Telomeres are repetitive 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 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 tumorigenesis. Telomere length of leukocyte is now measured routinely by gene amplification using robotic methodology provided by a Quiagen Quiagility and Rotor GeneQ;high throughput analysis is useful both in the laboratory 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 (also see below). Interesting molecular biology has also derived from the clinic. For example, we identified a pedigree in which short telomeres associated with aplastic anemia and pulmonary fibrosis, yet no mutation could be identified in either TERT or TERC. However, a mutation in the CCAAT box, a crucial transcriptional regulatory element, was discovered in the promoter of TERC in affected individuals, the first example of a CCAAT mutation that is pathogenic in humans. Telomere measurements have been applied to samples from large databases, including the NHLBI Womens Health Initiative and NIDDK HALT-C, which enroll patients with chronic hepatitis C infection at risk for hepatic cirrhosis. Telomere length measurements can be combined with high throughput sequencing for patients with candidate genes. Short telomeres in aplastic anemia represent the major risk factors, for the development of serious complications after immunosuppressive therapy, both relapse and malignancy evolution, separate from mutations in telomerase genes. In our analysis of almost two hundred patients followed serially for years following ATG therapy, those who had telomere lengths in the lowest quartile but within the normal range had approximately twice the risk of relapse and 5-7 fold increased risk of evolution to MDS/AML and monosomy 7. The mechanism for relapse is likely quantitative, a reduced number of hematopoietic stem cells at the outset of disease or increased replicative stress on the stem cell. Short telomeres at diagnosis associate with chromosome instability as measured in tissue culture using a variety of methods, including spectrakaryotyping and detection of telomere-free ends of chromosomes. Both naked chromosomes and characteristic chromosomal translocations and aneuploidy are detectable at a higher rate in patients with shorter telomeres compared to patients with normal to long telomeres, months to years prior to the development of clinical myelodysplasia. We propose that accelerated telomere attrition, either genetic or secondary to increased replicative stress, is the molecular link between inflammation and malignancy as occurs in chronic hepatitis and hepatocellular carcinoma, inflammatory bowel disease and colon cancer, Barretts esophagitis and esophageal cancer, with graft-vs-host disease and a variety of epithelial cancers, and others. In pedigrees in patients with aplastic anemia, individuals are often identified with diseases of other organs. Familial pulmonary fibrosis can also occur as a result of TERC and TERT mutation. We noted hepatitis cirrhosis in some of our pedigrees associating with telomerase gene mutations. In a systematic survey of patients with cirrhosis of various etiologies, we detected mutations in telomerase genes in a substantial minority, who were without prior family history suggested of an inherited basis. Patients with mutations had shorter telomeres. Similar work was reported concurrently with ours from Germany, suggesting that 5-7% of cirrhosis has telomere attrition due to genetic lesions as a contributing factor. We are interested in the relationship between sex hormones and telomerase gene regulation and telomere length maintenance, based on our findings in human primary cells that both androgens and estrogens up-regulate TERT expression, acting through estrogen response elements in the genes promoter. In preliminary experiments in a mouse model, we were unable to detect an effect of administered androgens on telomere length under steady state conditions in adult animals. Hypothesizing that telomerase might be more critical under circumstances of active tissue regeneration, we utilized a bone marrow transplant system to investigate the effect of sex hormones on telomere attrition with limited numbers of donor inoculums cells. Under these circumstances, androgens appeared to maintain or elongate telomeres in heterozygous +1-Terc animals, but not in either homozygous Terc 1- or in wild type animals. A clinical protocol is underway to test androgen effects on both hematologic and pulmonary disease in patients with short telomeres and/or telomerase gene mutations, as well as to determine their effect on telomere maintenance over a two year period. We have also used inducible pluripotent stem cells to study telomere maintenance in transformed fibroblasts. First, we have shown that IPSC can be derived from TERC and TERT heterozygous patient skin cells. Telomeres in these cells are short but are maintained after reversion to the embryonic state. Notably, there is considerable variability among clones for telomere maintenance, and the pattern of telomere elongation did not correlate with gene expression by members of the telomerase gene complex. These data suggest that IPSC is a suitable system to investigate the pathophysiology of dyskeratosis congenital of the telomerase gene complex mutations.

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Project End
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Budget End
Support Year
2
Fiscal Year
2011
Total Cost
$1,590,725
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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