Geraldine Aubert

Telomere Length Measurement - caveats and a critical assessment of the available technologies and tools

Studies of telomeres and telomere biology often critically rely on the detection of telomeric DNA and measurements of the length of telomere repeats in either single cells or populations of cells. Several methods are available that provide this type of information and it is often not clear what method is most appropriate to address a specific research question. The major variables that need to be considered are the material that is or can be made available and the accuracy of measurements that is required. The goal of this review is to provide a comprehensive summary of the most commonly used methods and discuss the advantages and disadvantages of each. Methods that start with genomic DNA include telomere restriction fragment (TRF) length analysis, PCR amplification of telomere repeats relative to a single copy gene by Q-PCR or MMQPCR and single telomere length analysis (STELA), a PCR-based approach that accurately measures the full spectrum of telomere lengths from individual chromosomes. A different set of methods relies on fluorescent in situ hybridization (FISH) to detect telomere repeats in individual cells or chromosomes. By including essential calibration steps and appropriate controls these methods can be used to measure telomere repeat length or content in chromosomes and cells. Such methods include quantitative FISH (Q-FISH) and flow FISH which are based on digital microscopy and flow cytometry, respectively. Here the basic principles of various telomere length measurement methods are described and their strengths and weaknesses are highlighted. Some recent developments in telomere length analysis are also discussed. The information in this review should facilitate the selection of the most suitable method to address specific research question about telomeres in either model organisms or human subjects.

Collapse of Telomere Homeostasis in Hematopoietic Cells Caused by Heterozygous Mutations in Telomerase Genes

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

Dyskeratosis congenita: The first NIH clinical research workshop

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure syndrome, characterized by abnormally short telomeres and mutations in telomere biology genes. The spectrum of telomere biology disorders is growing and the clinical management of these patients is complex. A DC‐specific workshop was held at the NIH on September 19, 2008; participants included physicians, patients with DC, their family members, and representatives from other support groups. Data from the UKs DC Registry and the NCIs DC cohort were described. Updates on the function of the known DC genes were presented. Clinical aspects discussed included androgen therapy, stem cell transplant, cancer risk, and cancer screening. Families with DC met for the first time and formed a family support group (http://www.dcoutreach.com/). Ongoing, open collaboration between the clinical, scientific, and family communities is required for continued improvement in our understanding of DC and the clinical consequences of telomeric defects. Pediatr Blood Cancer 2009;53:520–523.

Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC, we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular, and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells, although present at normal frequencies within the CD34(+) subset, were therefore absolutely decreased. In contrast, even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased, and the telomere lengths of these cells were also markedly reduced. Nevertheless, the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs.

The Luminal Progenitor Compartment of the Normal Human Mammary Gland Constitutes a Unique Site of Telomere Dysfunction

Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in <5% of basal cells. Paradoxically, 25% of LPs are still capable of exhibiting robust clonogenic activity in vitro. This may be partially explained by the elevated telomerase activity that was also seen only in LPs. Interestingly, this potential telomere salvage mechanism declines with age. Our findings thus reveal marked differences in the telomere biology of different subsets of primitive normal human mammary cells. The chronically dysfunctional telomeres unique to LPs have potentially important implications for normal mammary tissue homeostasis as well as the development of certain breast cancers.

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.

Cord Blood T Cells Retain Early Differentiation Phenotype Suitable for Immunotherapy After TCR Gene Transfer to Confer EBV Specificity

Adoptive T cell therapy can be effective for Epstein–Barr virus (EBV)‐associated posttransplant lymphoproliferative disease and melanoma. Transducing high‐affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor‐specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR‐transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV‐specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen‐driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57‐negative but were still capable of antigen‐specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation.

Defects in lymphocyte telomere homeostasis contribute to cellular immune phenotype in patients with cartilage-hair hypoplasia

Background: Mutations in the long noncoding RNA RNase component of the mitochondrial RNA processing endoribonuclease (RMRP) give rise to the autosomal recessive condition cartilage‐hair hypoplasia (CHH). The CHH disease phenotype has some overlap with dyskeratosis congenita, a well‐known “telomere disorder.” RMRP binds the telomerase reverse transcriptase (catalytic subunit) in some cell lines, raising the possibility that RMRP might play a role in telomere biology. Objective: We sought to determine whether a telomere phenotype is present in immune cells from patients with CHH and explore mechanisms underlying these observations. Methods: We assessed proliferative capacity and telomere length using flow–fluorescence in situ hybridization (in situ hybridization and flow cytometry) of primary lymphocytes from patients with CHH, carrier relatives, and control subjects. The role of telomerase holoenzyme components in gene expression and activity were assessed by using quantitative PCR and the telomere repeat amplification protocol from PBMCs and enriched lymphocyte cultures. Results: Lymphocyte cultures from patients with CHH display growth defects in vitro, which is consistent with an immune deficiency cellular phenotype. Here we show that telomere length and telomerase activity are impaired in primary lymphocyte subsets from patients with CHH. Notably, telomerase activity is affected in a gene dose‐dependent manner when comparing heterozygote RMRP carriers with patients with CHH. Telomerase deficiency in patients with CHH is not mediated by abnormal telomerase gene transcript levels relative to those of endogenous genes. Conclusion: These findings suggest that telomere deficiency is implicated in the CHH disease phenotype through an as yet unidentified mechanism. Graphical abstract Figure. No Caption available.

Correlation of Leukocyte Telomere Length Measurement Methods in Patients with Dyskeratosis Congenita and in Their Unaffected Relatives

Several methods have been employed to measure telomere length (TL) in human studies. It has been difficult to directly compare the results from these studies because of differences in the laboratory techniques and output parameters. We compared TL measurements (TLMs) by the three most commonly used methods, quantitative polymerase chain reaction (qPCR), flow cytometry with fluorescence in situ hybridization (flow FISH) and Southern blot, in a cohort of patients with the telomere biology disorder dyskeratosis congenita (DC) and in their unaffected relatives (controls). We observed a strong correlation between the Southern blot average TL and the flow FISH total lymphocyte TL in both the DC patients and their unaffected relatives (R2 of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (R2 of 0.54 in DC patients and of 0.43 in unaffected relatives). Similar results were noted when comparing the qPCR average TL and the flow FISH total lymphocyte TL (R2 of 0.49 in DC patients and of 0.42 in unaffected relatives). In conclusion, the strengths of the correlations between the three widely used TL assays (qPCR, flow FISH, and Southern blot) were significantly different. Careful consideration is warranted when selecting the method of TL measurement for research and for clinical studies.

Donor telomere length and causes of death after unrelated hematopoietic cell transplantation in patients with marrow failure

Previous studies have suggested that longer donor leukocyte telomere length (TL) is associated with improved survival after hematopoietic cell transplantation (HCT) in severe aplastic anemia (SAA). This study aimed to determine whether cell-specific lymphocyte TL is associated with certain post-HCT causes of death. We used flow cytometry and fluorescence in situ hybridization to measure TL in donor total lymphocytes and subsets: naïve enriched T cells (CD45RA+CD20-), memory enriched T cells (CD45RA-CD20-), natural killer (NK) fully differentiated T cells (CD45RA+CD57+), and B cells (CD45RA+CD20+). Competing risk survival regression was used for cause-specific death analyses. Clinical data and biospecimens were available from the Center for International Blood and Marrow Transplant Research database and biorepository. The study included 197 patients who underwent unrelated-donor HCT for SAA between 1988 and 2004. The median age at HCT was 15 years (range, 0.5-40 years), and the median follow-up was 5 years (range, <1 month to 20.7 years). Longer donor TL in all cell subsets was associated with lower risk of all-cause mortality (P < .01). In cause-specific mortality analyses, longer TL in B cells (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.46-0.87; P = .006) and possibly NK fully differentiated T cells (HR, 0.7; 95% CI, 0.51 to 0.97; P = .03) was associated with lower risk of infection-related death. Donor TL in other tested lymphocyte subsets was not statistically significantly associated with death resulting from graft-versus-host disease or graft failure (P > .05). However, a trend toward excess risk of graft-versus-host mortality was noted (HR for total lymphocyte TL, 1.26; P = .15). In conclusion, longer donor TL was associated with reduced rate of infection-related deaths after HCT for SAA.