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. In our laboratory and clinic, major resources have been devoted to the detection of short telomeres and mutations in patients who attend our clinic. For this purpose, we have purchased a Qiagility and Rotor Gene-Q for rapid performance of telomere length measurements as well as high throughput sequencing of telomerase genes (TERC, TERT and others) to increase. We have collected large pedigrees of patients with accelerated telomere attrition and clinical manifestations. One major result of this effort has been the association of telomere attrition and telomerase deficiency, due to known and novel mutations with severe liver disease. This association was suggested to us by a large Mennonite kindred in which four females suffered early severe liver diseases, in three cases fatal and in the fourth necessitated liver transplant for fulminant hepatic failure. Affected inficiduals in this pedigree have a novel mutation in the telomerase gene. Investigation of other families disclosed TERC and TERT mutations associated with cirrhosis and bone marrow failure. Such a clinical link between aplastic anemia and severe liver disease had been previously noted by us, without a satisfactory molecular explanation. Findings suggested the possibility that telomerase deficiency might underlie apparently sporadic cirrhosis, especially as a few reports from other groups indicated that patients with cirrhosis had short-for-age peripheral blood leukocyte telomeres. In screening almost 200 samples from patients with severe liver disease with usual clinical associations (hepatitis, alcohol, and steatosis), approximately 7% had mutations in telomerase genes, which decreased enzymatic activity in vitro. Thus, parallel to observation of the correlation between pulmonary fibrosis and telomerase mutations, cirrhosis, both familial and apparently sporadic, also is related to telomerase mutations. In further mechanistic studies, we have exploited a mouse model in which schistosomal infection of mice leads to either death or hepatic fibrosis. Utilizing this model in telomerase-deficient mice, preliminary data suggest that deficiency of Tert markedly accelerates fibrosis after infection. This is an excellent system to investigate the molecule biochemical changes that telomerase deficiency produces to make organs susceptible to fibrosis. TERT mutations were first described by us in patients with acquired aplastic anemia. In recent studies, we investigated whether telomere shortening (in almost all instances not due to mutations), affected the outcome of aplastic anemia. Almost 200 consecutive patients, treated at the Clinical Center between 2002 and 2008, were examined retrospectively;telomere length at diagnosis was correlated to response to immunosuppressive treatment, relapse, clonal evolution to myelodysplasia/acute myeloid leukemia, and survival. Only one patient was recognized later to have at TERT mutation. Short-for-age telomeres within the normal range did not affect the likelihood of response to immunosuppressive therapy. (In complimentary studies, we had observed that patients with telomerase mutations also can respond to immunosuppression and that their blood contains oligoclones of T cells similar to those observed idiopathic aplastic anemia see the report on aplastic anemia, HL002315-28). However, patients in the shortest-for-age telomere group length differed from the remaining aplastic anemia patients in several important respects. First, the likelihood of relapse was approximately doubled, consistent with a regenerative defect in tissue repair. Second, the rate of clonal evolution was markedly increased, 5-7 fold, as measured for both all myelodysplasia/AML and especially for the development of monosomy 7, the common and most severe manisfestation of clonal evolution. A parallel effect was seen for survival, as patients with short telomeres (but within the normal range) had a markedly lower survival;the combination of short telomeres and low numbers of reticulocytes demarcated a particularly poor prognosis group. In laboratory studies, bone marrow cells from patients shortly after treatment (at six months, when a sufficient number of cells were available for in vitro experiments) displayed abnormalities previously observed in mouse knock-out models of telomerase deficiency: increased telomere free ends by fluorescent in situ hybridization, end-to-end fusions of chromosomes, and aneuploidy after brief tissue culture. These abnormalities were present in patients months to years prior to the development of clinical manifestations of myelodysplasia/AML, suggesting that telomerase deficiency leads to critically short telomeres and, in some cells and some patients, predisposes to aneuploidy and malignancy. We have developed a clinical protocol to test whether male hormones, which increase TERT transcription and telomerase activity, can delay telomere attrition and enhance repair of effected organs, and prevent the development of cancer in patients with known telomerase deficiencies or with a consistent family history and short telomeres. In other laboratory studies, we have observed that erosion telomere signal stranded overhangs occur in patients with aplastic anemia with telomerase complex mutations;single strand overhang erosion is a critical event in tissue culture predisposing to malignant transformation, and it is not age-correlated. Observations in humans and in mice have also suggested that there is a telomeric set point. Telomere repair can occur in animals and man when they inherit short telomeres from one parent but have normal telomerase activity, and this combination does not appear to result in disease, emphasizing the importance of genetic investigations in individuals with short telomeres. To investigate the role of sex hormones in telomere stabilization, two studies have been initiated. In the mouse, preliminary data suggests that the administration of male hormones to animals over several months delays telomere attrition, except in animals which are telomerase deficient, as expected. We have also obtained cells from the Womens Health Initiative of NHLBI, which will allow us to determine whether hormone replacement affects telomere attrition over a 15 year span between sample collections. In concurrent experiments, we are examining samples from the HALT-C Study of progression to cirrhosis in patients with chronic hepatitis C viremia.

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