Clonal landscape and clinical outcomes of telomere biology disorders: somatic rescuing and cancer mutations.

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development.

The "telomereless" erythrocytes and telomere-length dependent erythropoiesis.

Approximately 25 trillion erythrocytes (red blood cells) circulate in the bloodstream of an adult human, surpassing the number of circulating leukocytes (white blood cells) by a factor of about 1000. Moreover, the erythrocyte turnover rate accounts for approximately 76% of the turnover rate of all circulating blood cells. This simple math shows that the hematopoietic system principally spends its telomere length-dependent replicative capacity on building and maintaining the erythrocyte blood pool. Erythropoiesis (red blood cell production) is thus the principal cause of telomere shortening with age in hematopoietic cells (HCs), a conclusion that holds significant implications for linking telomere length dynamics in HCs to health and lifespan of modern humans.

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.

Telomere-length dependent T-cell clonal expansion: A model linking ageing to COVID-19 T-cell lymphopenia and mortality.

BACKGROUND: Severe COVID-19 T-cell lymphopenia is more common among older adults and entails poor prognosis. Offsetting the decline in T-cell count during COVID-19 demands fast and massive T-cell clonal expansion, which is telomere length (TL)-dependent. METHODS: We developed a model of TL-dependent T-cell clonal expansion capacity with age and virtually examined the relation of T-cell clonal expansion with COVID-19 mortality in the general population. FINDINGS: The model shows that an individual with average hematopoietic cell TL (HCTL) at age twenty years maintains maximal T-cell clonal expansion capacity until the 6th decade of life when this capacity rapidly declines by more than 90% over the next ten years. The collapse in the T-cell clonal expansion capacity coincides with the steep increase in COVID-19 mortality with age. INTERPRETATION: Short HCTL might increase vulnerability of many older adults, and some younger individuals with inherently short HCTL, to COVID-19 T-cell lymphopenia and severe disease. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Short Telomeres and a T-Cell Shortfall in COVID-19: The Aging Effect.

The slow pace of global vaccination and the rapid emergence of SARS-CoV-2 variants suggest recurrent waves of COVID-19 in coming years. Therefore, understanding why deaths from COVID-19 are highly concentrated among older adults is essential for global health. Severe COVID-19 T-cell lymphopenia is more common among older adults, and it entails poor prognosis. Much about the primary etiology of this form of lymphopenia remains unknown, but regardless of its causes, offsetting the decline in T-cell count during SARS-CoV-2 infection demands fast and massive T-cell clonal expansion, which is telomere length (TL)-dependent. We have built a model that captures the effect of age-dependent TL shortening in hematopoietic cells and its effect on T-cell clonal expansion capacity. The model shows that an individual with average hematopoietic cell TL (HCTL) at age twenty years maintains maximal T-cell clonal expansion capacity until the 6th decade of life when this capacity plummets by more than 90% over the next ten years. The collapse coincides with the steep increase in COVID-19 mortality with age. HCTL metrics may thus explain the vulnerability of older adults to COVID-19. That said, the wide inter-individual variation in HCTL across the general population means that some younger adults with inherently short HCTL might be at risk of severe COVID-19 lymphopenia and mortality from the disease. SIGNIFICANCE STATEMENT: Declining immunity with advancing age is a general explanation for the increased mortality from COVID-19 among older adults. This mortality far exceeds that from viral illnesses such as the seasonal influenza, and it thus requires specific explanations. One of these might be diminished ability with age to offset the development of severe T-cell lymphopenia (a low T-cell count in the blood) that often complicates COVID-19. We constructed a model showing that age-dependent shortening of telomeres might constrain the ability of T-cells of some older COVID-19 patients to undertake the massive proliferation required to clear the virus that causes the infection. The model predicts that individuals with short telomeres, principally seniors, might be at a higher risk of death from COVID-19.

The age pattern of the male-to-female ratio in mortality from COVID-19 mirrors that of cardiovascular disease in the general population.

Males are at a higher risk of dying from COVID-19 than females. Older age and cardiovascular disease are also associated with COVID-19 mortality. To better understand how age and sex interact in contributing to COVID-19 mortality, we stratified the male-to-female (sex) ratios in mortality by age group. We then compared the age-stratified sex ratios with those of cardiovascular mortality and cancer mortality in the general population. Data were obtained from official government sources in the US and five European countries: Italy, Spain, France, Germany, and the Netherlands. The sex ratio of deaths from COVID-19 exceeded one throughout adult life, increasing up to a peak in midlife, and declining markedly in later life. This pattern was also observed for the sex ratio of deaths from cardiovascular disease, but not cancer, in the general populations of the US and European countries. Therefore, the sex ratios of deaths from COVID-19 and from cardiovascular disease share similar patterns across the adult life course. The underlying mechanisms are poorly understood and warrant further investigation.

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.

Telomeres and COVID-19.

The medical, public health, and scientific communities are grappling with monumental imperatives to contain COVID-19, develop effective vaccines, identify efficacious treatments for the infection and its complications, and find biomarkers that detect patients at risk of severe disease. The focus of this communication is on a potential biomarker, short telomere length (TL), that might serve to identify patients more likely to die from the SARS-CoV-2 infection, regardless of age. The common thread linking these patients is lymphopenia, which largely reflects a decline in the numbers of CD4/CD8 T cells but not B cells. These findings are consistent with data that lymphocyte TL dynamics impose a limit on T-cell proliferation. They suggest that T-cell lymphopoiesis might stall in individuals with short TL who are infected with SARS-CoV-2.

Association of Leukocyte Telomere Length With Mortality Among Adult Participants in 3 Longitudinal Studies.

Importance: Leukocyte telomere length (LTL) is a trait associated with risk of cardiovascular disease and cancer, the 2 major disease categories that largely define longevity in the United States. However, it remains unclear whether LTL is associated with the human life span. Objective: To examine whether LTL is associated with the life span of contemporary humans. Design, Setting, and Participants: This cohort study included 3259 adults of European ancestry from the Cardiovascular Health Study (CHS), Framingham Heart Study (FHS), and Women's Health Initiative (WHI). Leukocyte telomere length was measured in 1992 and 1997 in the CHS, from 1995 to 1998 in the FHS, and from 1993 to 1998 in the WHI. Data analysis was conducted from February 2017 to December 2019. Main Outcomes and Measures: Death and LTL, measured by Southern blots of the terminal restriction fragments, were the main outcomes. Cause of death was adjudicated by end point committees. Results: The analyzed sample included 3259 participants (2342 [71.9%] women), with a median (range) age of 69.0 (50.0-98.0) years at blood collection. The median (range) follow-up until death was 10.9 (0.2-23.0) years in CHS, 19.7 (3.4-23.0) years in FHS, and 16.6 (0.5-20.0) years in WHI. During follow-up, there were 1525 deaths (482 [31.6%] of cardiovascular disease; 373 [24.5%] of cancer, and 670 [43.9%] of other or unknown causes). Short LTL, expressed in residual LTL, was associated with increased mortality risk. Overall, the hazard ratio for all-cause mortality for a 1-kilobase decrease in LTL was 1.34 (95% CI, 1.21-1.47). This association was stronger for noncancer causes of death (cardiovascular death: hazard ratio, 1.28; 95% CI, 1.08-1.52; cancer: hazard ratio, 1.13; 95% CI, 0.93-1.36; and other causes: hazard ratio, 1.53; 95% CI, 1.32-1.77). Conclusions and Relevance: The results of this study indicate that LTL is associated with a natural life span limit in contemporary humans.

DNA methylation-based estimator of telomere length.

Telomere length (TL) is associated with several aging-related diseases. Here, we present a DNA methylation estimator of TL (DNAmTL) based on 140 CpGs. Leukocyte DNAmTL is applicable across the entire age spectrum and is more strongly associated with age than measured leukocyte TL (LTL) (r ~-0.75 for DNAmTL versus r ~ -0.35 for LTL). Leukocyte DNAmTL outperforms LTL in predicting: i) time-to-death (p=2.5E-20), ii) time-to-coronary heart disease (p=6.6E-5), iii) time-to-congestive heart failure (p=3.5E-6), and iv) association with smoking history (p=1.21E-17). These associations are further validated in large scale methylation data (n=10k samples) from the Framingham Heart Study, Women's Health Initiative, Jackson Heart Study, InChianti, Lothian Birth Cohorts, Twins UK, and Bogalusa Heart Study. Leukocyte DNAmTL is also associated with measures of physical fitness/functioning (p=0.029), age-at-menopause (p=0.039), dietary variables (omega 3, fish, vegetable intake), educational attainment (p=3.3E-8) and income (p=3.1E-5). Experiments in cultured somatic cells show that DNAmTL dynamics reflect in part cell replication rather than TL per se. DNAmTL is not only an epigenetic biomarker of replicative history of cells, but a useful marker of age-related pathologies that are associated with it.

Smoking does not accelerate leucocyte telomere attrition: a meta-analysis of 18 longitudinal cohorts.

Smoking is associated with shorter leucocyte telomere length (LTL), a biomarker of increased morbidity and reduced longevity. This association is widely interpreted as evidence that smoking causes accelerated LTL attrition in adulthood, but the evidence for this is inconsistent. We analysed the association between smoking and LTL dynamics in 18 longitudinal cohorts. The dataset included data from 12 579 adults (4678 current smokers and 7901 non-smokers) over a mean follow-up interval of 8.6 years. Meta-analysis confirmed a cross-sectional difference in LTL between smokers and non-smokers, with mean LTL 84.61 bp shorter in smokers (95% CI: 22.62 to 146.61). However, LTL attrition was only 0.51 bp yr-1 faster in smokers than in non-smokers (95% CI: -2.09 to 1.08), a difference that equates to only 1.32% of the estimated age-related loss of 38.33 bp yr-1. Assuming a linear effect of smoking, 167 years of smoking would be required to generate the observed cross-sectional difference in LTL. Therefore, the difference in LTL between smokers and non-smokers is extremely unlikely to be explained by a linear, causal effect of smoking. Selective adoption, whereby individuals with short telomeres are more likely to start smoking, needs to be considered as a more plausible explanation for the observed pattern of telomere dynamics.

Shortened leukocyte telomere length is associated with reduced pulmonary function and greater subsequent decline in function in a sample of World Trade Center responders.

The objective of this study was to examine whether shorter leukocyte telomere length (LTL) is associated with more rapid pulmonary function decline in a longitudinal study of World Trade Center (WTC) responders. WTC responders (N = 284) participating in a monitoring study underwent blood sampling and were followed prospectively for spirometric outcomes. A single blood sample was taken to measure LTL using southern blotting. Outcomes included percent-predicted one-second forced expiratory volume (FEV1%), forced vital capacity (FVC%), and the FEV1/FVC ratio. In a subset, percent-predicted diffusing capacity (DLCO%) was also measured. Longitudinal modeling examined prospectively collected information over five years since blood was banked was used to examine the rate of change in pulmonary functioning over time. Severity of WTC exposure was assessed. Shorter LTL was associated with lower FEV1% and FVC% at baseline. For example, 29.9% of those with LTL <6.5 kbps had FEV1% <80% whereas only 12.4% of those with LTL ≥6.5 had FEV1% <80% (RR = 2.53, 95%CI = [1.70-3.76]). Lower DLCO% was also significantly associated with shorter LTL. Longitudinal models identified a prospective association between shorter LTL and greater yearly rates of decline in FEV1% (0.46%/year, 95%CI = [0.05-0.87]) and in the FEV1/FVC ratio (0.19%/year, 95%CI = [0.03-0.36]). There were no associations between severity of exposure and either LTL or pulmonary function. Longitudinal analyses revealed that shorter LTL, but not severity of WTC exposures, was associated with poorer pulmonary functioning and with greater subsequent decline in pulmonary functioning over time. These findings are consistent with the idea that shortened LTL may act as a biomarker for enhanced pulmonary vulnerability in the face of acute severe toxic inhalation exposures.

DNA methylation GrimAge strongly predicts lifespan and healthspan.

It was unknown whether plasma protein levels can be estimated based on DNA methylation (DNAm) levels, and if so, how the resulting surrogates can be consolidated into a powerful predictor of lifespan. We present here, seven DNAm-based estimators of plasma proteins including those of plasminogen activator inhibitor 1 (PAI-1) and growth differentiation factor 15. The resulting predictor of lifespan, DNAm GrimAge (in units of years), is a composite biomarker based on the seven DNAm surrogates and a DNAm-based estimator of smoking pack-years. Adjusting DNAm GrimAge for chronological age generated novel measure of epigenetic age acceleration, AgeAccelGrim.Using large scale validation data from thousands of individuals, we demonstrate that DNAm GrimAge stands out among existing epigenetic clocks in terms of its predictive ability for time-to-death (Cox regression P=2.0E-75), time-to-coronary heart disease (Cox P=6.2E-24), time-to-cancer (P= 1.3E-12), its strong relationship with computed tomography data for fatty liver/excess visceral fat, and age-at-menopause (P=1.6E-12). AgeAccelGrim is strongly associated with a host of age-related conditions including comorbidity count (P=3.45E-17). Similarly, age-adjusted DNAm PAI-1 levels are associated with lifespan (P=5.4E-28), comorbidity count (P= 7.3E-56) and type 2 diabetes (P=2.0E-26). These DNAm-based biomarkers show the expected relationship with lifestyle factors including healthy diet and educational attainment.Overall, these epigenetic biomarkers are expected to find many applications including human anti-aging studies.

Rapid shortening of leukocyte telomeres is associated with poorer pulmonary function among healthy adults.

AIM: The study aimed to examine the association between leukocyte telomere length (LTL) attrition over 13 years (between mean age 30 and mean age 43) and lung function at mean age 50. MATERIALS & METHODS: In a longitudinal observational study LTL was determined twice on a population-based sample of 481 Jewish residents of Jerusalem at mean ages 30 and 43 years. Pulmonary function was determined at mean age 50 years. Multiple linear regression and multivariable ordinal logistic modeling were applied. Akaike's Information Criteria (AIC) was used for model selection. RESULTS: In unadjusted analysis, Forced Expiratory Volume in 1 s (FEV1%) was inversely associated with the LTL attrition rate (standardized beta = -0.110, P = 0.023) but not with the baseline LTL. Forced Vital Capacity (FVC%) was inversely associated with the LTL attrition rate (standardized beta = -0.108, P = 0.026). Multivariable adjustment mildly attenuated the association with the LTL attrition rate (standardized beta = -0.100, P = 0.034 for FEV1% and -0.093, P = 0.042 for FVC%). This would be consistent with a 3.3% [95% Confidence Interval (CI): 3.1-3.4%] decline in FEV1% and a 3.0% (95% CI:2.8-3.1%) decline in FVC% per year. In linear regression models the LTL-pulmonary function association did not differ by sex, social mobility, pack-years smoking exposure, or level of GlycA, a novel systemic inflammatory marker. CONCLUSIONS: Greater LTL attrition between mean age 30 and mean age 43 was associated with poorer lung function at mean age 50 years. The availability of longitudinal data on LTL attrition for the first time in the current study strengthens the case for LTL change preceding change in lung function.

An epigenetic biomarker of aging for lifespan and healthspan.

Identifying reliable biomarkers of aging is a major goal in geroscience. While the first generation of epigenetic biomarkers of aging were developed using chronological age as a surrogate for biological age, we hypothesized that incorporation of composite clinical measures of phenotypic age that capture differences in lifespan and healthspan may identify novel CpGs and facilitate the development of a more powerful epigenetic biomarker of aging. Using an innovative two-step process, we develop a new epigenetic biomarker of aging, DNAm PhenoAge, that strongly outperforms previous measures in regards to predictions for a variety of aging outcomes, including all-cause mortality, cancers, healthspan, physical functioning, and Alzheimer's disease. While this biomarker was developed using data from whole blood, it correlates strongly with age in every tissue and cell tested. Based on an in-depth transcriptional analysis in sorted cells, we find that increased epigenetic, relative to chronological age, is associated with increased activation of pro-inflammatory and interferon pathways, and decreased activation of transcriptional/translational machinery, DNA damage response, and mitochondrial signatures. Overall, this single epigenetic biomarker of aging is able to capture risks for an array of diverse outcomes across multiple tissues and cells, and provide insight into important pathways in aging.

Reflections on telomere dynamics and ageing-related diseases in humans.

Epidemiological studies have principally relied on measurements of telomere length (TL) in leucocytes, which reflects TL in other somatic cells. Leucocyte TL (LTL) displays vast variation across individuals-a phenomenon already observed in newborns. It is highly heritable, longer in females than males and in individuals of African ancestry than European ancestry. LTL is also longer in offspring conceived by older men. The traditional view regards LTL as a passive biomarker of human ageing. However, new evidence suggests that a dynamic interplay between selective evolutionary forces and TL might result in trade-offs for specific health outcomes. From a biological perspective, an active role of TL in ageing-related human diseases could occur because short telomeres increase the risk of a category of diseases related to restricted cell proliferation and tissue degeneration, including cardiovascular disease, whereas long telomeres increase the risk of another category of diseases related to increased proliferative growth, including major cancers. To understand the role of telomere biology in ageing-related diseases, it is essential to expand telomere research to newborns and children and seek further insight into the underlying causes of the variation in TL due to ancestry and geographical location.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.