Telomeres are repeated hexa nucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks;furthermore, the asymmetric replication of DNA would lead inevitably to a loss of genetic material, and telomerase, an enzymatic complex that adds telomeric sequences at mitosis, functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres and loss of both enzymatic activity: its consequences can be measured in vitro and in vivo. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in some cases of dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) occur in apparently acquired aplastic anemia and other diseases. Heterozygous mutations in TERT lead to defective telomere repair and short telomeres due to a mechanism of haploinsufficiency. Male hormones, long used to treat aplastic anemia, act by up regulating TERT transcription and telomerase activity, including in lymphocytes and hematopoietic progenitor cells. While critical telomere shortening often leads to either cell senescence or apoptosis, occasional cells become anneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for onco genesis. Telomere length of leukocyte is now measured routinely in our CLIA laboratory by gene amplification using robotic methodology provided by a Quiagen Quiagility and Rotor GeneQ;high throughput analysis is useful both for research and in the clinic, and our procedure is certified for patient data. Measurement of clinical samples is required for the adequate diagnosis of aplastic anemia and is predictive of late events after treatment with immunosuppression, and probably in other clinical circumstances. In the last year, we have focused on new methodologies to measure telomere length. For clinical specimens, we rely on a certified laboratory assessment using polymerase chain reaction in a robotic system. However, the disadvantage of PCR for telomere length is that an average value is obtained, while the biology of critical telomere shortening in tissue culture and animal models indicates that critically short telomere length of an individual chromosome may be the trigger for cell senescence, apoptosis, and, in the absence of check points, malignant transformation. We have adapted a published method called single telomere length analyses (STELA), which relies on amplification from chromosome specific sub-telomeric DNA sequences, allowing detection of critical telomere shortening in individual chromosomes. Beginning with the X chromosome, we now have STELA operational for five chromosomes. STELA has been applied to clinical samples obtained from patients with aplastic anemia, especially serial specimens in patients who progressed from marrow failure to myelodysplasia with monosomy 7. Patients in stable remissions have served as controls. STELA reveals critical telomere shortening (of the X chromosome) in patients with clonal evolution. In normal individuals, critically short telomeres also are detected with aging and in comparison of healthy controls to patients with telomerase mutation deficiencies, patients show many more short telomere fragments. STELA will be adapted also to in vitro experimentation, as, for example, to examine the consequences of stem cell exhaustion following CD34 cell stimulation in growth factor milieu. Methods related to STELA are being adapted for whole tissue histology in order to detect critical telomere shortening in organs such as the liver and lung. In the clinic, we continue to expand our database of patients with telomeropathies. Over 100 patients have been identified based on short telomere length of leukocytes and/or mutations in TERT and TERC. The hematology of telomere disease includes presentation with moderate thrombocytopenia or macrocytic anemia;association with liver or lung disease, often subclinical;the family history is positive in only 50% of patients. Patterns of disease suggests that liver and lung disease only rarely associate in a single pedigree, and further there may be TERT and TERC specific patterns of bone marrow failure. Deep clinical phenotyping should allow a fuller description of utility to practicing physicians in several subspecialties. In a clinical research protocol, we have treated patients with a synthetic androgen, Danazol, at maximum doses, intending a two year period of therapy to improve either hematologic parameters and/or pulmonary fibrosis. The biological endpoint is stabilization or improvement in telomere length. Remarkably, the majority of patients accrued to date have show hematologic improvement at three and six month time points, with very little major toxicity. We also have evidence of telomere stabilization increase in patients at six months. These results suggest that androgen therapy may have a high probability of success in selected patients with bone marrow failure, and the impact may be directly on telomerase and telomere repair. In animal experiments, we are also testing the effects of testosterone on telomere maintenance. In previous experiments, we were unable to show, either by testosterone treatment or castration, effects in steady state laboratory mice (which have very long telomeres in comparison to humans). However, under circumstances of hematopoietic stress, testosterone appears capable of ameliorating telomere attrition following stem cell transplantation in the mouse when telomerase deficient animals are used as donors, and in telomerase deficient animals subjected to intermittent irradiation. We are exploring the possibility of clinical assessment of patients following chemo- and radiation therapy in the long-term, the potential utility of hormone replacement in preventing secondary iatrogenic malignancies will be explored. Finally, in our collaboration with Lincoln Park Zoo in Chicago, we have measured telomere length by southern blot hybridization in Peromyscus mice bred for 20 generations for conservation purposes. Remarkably, a highly significant difference in telomere length was observed based on the breeding protocol with animals that were randomly bred having longer telomeres than those in a mixed kinship breeding protocol which is intended to preserve the genetic constitution of the original population and to avoid unintended bias from breeding in captivity. There was no relationship of telomere length to age (of adult animals), sex, and body mass. To complete these experiments, wild animals will be captured and their telomere lengths measured (further, the Tert gene in these animals has been sequenced and will be assessed for mutations in animals very long telomeres). Results to date suggestion that telomere elongation may have occurred in laboratory animals as a result of breeding and likely prolonged exposure to sex hormones in captivity compared to in the wild. In collaboration with Dr. Constantine Stratakis of NICHD, we have established a colony of Peromyscus in order to test breeding practices on telomere length in a prospective fashion, with correlation to sex hormone plasma levels.

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Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2012
Total Cost
$1,586,711
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
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Zhao, Xin; Tian, Xin; Kajigaya, Sachiko et al. (2016) Epigenetic landscape of the TERT promoter: a potential biomarker for high risk AML/MDS. Br J Haematol :
Townsley, Danielle M; Dumitriu, Bogdan; Liu, Delong et al. (2016) Danazol Treatment for Telomere Diseases. N Engl J Med 374:1922-31
Zhao, Xin; Ueda, Yasutaka; Kajigaya, Sachiko et al. (2015) Cloning and molecular characterization of telomerase reverse transcriptase (TERT) and telomere length analysis of Peromyscus leucopus. Gene 568:8-18
Chen, Jichun; Bryant, Mark A; Dent, James J et al. (2015) Hematopoietic lineage skewing and intestinal epithelia degeneration in aged mice with telomerase RNA component deletion. Exp Gerontol 72:251-60
Dumitriu, Bogdan; Feng, Xingmin; Townsley, Danielle M et al. (2015) Telomere attrition and candidate gene mutations preceding monosomy 7 in aplastic anemia. Blood 125:706-9
Balakumaran, Arun; Mishra, Prasun J; Pawelczyk, Edyta et al. (2015) Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders. Blood 125:793-802
Sellers, Stephanie E; Dumitriu, Bogdan; Morgan, Mary J et al. (2014) No impact of lentiviral transduction on hematopoietic stem/progenitor cell telomere length or gene expression in the rhesus macaque model. Mol Ther 22:52-8
Zhang, Yuwei; Calado, Rodrigo; Rao, Mahadev et al. (2014) Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas. PLoS One 9:e101010
Sun, Yu; Desierto, Marie J; Ueda, Yasutaka et al. (2014) Peromyscus leucopus mice: a potential animal model for haematological studies. Int J Exp Pathol 95:342-50
Ueda, Yasutaka; Calado, Rodrigo T; Norberg, Anna et al. (2014) A mutation in the H/ACA box of telomerase RNA component gene (TERC) in a young patient with myelodysplastic syndrome. BMC Med Genet 15:68

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