hours after caspase activation. These results reveal a new biologically relevant mechanism for telomerase down regulation through caspase-mediated cleavage of hTERT and expand the list of known caspase motifs.

Genetic Anticipation Is Associated with Telomere Shortening in Hereditary Breast Cancer

There is increasing evidence suggesting that short telomeres and subsequent genomic instability contribute to malignant transformation. Telomere shortening has been described as a mechanism to explain genetic anticipation in dyskeratosis congenita and Li-Fraumeni syndrome. Since genetic anticipation has been observed in familial breast cancer, we aimed to study telomere length in familial breast cancer patients and hypothesized that genetic defects causing this disease would affect telomere maintenance resulting in shortened telomeres. Here, we first investigated age anticipation in mother-daughter pairs with breast cancer in 623 breast cancer families, classified as BRCA1, BRCA2, and BRCAX. Moreover, we analyzed telomere length in DNA from peripheral blood leukocytes by quantitative PCR in a set of 198 hereditary breast cancer patients, and compared them with 267 control samples and 71 sporadic breast cancer patients. Changes in telomere length in mother-daughter pairs from breast cancer families and controls were also evaluated to address differences through generations. We demonstrated that short telomeres characterize hereditary but not sporadic breast cancer. We have defined a group of BRCAX families with short telomeres, suggesting that telomere maintenance genes might be susceptibility genes for breast cancer. Significantly, we described that progressive telomere shortening is associated with earlier onset of breast cancer in successive generations of affected families. Our results provide evidence that telomere shortening is associated with earlier age of cancer onset in successive generations, suggesting that it might be a mechanism of genetic anticipation in hereditary breast cancer.

Association between polymorphisms of metabolic genes and telomere length in workers exposed to polycyclic aromatic hydrocarbon

To investigate the association between the polymorphisms of metabolic genes and telomere length of genomic DNA in peripheral blood of workers exposed to polycyclic aromatic hydrocarbons (PAHs). One hundred and forty five coke-oven workers exposed to PAHs and sixty eight non-exposed medical staffs were recruited in this study. Urinary 1-hydroxypyrene (1-OHP) served as the internal exposure dose of PAHs for all subjects. Relative telomere length (RTL) of genomic DNA in peripheral blood was used as telomere length and measured by real-time PCR. Polymorphisms of metabolic genes were detected by PCR-based methods. Compared with control group, the exposure group shown a decreased RTL (1.10 +/- 0.75 vs 1.43 +/- 1.06, P < 0.05). In the coke-oven workers, after adjusting the sex, age, cigarettes per day and urinary 1-OHP, RTL (1.25 +/- 0.93) of workers with CT genotype at the CYP1A1 3801 T > C was significantly longer than that (0.93 +/- 0.51) of workers with TT genotype (P < 0.05). RTL (0.90 +/- 0.58) of individuals with the Tyr/His genotype at mEH Tyr113His was significantly shorter than that (1.24 +/- 0.90) of individuals with the Tyr/Tyr genotype (P < 0.05). RTL (1.02 +/- 0.64) of individuals with the CT genotype at AHR rs10250822 was significantly shorter than that (1.36 +/- 1.14) of individuals with the CC genotype (P < 0.05). RTL (0.93 +/- 0.54) of individuals with the AT genotype at AHR rs10247158 was significantly shorter than that (1.19 +/- 0.84) of individuals with the AA genotype (P < 0.05). The results of present study suggested that PAHs exposure could induce the shorted RTL, CYP1A1, mEH, AHR polymorphisms might influence the change of telomere length of genomic DNA in peripheral blood of workers exposed to PAHs.

A viable hypomorphic allele of the essential IMP3 gene reveals novel protein functions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis.

Telomere Shortening in Formerly Abused and Never Abused Women

Recent studies suggest that chronic psychological stress may accelerate aging at the cellular level. Telomeres are protective components that stabilize the ends of chromosomes and modulate cellular aging. Women exposed to intimate partner violence (IPV) experience chronic stress and report worse health. The purpose of this exploratory study was to examine telomeric DNA length in women who have experienced chronic stress related to IPV. We hypothesized that IPV exposure would be associated with shorter telomere length. The investigation used a cross-sectional design to study telomere length in women with a history of IPV exposure and control women who reported no prior exposure to IPV. Advertisements and public notices were used to recruit a convenience sample of healthy women. Mean leukocyte telomere length was measured in DNA samples from peripheral blood mononuclear cells (PBMCs) by a quantitative polymerase chain reaction assay (qPCR). Telomere length was significantly shorter in the 61 formerly abused women compared to the 41 controls (t = 2.4, p = .02). Length of time in the abusive relationship and having children were associated with telomere length after controlling for age and body mass index (BMI) (F(2, 99) = 10.23, p < .001). Numerous studies suggest that women who experience IPV have poorer overall health. It is often presumed that the stress of IPV may be causing greater morbidity. Findings from this descriptive study suggest a link between IPV exposure, duration of IPV-related stress, and telomere length molecular mechanisms that regulate cellular aging.

Measuring relative telomere length: Is tissue an issue?

Telomeres, the specialized structures at chromosome ends, consist of long stretches of protein-bound TTAGGG repeats [1]. In certain cell types, such as germ cells and stem cell populations, the length of telomeric DNA is maintained by the enzyme telo-merase. Most somatic cells, however, lack sufficient telomerase; consequently, telomeric DNA shortens progressively with each cell division. Upon reaching a critically short length, proliferation is halted and cells often enter a state of senescence. Thus, progressive telomere shortening is thought to serve as a molecular clock for cellular replicative aging. Based upon this fundamental tenet of telomere biology, an ever-increasing number of studies spanning a wide spectrum of human conditions have utilized telomere length, typically measured in buccal cells or blood mono-nuclear cells, as a biomarker for cellular replicative age. Individuals with relatively short age-adjusted telomere lengths, due to a combination of inherent factors and environmental stressors, are considered to have acceler-ated cellular replicative aging, potentially resulting in increased disease susceptibility [2]. Telomere length is also used as a diagnostic tool in diseases characterized by fundamental derangements of telomere biology, such as dyskeratosis congenita (DC). DC is a rare genetic disorder stemming from a defect in telomere maintenance. This defect results in a broad and highly variable clinical phenotype consisting of predisposition to bone marrow failure and malignancy, a triad of mucocutaneous features, and a number of less frequent manifestations such as pulmonary fibrosis and liver disease [3]. In nearly all reported cases, affect- ted individuals have severely shortened telomeres, which can be directly attributed to mutations in genes

RHPS4, An Anti-Telomere Agent, Specifically Targets the Subgroup of AML Cells with Loss or Gain of Parts of Chromosomes.

AbstractAbstract 1195Cytogenetic abnormalities vary greatly in AML and those with gains and losses of chromosome parts, terminal deletions and whole chromosome losses could theoretically derive from telomere dysfunction. Cells with these abnormalities are characterised by telomere shortening and abnormal function characterised by activation of both the negative telomere regulator TRF1 and also of hTERT. The G quadruplex ligand RHPS4 (Pharminox Ltd, Biocity, Nottingham, UK) binds telomeres and induces DNA damage at higher doses in the short term and critical telomere shortening in longer term growth assays. We have previously reported cytostatic and cytotoxic effects for this agent in AML cell lines, whilst others have found that normal haematopoietic progenitor cells are not targeted. We have now examined effects of RHPS4 on the in vitro growth and survival of primary AML cells, comprising 9 samples with normal and 8 with abnormal karyotypes (5 complex, one -7, one 5q- and one with additional material on chromosomes 2 and 9). Cells were incubated with RHPS4 in standard 14 day colony assays and for 7 and 21 days in a novel suspension culture medium supplemented with supernatant from cultured normal human osteoblasts. In the 7 day cultures, no effects were observed on cell survival. In the longer term growth assays, a submicromolar IC50 for RHPS4 was observed in 7/8 samples with aberrant cytogenetics in contrast with only 1/9 with normal cytogenetics (P=0.003, Fisher's exact test). Furthermore, RHPS4 significantly sensitised samples with abnormal cytogenetics to daunorubicin reducing the median IC50 from 27nM to 10nM (n=8, p<0.05).A qPCR method was used to measure telomere length of untreated DNA from 15 of the samples; the qPCR assay had previously been performed in a separate patient cohort alongside a Southern technique and comparable values were attained for the two different methods. There were no significant differences in telomere lengths between the normal and abnormal cytogenetic groups suggestive that telomere length is not a determinant of RHPS4 sensitivity in these assays. Samples with adverse cytogenetics have previously been associated with an up-regulation of basal DNA damage markers and may therefore be sensitive to compounds that can exacerbate levels of damage. We therefore studied the levels of p-ATM by immunocytochemistry in RHPS4-treated cells and found a significant increase compared to untreated cells - hence RHPS4 may be acting via a DNA damage mechanism.We conclude that telomere targeting has exciting potential as therapy for a subset of AML patients including those with adverse cytogenetics who have a poor response to conventional chemotherapeutics. Disclosures:No relevant conflicts of interest to declare.

Aneuploid Human Oocytes and Embryos Possess Significantly Reduced Quantities of Telomere DNA.

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading cause of miscarriage and developmental delay in newborns. Although maternal age is a well known risk factor, the underlying molecular etiology of human aneuploidy is unknown. Since telomere DNA content is associated with genomic instability and is naturally reset during mammalian preimplantation embryonic development, telomere DNA quantity may play a role in the development of aneuploidy commonly found in human embryos. To begin to characterize the potential association of telomere biology with development of human aneuploidy, we have characterized relative telomere DNA quantity at various stages of human preimplantation embryogenesis. First, we developed a method capable of accurate quantitation of telomere DNA from single cells. Whole genome amplification (WGA) of picogram amounts of DNA from cells with previously established telomere DNA quantities provided results equivalent to those obtained using large quantities of purified total DNA (Pearson correlation coefficient=0.97). This technology was then applied to human oocytes and embryos. Excess WGA DNA was available for telomere DNA analysis of oocyte and embryo biopsies as a result of previously performing SNP microarray based 24 chromosome aneuploidy screening on human in vitro fertilization derived samples. Paired analysis of aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. Embryo fragmentation rates, which were previously shown to correlate with telomere DNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Results demonstrated, for the first time, that aneuploid human polar bodies possess significantly less telomere DNA than euploid polar bodies (-3.07 fold, P=0.016). Since the first and second polar bodies are extruded upon completion of meiosis I and II, respectively, and reflect the chromosomal complement of the remaining oocyte, abnormalities observed in polar bodies are indicative of development of aneuploidy during meiosis of the oocyte. Therefore, the present results indicate that oocytes with reduced telomere DNA quantity are prone to development of aneuploidy during meiosis. We also show that aneuploid embryonic cells possess significantly less telomere DNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blastocyst stage (-1.18 fold, P=0.340). These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation between the cleavage and blastocyst stages of development. This further suggests that aneuploidy is not associated with failed resetting of telomere DNA length but is instead associated with failed telomere maintenance in the maternal genome during oogenesis and early embryo development. (platform)

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo-PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere-specific PNA probe conjugated with Cy5 fluorochrome, Cy5-OO-(CCCTAA)(3) . After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo-PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. According to our experience, oligo-PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.

Interspecific variability of telomeric DNA length in some siberian and endemic baikal planarians (Plathelminthes, Tricladida)

The length of the telomeric DNA in nine species of planarians inhabiting Lake Baikal and one Siberian species from Baikal rivers was determined using Southern hybridization. According to preliminary estimations, it varied in the range of 25-30 kb (Rimacephalus arecepta, Rimacephalus pulvinar, Sorocelis hepatizon, Sorocelis nigrofasciata, Protocotylus sp., Baikalobia guttata, Bdellocephala baikalensis, Phagocata sibirica) and 50 kb (Baikaloplana valida, Baikalobia copulatrix). It is the first estimation of the values of telomeric region lengths for Baikal free-living flat worms.

Telomere DNA is significantly reduced in aneuploid human oocytes and embryos

OBJECTIVE: Since telomere DNA (telDNA) content is associated with genomic instability and is naturally reset during mammalian preimplantation embryogenesis, we tested the hypothesis that telomere DNA quantity is associated with the development of aneuploidy. DESIGN: Paired analysis of telDNA quantity in aneuploid and euploid human polar bodies and embryo biopsies. MATERIALS AND METHODS: A telDNA real-time PCR method was developed to evaluate single cells after whole genome amplification (WGA). Results from picogram amounts of DNA were compared to results obtained using large quantities of purified total DNA from the same cell lines. WGA DNA was available for analysis from oocyte (n=18) and embryo (n=44) biopsies that had undergone microarray based 24 chromosome aneuploidy screening. Paired analysis of sibling aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. RESULTS: WGA of picograms of DNA from cells provided results equivalent to those obtained using large quantities of purified total DNA (Pearson R2=0.97). Embryo fragmentation rates, which were previously shown to correlate with telDNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Aneuploid human polar bodies displayed significantly less telDNA than euploid polar bodies (-3.07 fold, P=0.016). Aneuploid embryonic cells displayed significantly less telDNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blast stage (-1.18 fold, P=0.340). CONCLUSION: These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation. This further suggests that aneuploidy is not associated with resetting of telomere DNA length but is instead associated with telomere maintenance in the maternal genome during oogenesis and early embryo development.