Publications by authors named "Geraldine Aubert"

27 Publications

  • Page 1 of 1

Establishing a Framework for the Clinical Translation of Germline Findings in Precision Oncology.

JNCI Cancer Spectr 2020 Oct 29;4(5):pkaa045. Epub 2020 May 29.

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Inherited genetic variation has important implications for cancer screening, early diagnosis, and disease prognosis. A role for germline variation has also been described in shaping the molecular landscape, immune response, microenvironment, and treatment response of individual tumors. However, there is a lack of consensus on the handling and analysis of germline information that extends beyond known or suspected cancer susceptibility in large-scale cancer genomics initiatives. As part of the Personalized OncoGenomics program in British Columbia, we performed whole-genome and transcriptome sequencing in paired tumor and normal tissues from advanced cancer patients to characterize the molecular tumor landscape and identify putative targets for therapy. Overall, our experience supports a multidisciplinary and integrative approach to germline data management. This includes a need for broader definitions and standardized recommendations regarding primary and secondary germline findings in precision oncology. Here, we propose a framework for identifying, evaluating, and returning germline variants of potential clinical significance that may have indications for health management beyond cancer risk reduction or prevention in patients and their families.
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http://dx.doi.org/10.1093/jncics/pkaa045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583151PMC
October 2020

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study.

PLoS One 2020 10;15(2):e0228887. Epub 2020 Feb 10.

Department of Pediatrics, Baylor College of Medicine and Dan L. Duncan Cancer Center, Houston, TX, United States of America.

Background: Telomere length is associated with risk for thyroid subsequent malignant neoplasm in survivors of childhood cancer. Here, we investigated associations between thyroid subsequent malignant neoplasm and inherited variation in telomere maintenance genes.

Methods: We used RegulomeDB to annotate the functional impact of variants mapping to 14 telomere maintenance genes among 5,066 five-or-more year survivors who participate in the Childhood Cancer Survivor Study (CCSS) and who are longitudinally followed for incidence of subsequent cancers. Hazard ratios for thyroid subsequent malignant neoplasm were calculated for 60 putatively functional variants with minor allele frequency ≥1% in or near telomere maintenance genes. Functional impact was further assessed by measuring telomere length in leukocyte subsets.

Results: The minor allele at Protection of Telomeres-1 (POT1) rs58722976 was associated with increased risk for thyroid subsequent malignant neoplasm (adjusted HR = 6.1, 95% CI: 2.4, 15.5, P = 0.0001; Fisher's exact P = 0.001). This imputed SNP was present in three out of 110 survivors who developed thyroid cancer vs. 14 out of 4,956 survivors who did not develop thyroid cancer. In a subset of 83 survivors with leukocyte telomere length data available, this variant was associated with longer telomeres in B lymphocytes (P = 0.004).

Conclusions: Using a functional variant approach, we identified and confirmed an association between a low frequency intronic regulatory POT1 variant and thyroid subsequent malignant neoplasm in survivors of childhood cancer. These results suggest that intronic variation in POT1 may affect key protein binding interactions that impact telomere maintenance and genomic integrity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228887PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010302PMC
May 2020

From incomplete penetrance with normal telomere length to severe disease and telomere shortening in a family with monoallelic and biallelic PARN pathogenic variants.

Hum Mutat 2019 12 15;40(12):2414-2429. Epub 2019 Sep 15.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.

PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease.
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http://dx.doi.org/10.1002/humu.23898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874886PMC
December 2019

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method.

Cells 2018 Oct 24;7(11). Epub 2018 Oct 24.

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19⁻53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (² = 0.56, < 0.0001). In Bland-Altman analyses, individuals with the shortest (≤10th percentile) or longest (≥90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and -0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 5.8 kb, respectively, < 0.0001). Differences in annual TL attrition were also noted (31 13 bp/year, respectively, = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.
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http://dx.doi.org/10.3390/cells7110183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262465PMC
October 2018

Understanding the evolving phenotype of vascular complications in telomere biology disorders.

Angiogenesis 2019 02 25;22(1):95-102. Epub 2018 Aug 25.

Dyskeratosis Congenita Outreach, Inc., New York, NY, USA.

Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.
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http://dx.doi.org/10.1007/s10456-018-9640-7DOI Listing
February 2019

Correction: TERT promoter mutation in adult granulosa cell tumor of the ovary.

Mod Pathol 2019 04;32(4):593

Department of Molecular Oncology, British Columbia Cancer Agency, Vancouver, BC, Canada.

The original version of this Article omitted the author Hannah van Meurs from the Department of Gynecology, Center for Gynecologic Oncology Amsterdam, Academic Medical Center, 1100 DD Amsterdam, The Netherlands. This has been corrected in both the PDF and HTML versions of the article.
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http://dx.doi.org/10.1038/s41379-018-0085-8DOI Listing
April 2019

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

Blood 2018 05 9;131(21):2393-2398. Epub 2018 Apr 9.

Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI.

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 (CD45RACD20), memory enriched T cells (CD45RACD20), natural killer (NK) fully differentiated T cells (CD45RACD57), and B cells (CD45RACD20). 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 ( < .01). In cause-specific mortality analyses, longer TL in B cells (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.46-0.87; = .006) and possibly NK fully differentiated T cells (HR, 0.7; 95% CI, 0.51 to 0.97; = .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 ( > .05). However, a trend toward excess risk of graft-versus-host mortality was noted (HR for total lymphocyte TL, 1.26; = .15). In conclusion, longer donor TL was associated with reduced rate of infection-related deaths after HCT for SAA.
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http://dx.doi.org/10.1182/blood-2017-10-812735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969378PMC
May 2018

TERT promoter mutation in adult granulosa cell tumor of the ovary.

Mod Pathol 2018 07 15;31(7):1107-1115. Epub 2018 Feb 15.

Department of Molecular Oncology, British Columbia Cancer Agency, Vancouver, BC, Canada.

The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, which is a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified as gain-of-function mutations that promote transcriptional activation of TERT in multiple cancers, such as melanoma and glioblastoma. A recent study investigating TERT promoter mutations in ovarian carcinomas found C228T and C250T mutations in 15.9% of clear cell carcinomas. However, it is unknown whether these mutations are frequent in other ovarian cancer subtypes, in particular, sex cord-stromal tumors including adult granulosa cell tumors. We performed whole-genome sequencing on ten adult granulosa cell tumors with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that adult granulosa cell tumors with mutated TERT promoter have increased expression of TERT mRNA and exhibited significantly longer telomeres compared to those with wild-type TERT promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary adult granulosa cell tumors (22%), 24 of 58 recurrent adult granulosa cell tumors (41%), and 1 of 22 other sex cord-stromal tumors (5%). There was a significant difference in overall survival between patients with TERT C228T promoter mutation in the primary tumors and those without it (p = 0.00253, log-rank test). In seven adult granulosa cell tumors, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggest that TERT C228T promoter mutations may have an important role in progression of adult granulosa cell tumors.
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http://dx.doi.org/10.1038/s41379-018-0007-9DOI Listing
July 2018

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

Int J Mol Sci 2017 Aug 13;18(8). Epub 2017 Aug 13.

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

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 (² of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (² 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 (² 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.
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http://dx.doi.org/10.3390/ijms18081765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578154PMC
August 2017

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

J Allergy Clin Immunol 2017 Oct 23;140(4):1120-1129.e1. Epub 2017 Jan 23.

Section of Immunology, Allergy and Rheumatology, Texas Children's Hospital, Baylor College of Medicine, Houston, Tex. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jaci.2016.11.051DOI Listing
October 2017

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

Nat Genet 2016 10 29;48(10):1185-92. Epub 2016 Aug 29.

Department of Clinical Genetics, Liverpool Women's NHS Foundation Trust, Liverpool, UK.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045717PMC
http://dx.doi.org/10.1038/ng.3661DOI Listing
October 2016

Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies.

Clin Vaccine Immunol 2016 Apr 4;23(4):254-71. Epub 2016 Apr 4.

Repeat Diagnostics Inc., North Vancouver, British Columbia, Canada, and Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

Genetic defects of the immune system are referred to as primary immunodeficiencies (PIDs). These immunodeficiencies are clinically and immunologically heterogeneous and, therefore, pose a challenge not only for the clinician but also for the diagnostic immunologist. There are several methodological tools available for evaluation and monitoring of patients with PIDs, and of these tools, flow cytometry has gained prominence, both for phenotyping and functional assays. Flow cytometry allows real-time analysis of cellular composition, cell signaling, and other relevant immunological pathways, providing an accessible tool for rapid diagnostic and prognostic assessment. This minireview provides an overview of the use of flow cytometry in disease-specific diagnosis of PIDs, in addition to other broader applications, which include immune phenotyping and cellular functional measurements.
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http://dx.doi.org/10.1128/CVI.00001-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820507PMC
April 2016

Telomere dynamics and aging.

Authors:
Geraldine Aubert

Prog Mol Biol Transl Sci 2014 ;125:89-111

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

Telomeres consist of repetitive DNA-protein complexes that cap the ends of vertebrate linear chromosomes. Their capping function and dynamics both with regard to structure and length are carefully orchestrated by many regulatory mechanisms and factors, with likely more yet to be described. Telomere shortening has been shown to be a major measurable molecular characteristic of aging of cells in vitro and in vivo and is thought to have evolved as a tumor protection mechanism in long-lived species. Regulators and modifiers of telomere dynamics and dynamics with age together with the consequences of telomere shortening and telomere dysfunction in the context of aging and aging-related disorders are discussed in this chapter.
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http://dx.doi.org/10.1016/B978-0-12-397898-1.00004-9DOI Listing
April 2015

Paroxysmal nocturnal haemoglobinuria phenotype cells and leucocyte subset telomere length in childhood acquired aplastic anaemia.

Br J Haematol 2014 Mar 14;164(5):717-21. Epub 2013 Nov 14.

Division of Pediatric Hematology/Oncology/Bone Marrow Transplantation, British Columbia Children's Hospital, University of British Columbia, Vancouver, BC, Canada.

The significance of paroxysmal nocturnal haemoglobinuria (PNH(pos) ) cells and leucocyte subset telomere lengths in paediatric aplastic anaemia (AA) is unknown. Among 22 children receiving immunosuppressive therapy (IST) for AA, 73% (16/22) were PNH(pos) , of whom 94% achieved at least a partial response (PR) to IST; 11/16 (69%) achieved complete response (CR). Only 2/6 (33%) PNH(neg) patients achieved PR. PNH(pos) patients were less likely to fail IST compared to PNH(neg) patients (odds ratio 0·033; 95% confidence interval 0·002-0·468; P = 0·012). Children with AA had short granulocyte (P = 7·8 × 10(-9) ), natural killer cell (P = 6·0 × 10(-4) ), naïve T lymphocyte (P = 0·002) and B lymphocyte (P = 0·005) telomeres compared to age-matched normative data.
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http://dx.doi.org/10.1111/bjh.12656DOI Listing
March 2014

The luminal progenitor compartment of the normal human mammary gland constitutes a unique site of telomere dysfunction.

Stem Cell Reports 2013 4;1(1):28-37. Epub 2013 Jun 4.

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.

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.
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http://dx.doi.org/10.1016/j.stemcr.2013.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757746PMC
May 2015

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

PLoS Genet 2012 17;8(5):e1002696. Epub 2012 May 17.

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

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.
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http://dx.doi.org/10.1371/journal.pgen.1002696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355073PMC
September 2012

The First AACR special conference on stem cells, development, and cancer: some of these cells are not like the others.

Cancer Res 2011 Sep;71(17):5616-20

Department of Haematology, Cambridge Institute for Medical Research, Cambridge, UK.

The American Association for Cancer Research (AACR) held an exciting conference on Stem Cells, Development, and Cancer in Vancouver, British Columbia, Canada (March 3-6, 2011). The meeting was cochaired by Geoffrey Wahl, Connie Eaves, and Hans Clevers and was attended by 250 international researchers, 40% of whom were young investigators. Three key themes emerged: (i) heterogeneity in stem cells and cancer, (ii) solid tissue cancer stem cells, and (iii) lessons from development. The interdisciplinary foundation of this meeting was central to its success and appeal, underscoring the value of juxtaposing and interrelating work from the three topics addressed.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-1310DOI Listing
September 2011

Telomere length measurement-caveats and a critical assessment of the available technologies and tools.

Mutat Res 2012 Feb 12;730(1-2):59-67. Epub 2011 Jun 12.

Terry Fox Laboratory, BC Cancer Agency, Vancouver BC, Canada.

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.
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http://dx.doi.org/10.1016/j.mrfmmm.2011.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460641PMC
February 2012

Dyskeratosis congenita: the first NIH clinical research workshop.

Pediatr Blood Cancer 2009 Sep;53(3):520-3

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, Maryland 20892, USA.

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 UK's DC Registry and the NCI's 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.
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http://dx.doi.org/10.1002/pbc.22061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739803PMC
September 2009

Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses.

Cancer Immunol Immunother 2009 Sep 5;58(9):1459-70. Epub 2009 May 5.

Anthony Nolan Research Institute, The Royal Free Hospital, University College of London, Hampstead, London NW3 2QG, UK.

Soluble MHC-peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8(+) T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens.
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http://dx.doi.org/10.1007/s00262-009-0711-xDOI Listing
September 2009

Telomeres and aging.

Physiol Rev 2008 Apr;88(2):557-79

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

Telomeres play a central role in cell fate and aging by adjusting the cellular response to stress and growth stimulation on the basis of previous cell divisions and DNA damage. At least a few hundred nucleotides of telomere repeats must "cap" each chromosome end to avoid activation of DNA repair pathways. Repair of critically short or "uncapped" telomeres by telomerase or recombination is limited in most somatic cells and apoptosis or cellular senescence is triggered when too many "uncapped" telomeres accumulate. The chance of the latter increases as the average telomere length decreases. The average telomere length is set and maintained in cells of the germline which typically express high levels of telomerase. In somatic cells, telomere length is very heterogeneous but typically declines with age, posing a barrier to tumor growth but also contributing to loss of cells with age. Loss of (stem) cells via telomere attrition provides strong selection for abnormal and malignant cells, a process facilitated by the genome instability and aneuploidy triggered by dysfunctional telomeres. The crucial role of telomeres in cell turnover and aging is highlighted by patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Short telomeres in such patients are implicated in a variety of disorders including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, and cancer. Here the role of telomeres and telomerase in human aging and aging-associated diseases is reviewed.
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http://dx.doi.org/10.1152/physrev.00026.2007DOI Listing
April 2008

Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita.

Blood 2008 May 29;111(9):4523-31. Epub 2008 Feb 29.

Department of Pediatrics, Division of Hematology/Oncology, University of Iowa Children's Hospital, Iowa City 52242, USA.

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.
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http://dx.doi.org/10.1182/blood-2007-10-120204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2343591PMC
May 2008

Hematopoietic stem-cell behavior in nonhuman primates.

Blood 2007 Sep 25;110(6):1806-13. Epub 2007 May 25.

Department of Biostatistics, Vanderbilt University, Nashville, TN, USA.

Little is known about the behavior of hematopoietic stem cells (HSCs) in primates because direct observations and competitive-repopulation assays are not feasible. Therefore, we used 2 different and independent experimental strategies, the tracking of transgene expression after retroviral-mediated gene transfer (N = 11 baboons; N = 7 rhesus macaques) and quantitation of the average telomere length of granulocytes (N = 132 baboons; N = 14 macaques), together with stochastic methods, to study HSC kinetics in vivo. The average replication rate for baboon HSCs is once per 36 weeks according to gene-marking analyses and once per 23 weeks according to telomere-shortening analyses. Comparable results were derived from the macaque data. These rates are substantially slower than the average replication rates previously reported for HSCs in mice (once per 2.5 weeks) and cats (once per 8.3 weeks). Because baboons and macaques live for 25 to 45 years, much longer than mice ( approximately 2 years) and cats (12-18 years), we can compute that HSCs undergo a relatively constant number ( approximately 80-200) of lifetime replications. Thus, our data suggest that the self-renewal capacity of mammalian stem cells in vivo is defined and evolutionarily conserved.
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http://dx.doi.org/10.1182/blood-2007-02-075382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976353PMC
September 2007

Large numbers of dysfunctional CD8+ T lymphocytes bearing receptors for a single dominant CMV epitope in the very old.

J Clin Immunol 2003 Jul;23(4):247-57

Tuebingen Ageing and Tumour Immunology Group, Section for Transplantation-Immunology and Immunohematology, University of Tübingen, Tübingen, Germany.

Longitudinal studies suggest that a set of immune parameters including high percentages of peripheral CD8+, CD28-, CD57+ T lymphocytes, low CD4 and B cell counts, and poor T cell proliferative responses to mitogens is associated with decreased remaining longevity in the free-living very elderly (> 85 years). This combination of immune parameters was also significantly associated with an inverted CD4/CD8 ratio and cytomegalovirus seropositivity. Here, using tetramer technology, we show markedly increased numbers of CD8+ T cells bearing receptors for one single CMV epitope in the very elderly. Moreover, the fraction of these tetramer-reactive cells secreting interferon-gamma after specific antigenic stimulation was significantly lower in the old than in the young, as was the percentage of CD28-positive cells in this population. Therefore, we conclude that marked expansions of CMV-specific CD8+ T cells have occurred and that the obsession of a large fraction of the entire CD8+ T cell subset with one single viral epitope may contribute to the increased incidence of infectious disease in the elderly by shrinking the T cell repertoire available for responses to other antigens.
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http://dx.doi.org/10.1023/a:1024580531705DOI Listing
July 2003

An age-related increase in the number of CD8+ T cells carrying receptors for an immunodominant Epstein-Barr virus (EBV) epitope is counteracted by a decreased frequency of their antigen-specific responsiveness.

Mech Ageing Dev 2003 Apr;124(4):477-85

Tuebingen Ageing and Tumour Immunology Group, Center for Medical Research, ZMF, Waldhoernlestr. 22, 72072, Tuebingen, Germany.

The aim of this study was to provide a basis for investigating the effects of one very common environmental factor, Epstein-Barr virus (EBV), on age-related changes in the immune system. To this end, the frequency of CD8(+) T cells carrying receptors for an immunodominant EBV lytic epitope was assessed by direct staining with HLA-peptide tetrameric complexes in 19 very old (>87 years) and 12 young (20-40 years) EBV carriers. The frequency of EBV-tetramer-positive cells within the CD8(+) subset was significantly greater in the old compared to the young group (P=0.001). However, the frequency of EBV antigen-specific IFN-gamma producing T cells, as determined by ELISPOT, was significantly lower in the old (P=0.001). Therefore, the absolute number of functional EBV-specific T cells in the elderly and the young was probably similar. These data suggest CD8 clonal expansions in the elderly, resulting in an accumulation of dysfunctional EBV-specific cells which possibly fill the 'immunological space' and could lead to a shrinking of the T cell repertoire for other novel antigens. This may help to explain the increased incidence and case-fatality caused by viruses and intracellular pathogens in the elderly.
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http://dx.doi.org/10.1016/s0047-6374(03)00026-5DOI Listing
April 2003