T-cell count and T-cell telomere length in patients with severe COVID-19.

Lymphocyte telomere length (TL) is highly variable and shortens with age. Short telomeres may impede TL-dependent T-cell clonal expansion with viral infection. As SARS-CoV-2 infection can induce prolonged and severe T-cell lymphopenia, infected adults, and particularly older adults with short telomeres, may display severe T-cell lymphopenia. To examine the relationship between T-cell TL parameters and T-cell counts, we studied 40 patients hospitalized with severe COVID-19. T-cells were isolated from lymphocytes, counted using flow cytometry, and their TL parameters were measured using the Telomere Shortest Length Assay. The cohort (median age = 62 years, 27% female) was racially and ethnically diverse (33% White, 35% Black, and 33% Other). On intensive care unit study day 1, T-cell count (mean=1.03 x109/L) was inversely related to age (p=0.007) and higher in females than males (p=0.025). Mean TL was 3.88 kilobases (kb), and 45.3% of telomeres were shorter than 3 kb. Using multiple regression analysis and adjusting for age and sex, T-cell count decreased with increased proportion of T-cell telomeres shorter than 3 kb (p=0.033) and increased with mean TL (p=0.052). Our findings suggest an association between the buildup of short telomeres within T-cells and explain in part reduced peripheral blood T-cell counts in patients with severe COVID-19. Shortened T-cell telomeres may be a risk factor for COVID-19-associated T-cell lymphopenia.

The distribution and accumulation of the shortest telomeres in telomere biology disorders.

Individuals with telomere biology disorders (TBDs) have very short telomeres, high risk of bone marrow failure (BMF), and reduced survival. Using data from TBD patients, a mean leukocyte Southern blot telomere length (TL) of 5 kilobases (kb) was estimated as the 'telomere brink' at which human survival is markedly reduced. However, the shortest telomere, not the mean TL, signals replicative senescence. We used the Telomere Shortest Length Assay (TeSLA) to tally TL of all 46 chromosomes in blood-derived DNA and examined its relationship with TBDs. Patients (n = 18) had much shorter mean TL (TeSmTL) (2.54 ± 0.41 kb vs. 4.48 ± 0.52 kb, p < 0.0001) and more telomeres <3 kb than controls (n = 22) (70.43 ± 8.76% vs. 33.05 ± 6.93%, p < 0.0001). The proportion of ultrashort telomeres (<1.6 kb) was also higher in patients than controls (39.29 ± 10.69% vs. 10.40 ± 4.09%, p < 0.0001). TeS <1.6 kb was associated with severe (n = 11) compared with non-severe (n = 7) BMF (p = 0.027). Patients with multi-organ manifestations (n = 10) had more telomeres <1.6 kb than those with one affected organ system (n = 8) (p = 0.029). Findings suggest that TBD clinical manifestations are associated with a disproportionately higher number of haematopoietic cell telomeres reaching a telomere brink, whose length at the single telomere level is yet to be determined.

Buildup from birth onward of short telomeres in human hematopoietic cells.

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere-Shortest-Length-Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age-dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth-89 years) from the general population, and 18 patients with dyskeratosis congenita-telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL-mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans.

Decoupling blood telomere length from age in recipients of allogeneic hematopoietic cell transplant in the BMT-CTN 1202.

The age of allogeneic hematopoietic cell transplant (HCT) donors and their hematopoietic cell telomere length (TL) might affect recipients' outcomes. Our goals were to examine the possible effect of these donors' factors on the recipients' hematopoietic cell TL and quantify hematopoietic cell TL shortening in the critical first three-month post-HCT. We measured hematopoietic cell TL parameters in 75 recipient-donor pairs, from the Blood and Marrow Transplant Clinical Trials Network (protocol#1202), by Southern blotting (SB), the Telomeres Shortest Length Assay (TeSLA), and quantitative PCR (qPCR). Recipients' hematopoietic cell TL parameters post-HCT correlated with donors' age (p<0.001 for all methods), but not recipients' own age, and with donors' pre-HCT hematopoietic cell TL (p<0.0001 for all). Multivariate analyses showed that donors' hematopoietic cell TL pre-HCT, independent of donors' age, explained most of the variability in recipients' hematopoietic cell TL post-HCT (81% for SB, 56% for TeSLA, and 65% for qPCR; p>0.0001 for all). SB and TeSLA detected hematopoietic cell TL shortening in all recipients post-HCT (mean=0.52kb and 0.47kb, respectively; >15-fold the annual TL shortening in adults; p<0.00001 for both), but qPCR detected shortening only in 57.5% of recipients. TeSLA detected a buildup of post-HCT of telomeres <3 kb in 96% of recipients (p<0.0001). In conclusion, HCT decouples hematopoietic cell TL in the recipients from their own age to reflect the donors' age. The potential donors' age effect on outcomes of HCT might be partially mediated by short hematopoietic cell TL in older donors. qPCR-based TL measurement is suboptimal for detecting telomere shortening post-HCT.

Number and Replating Capacity of Endothelial Colony-Forming Cells are Telomere Length Dependent: Implication for Human Atherogenesis.

Background Short leukocyte telomere length (TL) is associated with atherosclerotic cardiovascular disease. Endothelial repair plays a key role in the development of atherosclerosis. The objective was to examine associations between TL and proliferative dynamics of endothelial colony-forming cells (ECFCs), which behave as progenitor cells displaying endothelial repair activity. Methods and Results To isolate ECFCs, we performed a clonogenic assay on blood samples from 116 participants (aged 24-94 years) in the TELARTA (Telomere in Arterial Aging) cohort study. We detected no ECFC clones in 29 (group 1), clones with no replating capacity in other 29 (group 2), and clones with replating capacity in the additional 58 (group 3). Leukocyte TL was measured by Southern blotting and ECFCs (ECFC-TL). Age- and sex-adjusted leukocyte TL (mean±SEM) was the shortest in group 1 (6.51±0.13 kb), longer in group 2 (6.69±0.13 kb), and the longest in group 3 (6.78±0.09 kb) (P<0.05). In group 3, ECFC-TL was associated with the number of detected clones (P<0.01). ECFC-TL (7.98±0.13 kb) was longer than leukocyte TL (6.74±0.012 kb) (P<0.0001) and both parameters were strongly correlated (r=0.82; P<0.0001). Conclusions Individuals with longer telomeres display a higher number of self-renewing ECFCs. Our results also indicate that leukocyte TL, as a proxy of TL dynamics in ECFCs, could be used as a surrogate marker of endothelial repair capacity in clinical and laboratory practice because of easy accessibility of leukocytes. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02176941.

Telomeres and replicative cellular aging of the human placenta and chorioamniotic membranes.

Recent hypotheses propose that the human placenta and chorioamniotic membranes (CAMs) experience telomere length (TL)-mediated senescence. These hypotheses are based on mean TL (mTL) measurements, but replicative senescence is triggered by short and dysfunctional telomeres, not mTL. We measured short telomeres by a vanguard method, the Telomere shortest length assay, and telomere-dysfunction-induced DNA damage foci (TIF) in placentas and CAMs between 18-week gestation and at full-term. Both the placenta and CAMs showed a buildup of short telomeres and TIFs, but not shortening of mTL from 18-weeks to full-term. In the placenta, TIFs correlated with short telomeres but not mTL. CAMs of preterm birth pregnancies with intra-amniotic infection showed shorter mTL and increased proportions of short telomeres. We conclude that the placenta and probably the CAMs undergo TL-mediated replicative aging. Further research is warranted whether TL-mediated replicative aging plays a role in all preterm births.

Imaging assay to probe the role of telomere length shortening on telomere-gene interactions in single cells.

Telomeres are repetitive non-coding nucleotide sequences (TTAGGGn) capping the ends of chromosomes. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Here, we describe an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. We examined two human genes, ISG15 and TERT, for which TPE-OLD has been described before. For both genes, we found less interaction with the telomere on the same chromosome in old cells compared to young cells; and experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.

Measurement of Telomere Length for Longitudinal Analysis: Implications of Assay Precision.

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r = 0.90) and follow-up (r = 0.85), yet their correlation for TL change was poor (r = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis.

The Nexus Between Telomere Length and Lymphocyte Count in Seniors Hospitalized With COVID-19.

Profound T-cell lymphopenia is the hallmark of severe coronavirus disease 2019 (COVID-19). T-cell proliferation is telomere length (TL) dependent and telomeres shorten with age. Older COVID-19 patients, we hypothesize, are, therefore, at a higher risk of having TL-dependent lymphopenia. We measured TL by the novel Telomere Shortest Length Assay (TeSLA), and by Southern blotting (SB) of the terminal restriction fragments in peripheral blood mononuclear cells of 17 COVID-19 and 21 non-COVID-19 patients, aged 87 ± 8 (mean ± SD) and 87 ± 9 years, respectively. TeSLA tallies and measures single telomeres, including short telomeres undetected by SB. Such telomeres are relevant to TL-mediated biological processes, including cell viability and senescence. TeSLA yields 2 key metrics: the proportions of telomeres with different lengths (expressed in %) and their mean (TeSLA mTL), (expressed in kb). Lymphocyte count (109/L) was 0.91 ± 0.42 in COVID-19 patients and 1.50 ± 0.50 in non-COVID-19 patients (p < .001). In COVID-19 patients, but not in non-COVID-19 patients, lymphocyte count was inversely correlated with the proportion of telomeres shorter than 2 kb (p = .005) and positively correlated with TeSLA mTL (p = .03). Lymphocyte count was not significantly correlated with SB mTL in either COVID-19 or non-COVID-19 patients. We propose that compromised TL-dependent T-cell proliferative response, driven by short telomere in the TL distribution, contributes to COVID-19 lymphopenia among old adults. We infer that infection with SARS-CoV-2 uncovers the limits of the TL reserves of older persons. Clinical Trials Registration Number: NCT04325646.

A Mechanism for Severity of Disease in Older Patients with COVID-19: The Nexus between Telomere Length and Lymphopenia.

BACKGROUND: Lymphopenia due to a plummeting T-cell count is a major feature of severe COVID-19. T-cell proliferation is telomere length (TL)-dependent and TL shortens with age. Older persons are disproportionally affected by severe COVID-19, and we hypothesized that those with short TL have less capacity to mount an adequate T-cell proliferative response to SARS-CoV-2. This hypothesis predicts that among older patients with COVID-19, shorter telomeres of peripheral blood mononuclear cells (PBMCs) will be associated with a lower lymphocyte count. METHODS: Our sample comprised 17 COVID-19 and 21 non-COVID-19 patients, aged 87(8) (mean(SD)) and 87 (9) years, respectively. We measured TL by the Telomere Shortest Length Assay, a novel method that measures and tallies the short telomeres directly relevant to telomere-mediated biological processes. The primary analysis quantified TL as the proportion of telomeres shorter than 2 kilobases. For comparison, we also quantified TL by Southern blotting, which measures the mean length of telomeres. RESULTS: Lymphocyte count (109/L) was 0.91 (0.42) in COVID-19 patients and 1.50(0.50) in non-COVID-19 patients (P < 0.001). In COVID-19 patients, but not in non-COVID-19 patients, lymphocyte count was inversely correlated with the proportion of telomeres shorter than 2 kilobases (P = 0.005) and positively correlated with the mean of telomeres measured by TeSLA (P = 0.03). Lymphocyte counts showed no statistically significant correlations with Southern blotting results in COVID-19 or non-COVID-19 patients. CONCLUSIONS: These results support the hypothesis that a compromised TL-dependent T-cell proliferative response contributes to lymphopenia and the resulting disproportionate severity of COVID-19 among old adults. We infer that infection with SARS-CoV-2 uncovers the limits of the TL reserves of older persons.

Determinants of telomere length across human tissues.

Telomere shortening is a hallmark of aging. Telomere length (TL) in blood cells has been studied extensively as a biomarker of human aging and disease; however, little is known regarding variability in TL in nonblood, disease-relevant tissue types. Here, we characterize variability in TLs from 6391 tissue samples, representing >20 tissue types and 952 individuals from the Genotype-Tissue Expression (GTEx) project. We describe differences across tissue types, positive correlation among tissue types, and associations with age and ancestry. We show that genetic variation affects TL in multiple tissue types and that TL may mediate the effect of age on gene expression. Our results provide the foundational knowledge regarding TL in healthy tissues that is needed to interpret epidemiological studies of TL and human health.

Genetics and geography of leukocyte telomere length in sub-Saharan Africans.

Leukocyte telomere length (LTL) might be causal in cardiovascular disease and major cancers. To elucidate the roles of genetics and geography in LTL variability across humans, we compared LTL measured in 1295 sub-Saharan Africans (SSAs) with 559 African-Americans (AAms) and 2464 European-Americans (EAms). LTL differed significantly across SSAs (P = 0.003), with the San from Botswana (with the oldest genomic ancestry) having the longest LTL and populations from Ethiopia having the shortest LTL. SSAs had significantly longer LTL than AAms [P = 6.5(e-16)] whose LTL was significantly longer than EAms [P = 2.5(e-7)]. Genetic variation in SSAs explained 52% of LTL variance versus 27% in AAms and 34% in EAms. Adjustment for genetic variation removed the LTL differences among SSAs. LTL genetic variation among SSAs, with the longest LTL in the San, supports the hypothesis that longer LTL was ancestral in humans. Identifying factors driving LTL variation in Africa may have important ramifications for LTL-associated diseases.

TERC Variants Associated with Short Leukocyte Telomeres: Implication of Higher Early Life Leukocyte Telomere Attrition as Assessed by the Blood-and-Muscle Model.

Short leukocyte telomere length (LTL) is associated with atherosclerotic cardiovascular disease (ASCVD). Mendelian randomisation studies, using single nucleotide polymorphisms (SNPs) associated with short LTL, infer a causal role of LTL in ASCVD. Recent results, using the blood-and-muscle model, indicate that higher early life LTL attrition, as estimated by the ratio between LTL and skeletal muscle telomere length (MTL), rather than short LTL at conception, as estimated by MTL, should be responsible of the ASCVD-LTL connection. We combined LTL and MTL measurements and SNPs profiling in 402 individuals to determine if 15 SNPs classically described as associated with short LTL at adult age were rather responsible for higher LTL attrition during early life than for shorter LTL at birth. Two of these SNPs (rs12696304 and rs10936599) were associated with LTL in our cohort (p = 0.027 and p = 0.025, respectively). These SNPs, both located on the TERC gene, were associated with the LTL/MTL ratio (p = 0.007 and p = 0.037, respectively), but not with MTL (p = 0.78 and p = 0.32 respectively). These results suggest that SNPs located on genes coding for telomere maintenance proteins may contribute to a higher LTL attrition during the highly replicative first years of life and have an impact later on the development of ASCVD.

Telomere length tracking in children and their parents: implications for adult onset diseases.

Adults with comparatively short or long leukocyte telomere length (LTL) typically continue to display comparatively short or long LTL throughout life. This LTL tracking stems from the inability of person-to-person variation in age-dependent LTL shortening during adulthood to offset the wide interindividual LTL variation established prior to adult life. However, LTL tracking in children is unstudied. This study aimed to examine LTL shortening rates and tracking in children and their parents. Longitudinal study in children (n = 67) and their parents (n = 99), whose ages at baseline were 11.4 ± 0.3 and 43.4 ± 0.4 yr, respectively. LTL was measured by Southern blotting at baseline and ∼14 yr thereafter. LTL displayed tracking in both children [intraclass correlation coefficient (ICC) = 0.905, P < 0.001] and their parents (ICC = 0.856, P < 0.001). The children's rate of LTL shortening was twice that of their parents (40.7 ± 2.5 bp/yr; 20.3 ± 2.1 bp/yr, respectively; P < 0.0001). LTL tracking applies not only to adulthood but also to the second decade of life. Coupled with previous work showing that the interindividual variation in LTL across newborns is as wide as in their parents, these findings support the thesis that the LTL-adult disease connection is principally determined before the second decade of life, perhaps mainly at birth.-Benetos, A., Verhulst, S., Labat, C., Lai, T.-P., Girerd, N., Toupance, S., Zannad, F., Rossignol, P., Aviv, A. Telomere length tracking in children and their parents: implications for adult onset diseases.

Transient introduction of human telomerase mRNA improves hallmarks of progeria cells.

Hutchinson-Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra-individual heterogeneity of expression of telomere-associated proteins DNA PKcs/Ku70/Ku80, with low-expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere-associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by ß-galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.

Telomere length and telomerase activity in T cells are biomarkers of high-performing centenarians.

It is generally recognized that the function of the immune system declines with increased age and one of the major immune changes is impaired T-cell responses upon antigen presentation/stimulation. Some "high-performing" centenarians (100+ years old) are remarkably successful in escaping, or largely postponing, major age-related diseases. However, the majority of centenarians ("low-performing") have experienced these pathologies and are forced to reside in long-term nursing facilities. Previous studies have pooled all centenarians examining heterogeneous populations of resting/unstimulated peripheral blood mononuclear cells (PBMCs). T cells represent around 60% of PBMC and are in a quiescence state when unstimulated. However, upon stimulation, T cells rapidly divide and exhibit dramatic changes in gene expression. We have compared stimulated T-cell responses and identified a set of transcripts expressed in vitro that are dramatically different in high- vs. low-performing centenarians. We have also identified several other measurements that are different between high- and low-performing centenarians: (a) The amount of proliferation following in vitro stimulation is dramatically greater in high-performing centenarians compared to 67- to 83-year-old controls and low-performing centenarians; (b) telomere length is greater in the high-performing centenarians; and (c) telomerase activity following stimulation is greater in the high-performing centenarians. In addition, we have validated a number of genes whose expression is directly related to telomere length and these are potential fundamental biomarkers of aging that may influence the risk and progression of multiple aging conditions.

NOVA1 regulates hTERT splicing and cell growth in non-small cell lung cancer.

Alternative splicing is dysregulated in cancer and the reactivation of telomerase involves the splicing of TERT transcripts to produce full-length (FL) TERT. Knowledge about the splicing factors that enhance or silence FL hTERT is lacking. We identified splicing factors that reduced telomerase activity and shortened telomeres using a siRNA minigene reporter screen and a lung cancer cell bioinformatics approach. A lead candidate, NOVA1, when knocked down resulted in a shift in hTERT splicing to non-catalytic isoforms, reduced telomerase activity, and progressive telomere shortening. NOVA1 knockdown also significantly altered cancer cell growth in vitro and in xenografts. Genome engineering experiments reveal that NOVA1 promotes the inclusion of exons in the reverse transcriptase domain of hTERT resulting in the production of FL hTERT transcripts. Utilizing hTERT splicing as a model splicing event in cancer may provide new insights into potentially targetable dysregulated splicing factors in cancer.

Telomerase-Mediated Strategy for Overcoming Non-Small Cell Lung Cancer Targeted Therapy and Chemotherapy Resistance.

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.

Comparison of telomere length measurement methods.

The strengths and limitations of the major methods developed to measure telomere lengths (TLs) in cells and tissues are presented in this review. These include Q-PCR (Quantitative Polymerase Chain Reaction), TRF (Terminal Restriction Fragment) analysis, a variety of Q-FISH (Quantitative Fluorescence In Situ Hybridization) methods, STELA (Single TElomere Length Analysis) and TeSLA (Telomere Shortest Length Assay). For each method, we will cover information about validation studies, including reproducibility in independent laboratories, accuracy, reliability and sensitivity for measuring not only the average but also the shortest telomeres. There is substantial evidence that it is the shortest telomeres that trigger DNA damage responses leading to replicative senescence in mammals. However, the most commonly used TL measurement methods generally provide information on average or relative TL, but it is the shortest telomeres that leads to telomere dysfunction (identified by TIF, Telomere dysfunction Induced Foci) and limit cell proliferation in the absence of a telomere maintenance mechanism, such as telomerase. As the length of the shortest telomeres is a key biomarker determining cell fate and the onset of senescence, a new technique (TeSLA) that provides quantitative information about all the shortest telomeres will be highlighted.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

A method for measuring the distribution of the shortest telomeres in cells and tissues.

Improved methods to measure the shortest (not just average) telomere lengths (TLs) are needed. We developed Telomere Shortest Length Assay (TeSLA), a technique that detects telomeres from all chromosome ends from <1 kb to 18 kb using small amounts of input DNA. TeSLA improves the specificity and efficiency of TL measurements that is facilitated by user friendly image-processing software to automatically detect and annotate band sizes, calculate average TL, as well as the percent of the shortest telomeres. Compared with other TL measurement methods, TeSLA provides more information about the shortest telomeres. The length of telomeres was measured longitudinally in peripheral blood mononuclear cells during human aging, in tissues during colon cancer progression, in telomere-related diseases such as idiopathic pulmonary fibrosis, as well as in mice and other organisms. The results indicate that TeSLA is a robust method that provides a better understanding of the shortest length of telomeres.