Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

Telomere length is associated with disease severity and declines with age in dyskeratosis congenita

Dyskeratosis congenita is a cancer-prone bone marrow failure syndrome caused by aberrations in telomere biology.

Telomere length in paroxysmal nocturnal hemoglobinuria correlates with clone size

OBJECTIVE: To study if telomere length can be used as a surrogate marker for the mitotic history in normal and affected hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The telomere length was measured by automated multicolor flow fluorescence in situ hybridization in glycosyl-phosphatidyl-inositol anchored protein (GPI)-negative and GPI-positive peripheral blood leukocytes. Eleven patients were studied, two with predominantly hemolytic PNH and nine with PNH associated with marrow failure. RESULTS: Telomere length in GPI-negative cells was significantly shorter than in GPI-positive cells of the same patient (p < 0.01, n = 11). The difference in telomere length (telomere length in GPI-positive minus telomere length in GPI-negative cells) correlated with the percentage of GPI-negative white blood cells. CONCLUSION: Our results support the hypothesis that telomere length is correlated to the replicative history of GPI-positive and GPI-negative cells and warrant further studies of telomere length in relation to disease progression in PNH.

Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in stem cell turnover around 1 year of age

Stem cells of various tissues are typically defined as multipotent cells with 'self-renewal' properties. Despite the increasing interest in stem cells, surprisingly little is known about the number of times stem cells can or do divide over a lifetime. Based on telomere-length measurements of hematopoietic cells, we previously proposed that the self-renewal capacity of hematopoietic stem cells is limited by progressive telomere attrition and that such cells divide very rapidly during the first year of life. Recent studies of patients with aplastic anemia resulting from inherited mutations in telomerase genes support the notion that the replicative potential of hematopoietic stem cells is directly related to telomere length, which is indirectly related to telomerase levels. To revisit conclusions about stem cell turnover based on cross-sectional studies of telomere length, we performed a longitudinal study of telomere length in leukocytes from newborn baboons. All four individual animals studied showed a rapid decline in telomere length (approximately 2-3 kb) in granulocytes and lymphocytes in the first year after birth. After 50-70 weeks the telomere length appeared to stabilize in all cell types. These observations suggest that hematopoietic stem cells, after an initial phase of rapid expansion, switch at around 1 year of age to a different functional mode characterized by a markedly decreased turnover rate.

Telomere length in human natural killer cell subsets

Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. In the elderly, the cytotoxic function of NK cells is often compromised. Telomeres progressively shorten with each cell division and with age in most somatic cells eventually leading to chromosomal instability and cellular senescence. We studied the telomere length in NK cell subsets isolated from peripheral blood using "flow FISH," a method in which the hybridization of telomere probe in cells of interest is measured relative to internal controls in the same tube. We found that the average telomere length in human NK cells decreased with age as was previously found for human T lymphocytes. Separation of adult NK cells based on CD56 and CD16 expression revealed that the telomere length was significantly shorter in CD56(dim)CD16(+) (mature) NK cells compared to CD56(bright)CD16(-) (immature) NK cells from the same donor. Furthermore, sorting of NK cells based on expression of activation markers, such as NKG2D and LFA-1, revealed that NK cells expressing these markers have significantly shorter telomeres. Telomere fluorescence was very heterogeneous in NK cells expressing CD94, killer inhibitory receptor (KIR), NKG2A, or CD161. Our observations indicate that telomeric DNA in NK cells is lost with cell division and with age similar to what has been observed for most other hematopoietic cells. Telomere attrition in NK cells is a plausible cause for diminished NK cell function in the elderly.

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.

Comparison of telomere length in black and white teachers from South Africa: the sympathetic activity and ambulatory blood pressure in Africans study

OBJECTIVE Telomere length is a marker of biological aging that has been linked to cardiovascular disease risk. The black South African population is witnessing a tremendous increase in the prevalence of cardiovascular disease, part of which might be explained through urbanization. We compared telomere length between black South Africans and white South Africans and examined which biological and psychosocial variables played a role in ethnic difference in telomere length. METHODS We measured leukocyte telomere length in 161 black South African teachers and 180 white South African teachers aged 23 to 66 years without a history of atherothrombotic vascular disease. Age, sex, years having lived in the area, human immunodeficiency virus (HIV) infection, hypertension, body mass index, dyslipidemia, hemoglobin A1c, C-reactive protein, smoking, physical activity, alcohol abuse, depressive symptoms, psychological distress, and work stress were considered as covariates. RESULTS Black participants had shorter (median, interquartile range) relative telomere length (0.79, 0.70-0.95) than did white participants (1.06, 0.87-1.21; p < .001), and this difference changed very little after adjusting for covariates. In fully adjusted models, age (p < .001), male sex (p = .011), and HIV positive status (p = .023) were associated with shorter telomere length. Ethnicity did not significantly interact with any covariates in determining telomere length, including psychosocial characteristics. CONCLUSIONS Black South Africans showed markedly shorter telomeres than did white South African counterparts. Age, male sex, and HIV status were associated with shorter telomere length. No interactions between ethnicity and biomedical or psychosocial factors were found. Ethnic difference in telomere length might primarily be explained by genetic factors.

Telomere length increase after weight loss induced by bariatric surgery: results from a 10 years prospective study

BACKGROUND/OBJECTIVES Obesity contributes to telomere attrition. Studies focusing on short-term effects of weight loss have been unable to identify protection of telomere length. This study investigates long-term effects of pronounced weight loss induced by bariatric surgery on telomere length. SUBJECTS/METHODS One hundred forty-two patients were recruited in a prospective, controlled intervention study, follow-up investigations were done after 10.46±1.48 years. A control group of normal weight participants was recruited and followed from 1995 to 2005 in the Bruneck Study. A total of 110 participants from each study was matched by age and sex to compare changes in telomere length. Quantitative PCR was used to determine telomere length. RESULTS Telomere length increased significantly by 0.024±0.14 (P=0.047) in 142 bariatric patients within 10 years after surgery. The increase was different from telomere attrition in an age- and sex-matched cohort population of the Bruneck Study (-0.057±0.18; β=0.08; P=0.003). Significant changes in telomere length disappeared after adjusting for baseline body mass index (BMI) because of general differences in BMI and telomere length between the two study populations (β=0.07; P=0.06). Age was proportional to telomere length in matched bariatric patients (r=0.188; P=0.049) but inversely correlated with telomere length in participants of the Bruneck Study (r=-0.197; P=0.039). There was no association between percent BMI/excess weight loss and telomere attrition in bariatric patients. Baseline telomere length in bariatric patients was inversely associated with baseline plasma cholesterol and triglyceride concentrations. Telomere shortening was associated with lower high-density lipoprotein cholesterol and higher fasting glucose concentration at baseline in bariatric patients. CONCLUSIONS Increases in relative telomere length were found after bariatric surgery in the long term, presumably due to amelioration of metabolic traits. This may overrule the influence of age and baseline telomere length and facilitate telomere protection in patients experiencing pronounced weight loss.

Longitudinal Telomere Erosion in Lymphocyte Subsets of Patients with Atherosclerotic Peripheral Arterial Disease (PAD).

Telomere attrition has been linked to accelerate vascular ageing and seems to predispose for vascular disease. Our aim was to study the telomere length dynamics over time and in subsets of leukocytes from 15 patients with peripheral arterial disease (PAD). The mean telomere length in subsets of leukocytes of patients with PAD was in the normal range of age-related telomere length values from healthy individuals. However, we found significant higher telomere attrition for T-cells from patients with PAD over a time period of six months when compared to the controls. The higher telomere loss in T-cells of patients with PAD most likely reflects a higher cell turnover of this leukocyte subset, which is involved in the process of chronic inflammatory disease underlying vascular disease. Further studies are needed to confirm these data and to assess how far this T-cell telomere attrition will correlate to the extent of the disease.

Telomeres and prognosis in patients with chronic lymphocytic leukaemia

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length ( TL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold TL of -4.2 kb was the best predictor for progression-free and overall survival. TL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the TL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the TL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

Disabling c-Myc in childhood medulloblastoma and atypical teratoid/rhabdoid tumor cells by the potent G-quadruplex interactive agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex-forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC(50), 0.25-0.39 micromol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc-dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors.