Relative Telomere Length Measurement Research Articles

Sex-specific differences in telomere length of patients with primary knee osteoarthritis.

Accelerated telomere shortening is associated with age-related diseases, including osteoarthritis (OA). We aimed to determine the relative telomere length (TL) in leukocytes and cartilage of patients with primary knee OA and to investigate factors that may affect TL in OA. Relative TL measurements were performed using qPCR in leukocytes of 612 individuals (310 patients with primary knee OA undergoing total knee arthroplasty (TKA) and 302 unaffected controls). We also analysed cartilage in 57 of the 310 OA patients, measuring relative TL in severely affected and less affected (control) cartilage collected from the same knee. Cartilage TLs were compared to leukocyte TLs in all 57 patients. A significant sex-by-disease-status interaction was found in regard to relative TL. Controlling for age, the average difference of leukocyte TL between female OA patients versus female controls was 0.217 units greater than that between male OA patients versus male controls (95% CI; [0.014, 0.421]). Relative TL comparison of severely and less affected cartilage samples from the same joint showed attrition of telomeres corresponding to disease severity (0.345 mean TL difference with 95% CI of [0.151, 0.539]) in the joint. We also noted that both severely and less affected cartilage had shorter telomeres than leukocytes collected from the same patient. Severe and moderate pain in OA patients was associated with shorter TL in leukocytes, but there was no association with depression or smoking in leukocytes and cartilage. Our study indicates that sex is an important factor in OA contributing to leukocyte and cartilage TL and that pain in OA shows an inverse association only with leukocyte TL.

P-175 Human cumulus cell telomere length and its association with assisted reproduction outcomes

Abstract Study question Is there any relationship between the relative telomere length (RTL) within cumulus cells (CCs) and the outcome of assisted reproductive treatment using the corresponding oocyte? Summary answer Lower RTLs in CCs were significantly associated with embryos chosen for transfer or cryopreservation. In contrast, embryos considered non-viable (discarded) tended to have higher RTLs. What is known already Cumulus cells fulfil vital roles in support of oocyte development, including the transduction of external signals and the provision of resources via transzonal projections. Given their essential role in the acquisition of oocyte developmental competence, the biology of CCs is of clinical relevance. Telomeres are specialised structures protecting the ends of chromosomes, composed of repetitive DNA sequences and associated proteins. Telomeres shorten with each mitotic division, as well as due to oxidative damage, eventually reaching a critical threshold at which point cellular senescence occurs. Currently, published data on CCs telomere length and relationship with oocyte potential are conflicting. Study design, size, duration The study involved 182 human CC samples collected from 52 IVF patients. Quantitative PCR (qPCR) was used to measure the relative telomere length in each of the CC samples. Telomere lengths were assessed for associations with various patient characteristics (e.g. age, body mass index, infertility diagnosis). Additionally, potential relationships with clinically relevant oocyte/embryo features were investigated (fertilisation; development/morphology), as well as the eventual fate of the associated embryo (transferred; cryopreserved for potential future use; discarded). Participants/materials, setting, methods Real-time quantitative PCR was carried out using PCR primers specific for the telomere repeat. A single-copy gene was also amplified from each CC sample. Quantification of this gene was used for normalisation of the telomere data, allowing control for variation in the number of cumulus cells in each sample. Associations between RTL in CCs and patient, embryonic, and clinical factors were assessed using various statistical methods, with P-values <0.05 considered significant. Main results and the role of chance No associations were identified between RTL and any patient characteristics, except for BMI. The amount of CC telomeric DNA tended to be greater for patients with higher BMI (P = 0.002). When considering links between RTL and oocyte or embryonic factors, a significant relationship was detected between the quantity of telomeric DNA in CCs and whether the corresponding embryo was considered non-viable (discarded) or whether it was transferred or cryopreserved (P = 0.019). This finding raises the possibility that measurement of RTL in CCs could provide a pre-conception, non-invasive assessment of oocyte quality. In the context of fertility preservation, RTL measurement could assist in evaluating a cohort of oocytes, indicating whether the cryopreserved eggs are likely to be sufficient or whether additional cycles to generate more would be advisable. If future studies confirm that CC RTL has a strong predictive value, the possibility of limiting fertilisation to oocytes considered to have high likelihood of viability could also be considered for routine IVF cycles. It is unclear why shorter CC telomeres might be associated with oocytes of superior potential, but one possibility is that the cells may have undergone a greater proliferation (more mitoses), resulting in a more extensive cumulus mass supporting the enclosed oocyte. Limitations, reasons for caution Before drawing definitive conclusions, confirmation of the findings within a larger, independent data set is necessary. Even if confirmed, determination of the true clinical value of telomere assessment will require further, appropriately designed studies. The current study was not powered to evaluate relationships between CC telomere lengths and IVF outcomes. Wider implications of the findings Currently, simplistic morphological evaluation is the only method for assessing oocyte competence prior to fertilisation. If CCs telomere measurement is confirmed to have predictive value, a preconception test of oocyte potential could be offered. This would be extremely valuable for patients cryopreserving oocytes for fertility preservation and for donor banks. Trial registration number NA

Dynamics of cellular senescence markers after HCV elimination spontaneously or by DAAs in people living with HIV

BackgroundWe identified that acute or chronic Hepatitis C (HCV) infection in people living with HIV (PLWHIV) results in different senescence profiles. However, variations in these profiles after HCV elimination, spontaneously or with direct-acting antivirals (DAAs), remain unclear. MethodsLongitudinal observational study (48 weeks) in 70 PLWHIV: 23 PLWHIV with active HCV-chronic infection (CHC) before and after HCV eradication with DAAs, 12 PLWHIV who spontaneously clarify the HCV (SC), and 35 controls (HIV). Oxidative stress was quantified at DNA, lipid, protein, and nitrate levels, as well as the antioxidant capacity and glutathione enzyme. The replicative senescence was evaluated by relative telomere length measurement by PCR and twenty-six factors related to Senescence-Associated Secretory Phenotype (SASP) were characterized by Luminex. Differences in senescence markers was evaluated by generalized linear models. ResultsDuring follow-up, the SC group achieved a significant improvement in glutathione enzyme and lipid peroxidation. The secretion of SASP markers increased but was still lower than that of the HIV group. Overall, the CHC group reduced the levels of oxidative stress and SASP markers to levels like those of the HIV group. No significant differences in telomere shortening were observed between groups. ConclusionsAs the time since spontaneous resolution of HCV infection increased, patients had an improved senescence profile compared to the HIV group. Elimination of chronic HCV infection by DAAs led to a partial improvement of the senescent profile by restoring oxidative stress levels. However, although some SASP markers reached levels like those of the HIV group, others remained altered.

SLC38A10 Deficiency in Mice Affects Plasma Levels of Threonine and Histidine in Males but Not in Females: A Preliminary Characterization Study of SLC38A10-/- Mice.

Solute carriers belong to the biggest group of transporters in the human genome, but more knowledge is needed to fully understand their function and possible role as therapeutic targets. SLC38A10, a poorly characterized solute carrier, is preliminary characterized here. By using a knockout mouse model, we studied the biological effects of SLC38A10 deficiency in vivo. We performed a transcriptomic analysis of the whole brain and found seven differentially expressed genes in SLC38A10-deficient mice (Gm48159, Nr4a1, Tuba1c, Lrrc56, mt-Tp, Hbb-bt and Snord116/9). By measuring amino acids in plasma, we found lower levels of threonine and histidine in knockout males, whereas no amino acid levels were affected in females, suggesting that SLC38A10-/- might affect sexes differently. Using RT-qPCR, we investigated the effect of SLC38A10 deficiency on mRNA expression of other SLC38 members, Mtor and Rps6kb1 in the brain, liver, lung, muscle, and kidney, but no differences were found. Relative telomere length measurement was also taken, as a marker for cellular age, but no differences were found between the genotypes. We conclude that SLC38A10 might be important for keeping amino acid homeostasis in plasma, at least in males, but no major effects were seen on transcriptomic expression or telomere length in the whole brain.

Novel Germline POT1 Variant Predisposes to Childhood Acute Myeloid Leukemia

Introduction: Current studies indicate a contribution of germline predisposition in the development of approximately 8.5% of childhood cancers (Zhang J. et al., N Engl J Med, 2015), although their apparent rate is estimated to be much higher. Understanding tumor evolution based on a predisposed cell can open unknown doors for prevention and therapy of childhood cancer e.g., leukemia. Here we present a novel rare (MAF<0.1%) germline POT1 variant (Q199*) predisposing to acute myeloid leukemia (AML). POT1 as part of the telomeric shelterin complex is known to play an important role in DNA damage protection, telomere length maintenance and chromosomal stability (Calvete O. et al., Nat. Commun., 2015). POT1 variants are associated with a broad range of cancer, including myeloid and lymphoid neoplasms in adults (Lim T.L. et al., Leukemia, 2021), but not yet described for myeloid malignancies in childhood.Methods: Whole exome sequencing (WES) was implemented to identify germline variants. To assess the effect of POT1 p.Q199*, patient's fibroblast and stably transfected HEK293T cells were used as cell models. The variant's functional impact was experimentally tested performing yH2AX and 53BP1 immunofluorescence assays for DNA damage detection, qRT-PCR for telomere length measurement and telomere FISH to assess chromosomal instability.Results: Utilizing WES to detect variants within shelterin complex genes we analyzed genomic data of an unselected German parent-child cohort of children with cancer (n=60, TRIO-DD), as well as a recently published parent-child pediatric cancer cohort (n=158, TRIO-D) (Wagener R. et al., Eur. J. Hum. Genet, 2021). Here, we identified a novel germline POT1 variant in a boy affected with Myelodysplastic syndrome (MDS) and secondary AML (7q-). This novel germline variant constitutes a stop-gain mutation causing a substitution of the amino acid Glutamine by a stop codon (p.Q199*).QRT-PCR analysis within the patient's fibroblasts showed a significant (student's t-test p=0.0037) reduction of POT1 mRNA expression to ≈0.5 compared to POT1 wildtype. Western Blot analysis revealed reduced POT1 levels, confirming the loss of one POT1 allele mediated by p.Q199*. Thereupon, POT1 p.Q199* cloning and stable transfection into Hek293T cells was performed to test the variant's cooperative functionality in a controlled environment. Subsequently, POT1 p.Q199* lead to a drastically significant (student's t-test p=<0.001) increase of DNA double strand breaks in transfected Hek293T cells determined by yH2AX and 53BP1 immunofluorescence assays, which is in line with a deregulated DNA damage response and inappropriate repair by non-homologous end joining.In addition, we detected dysregulation of telomere length maintenance. Here, relative telomere length measurement by means of qRT-PCR indicated significant (student's t-test p=0.019) telomere elongation in POT1 p.Q199* fibroblast cells. Furthermore telomere FISH on metaphase chromosomes was performed to analyse chromosomal stability. In POT1 p.Q199* Hek293T cells we identified a significant (student's t-test p=0.002) increase in telomere fragility compared to POT1 WT cells.Conclusion: Taken together, we present the functional effects of POT1 p.Q199* leading to a significant increase of DNA damage, telomere length and chromosomal instability. Our results on functional dysregulation strengthen a potential genetic predisposition to childhood AML mediated by germline POT1 variants. DisclosuresNo relevant conflicts of interest to declare.

Evaluating The Utility Of Telomere Length Measurement By Qpcr As a Diagnostic Test For Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (IBMFS) classically diagnosed by the triad of dysplastic nails, reticular skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure (BMF), cancer, pulmonary fibrosis, and other medical problems. Germline mutations in key telomere biology genes cause DC (DKC1, TERC, TERT, TINF2, NOP10, NHP2, WRAP53, CTC1 or RTEL1), although about 30% of patients lack a known mutation. Telomere length (TL) below the1st percentile for age in leukocyte subset, measured by flow cytometry with in situhybridization (flow FISH), is diagnostic of DC with a sensitivity and specificity of more than 95%. However, flow FISH requires fresh or cryopreserved blood, and thus is relatively low-throughput and costly. Quantitative PCR (QPCR) of telomeric DNA content, expressed as the ratio of the telomeric product (T) to a single copy gene (S) product, is a high-throughput, inexpensive method of determining relative TL (RTL) that is being used in numerous studies.We evaluated the clinical utility of QPCR RTL measurement as a diagnostic test for DC in the NCI's IRB-approved longitudinal cohort study of IBMFS. Patients were classified as DC (n=31) if they had a mutation in one of the nine known causative genes and/or met the clinical criteria for DC including at least two features of the diagnostic triad with other clinical findings consistent with DC. Fifty-one mutation-negative, healthy relatives of DC patients served as controls. Flow FISH lymphocyte TL was less than or equal to the 1st percentile for age in 29/31 (94%) DC patients and above 1stpercentile in all but one mutation-negative relative. Monoplex QPCR of DNA derived from whole blood was used to determine the RTL (T/S ratio). QPCR RTL data were log transformed and standardized for age using percentile curves and z-scores calculated from 477 healthy age-matched controls. Control samples from children were derived from the control arm of the eating behavior and obesity studies at the National Institute of Child Health and Human Development, NIH, and adult samples were from healthy donors in the National Marrow Donor Program research sample repository. We used Mann-Whitney and Kruskal-Wallis tests to compare z-scores of TL between categories and Receiver Operating Curve (ROC) analysis to calculate the sensitivity and specificity of QPCR RTL at different percentile cut-offs.DC patients had significantly shorter QPCR RTL than relatives (median age adjusted z-score -1.9 vs. 0.09, p<0.0001). Patients with a mutation in TINF2, or DKC1had shorter QPCR relative TL than those with other mutations (p=0.07). DC patients who had at least two of the three features of the diagnostic triad had shorter QPCR relative TL than those who had one or zero features (p=0.05), but there was no difference in QPCR TL between individuals based on severity of bone marrow failure (p=0.2).QPCR RTL was not correlated with flow FISH TL in DC patients (r=0.09, p=0.64). QPCR RTL was only modestly correlated with flow FISH TL in unaffected relatives (r=0.47, p=0.001). QPCR RTL less than the 1st percentile for age missed diagnosing 60% of the known DC patients as having DC (sensitivity=38.7%, and specificity=98%). Only 75% of DC patients had QPCR relative TL below the 10thpercentile for age (sensitivity=74%), as did 12% of the unaffected relatives (specificity=88%).In summary, QPCR RTL measurement less than 1st percentile for age had low sensitivity for identifying patients with DC. Raising the cutoff to the 10th percentile increased the sensitivity, but 12% of healthy individuals were also falsely diagnosed with DC. Our findings raise doubt about using QPCR for clinical diagnosis of DC. This has implications in the interpretation of studies utilizing different TL measurement methods in studies of DC, bone marrow failure, and related telomere biology disorders. With the current limitations of QPCR RTL in identifying DC patients, 6-panel flow FISH TL continues to be the recommended diagnostic test for DC. Disclosures:No relevant conflicts of interest to declare.

Patterns of Relative Telomere Length is Associated With hTERT Gene Expression in the Tissue of Patients With Breast Cancer

BackgroundHomeostasis of telomere in breast cancer might be altered as a result of cumulative effects of various factors causing genomic instability and affecting prognosis. This study aimed to compare the relative telomere length (RTL) and hTERT mRNA expression in the tissue of patients with breast cancer along with the clinicopathologic parameters. Patients and MethodsFrozen tumor tissues and adjacent normal breast tissue from 98 patients with invasive ductal breast cancer were used for the analysis. RTL and hTERT mRNA expression were measured using quantitative real time polymerase chain reaction. ResultsAmong the 98 cases, 51% had an early-stage carcinoma, 66% were tumor size < 5 cm, 30% were node-negative, and 20% were low-grade tumors. In this study, 63% of cases showed higher hTERT gene expression with an odds ratio of 2.77 (P = .02). The median RTL for elongated telomere was 3.49, and the value was significantly elevated when compared with the shorter telomere. Shortened RTL was present in 60% of early-stage cancer cases, 55% where the tumor size was < 5 cm, 72% of the lymph node-negative cases, and 68% of low-grade carcinoma. Significantly elongated RTL, with median 4.22, 3.19, 3.17, and 3.28 was observed (P < .05) in the advanced stage, larger tumor size, node-positive, and high-grade cases respectively. ConclusionIn this study, shortened telomere was observed in early-stage cancer, and elongated telomere was found in advanced diseases. However, 13% of patients with lower hTERT gene expression showed elongated telomeres, indicating relative telomere length measurement in tissue is different from blood leukocyte, showing the dynamic process of tumorigenesis in tissue.

Dyskeratosis congenita with a novel genetic variant in the DKC1 gene: a case report

BackgroundDyskeratosis congenita (DC) is a rare genetic disorder of bone marrow failure inherited in an X-linked, autosomal dominant or autosomal recessive pattern. It has a wide array of clinical features and patients may be cared for by many medical sub specialties. The typical clinical features consist of lacy reticular skin pigmentation, nail dystrophy and oral leukoplakia. As the disease advances, patients may develop progressive bone marrow failure, pulmonary fibrosis, oesophageal stenosis, urethral stenosis, liver cirrhosis as well as haematological and solid malignancies. Several genes have been implicated in the pathogenesis of dyskeratosis congenita, with the dyskerin pseudouridine synthase 1 (DKC1) gene mutations being the X-linked recessive gene.Case presentationHerein, we report a 31-year-old male with history of recurrent febrile episodes who was found to have reticulate skin pigmentation interspersed with hypopigmented macules involving the face, neck and extremities, hyperkeratosis of palms and soles, nail dystrophy, leukoplakia of the tongue, premature graying of hair, watery eyes and dental caries. Several of his male relatives, including two maternal uncles and three maternal cousins were affected with a similar type of disease condition. Pedigree analysis suggested a possible X-linked pattern of inheritance. Genetic testing in the proband showed a novel hemizygous, non-synonymous likely pathogenic variant [NM_001363.4: c.1054A > G: p.Thr352Ala] in the PUA domain of the DKC1 gene. Quantitative polymerase chain reaction for relative telomere length measurements performed in the proband showed that he had very short telomeres [0.38, compared to a control median of 0.71 (range 0.44–1.19)], which is consistent with the DC diagnosis. Co-segregation analysis of the novel mutation and telomere length measurements in the extended family members could not be performed as they were unwilling to provide consent for testing.ConclusionsThe novel variant detected in the DKC1 gene adds further to the existing scientific literature on the genotype-phenotype correlation of DC, and has important implications for the clinical and molecular characterization of the disease.

Development of a New Monochrome Multiplex qPCR Method for Relative Telomere Length Measurement in Cancer

Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.

Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescence

ObjectivePin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. MethodsImmunohistochemistry and Western blotting were used to assess Pin1 protein level in human normal and atherosclerotic tissues. β-galactosidase staining, cumulative population doubling level, telomerase activity, and relative telomere length measurement were used to confirm VSMC senescence. The expressions of Pin1 and other genes involved in this research were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting in VSMCs. Apolipoprotein E gene-deleted mice (ApoE−/−) fed a high-fat diet were treated with juglone or 10% ethanol, respectively, for 3 weeks. The extent of atherosclerosis was evaluated by Oil Red O, Masson trichrome staining, and immunohistology. ResultsPin1 protein level decreased in human atherosclerotic tissues and VSMCs, synchronously with increased VSMC senescence. Adenoviral-mediated Pin1 overexpression rescued cellular senescence in atherosclerotic VSMCs, with concurrent down-regulation of P53, p21, growth arrest and DNA-damage-inducible protein 45-alpha (Gadd45a), phosphorylated retinoblastoma (p-pRb), p65 and upregulation of cyclin subfamilies (cyclin B, D, and E), and cyclin-dependent kinase subfamilies (2, 4, and 6), whereas Pin1 knockdown resulted in the converse effects, indicating that VSMC senescence mediated by Pin1 is an integrated response to diverse signals. In vivo data from ApoE−/− mice showed that treatment of juglone led to accelerated atherosclerosis development. ConclusionsAltogether this work supports a role for Pin1 as a vital modulator of VSMC senescence, thereby providing a novel target for regulation and control of atherosclerosis.

Effect of pre-analytic variables on the reproducibility of qPCR relative telomere length measurement.

Telomeres, long nucleotide repeats and a protein complex at chromosome ends, shorten with each cell division and are susceptible to oxidative damage. Quantitative PCR (qPCR) is a widely-used technique to measure relative telomere length (RTL) in DNA samples but is challenging to optimize and significant lab-to-lab variability has been reported. In this study, we evaluated factors that may contribute to qPCR RTL measurement variability including DNA extraction methods, methods used for removing potential residual PCR inhibitors, sample storage conditions, and sample location in the PCR plate. Our results show that the DNA extraction and purification techniques, as well as sample storage conditions introduce significant variability in qPCR RTL results. We did not find significant differences in results based on sample location in the PCR plate or qPCR instrument used. These data suggest that lack of reproducibility in published association studies of RTL could be, in part, due to methodological inconsistencies. This study illustrates the importance of uniform sample handling, from DNA extraction through data generation and analysis, in using qPCR to determine RTL.

Telomere Length and Accelerated Biological Aging in the China Suboptimal Health Cohort: A Case-Control Study.

Suboptimal health status (SHS) has been linked to cardiovascular risk factors, psychosocial stress, and unhealthy lifestyle. These factors also contribute to the shortening of telomere length (TL). A case-control study was conducted to examine the association between subjective health measures of SHS from the behavior perspective and also objective measures of TL at molecular level. SHS (cases = 294) was matched by age, sex, and body mass index with ideal health (controls = 294) using a propensity score matching method. Suboptimal health status questionnaire-25 (SHSQ-25) was used in the community-based health survey. A quantitative polymerase chain reaction was used to measure relative telomere length (RTL). Shorter RTL was found among the SHS group compared to the ideal health group (p < 0.05). SHS was almost four times likely to be in the first quartile (odds ratio [OR] = 3.81; 95% confidence interval [CI] 2.21-6.56), almost thrice in second quartile (OR = 2.84; 95% CI 1.65-4.90), and almost twice likely to be in the third quartile (OR = 1.71; 95% CI 1.00-2.94) compared to the fourth quartile (the longest) of RTL after adjusting for socioeconomic, dietary intake, anthropometric, blood pressure, and biochemistry variables (p < 0.05). Notably, SHS score was negatively correlated with RTL (r = -0.218, p < 0.05). Our study confirms an association between SHS and short RTL. Combination of subjective (SHS) and objective (RTL) measures is a novel tool for health aging investigation. Therefore, SHSQ-25 could be used as a screening tool for measuring biological aging in low-income countries at community level where the expensive technique for RTL measurement is not applicable.

Telomere length measurement by qPCR in birds is affected by storage method of blood samples

Given the potential role of telomeres as biomarkers of individual health and ageing, there is an increasing interest in studying telomere dynamics in a wider range of taxa in the fields of ecology and evolutionary biology. Measuring telomere length across the lifespan in wild animal systems is essential for testing these hypotheses, and may be aided by archived blood samples collected as part of longitudinal field studies. However, sample collection, storage, and DNA extraction methods may influence telomere length measurement, and it may, therefore, be difficult to balance consistency in sampling protocol with making the most of available samples. We used two complementary approaches to examine the impacts of sample storage method on measurements of relative telomere length (RTL) by qPCR, particularly focusing on FTA (Flinders Technology Associates) cards as a long-term storage solution. We used blood samples from wandering albatrosses collected over 14 years and stored in three different ways (n = 179), and also blood samples from captive zebra finches (n = 30) that were each stored using three different methods. Sample storage method influenced RTL in both studies, and samples on FTA cards had significantly shorter RTL measurements. There was no significant correlation between RTL measured in zebra finch blood on FTA cards and the same samples stored either as frozen whole blood or as extracted DNA. These results highlight the importance of consistency of sampling protocol, particularly in the context of long-term field studies, and suggest that FTA cards should not be used as a long-term storage solution to measure RTL without validation.

Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.

Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.

Influence of DNA extraction methods on relative telomere length measurements and its impact on epidemiological studies.

Measurement of telomere length is widely used in epidemiologic studies. Insufficient standardization of the measurements processes has, however, complicated the comparison of results between studies. We aimed to investigate whether DNA extraction methods have an influence on measured values of relative telomere length (RTL) and whether this has consequences for epidemiological studies. We performed four experiments with RTL measurement in quadruplicate by qPCR using DNA extracted with different methods: 1) a standardized validation experiment including three extraction methods (magnetic-particle-method EZ1, salting-out-method INV, phenol-chloroform-isoamyl-alcohol PCI) each in the same 20 samples demonstrated pronounced differences in RTL with lowest values with EZ1 followed by INV and PCI-isolated DNA; 2) a comparison of 307 samples from an epidemiological study showing EZ1-measurements 40% lower than INV-measurements; 3) a matching-approach of two similar non-diseased control groups including 143 pairs of subjects revealed significantly shorter RTL in EZ1 than INV-extracted DNA (0.844 ± 0.157 vs. 1.357 ± 0.242); 4) an association analysis of RTL with prevalent cardiovascular disease detected a stronger association with INV than with EZ1-extracted DNA. In summary, DNA extraction methods have a pronounced influence on the measured RTL-values. This might result in spurious or lost associations in epidemiological studies under certain circumstances.

Leucocyte telomere length and risk of type 2 diabetes mellitus: new prospective cohort study and literature-based meta-analysis.

BackgroundShort telomeres have been linked to various age-related diseases. We aimed to assess the association of telomere length with incident type 2 diabetes mellitus (T2DM) in prospective cohort studies.MethodsLeucocyte relative telomere length (RTL) was measured using quantitative polymerase chain reaction in 684 participants of the prospective population-based Bruneck Study (1995 baseline), with repeat RTL measurements performed in 2005 (n = 558) and 2010 (n = 479). Hazard ratios for T2DM were calculated across quartiles of baseline RTL using Cox regression models adjusted for age, sex, body-mass index, smoking, socio-economic status, physical activity, alcohol consumption, high-density lipoprotein cholesterol, log high-sensitivity C-reactive protein, and waist-hip ratio. Separate analyses corrected hazard ratios for within-person variability using multivariate regression calibration of repeated measurements. To contextualise findings, we systematically sought PubMed, Web of Science and EMBASE for relevant articles and pooled results using random-effects meta-analysis.ResultsOver 15 years of follow-up, 44 out of 606 participants free of diabetes at baseline developed incident T2DM. The adjusted hazard ratio for T2DM comparing the bottom vs. the top quartile of baseline RTL (i.e. shortest vs. longest) was 2.00 (95% confidence interval: 0.90 to 4.49; P = 0.091), and 2.31 comparing the bottom quartile vs. the remainder (1.21 to 4.41; P = 0.011). The corresponding hazard ratios corrected for within-person RTL variability were 3.22 (1.27 to 8.14; P = 0.014) and 2.86 (1.45 to 5.65; P = 0.003). In a random-effects meta-analysis of three prospective cohort studies involving 6,991 participants and 2,011 incident T2DM events, the pooled relative risk was 1.31 (1.07 to 1.60; P = 0.010; I 2 = 69%).Conclusions/InterpretationLow RTL is independently associated with the risk of incident T2DM. To avoid regression dilution biases in observed associations of RTL with disease risk, future studies should implement methods correcting for within-person variability in RTL. The causal role of short telomeres in T2DM development remains to be determined.

Complete sequence analysis of mitochondrial DNA and telomere length in aplastic anemia.

The present study was primarily undertaken to examine the hypothesis that mitochondrial DNA (mtDNA) mutations and telomere length may be associated with aplastic anemia(AA). Our study included a single institution analysis of 40patients presenting with AA first diagnosed at the Affiliated Hospital of Shandong, University of Traditional Chinese Medicine between 2010 and 2013. Bone marrow and oral epithelial samples were collected from patients with AA (n=40) for mtDNA mutation and telomere length determinations. Bone marrow specimens were collected from 40healthy volunteers as controls for the examination of telomere length. The mitochondrial genome was amplified by polymerase chain reaction (PCR), and the products were used for sequencing and analysis. We detected 146heteroplasmic mutations in 18genes from 40patients with AA, including 39silent mutations and 28frameshift mutations. We used the gamma globin gene (HBG) as the control gene in real-time PCR to survey the relative telomere length measurements of the patients with AA and the healthy volunteers. Telomere length was expressed as the relative T/Svalue. We observed a negative correlation between the mtDNA non-silent mutation and the white blood cell (WBC) count, hemoglobin and platelet count. Of note, there was a positive correlation between the relative T/Svalue and WBC count, hemoglobin and platelet count, and a negative correlation between the non-silent mutation and the relative T/Svalue. We conclude that the functional impairment of the mitochondrial respiratory chain induced by mutation and telomere length shortening may play an important role in the process of hematopoietic failure in patients with AA. Additionally, mtDNA mutations and telomere length shortening influenced each other.

Blood and Dried Blood Spot Telomere Length Measurement by qPCR: Assay Considerations

Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types.

60 TELOMERE LENGTH DYNAMICS DURING BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT

The establishment of telomere length (TL) during embryogenesis determines telomere reserves in newborn mammals. However, limited information is available on TL dynamics during preimplantation in contrast to the extensive existing data on telomerase activity in germ cells and embryogenesis. Telomerase activity is high in the male germ line, low or absent in oocytes and early stage embryos and high in blastocysts (Bl). Mechanisms allowing early embryos to reset TL remain poorly understood. The documented telomere lengthening at the morula/Bl transition in mice and bovines is dependent on telomerase activity. A recombinant-based mechanism termed alternative lengthening of telomeres (ALT) has been postulated to be responsible for the lengthening of telomeres in early embryos. The aims of the present study were to analyse the telomere dynamics during preimplantation embryo development in 2 species of known different TL: mice and bovines and the relative expression of 2 components of telomerase [telomerase reverse transcriptase (Tert; the key factor that controls the activity of the telomerase) and telomerase RNA component (Terc)]. Twenty samples for each developmental stage with an equivalent number of cells (matured oocytes/zygotes: 8 and 32; 2-cell embryos: 4 and 16; 4-cell embryos: 2 and 8; 8-cell embryos: 1 and 4; 16-cell embryos: 2 only for bovine; morulae: 1 and 1; and Bl: 1 and 1 for mice and bovines, respectively) were analysed for relative TL measurement using the real-time quantitative PCR method described previously (Bermejo-Alvarez et al. 2008 Physiol. Genomics 32, 264272). For measuring the mRNA, 3 groups of 10 oocytes/embryos for each developmental stage were used. Data were analysed by 1-way ANOVA. In mice, matured oocytes had the shortest telomeres of all stages examined (P &lt; 0.01); a slight increase up to the 4-cell stage and a decrease at the 8-cell and morula stages was noted (P &lt; 0.05), while a marked increase was observed in Bl, as expected (P &lt; 0.01). In contrast, bovine matured oocytes had longer telomeres than zygotes and this length gradually decreased up to the 4-cell stage and increased again at the 16-cell stage (P &lt; 0.05). Then, telomeres shortened at the morula stage (P &lt; 0.05) and a significant increase was observed at the Bl stage like in mice (P &lt; 0.01). The relative abundance of mTerc increased throughout development with a marked up-regulation at the morula and Bl stages (P &lt; 0.01). On the other hand, the relative abundance of mTert was significantly higher in the mature oocytes and zygotes compared to later stages (P &lt; 0.01); however, it should be noted that there was a gradual increase from the 2-cell stage up to Bl. In conclusion, in contrast to mice, bovine oocytes have longer telomeres than zygotes. Knowing that the telomerase activity is low or absent until the Bl stage (indicated by the low expression of Tert), the TL increase detected in 16-cell bovine embryos indicates an alternative mechanism for telomere elongation during early development, like that observed in mice. Understanding how telomeres reset during early embryo development has implications for the study of stem cells and regenerative biology.