Lower Telomere Length Research Articles

Decreased telomere length in a subgroup of young individuals with bipolar disorders: replication in the FACE-BD cohort and association with the shelterin component POT1

IntroductionA 10-15 years decrease in life expectancy has been observed in individuals with bipolar disorder (BD) and has been associated with premature cellular aging, but mechanisms involved remain unclear. Our team recently identified a subgroup of young individuals with prematurely shortened telomere length (TL).ObjectivesThe aims of the present study were to replicate this observation in a larger sample and to analyze the expression levels of genes associated with age or TL in a subsample of these individuals.Methods TL was measured by qPCR using peripheral blood DNA from 542 individuals with BD. Clustering analyzes were performed with age and TL as classification variables to identify similar groups.Gene expression of 29 genes, including 20 associated with age and 9 with TL, was analyzed by RT-qPCR using peripheral blood RNA in a subgroup of 129 individuals. Gene expressions were compared between groups obtained from the previous clustering analyzes by Kruskal-Wallis and Mann-Whitney tests.ResultsClustering analyzes identified 3 subgroups and replicated the clustering previously described: a subgroup of aged individuals with a low TL (mean age : 51.73 years ; mean TL : 2), a subgroup of young individuals with a high TL (mean age : 29.02 years ; mean TL : 4.36) and a subgroup of young individuals but with a low TL (mean age : 29.64 years ; mean TL : 1.96). None of the tested clinical variables were significantly associated with this subgroup.Furthermore, gene expression level analyzes showed that only POT1 expression was different between the two subgroups of young individuals, with a downregulation of POT1 expression in the subgroup with a lower TL level. POT1 is a protein involved in the maintenance of TL. POT1 binds to another protein TPP1 allowing the recruitment of telomerase, the enzyme which extends TL. Our hypothesis is that in the subgroup presenting a lower POT1 expression, the POT1-TPP1 complex cannot form and thus prevents telomerase recruitment and TL elongation.ConclusionsThis study confirms, on a larger sample, the existence of a subgroup of young individuals with BD presenting accelerated cellular aging. The observed decrease of POT1 expression level suggests a newly described cellular mechanism in individuals with BD, that may contribute to telomere shortening.Disclosure of InterestNone Declared

Bone Marrow Failure Associated with Short Telomeres and Digenic Variants of Uncertain Significance in Telomere Biology Genes

DISCUSSION Telomere Biology Disorders (TBD) are a group of heritable disorders characterized by abnormally short telomeres. There are at least 15 genes associated with TBD including ACD, CTC1, DKC1, NHP2, NOP10, PARN, POT1, RTEL1, STN1, TERC, TERT, TINF2, WRAP53 NAF1 and ZCCHC8, which can be inherited in autosomal dominant (AD), autosomal recessive (AR), or X-linked recessive patterns. Genetic anticipation has been described in some families with more severe symptoms appearing in each successive generation. We report two patients that presented with bone marrow failure, who were subsequently identified to have short telomeres and variants of uncertain significance (VUS) in two different telomere genes inherited from their parents. These cases suggest a potential for digenic inheritance of TBD. CASE 1: At age 6, patient was diagnosed with stage 4 neuroblastoma. He was treated with chemotherapy, surgery, immunotherapy, and autologous stem cell rescue. At age 10, he developed frequent and severe epistaxis and was found to have pancytopenia. He underwent a bone marrow aspirate and biopsy which revealed a hypocellular marrow and der (1;7), which is associated with myelodysplastic syndrome (MDS). Genetic evaluation revealed one pathogenic variant in DNAJC21 (from father) and VUS in NAF1 (c.1375C>T) (from father) and RTEL1 (c.533T>C) (from mother). Because NAF1 and RTEL1 are related to AD or AR TBD, telomere length testing was performed. Patient was found to have low telomere length, < 1st percentile, in granulocytes, lymphocytes, memory T cells and NK cells (Figure 1). The patient's mother was found to have borderline low telomere length >=1 and <10 th percentiles in her lymphocytes, naïve T cells, memory T cells, and B cells, and low telomere length in her granulocytes. Father had borderline low telomere length >=1 and <10 th percentiles in granulocytes but normal telomere length in all other subsets. Neither parent reported any symptoms that would be consistent with TBD. We interpreted these findings as consistent with the patient having TBD and the patient's mother either also having TBD or being a carrier, and the potential that genetic anticipation may be involved. The NAF1 variant appears likely to be benign as father's telomeres were normal in length except for low telomere length in his granulocyte compartment. It is possible that the patient's prior treatment for neuroblastoma may have contributed to the shortened telomeres. Over the course of a year, patient developed worsening bone marrow failure. He then underwent reduced intensity conditioning using busulfan, fludarabine and anti-thymocyte globulin for a 4/6 matched cord stem cell transplant. He engrafted on day +10. CASE 2: A 15-year-old male presented with a depressed skull fracture and scalp hematoma following a sports-related head injury. He was incidentally found to have a platelet count of 69 x 10 3/microliter, mild anemia with macrocytosis, and mild leukopenia. Bone marrow biopsy revealed a hypocellular marrow with multilineage maturation, mild fibrosis, and no cytogenetic abnormalities. The patient's telomere lengths were low, below the 1 st percentile, in all 6 white blood cell subsets (Figure 2). He underwent clinical trio exome sequencing along with his parents. He was found to have VUSs in TERT (c.3158-80G>A) (from mother), TINF2 (c.814T>C) (from mother) and SRP72 (c.1928C>T) (from father). Mother was also found to have low telomere length (<1 st percentile) in all 6 subsets. Both parents have normal blood counts while patient continues to have pancytopenia and is transfusion independent. It is presently unclear which of the VUSs are contributing to the patient's phenotype as the TERT and TINF2 variants are both associated with bone marrow failure and heterozygous germline variants in SRP72 is associated with familial MDS. Genes that cause TBD can be inherited through various modes of inheritance. These two patients who presented with bone marrow failure, identified to have VUSs in more than one TBD-associated genes and functional evidence of shortened telomeres, highlight the potential for a digenic mode of inheritance underlying TBD. Synergy between two variants contributes to a penetrant phenotype and can result in earlier or more severe onset of disease. Our patients' parents were asymptomatic or mildly affected, compared to our patients. Larger cohort studies are needed to determine the prevalence of this mode of inheritance.

Blood Count Recovery and Telomere Elongation in a TINF2 Patient Under Treatment with a Gonadotropin-Releasing Hormone (GnRH) Agonist

Telomere biology disorders (TBDs) caused by germline variants in TINF2 are a common cause of severe disease presenting in early childhood. Progressive bone marrow failure in patients with TBDs is often treated with androgens, known to improve hematopoiesis but with a controversial impact on telomere elongation. Here, we report a unique case of a young TINF2 male patient that had a striking recovery of blood counts and robust telomere elongation in peripheral blood cells during treatment with histrelin, a gonadotropin-releasing hormone (GnRH) agonist and inhibitor of testosterone production. We investigated whether underlying clonal events were linked to blood counts and telomere length (TL) dynamics. We extracted medical information and collected peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) over an 10-yr period beginning at diagnosis (age 4), which was based on cytopenias, marked marrow hypocellularity, very low TL in all leukocyte subsets by Flow-FISH, and a de novo TINF2 p.K302Rfs*17 germline variant. Following 2 yrs of observation, he was started on danazol for progressive cytopenias but it was discontinued after 21 months due to the onset of danazol-related precocious puberty, for which histrelin was administered via subcutaneous implants over 4.7 yrs. Hemoglobin levels normalized while on danazol and remained stable at last follow-up. Although platelet counts increased and stabilized at ~ 40,000 on danazol, they steadily increased after initiation of histrelin up to 156,000; they then gradually declined to 108,000 over the 18 months following removal of the last histrelin implant (Fig. 1A). The absolute neutrophil count normalized while on histrelin and has remained in normal range. While TLs remained very low in all leukocyte subsets while on danazol, they either stabilized or minimally changed (+/- 0.2 kb) in lymphocytes, CD45RA+, CD45RA-, and CD57+ cells or increased by 2.8 kb or 1 kb in granulocytes and B cells, respectively. While on histrelin, TL further increased in the lymphocytes, granulocytes, and B cells to max gains of 0.4 kb, 3 kb, and 2 kb, respectively. Fourteen months after discontinuing histrelin, TL shortened in all subsets. Screening of PBMC at 8.5 yrs of follow-up for clonal hematopoiesis (CH) in myeloid and telomere-related genes by error-corrected DNA sequencing identified POT1 p.D291Y and TINF2 p.W20* variants at variant allele frequencies (VAFs) of 20% and 4%, respectively, as likely somatic genetic rescue (SGR) mechanisms (Fig. 1B). POT1 D291Ywas retrospectively tracked at a VAF of 0.7% in total DNA before any treatment and found at a VAF of 8% in PBMC collected during danazol treatment, 27% during histrelin treatment, and 14% after histrelin discontinuation. TINF2 W20* emerged while on histrelin at a VAF of 1% in PBMC; this mutation VAF slightly increased during histrelin treatment but robust clonal expansion up to a VAF of 21% was not seen until off histrelin. Hematologic improvement, particularly thrombocytopenia, and continued telomere elongation in granulocytes and B cells (CD20+) coincided with clonal expansion of the POT1 D291Y variant. Single-cell proteogenomic sequencing of BMMNCs evidenced POT1 D291Y and TINF2 W20* as independent clonal events that emerged in CD34+ hematopoietic stem cells (HSC) and were present both in the megakaryocytic and erythroid, and myeloid and lymphoid progenitor cells. During histrelin treatment, POT1 D291Y dominated the landscape and associated with increased lymphocyte counts, being present in 30% of HSC and progenitor cells, myeloid cells, and T lymphocytes. When histrelin was discontinued, hematopoiesis greatly shifted toward myeloid production, which coincided with TINF2 W20*clonal expansion and a substantial reduction of progenitors and CD8+ cells. Single-cell RNAseq of BM HSC revealed similar transcriptional profiles while on danazol and histrelin compared to baseline, with up and downregulation of inflammatory pathways and MYC targets, respectively; these programs were enhanced while on histrelin compared to danazol. In summary, we characterized a unique combination of SGR events in a patient with a TINF2 germline mutation, which can guide future research on new therapeutic strategies. As opposed to androgens, histrelin promoted the specific expansion of POT1 clones with proliferation of HSC and progenitor cells, improved hematopoiesis, and increased TL.

The association between telomere length and non-alcoholic fatty liver disease: a prospective study

BackgroundResearch on the association between telomere length (TL) and incident non-alcoholic fatty liver disease (NAFLD) is limited. This study examined this association and further assessed how TL contributes to the association of NAFLD with its known risk factors.MethodsQuantitative PCR (polymerase chain reaction) was employed to assess leucocyte telomere length. Polygenic risk score (PRS) for NAFLD, air pollution score, and lifestyle index were constructed. Cox proportional hazard models were conducted to estimate the hazard ratios (HRs) and 95% confidence intervals.ResultsAmong 467,848 participants in UK Biobank, we identified 4809 NAFLD cases over a median follow-up of 12.83 years. We found that long TL was associated with decreased risk of incident NAFLD, as each interquartile range increase in TL resulted in an HR of 0.93 (95% CI 0.89, 0.96). TL partly mediated the association between age and NAFLD (proportion mediated: 15.52%). When assessing the joint effects of TL and other risk factors, the highest risk of NAFLD was found in participants with low TL and old age, low TL and high air pollution score, low TL and unfavorable lifestyle, and low TL and high PRS, compared to each reference group. A positive addictive interaction was observed between high PRS and low TL, accounting for 14.57% (2.51%, 27.14%) of the risk of NAFLD in participants with low telomere length and high genetic susceptibility.ConclusionsLong telomere length was associated with decreased risk of NAFLD incidence. Telomere length played an important role in NAFLD.Graphical

Telomere Length Testing of Lymphocytes Alone By Flow-FISH Is a Highly Sensitive and Specific Test to Screen for a Telomere Biology Disorder in a Cohort of Children and Very Young Adults with Bone Marrow Failure

Background Uncovering an underlying telomere biology disorder (TBD) in patients with bone marrow failure (BMF) has crucial implications for treatment and outcomes, including hematopoietic cell transplantation and malignancy risk. Telomere length (TL) testing of peripheral blood leukocytes by clinical Flow-FISH has become an important test in diagnosing TBDs. In seminal studies, Alter et al. compared age-adjusted TL lengths (< or ≥1 st percentile) in lymphocytes (lymphs), granulocytes (grans), and four lymph subsets in individuals with a known TBD and their unaffected relatives. They found that very low (VL) TL (<1 st percentile) in lymphs had a sensitivity (Sens) of 97%, specificity (Spec) of 91%, and positive predictive value (PPV) of 85% for a TBD diagnosis. VL TL in 3 or 4 lymph subsets had slightly better test performance with a Sens of 98%, Spec of 94%, and PPV of 89%. We hypothesized that test performance characteristics of flow FISH would be different if applied to a ‘real-world’ cohort, which could inform the use of TL in lymphs alone versus in the four lymph subsets for different clinical indications. Methods We performed a search of the electronic medical record to identify all individuals who had Flow-FISH performed at Texas Children's Hospital from 2007 - 2022 (Figure 1). We excluded those who had the 2-panel test (lymphs and grans) or family TL cascade testing. We collected the indication for and results of Flow-FISH, demographic and clinical characteristics, and genetic testing information via chart review. A composite score (Comp Score) was created to define TBD (Comp Score of 1 or 2) vs. Not TBD (Comp Score <1). Performance of lymph TL alone and TL in 3 or 4 lymph subsets in predicting TBD was evaluated using Sens, Spec, PPV, NPV, area under ROC curve (AUC), and Cohen's Kappa statistic (k). Results Our final cohort included 433 individuals who received the 6-panel test (lymphs, grans, and four lymph subsets) with a median age of 9.7 years, range 0.06-22.6. Top indications for Flow-FISH were severe aplastic anemia (SAA) (n=106), mild aplastic anemia (AA) (i.e., not fulfilling criteria for MAA or SAA) (103), cytopenias without known or documented bone marrow hypoplasia or dysplasia (70), moderate aplastic anemia (MAA) (37), non-therapy-related myelodysplastic syndrome (26), and an incidental TBD gene variant identified through comprehensive sequencing (17). Twenty-two subjects (5.1%) had a TBD, 17 (3.9%) of which had a pathogenic/likely pathogenic variant (P/LPV) in a known TBD gene (Figure 1). Seven of those with SAA (6.6%) had VL TL in lymphs, 5 (4.7%) of which also had VL TL in 3 or 4 of the lymph subsets, and only 1 (0.9%) had a TBD based on Comp Score. Importantly, none had VL TL in 3 or 4 of the lymph subsets without VL TL in lymphs. Five of those with MAA (13.5%) had VL TL in lymphs, all of which had VL TL in 3 or 4 lymph subsets. Three had a TBD based on Comp Score, and each had a PV in a TBD gene; a TBD variant was not identified in two despite exome sequencing and chromosomal microarray testing. Eighteen of those with mild AA (17.5%) had VL TL in lymphs, 15 (14.6%) also had VL TL in 3 or 4 lymph subsets, 9 (8.7%) had a TBD based on Comp Score, and 7 (6.8%) had a P/LPV in a TBD gene. The Sens of TL in lymphs < vs. ≥ 1 st percentile and TL < 1 st percentile in 3 or 4 vs. < 3 lymph subsets against TBD based on Comp Score in those with AA regardless of severity was 100%, and the Spec ranged from 90.4% (lymph TL testing alone in mild AA) to 96.2% (lymph subsets testing in SAA) (Table 1). The PPV for both test approaches was low (≤20%) in SAA due to the low prevalence of TBD in this population. PPV was between 50-60% in the other AA groups. The AUC for detecting a TBD in the various AA groups was comparably high for both test approaches (0.95-0.98). The two tests had a substantial to almost perfect agreement (k=0.82, p<0.001). Conclusions This single institutional cohort demonstrates that the prevalence of TBD in young patients presenting with SAA is remarkably low, and the Sens and Spec of TL testing in lymphs alone are sufficiently high to detect the rare TBD patient and prompt additional testing (e.g., genetics) in few patients due to false positive testing. The prevalence of TBD is higher in those with MAA or mild AA, yet both test approaches detect all TBD patients. Spec level in mild AA is marginally higher with lymph subset TL testing but, at ~94%, underscores the need for other clinical parameters (e.g., genetics) to establish a TBD diagnosis in a small percentage of patients.

Peripheral blood mononuclear cell mitochondrial dysfunction in acute alcohol-associated hepatitis.

Patients with acute alcohol-associated hepatitis (AH) have immune dysfunction. Mitochondrial function is critical for immune cell responses and regulates senescence. Clinical translational studies using complementary bioinformatics-experimental validation of mitochondrial responses were performed in peripheral blood mononuclear cells (PBMC) from patients with AH, healthy controls (HC), and heavy drinkers without evidence of liver disease (HD). Feature extraction for differentially expressed genes (DEG) in mitochondrial components and telomere regulatory pathways from single-cell RNAseq (scRNAseq) and integrated 'pseudobulk' transcriptomics from PBMC from AH and HC (n=4 each) were performed. After optimising isolation and processing protocols for functional studies in PBMC, mitochondrial oxidative responses to substrates, uncoupler, and inhibitors were quantified in independent discovery (AH n=12; HD n=6; HC n=12) and validation cohorts (AH n=10; HC n=7). Intermediary metabolites (gas-chromatography/mass-spectrometry) and telomere length (real-time PCR) were quantified in subsets of subjects (PBMC/plasma AH n=69/59; HD n=8/8; HC n=14/27 for metabolites; HC n=13; HD n=8; AH n=72 for telomere length). Mitochondrial, intermediary metabolite, and senescence-regulatory genes were differentially expressed in PBMC from AH and HC in a cell type-specific manner at baseline and with lipopolysaccharide (LPS). Fresh PBMC isolated using the cell preparation tube generated optimum mitochondrial responses. Intact cell and maximal respiration were lower (p≤.05) in AH than HC/HD in the discovery and validation cohorts. In permeabilised PBMC, maximum respiration, complex I and II function were lower in AH than HC. Most tricarboxylic acid (TCA) cycle intermediates in plasma were higher while those in PBMC were lower in patients with AH than those from HC. Lower telomere length, a measure of cellular senescence, was associated with higher mortality in AH. Patients with AH have lower mitochondrial oxidative function, higher plasma TCA cycle intermediates, with telomere shortening in nonsurvivors.

Absolute telomere length in peripheral blood lymphocytes of workers exposed to construction environment

ABSTRACT Construction environment is composed of various substances classified as carcinogens. Thus, workers exposed in this environment can be susceptible to genomic instability that can be evaluated by absolute telomere length (TL). In this work, we evaluated TL in construction workers compared to a non-exposed group performed by qPCR assay. The TL was evaluated in 59 men exposed to the construction environment (10 years of exposure) and 49 men non-exposed. Our data showed that individuals exposed to the construction environment exhibited a significantly lower TL in relation to non-exposed group (p = 0.009). Also, on the multiple linear regression model, we observed that TL was significantly influenced by the construction environment exposure (p ≤ 0.001). Additionally, the arsenic exposure is associated to a shortening telomere (p ≤ 0.001), and the lead exposure caused an increase in TL (p ≤ 0.001). Thus, our findings suggest a modulation in TL by construction environment exposure, mainly by arsenic and lead exposure.

Telomere length and mitochondrial DNA copy number in bipolar disorder: identification of a subgroup of young individuals with accelerated cellular aging

The 10–15-years decrease in life expectancy observed in individuals with bipolar disorder (BD) has been linked to the concept of accelerated cellular aging. Telomere length (TL) and mitochondrial DNA copy number (mtDNAcn) have been proposed as markers of cellular aging and comparisons between individuals with BD and healthy controls (HC) sometimes led to conflicting results. Previous studies had moderate sample sizes and studies combining these two markers into a single analysis are scarce. Using quantitative polymerase chain reaction, we measured both TL and mtDNAcn in DNA (peripheral blood) in a sample of 130 individuals with BD and 78 HC. Regression analyses, receiver operating characteristic (ROC), and clustering analyses were performed. We observed significantly lower TL and mtDNAcn in individuals with BD as compared to HC (respective decrease of 26.5 and 35.8%). ROC analyses showed that TL and mtDNAcn highly discriminated groups (AUC = 0.904 for TL and AUC = 0.931 for mtDNAcn). In the whole population, clustering analyses identified a group of young individuals (age around 36 years), with accelerated cellular aging (both shorter TL and lower mtDNAcn), which consisted mostly of individuals with BD (85.5%). The subgroup of patients with young age but accelerated aging was not characterized by specific clinical variables related to the course of BD or childhood maltreatment. However, patients in this subgroup were more frequently treated with anticonvulsants. Further characterization of this subgroup is required to better understand the molecular mechanisms and the risk factors of accelerated cellular aging in BD.

Endotoxemia, vitamin D and premature biological ageing in Arab adults with different metabolic states

There are limited studies on the association of endotoxin, a potent mediator of gut-derived inflammation and telomere length (TL). We investigated (1) the influence of adiposity on endotoxin and TL amongst Saudi adults according to type 2 diabetes mellitus (T2DM) status and (2) the influence vitamin D may have on TL attrition. Anthropometric data and fasting blood samples were taken from 775 Saudi adults visiting different primary care centers in Riyadh [387 T2DM and 388 non-T2DM]. TL, derived from peripheral blood mononuclear cells, was analyzed by Quantitative real-time polymerase chain reaction and circulating endotoxin levels by Limulus Amebocyte Lysate assay. Subjects were stratified based on obesity and T2DM status. A significant lower TL was observed in the non-obese T2DM group as compared with their non-obese, non-T2DM counterparts (p = 0.002). Significant inverse associations between TL, endotoxin and endotoxin activity were observed in the cohort with obesity. Regression analysis showed that endotoxin was a significant predictor for TL in all subjects and even after stratification according to subgroups; with variances perceived in circulating TL stronger among non-T2DM obese (10%; p = 0.003) than non-T2DM non-obese (12%; p = 0.007). Also, in the non-T2DM group, TL and HDL-cholesterol predicted 29% of the variances perceived in 25(OH)D (p < 0.001). Taken together these findings show that circulating endotoxin and 25(OH)D are associated with premature biological ageing influenced by adiposity and metabolic state; suggesting future intervention studies to manipulate gut microbiome and or vitamin D levels may offer ways to mitigate premature TL attrition.

Targeting Telomere Biology in Acute Lymphoblastic Leukemia.

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target; hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.

Telomere length and uric acid in patients with new cases of hypertension according to the 10-year prospective study in urban randomized population

Abstract Objective To reveal the association between lymphocyte telomere length and serum uric acid level in 10-year prospective randomized study of Belarusian urban population. Design and method In 2007/2008 a 10-year prospective study of 3500 persons living in a district of a belarussian city and selected by the random number method started (coverage of the survey was 97.9%). In 2012/2013 according to the results of the second screening 2888 people were examined (coverage of the survey was 84.3%). A 10-year prospective analysis was provided in 2017/2018 and included 145 randomized subjects with hypertension diagnosed in the second screening. Examination of these patients consisted of standard cardiovascular risk factors questionnaires, measurements of blood pressure, blood chemistry, electrocardiography, echocardiography, ultrasound examination of brachiocephalic vessels, real time PCR for the detection of telomere length of peripheral blood lymphocytes. Results 2171 persons with normal blood pressure and 1257 persons with hypertension were examined in 2007/2008. In the second screening we established 286 new cases of hypertension (14,4% of men and 12,2% of women). According to multifactorial regression analysis, adjusted for age and sex, a significant positive relationship between new cases of hypertension and IV quartile of uric acid level (339–527 μmol/l) was revealed in 5 years (df=1; χ2 Wald=5,1; p&amp;lt;0,05). Regression analysis of 145 randomized subjects of 286 new cases of hypertension showed that, adjusted for age and sex, the IV quartile of uric acid level (339–527 μmol/l), determined in the first screening, was also associated with the I quartile of telomere length in 10 years (df=1; χ2 Wald=4,5; p&amp;lt;0,05). It was investigated that the level of telomere length had a significant negative association with BMI (p&amp;lt;0.05), R wave in aVL (p&amp;lt;0.05), IL-6 level (p&amp;lt;0,05) in these subjects in cross-sectional study in 2017/2018. Conclusions Regardless of age and sex, level of serum uric acid ≥339 μmol/l was a risk factor for development of hypertension in randomized population in 5-year prospective analysis and predicted lower telomere length in patients with new cases of hypertension in 10-year prospective analysis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): State scientific and technical program “Medical and diagnostic technologies”, subprograms “Heart and blood vessels”

PF194 EVALUATION OF TELOMERE LENGTH AND TELOMERASE ACTIVITY AS PROGNOSTIC MARKERS IN A PEDIATRIC PATIENT COHORT WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Telomere length maintenance is tightly regulated with cell proliferation and senescence. To overcome physiologic telomere attrition with cell division, long‐term repopulating cells like stem and progenitor cells upregulate telomerase. Pathophysiologically, over 90% of cancer cells reactivate telomerase enabling replicative immortality. Acute lymphoblastic leukemia (ALL) is the most common type of acute leukemias in pediatric patients (pts) and a neoplastic disease with a rapid clonal expansion.Aims:We aimed to assess the mitotic history of the malignant ALL clone by telomere length (TL) and the potential of telomerase activity (TA) as a prognostic marker.Methods:Peripheral blood samples collected before treatment initiation from 9 pediatric pts (4 females/ 5 males) with newly diagnosed pre‐B cell ALL according to WHO 2016 criteria were analyzed for TL and TA. All pts were treated according to the international collaborative protocol (AIEOP‐BFM ALL 2009 Registry) for children and adolescents with ALL. Based on (cyto)genetics and treatment response, 6 pts are in the intermediate and 3 pts are in the high‐risk prognostic group. TL was measured in subsets of leukocytes by automated multicolor flow‐FISH and compared to TL percentiles of the reference range obtained from over 400 healthy individuals. TA in leukocytes was analyzed by Telomere Repeat Amplification Protocol.Results:The median age of the patient (pt) cohort was 10.8 years (range: 2.8–17.8) at diagnosis and study entry. The median leukocyte count was 9.8 G/L (1.4–38.6) with a median lymphocyte count of 2.0 G/L (0.6–8.1) and a median lymphoblast count of 6.37 G/L (0.2–31.27). The median TL value in blasts was with 5.2 kb (1.9–8.3) significantly (p = 0.016) lower than 7.5 kb (6.9–9.3) for the B‐cell subset. In 6/9 pt samples the TL values for blasts were below the 1st percentile of the TL reference range for lymphocytes whereas 3 pts had values between the 1st and 50th percentile. The median age‐adjusted difference in TL (dTL = difference of TL to the 50th percentile for respective age) was −4.5 kb (−7.1–1.1) in blasts which was significantly higher than median dTL values in lymphocytes (−2.1 kb; range −6.2–0) and B‐cells (−1.9 kb, range −2.6–0.4). Median TA in the mononuclear cell fraction containing blasts was 2.4 telomere‐elongated products (TP)/cell (0.3–11.1), whereas no TA was detected in leukocytes from healthy donors. Notably, pediatric ALL pts with a high risk score (n = 3) had a higher median TA of 5.5 TP/cell (2.2–11.1) than those with an intermediate risk score (n = 6) with 1.8 TP/cell (0.3–3.3).Summary/Conclusion:The significantly lower TL values in ALL blast cells from newly diagnosed pediatric pts reflect the mitotic history of the malignant clone with a high number of cell divisions. Despite measurable TA in ALL blasts, attrition of TL seemed not compensated and therefore, the extent of clonal proliferation might even be underestimated. Importantly, the higher median TA in blast cells of pts in the high‐risk prognostic group indicates that TA could be an attractive prognostic marker. Further data on TL and TA on serial pt samples will identify if TA could also serve as predictive marker and therapeutic target.

Profiles of Telomere Length Adult and Childhood Myelodysplastic Syndromes: A Comparison with Hematologic Diseases

Background: Telomere is a repeatitive sequence at the chromosome end, functioning as a cap, and the length of telomere becomes shortened after each cell devision, eventually going to senescence. Thus, the length of telomere reflects the proliferative capacity of cell and cellular age. Inherited mutation of telomerase gene results in dyskeratosis congenita characterized by telomere shortening and multi-organ stem cell damage. In contrast, stem cells or tumor cells maintain their telomere length by telomerase or alternative telomere lengthening. To investigate the profiles of telomere length among hematologic malignancies, we measured mean telomere length and heterogeneity of telomere length in various hematologic diseases, in comparison with bone marrow failure syndrome, aplastic anemia (AA) and myelodysplastic syndrome (MDS).Methods: Telomere length was measured by interphase fluorescent in situ hybridization. A total of 153 patients were enrolled; adult MDS (n=53), childhood MDS (n=17), adult AA (n=20), childhood AA (n=46), acute myelogenous leukemia (AML) (n=5), Fanconi anemia (FA) (n=9) and normal control (n=72: normal bone marrow n=36), and normal peripheral blood of children n=36) as a control group. Telomere length was expressed as T/C ratio with adjustment of fluorescence intensity of centromeric signal in chromosome 2. Mean length of telomere and distribution width (SD: standard deviation), were compared to those of normal cells.Results: Mean telomere length (T/C ratio) was 6.7 (adult AA), 5.9 (childhood AA), 5.0 (adult MDS), 4.4 (childhood MDS), 2.1 in FA, 9.4 in AML, and 19.0 in normal control. Heterogeneity of telomere length expressed as telomere length SD was 4.6 in adult AA, 3.7 in childhood AA, 3.5 in adult MDS, 2.5 in childhood MDS, 1.4 in FA and 4.1 in AML. Cell population below 5th percentile of normal control expressed as percentage among total cells, was 75.0% in adult AA, 4.8% in childhood AA, 12.5% in childhood MDS, 87.0% in adult MDS and 0% in AML. When adult MDS patients were divided into two groups, high (¡Ã average) or low (<average), according to the interphase telomere length among MDS patients. The mean survival time for MDS patients with high telomere length group (n=16) was 53.6 months, and 88.5 months in the low telomere length group (n=37). The overall survival rate of the high telomere length group was higher than the low group with minimal statistical significance by Kaplan Meier survival analysis (P =0.056). The multivariate Cox regression analysis showed that higher age, transformation to acute myeloid leukemia, the IPSS subtypes and the lower interphase telomere length were independent adverse prognostic factors. The hazard ratio for higher age was 1.035, 3.678 for transformation to AML, and 5.169 for the low interphase telomere length).Conclusion: Short telomere was signature of MDS both in adult and childhood MDS and associated with adverse prognosis. However, telomere length of patients with childhood bone marrow failure syndrome was shortest. Heterogeneity in telomere length of patients with MDS was less prominent than those in patients with AA. DisclosuresNo relevant conflicts of interest to declare.

Spermatozoa telomeres determine telomere length in early embryos and offspring.

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretus spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.

Can the TLR-4-mediated signaling pathway be "a key inflammatory promoter for sporadic TAA"?

Thoracic aorta shows with advancing age various changes and a progressive deterioration in structure and function. As a result, vascular remodeling (VR) and medial degeneration (MD) occur as pathological entities responsible principally for the sporadic TAA onset. Little is known about their genetic, molecular, and cellular mechanisms. Recent evidence is proposing the strong role of a chronic immune/inflammatory process in their evocation and progression. Thus, we evaluated the potential role of Toll like receptor- (TLR-) 4-mediated signaling pathway and its polymorphisms in sporadic TAA. Genetic, immunohistochemical, and biochemical analyses were assessed. Interestingly, the rs4986790 TLR4 polymorphism confers a higher susceptibility for sporadic TAA (OR = 14.4, P = 0.0008) and it represents, together with rs1799752 ACE, rs3918242 MMP-9, and rs2285053 MMP-2 SNPs, an independent sporadic TAA risk factor. In consistency with these data, a significant association was observed between their combined risk genotype and sporadic TAA. Cases bearing this risk genotype showed higher systemic inflammatory mediator levels, significant inflammatory/immune infiltrate, a typical MD phenotype, lower telomere length, and positive correlations with histopatological abnormalities, hypertension, smoking, and ageing. Thus, TLR4 pathway should seem to have a key role in sporadic TAA. It might represent a potential useful tool for preventing and monitoring sporadic TAA and developing personalized treatments.

Abstract C22: Telomere length variation and frequency of short telomeres in blood lymphocytes: Novel biomarkers for lung cancer risk

Abstract Background: Although it is widely recognized that telomere dysfunction plays an important role in cancer, the relationship between telomere function in somatic cells and lung cancer risk is not well defined. In a case-control study of non-small cell lung cancer, we examined relationship between several key telomere features and lung cancer risk. Method: Telomere fluorescent in situ hybridization was used to measure telomere features using short-term cultured blood lymphocytes. Logistic regression was used to estimate the strength of association between telomere features and lung cancer risk. Results: Telomere length variation across all chromosomal ends was significantly associated with lung cancer risk, with adjusted odds ratios of 4.67 [95% confidence interval (CI) = 1.46 – 14.9] and 0.46 (95% CI = 0.25 – 0.84) for young (age &amp;lt; 60) and old (age &amp;gt; 60) individuals, respectively. Similar strength and direction of associations between percentage of short telomeres and lung cancer risk were also observed. Telomere length variation and average telomere length jointly affect lung cancer risk. When comparing individuals with short telomere and high telomere length variation to those with long telomere and low telomere length variation, adjusted odd ratios were 8.21 (95% CI = 1.71 – 39.5) and 0.33 (95% CI = 0.15 – 0.72) for young and old individuals, respectively. Conclusion: Telomere length variation and percentage of short telomeres in blood lymphocytes are significantly associated with lung cancer risk and the associations are modulated by age. Telomere length variation in combination with average telomere length may be a promising tool for lung cancer risk assessment. Citation Format: Bing Sun, Ying Wang, Krishna Kota, Yaru Shi, Salaam Motlek, Kepher Makambi, Christopher A. Loffredo, Peter G. Shields, Qin Yang, Curtis C. Harris, Yun-Ling Zheng. Telomere length variation and frequency of short telomeres in blood lymphocytes: Novel biomarkers for lung cancer risk. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C22.

Inverse association between adiposity and telomere length: The fels longitudinal study

To assess the relationship between telomere length and adiposity, using dual-energy X-ray absorptiometry (DXA) and magnetic resonance imaging (MRI), in addition to conventional anthropometric proxies including body mass index (BMI) and cardiovascular disease risk factors. A cross-sectional sample of 309 non-Hispanic white participants in the Fels Longitudinal Study aged 8 to 80 yr (52% female) was included. Average telomere length was measured by quantitative PCR. Telomere length was negatively correlated with age (r = -0.32, P < 0.0001) and had numerous significant correlations with established cardiovascular disease risk factors including waist circumference (r = -0.33), apolipoprotein B (r = -0.26), systolic blood pressure (r = -0.28), and fasting serum glucose (r = -0.15); all P < 0.0025. In backward selection linear regression models of telomere length, adiposity measures were consistently retained in the best models; BMI, waist circumference, hip circumference, total body fat, and visceral adipose tissue volume were all inversely associated with telomere length at the nominal P < 0.05 level or lower, independent of age, sex, systolic blood pressure, and fasting serum lipid, lipoprotein, and glucose concentrations. The negative association of BMI with telomere length was stronger among younger than older participants (P for interaction, 0.03). Individuals with higher total and abdominal adiposity have lower telomere length, a marker of cellular senescence, suggesting obesity may hasten the aging process. Longitudinal studies are required to establish the causal association of early life adiposity with biological aging.

Study of telomere length and different markers of oxidative stress in patients with Parkinson's disease

Many studies have shown that short telomere length (TL) is associated with high oxidative stress and various age-related diseases. Parkinson's disease (PD) is an age-related disease, and although its pathogenic mechanism is uncertain, oxidative stress is believed to be implicated in this pathology. The aim of this case-control study was to assess both TL and the different markers of oxidative stress in elderly patients with PD compared to age control subjects. 20 PD patients and 15 age-matched controls, >65 years were studied. TL was measured by Southern blotting from DNA samples extracted from white blood cells. Superoxide dismutase (SOD) activity and plasma levels of total glutathione and protein carbonyls were determined. There was a trend for lower TL in PD patients: 6.06 ± 0.81 kb in PD versus 6.45 ± 0.73 kb in controls (p = 0.08). No significant difference was found between the two groups in terms of oxidative stress markers. In controls, age was the main determinant of telomere shortening (r = -0.547; p = 0.03) whereas, in PD patients, telomere shortening was mainly dependent on plasmatic concentrations of carbonyl proteins (r= -0.544; p=0.044). In PD patients, a negative association was observed between plasma carbonyl protein levels and SOD activity (r= -0.622, p=0.004). In PD, TL is shorter in presence of high oxidative stress as measured by carbonyl protein levels. The absence of telomere attrition with age among patients with PD could reflect a telomere regulation by mechanisms other than age.

Shorter telomere length in peripheral blood lymphocytes of workers exposed to polycyclic aromatic hydrocarbons

Shorter telomere length (TL) in peripheral blood lymphocytes (PBLs) is predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens that cause chromosome instability. Whether PAH exposure and its molecular effects are linked with shorter TL has never been evaluated. In the present study, we investigated the effect of chronic exposure to PAHs on TL measured in PBLs of Polish male non-current smoking cokeoven workers and matched controls. PAH exposure and molecular effects were characterized using measures of internal dose (urinary 1-pyrenol), effective dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)-DNA adduct], genetic instability (micronuclei, MN) and DNA methylation [p53 promoter and Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements, as surrogate measures of global methylation] in PBLs. TL was measured by real-time polymerase chain reaction. Cokeoven workers were heavily exposed to PAHs (79% exceeded the urinary 1-pyrenol biological exposure index) and exhibited lower TL (P = 0.038) than controls, as well as higher levels of genetic and chromosomal alterations [i.e. anti-BPDE-DNA adduct and MN (P < 0.0001)] and epigenetic changes [i.e. p53 gene-specific promoter and global methylation (P <or= 0.001)]. TL decreased with longer duration of work as cokeoven worker (P = 0.039) and in all subjects with higher levels of anti-BPDE-DNA adduct (P = 0.042), p53 hypomethylation (P = 0.005) and MN (P = 0.009). In multivariate analysis, years of work in cokery (P = 0.008) and p53 hypomethylation (P = 0.001) were the principal determinants of shorter TL. Our results indicate that shorter TL is associated with chronic PAH exposure. The interrelations with other genetic and epigenetic mechanisms in our data suggest that shorter TL could be a central event in PAH carcinogenesis.

Telomerase activity and telomere length as prognostic factors of adult T-cell leukemia

For the oncogenesis of many malignancies, it is crucial to prevent the shortening of the telomeres by the action of telomerase. In this study, clinical data and disease outcomes were analyzed in conjunction with the telomerase activity (TA) and telomere length (TL) of peripheral blood mononuclear cells. The study was carried out in 22 patients with adult T-cell leukemia (ATL) (7 chronic and 15 acute types) and in 13 asymptomatic human T-lymphotropic virus type 1 (HTLV-1) carriers. The mean values of TA in acute and chronic type patients were 13.8 and 1.6 total product generated (TPG) units, respectively, as determined by telomeric repeat amplification assays. The mean TA values in HTLV-1 carriers and healthy volunteers were 1.8 and 0.7 TPG, respectively. The mean TA value in acute type patients was significantly higher than in the three other subject groups. The mean TL values in patients with acute and chronic types were 5.39 and 4.38 Kb, respectively, while the mean TL values in HTLV-1 carriers and healthy volunteers were 7.69 and 7.06 Kb, respectively. The mean TL values in all ATL patients and in non-ATL subjects were 5.2 and 7.3 Kb, respectively. The former value is significantly shorter than the latter (p < 0.01). Neither TA nor TL of ATL cells showed any significant association with the number of ATL cells, serum soluble interleukin-2 receptor, or serum lactate dehydrogenase in the peripheral blood of acute type patients. This suggests that the levels of TA and TL did not reflect the ATL tumor load. The median survival period of acute ATL patients with high TA and shortened TL was 0.47 years, however, which was significantly shorter than that of acute ATL patients with low TA and normal TL (4.21 years) (p < 0.002). These data suggest that high TA and shortened TL were associated with poorer prognosis, and that TA and TL may be novel markers for the prognosis of ATL patients.