Terminal Restriction Fragment Length Research Articles

Chronic Lymphocytic Leukemia in African Ancestry Population Is Characterized By Increased Telomere Erosion

Background: Chronic lymphocytic leukemia (CLL), a neoplastic disease of relatively mature B-cells, is characterized by a variable clinical course. Although initial studies suggesting more aggressive disease phenotype and inferior clinical outcomes among CLL patients of African ancestry (AA) were published a decade ago, biological features contributing towards this augmented disease severity remain largely unknown. Telomere erosion in a cohort of CLL patients of European Ancestry (EA) has been previously associated with high-risk molecular markers including unmutated immunoglobulin heavy-chain variable region (u-IGHV) status, and genetic alterations such as del(17p) and del(11q) by FISH. Here we characterized telomere erosion in AA CLL patients compared to CLL patients of EA. Methods: We extracted DNA from CD5+/CD19+ cells peripheral blood mononuclear cells and utilized it to measure the average telomere length through quantitative-PCR (qPCR). Briefly, primers for telomere and the single copy gene (SCG) HBB were used for amplification of 20 ng of DNA in a total volume of 20 µL in triplicates using the KAPA SYBR® FAST optimized for LightCycler®480 and analyzed on a LightCycler®480. Absolute telomere lengths were then obtained by generating standard curves using synthetic oligonucleotide PCR standards for telomere and the SCG gene. The qPCR results were validated by terminal restriction fragment length (TRF) analysis and Southern hybridization (N=28; r=0.8531; p<0.0001) using the TeloTTAGGG Telomere Length Assay. TRF was used as the gold standard of telomere length quantification; however, the high quantity of genomic DNA required for the assay (2µg) limited its broad use. Five CLL samples with known telomere lengths through TRF analysis were included in every qPCR plate in order to perform a linear regression to account for variability between qPCR plates as described previously. Results: Telomere erosion was evaluated in a cohort of 133 AA (mean age at sample collection 62.7 years ±13.6) and 147 age-matched EA CLL patients (mean age at sample collection 61.6 years ±10.2). The AA samples were collected from 95 (71%) male patients and 71% presented u-IGHV, while EA samples were collected from 110 (77%) male patients and 66% u-IGHV. As expected, we observed shorter telomere length in u-IGHV CLL (median 3.7 kb; range 0.8-44.9) compared to mutated immunoglobulin heavy-chain variable region (m-IGHV) CLL (median 6.8 kb; range 1.8-44.7; p<0.0001) in the full cohort. AA CLL presented shorter telomere lengths (median 4.2 kb; range 0.6-13.1) when compared to EA CLL (median 4.7 kb; range 1.1-44.9; p=0.005; Figure 1A). We highlight that the differences in telomere length observed were restricted to the CD5+/CD19+ cells, with no difference observed in germline samples collected from AA and EA patients. When stratifying by IGHV mutation status, shorter telomere length was observed in u-IGHV when compared to m-IGHV among both AA CLL (median 3.5 kb and 6.3, respectively; p<0.0001) and EA CLL (median 3.9 kb and 7.5, respectively; p<0.0001; Figure 1B). The significant shorter telomere lengths in AA CLL remained significant (p=0.006) after accounting for IGHV status, age, sex, number of mutated CLL driver genes, and Rai stage in multivariate analyses. Conclusions: Our results demonstrated increased telomere erosion in AA CLL compared to EA CLL, even after accounting for other known prognostic factors. Telomere length has been described as an independent prognostic factor in multiple cancers. In CLL, telomere shortening can lead to a selective pressure for loss of function of DNA damage checkpoint genes, becoming permissive to further cell proliferation and telomere shortening. Future studies are needed to see if these findings contribute towards the severe clinical outcomes in AA CLL compared to EA CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Leukocyte Telomere Length Correlates with Extended Female Fertility.

Current social trends of delayed reproduction to the fourth and fifth decade of life call for a better understanding of reproductive aging. Demographic studies correlated late reproduction with general health and longevity. Telomeres, the protective ends of eukaryotic chromosomes, were implicated in various aging-associated pathologies and longevity. To examine whether telomeres are also associated with reproductive aging, we measured by Southern analysis the terminal restriction fragments (TRF) in leukocytes of women delivering a healthy infant following a spontaneous pregnancy at 43–48 years of age. We compared them to age-matched previously fertile women who failed to conceive above age 41. The average TRF length in the extended fertility group (9350 bp) was significantly longer than in the normal fertility group (8850 bp; p-value = 0.03). Strikingly, excluding women with nine or more children increased the difference between the groups to over 1000 bp (9920 and 8880 bp; p-value = 0.0009). Nevertheless, we observed no apparent effects of pregnancy, delivery, or parity on telomere length. We propose that longer leukocyte telomere length reflects higher oocyte quality, which can compensate for other limiting physiological and behavioral factors and enable successful reproduction. Leukocyte telomere length should be further explored as a novel biomarker of oocyte quality for assessing reproductive potential and integrating family planning with demanding women’s careers.

Peripheral airways type 2 inflammation, neutrophilia and microbial dysbiosis in severe asthma.

BackgroundIL‐13 is considered an archetypal T2 cytokine central to the clinical disease expression of asthma. The IL‐13 response genes, which are upregulated in central airway bronchial epithelial of asthma patients, can be normalized by high‐dose inhaled steroid therapy in severe asthma. However, this is not the case within the peripheral airways. We have sought to further understand IL‐13 in the peripheral airways in severe asthma through bronchoalveolar analysis.MethodsBronchoalveolar lavage samples were collected from 203 asthmatic and healthy volunteers, including 78 with severe asthma. Inflammatory mediators were measured using a multiple cytokine immunoassay platform. This analysis was replicated in a further 59 volunteers, in whom 16S rRNA analysis of BAL samples was undertaken by terminal restriction fragment length polymorphism.ResultsSevere asthma patients with high BAL IL‐13, despite treatment with high‐dose inhaled corticosteroids, had more severe lung function and significantly higher BAL neutrophil percentages, but not BAL eosinophils than those with normal BAL‐13 concentrations. This finding was replicated in the second cohort, which further associated BAL IL‐13 and neutrophilia with a greater abundance of potentially pathogenic bacteria in the peripheral airways.ConclusionOur findings demonstrate a steroid unresponsive source of IL‐13 that is associated with BAL neutrophilia and bacterial dysbiosis in severe asthma. Our findings highlight the biological complexity of severe asthma and the importance of a greater understanding of the innate and adaptive immune responses in the peripheral airways in this disease.

Abstract P017: Biomarkers Representing Aging-Related Biological Pathways Are Associated With All-Cause Mortality: The Framingham Study

Background: Chronological aging is the result of different biological processes and molecular mechanisms that, if identified, may increase our ability to mitigate aging-related decline in body functions. We hypothesized that aging-related biomarkers reflecting multiple biological pathways are associated with all-cause mortality. Methods and Results: We related leukocyte telomere length (LTL) expressed by terminal restriction fragment (TRF) length, urinary concentrations of creatinine-corrected isoprostanes, circulating concentrations of insulin-like growth factor (IGF)-1, and asymmetrical dimethylarginine (ADMA) in up to 2314 Framingham Offspring Study participants attending routine examination cycles (mean age 58 yrs, 55% women) to all-cause mortality using Cox proportional hazards regression models. Biomarkers were modeled individually and in combination, adjusting for age, sex, body mass index, systolic blood pressure, diabetes, smoking status, hypertension medication, total cholesterol/HDL. We created also a biomarker score as a composite of biomarkers associated with mortality risk. Kaplan Meier curves were created ( Figure ) to graphically present the survival time as a function of tertiles of the biomarker score. There were 593 deaths (274 women) during a median follow-up of 20 years. TRF and IGF-1 values were inversely related to all-cause mortality (multivariable-adjusted hazard ratios [HR] per SD increase, 0.86, 95% confidence interval [CI], 0.75-0.998 and 0.89, 95% CI 0.81-0.97 for TRF and IGF-1, respectively). Isoprostanes and ADMA values were positively related to all-cause mortality (multivariable-adjusted HR per SD increase, 1.17, 95% CI, 1.11-1.23, and 1.09, 95% CI, 1.01-1.18, respectively). Conclusion: In our prospective community-based study, aging-related biomarkers were associated with all-cause mortality, supporting the concept that molecular pathways represented by these biomarkers may reflect the processing of aging in the community.

Telomere length is independently associated with age, oxidative biomarkers, and sport training in skeletal muscle of healthy adult males

In skeletal muscle, which mainly contains postmitotic myonuclei, it has been suggested that telomere length remains roughly constant throughout adult life, or shortens in response to physiopathological conditions in muscle diseases or in the elderly. However, telomere length results from both the replicative history of a specific tissue and the exposure to environmental, DNA damage-related factors, therefore the predictive biological significance of telomere measures should combine the analysis of the various interactive factors. In the present study, we analysed any relationship between telomere length [mean and minimum terminal restriction fragment (TRF) length] chronological age, oxidative damage (4-HNE, protein carbonyls), catalase activity, and heat shock proteins expression (αB-crystallin, Hsp27, Hsp90) in semitendinous muscle biopsies of 26 healthy adult males between 20 and 50 years of age, also exploring the influence of regular exercise participation. The multiple linear regression analysis identified age, 4-HNE, catalase, and training status as significant independent variables associated with telomere length and jointly accounting for ∼30–36% of interindividual variation in mean and/or minimum TRF length. No association has been identified between telomere length and protein carbonyl, αB-crystallin, Hsp27, and Hsp90, as well as between age and the variables related to stress response. Our results showed that skeletal muscle from healthy adults displays an age-dependent telomere attrition and that oxidised environment plays an age-independent contribution, partially influenced by exercise training.

Long-term dynamics of the bacterial community in a Swedish full-scale wastewater treatment plant

ABSTRACTThe operational efficiency of activated sludge wastewater treatment plants depends to a large extent on the microbial community structure of the activated sludge. The aims of this paper are to describe the composition of the bacterial community in a Swedish full-scale activated sludge wastewater treatment plant, to describe the dynamics of the community and to elucidate possible causes for bacterial community composition changes. The bacterial community composition in the activated sludge was described using 16S rRNA gene libraries and monitored for 15 months by a terminal restriction fragment (T-RF) length polymorphism (T-RFLP) analysis of the 16S rRNA gene. Despite variable environmental conditions, a large fraction of the observed T-RFs were present at all times, making up at least 50% in all samples, possibly representing a relatively stable core fraction of the bacterial community. However, the proportions of the different T-RFs in this fraction as well as the T-RFs in the more variable fraction showed a significant variation over time and temperature. The difference in community composition between summer and winter coincided with observed differences in floc structure. These observations suggest a relationship between floc properties and bacterial community composition, although additional experiments are required to determine causality.

Repeatability of a terminal restriction fragment telomere length assay on red drum fin tissue

Telomere length measurements are becoming increasingly popular in biology as a non-lethal tool for estimating organisms’ biological conditions. One particularly appealing application of telomere length measurements for conservation biologists is genetic age estimation. Most age estimation methods require animals to be sacrificed followed by the dissection and sectioning of calcified structures. Because telomeres degrade over time, telomere lengths may provide non-lethal and minimally invasive tool for estimating age. Furthermore, telomere length may also be useful for predicting longevity and fitness. Traditionally, telomere lengths have been measured using terminal restriction fragment (TRF) methodology; however, there is no one standard laboratory procedure to estimate the size of these fragments, and few studies supply repeatability data for the respective methods. While TRF telomere length assays are applicable to all organisms with known telomere repeat motif sequences, inconsistencies in TRF methodology include template DNA treatment, restriction enzymes used, gel electrophoresis methods, and probe/detection methods. Here we present a study documenting repeatability data for a specific laboratory procedure for estimating telomere length using two common restriction enzymes, denaturing pulsed field gel electrophoresis, and a chemiluminescent detection system for three replicates of 16 individual red drum collected in South Carolina. The data were analyzed following traditional methods using an average across the entire TRF distribution, which represents all TRF lengths found in the sample; and following non-traditional methods, where only a subsample of the TRF distributions are analyzed. Our results demonstrate a strong correlation among average TRF lengths between replicates while analyzing the entire TRF distribution; however, our results show a decreasing trend of consistency between replicates as smaller portions of the TRF distribution were analyzed. These results suggest our method provides a repeatable estimate of average TRF length in a sample, and we urge that caution should be exercised when subsampling TRF distributions for telomere length analysis. Repeatability data not only validate the assay producing the data, but also may allow researchers to develop standard optimal criteria to balance cost and extent of their study design.

Changes in telomere length distribution in low-dose X-ray-irradiated human umbilical vein endothelial cells.

Ionizing radiation (IR) is known to be a cause of telomere dysfunction in tumor cells; however, very few studies have investigated X-ray-related changes in telomere length and the telomerase activity in normal human cells, such as umbilical vein endothelial cells (HUVECs). The loss of a few hundred base pairs from a shortened telomere has been shown to be important with respect to cellular senescence, although it may not be detected according to traditional mean telomere length [assessed as the terminal restriction fragment (TRF)] analyses. In the present study, a continuous time window from irradiation was selected to examine changes in the telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, apoptotic rate, and survival rate in HUVECs from the first day to the fourth day after the administration of a 0.5-Gy dose of irradiation. The mean TRF length in the irradiated HUVECs showed shorter telomere length in first 3 days, but they were not statistically significant. On the other hand, according to the percentage analysis of the telomere length, a decreasing tendency was noted in the longer telomere lengths (9.4-4.4 kb), with a significant increase in the shortest telomeres (4.4-2.3 kb) among the irradiated cells versus the controls from the first day to the third after irradiation; no significant differences were noted on the fourth day. These results suggest that the shortest telomeres are sensitive to the late stage of radiation damage. The proliferation of irradiated cells was suppressed after IR in contrast to the non-irradiated cells. The apoptotic rate was elevated initially both in IR- and non-IR-cells, but that of IR-cells was maintained at an elevated level thereafter in contrast to that of non-IR-cells decreasing promptly. Therefore, a 0.5-Gy dose of IR induces persistent apoptosis leading to an apparent growth arrest of the normal HUVECs.

The study on telomere length for age estimation in a Thai population.

Age is one of the key parameters in establishing a physical characteristic profile of an individual. For biological evidence left in crime scenes such as blood, saliva, hair, etc, the evidence owner's age can be determined only by DNA extracted from these materials. Previous researches have found that there are certain DNA regions with specialized characteristic and function called telomere being able to predict age. The present study was to determine the correlation between telomere length and age as well as the effect of sex on the correlation and to create linear regression equation for age estimation in Thai population for forensic purposes. Blood samples obtained from unrelated healthy Thai fresh cadavers without anatomical organ abnormalities were used in this study. All cadaver subjects underwent the postmortem examination in jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital. Fifty blood samples from both sexes of all ages divided into 6 groups for equal age distribution (0-11, 12-23, 24-35, 36-47, 48-59, and 60 years old and older) were collected for a total of 100 samples. The extracted genomic DNA samples were then subjected to telomere length estimation by terminal restriction fragment (TRF) assay. The results showed that the mean TRF length was inversely correlated with age (r = -0.625), and sex did not have a statistically significant influence on the association between age and mean TRF length (P > 0.05). The obtained linear regression equation was y = 113.538 ± 9.604 - 0.012 × (R = 0.391; P < 0.001). However, the correlation was too low to be used for age estimation with high certainty and a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood, and other biological factors such as ethnicity and DNA sources. Roughly, those individuals who had a mean TRF length longer than 6.3 kilobase (kb), between 5.5 and 6.3 kb, and shorter than 5.5 kb aged younger than 28 years, 30 to 44 years, and older than 46 years, respectively (P < 0.01). As a preliminary study, this study highlighted that telomere length could act as a useful biomarker of aging in human population and might be used for rough age estimation in a Thai population. However, further studies with a larger sample size and/or in living human bloods as well as other cell types are recommended to support the results of this study.

Estimating telomere length from whole genome sequence data

Telomeres play a key role in replicative ageing and undergo age-dependent attrition in vivo. Here, we report a novel method, TelSeq, to measure average telomere length from whole genome or exome shotgun sequence data. In 260 leukocyte samples, we show that TelSeq results correlate with Southern blot measurements of the mean length of terminal restriction fragments (mTRFs) and display age-dependent attrition comparably well as mTRFs.

Differential Sensitivity of Telomerase from Human Hematopoietic Stem Cells and Leukemic Cell Lines to Mild Hyperthermia

We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0-4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.

Telomere Shortening in the Colonial CoralAcropora digitiferaDuring Development

To test whether telomere length can be used in estimating the age of colonial corals, we used terminal restriction fragment (TRF) length analysis to compare the telomere lengths of the coral Acropora digitifera at three developmental stages: sperm, planula larvae, and polyps of adult colonies. We also compared the mean TRF lengths between branches at the center and periphery of tabular colonies of A. digitifera. A significant difference was observed in the mean TRF lengths in sperm, planulae, and polyps. The mean TRF length was longest in sperm and shortest in polyps from adult colonies. These results suggest that telomere length decreases during coral development and may be useful for estimating coral age. However, the mean TRF length of branches at the center of a table-form colony tended to be longer than that of peripheral branches, although this difference was not statistically significant. This suggests that both the chronological age of polyps and cell proliferation rate influence telomere length in polyps, and that estimating coral age based on telomere length is not a simple endeavor.

Correlation between terminal restriction fragments and flow‐FISH measures in samples over wide range telomere lengths

Terminal restriction fragment (TRF) analysis of human telomeres was used to calibrate flow-fluorescence in situ hybridization (FF) measures of telomere lengths to expand the range of measures and increase power of resolution of our previously published protocol. TRF data used as the gold standard should be obtained by electrophoresis with suitable resolution applied to appropriately isolated genomic DNA. When we considered TRF attained by correct methods, we found our method to be insufficiently accurate, thus we have reviewed our previously published FF protocol to obtain the best coefficient of determination (r(2)) between our experimental results and valid TRF lengths. Using human telomere-specific PNA probe, Cy5-OO-(CCCTAA)3 , we measured telomere lengths of continuous cell line and of peripheral blood lymphocytes by FF. We modified hybridization, stringency, negative control handling, stoichiometric DNA staining and telomere fluorescence assessment of the protocol. We realized a procedure with increased power of resolution, improved TRF versus FF r(2) values that allowed simultaneous analysis of DNA and telomere duplication. Notwithstanding multiple steps in formamide sampling, recovery was satisfactory. The reviewed FF protocol appeared at least as suitable as the TRF method. Measures obtained by TRF can be affected by chromosome end variability, DNA fragmentation, incomplete digestion and unsuitable electrophoresis. In contrast, the FF technique analyses telomeric sequences confined to preserved nuclei thus overcome most previous limitations. As yet, however, the FF telomere measure cannot be performed together with immunophenotyping and/or generation study by the dye dilution method.

Telomere Length Differences upon Keratinization and its Variations in Normal Human Epidermal Keratinocytes

Telomeres are the physical ends of chromosomes, and are cleaved with each cell division. Telomere loss results from not only incomplete DNA replication (“end replication problem”), followed by the processing of chromosome, but also exposure to various DNA damaging agents. Human epidermis is constantly replaced through replication at the basal layer, followed by upward movement and terminal differentiation at the suprabasal layer. The objective of this study was to assess differences in telomere length between basal and suprabasal keratinocytes, and variations of telomere lengths among basal cells. We investigated terminal restriction fragment (TRF) length in fractions of epidermal cells by Southern blot analysis. Time-dependent trypsin treatment to epidermis divided into basal cell rich and suprabasal cell rich fractions. In the five skin samples from various sites of the body, the mean TRF length were 5,932 ± 470 (range 5,182-6,630) bp in basal cell rich fraction showing 21.5% ± 5.8% (range 12.6-28.6%) of keratin 10 (K10)-positivity, which is marker of keratinizing suprabasal cell, and 5,320 ± 640 (range 4,495-6,212) bp in suprabasal cell rich fraction showing 78.8% ± 4.0% (range 72.8-83.0%) of K10-positivity. It was calculated that K10 positive cells have 1,091 ± 963 (range 248-2,650) bp shorter telomeres compared to K10 negative cells. In addition, fluorescence in situ hybridization (FISH) study showed that columnar groups of keratinocytes with statistically longer telomere in each nucleus than the other epidermal cells in the vicinity occupied occasionally between the tip of dermal papilla and the bottom of rete ridge. The present study showed that the keratinizing suprabasal cells have shorter telomeres compared to basal cells. FISH study showed that some groups of epidermal cells having different replicative histories among the groups of epidermal cells in the vicinity.

Fatty acid oxidation products (‘green odour’) released from perennial ryegrass following biotic and abiotic stress, potentially have antimicrobial properties against the rumen microbiota resulting in decreased biohydrogenation

In this experiment, we investigated the effect of 'green odour' products typical of those released from fresh forage postabiotic and biotic stresses on the rumen microbiota and lipid metabolism. Hydroperoxyoctadecatrienoic acid (HP), a combination of salicylic and jasmonic acid (T), and a combination of both (HPT) were incubated in vitro in the presence of freeze-dried ground silage and rumen fluid, under rumen-like conditions. 16S rRNA (16S cDNA) HaeIII-based terminal restriction fragment length polymorphism-based (T-RFLP) dendrograms, canonical analysis of principal coordinates graphs, peak number and Shanon-Weiner diversity indices show that HP, T and HPT likely had antimicrobial effects on the microbiota compared to control incubations. Following 6 h of in vitro incubation, 15.3% of 18:3n-3 and 4.4% of 18:2n-6 was biohydrogenated in control incubations, compared with 1.3, 9.4 and 8.3% of 18:3n-3 for HP, T and HPT treatments, respectively, with negligible 18:2n-6 biohydrogenation seen. T-RFLP peaks lost due to application of HP, T and HPT likely belonged to as yet uncultured bacteria within numerous genera. Hydroperoxyoctadecatrienoic acid, T and HPT released due to plant stress potentially have an antimicrobial effect on the rumen microbiota, which may explain the decreased biohydrogenation in vitro. These data suggest that these volatile chemicals may be responsible for the higher summer n-3 content of bovine milk.

Leukocyte telomere dynamics in the elderly

Limited data suggest that leukocytes of the elderly display ultra-short telomeres. It was reported that in some elderly persons leukocyte telomere length (LTL) shows age-dependent elongation. Using cross-sectional and longitudinal models, we characterized LTL dynamics in participants of the Longitudinal Study of Aging Danish Twins. We measured LTL by Southern blots of the terminal restriction fragment length (TRFL) in 476 individuals (73–94 years) in a cross-sectional evaluation and in a subset of this cohort comprising 80 individuals (73–81 years at baseline) who were followed–up for approximately 10 years. Based on the mean TRFL, we found that a) the average rate of LTL attrition was respectively, 27 bp/year (P < 0.001) and 31 bp/year (P < 0.001) for the cross-sectional and longitudinal evaluations, and b) mean TRFL was 180 bp (95 % CI 43, 320) longer in females than males (P < 0.010). For the TRFL distribution, which captures telomeres of all lengths in the DNA sample, we observed significant shifts with age toward shorter telomeres. Based on the measurement error of the TRFLs, we computed that in the longitudinal evaluation 10.6 % of individuals would manifest LTL elongation over 10 years, assuming a 340 bp attrition during this period. This was not significantly different from the empirical observation of 7.5 % of individuals showing LTL elongation. We conclude that accumulation of ultra-short telomeres in leukocytes of the elderly reflects a shift toward shorter telomeres in the entire telomere distribution. Measurement error is the probable explanation for LTL elongation in longitudinal studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10654-013-9780-4) contains supplementary material, which is available to authorized users.

Telomere Length in Mantle Cell Lymphoma.

AbstractAbstract 2509Mantle cell lymphoma (MCL) is a specific subtype of non-Hodgkin lymphoma with a median overall survival of approximately 4–5 years with significant heterogeneity. MCL shows a relatively high number of genomic alterations per case and one of the known causes of genomic instability in somatic cells is telomere dysfunction. Short, dysfunctional telomeres lead to an increase in chromosomal end-to-end fusions which predispose the cells to chromosomal aberrations, genomic complexity and malignant transformation. Here we investigated telomere length in MCL to evaluate associations with biological and clinical disease characteristics as well as outcome. We included a CLL cohort as a control group and for comparison with MCL.A well characterized cohort of 73 MCL and 55 CLL patients treated at the Universities of Ulm, Würzburg and Heidelberg was used for the study. Telomere length was measured using a Q-PCR-based technique with 25ng of DNA used per reaction in triplicates. Three telomere length controls were included in every plate to control for plate to plate variations. The technique was validated by terminal restriction fragment length (TRF) analysis (R2=0.8516). The TRF value of telomere length for each sample was calculated from the linear regression of Q-PCR vs. TRF. Samples were selected for tumor loads of >70% and were analyzed for correlation with biological and clinical factors.Telomere length in MCL had no significant association with IGHV (P=0.4966) and TP53(P=0.4225) mutation status which was in marked contrast to CLL where a significant correlation of short telomeres with unmutated IGHV(P<0.0001) and mutant TP53(P=0.0062) was confirmed (Figure 1). Moreover, no significant variations in median telomere length were observed among the MCL subgroups with the different genomic aberrations (del(9p21), del(11q22-q23), del(13q14), del(13qTerminal) and del(17p13)), while in CLL, short telomeres were found to be associated with high risk genomic aberrations del(17p13) (P=0.0001) and del(11q22-q23) (P=0.0037). Since telomere dysfunction is known to confer genomic complexity, we analyzed the correlation of telomere length with the number of genomic aberrations per case. A weak but significant (R2=0.38, P=0.0106) association of telomere shortening with increasing number of genomic aberrations was observed in the younger (age<median) MCL subgroup, whereas no correlation (R2=0.0079, P=0.7516) was observed in the older patient subgroup (age>median). No correlation of telomere length with the proliferation index (Ki67) was observed in the MCL samples, which might be due to variable telomerase activity among the different MCL subsets. Telomere length in MCL also did not correlate with blastoid subtype, age, LDH levels, WBC or with MIPI groups. We studied the impact of telomere length on outcome in MCL and no significant difference in overall survival (median 48 vs. 50 months, p=0.7997) (Figure 2) or progression-free survival (median 26.4 vs. 21.7, p=0.9178) was observed between the long (>median) and short (<median) subgroups. In contrast, when evaluating the impact of established prognostic factors on outcome to verify that our cohort was representative, we observed the expected correlations of Ki67 (p=0.0001), MIPI index (p=0.007) and B symptoms (p=0.0119) with disease outcome.In conclusion, telomere length in MCL was not associated with any of the established clinical or biological factors relevant for pathogenesis or prognostication and there was no correlation with disease outcome. This is in stark contrast to CLL where telomere length has been significantly associated with biological disease characteristics, pathogenic mechanisms and survival. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Telomere maintenance mechanisms in malignant peripheral nerve sheath tumors: expression and prognostic relevance

This study investigated the prevalence and the prognostic relevance of the 2 known telomere maintenance mechanisms (TMMs), telomerase activity (TA) and alternative lengthening of telomeres (ALT), in malignant peripheral nerve sheath tumors (MPNST). In 57 specimens from 49 patients with MPNST (35 sporadic, 14 neurofibromatosis type 1-related), TA was determined using the telomeric repeat amplification protocol, and ALT was detected by assaying ALT-associated promyelocytic leukemia bodies (APB) and terminal restriction fragment (TRF) length distribution. TA or ALT (defined on the basis of APB) alone was found in 24.6% or 26.3% of the lesions, respectively, whereas 6 cases (10.5%) were TA+/ALT+. A concordance between APB and TRF results in defining the ALT status was observed in 44 of 57 cases (77.2%; P < .0001). TA was more frequently expressed in samples from patients with neurofibromatosis type 1 than in those with sporadic disease (60% vs 29.4%, P = 0.087). In the overall series, TA proved to be prognostic for 5-year disease-specific death (hazard ratio, 3.78; 95% confidence interval [CI], 1.60-8.95; P = .002), even when adjusted for the presence of neurofibromatosis type 1 (hazard ratio, 4.22; 95% CI, 1.804-9.874; P = .001) and margin status after surgery (hazard ratio, 5.78; 95% CI, 2.19-15.26; P < .001). Conversely, ALT did not significantly affect clinical outcome of MPNST using either APB expression (hazard ratio, 1.25; 95% CI 0.54-2.89; P = 0.605) or TRF distribution (hazard ratio, 0.57; 95% CI, 0.17-1.96; P = .375) as the detection approach. Our results indicate for the first time that both TMMs, TA and ALT, are present in MPNST and differentially affect patient prognosis.

Telomere length is a prognostic factor for overall survival in colorectal cancer

The aim of this study was to determine whether telomere length is an independent prognostic factor for the prevention and survival of colorectal cancer. Terminal restriction fragment (TRF) length was determined by Southern blot in tumours and paired normal tissue samples from 147 patients with sporadic colorectal cancer who had undergone surgery. The TRF length ratio (TRFLR) was determined as the ratio between the length of the patient's tumour and normal tissue.The classification and regression tree technique was used to determine optimal cut-off values (≤ 1 or > 1). Mean TRF length was 6.79 Kbp (1.19-13.99) in tumour tissue and 7.81 Kbp (3.63-15.70) in normal mucosa (P < 0.001). Mean TRFLR was 0.88. Telomere length and telomere length ratio were not correlated with any clinicopathological factors. In univariate analysis, overall survival was related to N stage (lymph node +/-; P = 0.002), TNM classification (P = 0.019) and TRFLR (≤ 1 or > 1; P = 0.014). In multivariate analysis, overall survival was significantly associated with TRFLR and N stage. Colorectal cancer patients with TRFLR ≤ 1 and negative lymph node involvement had a higher overall survival rate. Telomere length ratio is an independent prognostic factor for survival in colorectal cancer patients, and the telomere lengths in the normal and tumour mucosa of the same patient present with parallel behaviour.

Telomere maintenance in wilms tumors: First evidence for the presence of alternative lengthening of telomeres mechanism

Unlimited proliferative potential is a hallmark of cancer, and can be achieved through the activation of telomere maintenance mechanisms (TMMs). Most tumors activate telomerase, but a significant minority, mainly of mesenchymal origin, uses a recombination-based, alternative lengthening of telomeres (ALT) mechanism. We investigated the presence of ALT in 34 Wilms tumor (WT) samples from 30 patients by using two approaches: (i) the detection of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization and (ii) the assessment of terminal restriction fragment (TRF) length distribution by pulsed field gel electrophoresis. In parallel, telomerase activity (TA) was determined by the telomeric repeat amplification protocol (TRAP) assay. Based on APB expression, ALT was detectable in five samples as the sole TMM and in six samples in association with telomerase. Seventeen samples only expressed TA and in six cases no known TMM was appreciable. Results of TRF length distribution were available in 32 cases, and a concordance between APB and TRF data in defining the ALT phenotype was found in 26/32 cases (81%). The study provides the first evidence of the presence of ALT in WT, and indicates that in a small but defined fraction of cases (about 15%) ALT is the only TMM that supports the development of WT.