Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks. The asymmetric replication of DNA would lead inevitably to a loss of genetic material, and telomerase, functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) and in TERC occur in apparently acquired aplastic anemia and other diseases. Male hormones, long used to treat aplastic anemia, act to regulate TERT transcription and telomerase activity. While critical telomere shortening leads to either cell senescence or apoptosis, occasional cells become aneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for oncogenesis, in which chromosome instability rather than the cumulative acquisition of somatic mutations in specific genes is etiologic. We measure telomere length of leukocytes in our CLIA laboratory by gene amplification; high throughput analysis is useful both for research and in the clinic, and our procedure is certified for patient data. We confirm qpcr results with measurement of telomeres by Flow-FISH, which allows measurement of telomere length individual cells in suspension. Measurement of telomere length in clinical samples is required for the adequate diagnosis of aplastic anemia. Telomere length and the rate of loss of telomere are predictive of late events after treatment with immunosuppression, and probably in other clinical circumstances. We also have established single telomere length analysis (STELA), which relies on amplification using chromosome specific sub-telomeric DNA sequence to detect critical telomere shortening in individual chromosomes. We have continued to examine the role of telomerase in aging using genetically modified mice. For example, we have shown in Terc-/- mice that lack of the RNA component results in both hematpoietic lineage skewing towards myelopoiesis, away from erythropoiesis and lymphocytopoiesis, and also histologic changes in the intestinal epothelium. We have examined the TERT promoter, in which both mutations and epigenetic changes have been implicated in a wide variety of cancers. We found that epigenetic changes in regulatory regions of the TERT gene correlated with clinical outcomes in acute myeloid leukemia. Regions of the TERT genes were highly methylated in all cell lines and primary leukemia cells showed diverse methylation profiles; most patient samples also showed hypermethylated regions upstream, associated with inferior patient survival. Methylation status could also be correlated with cytotoxicity with a chemical inhibitor of telomerase. the combination of the inhibitor with a hypermethylating agent was synergistic. We have now published our study of the effects of high doses of danazol, a synthetic androgenic sex hormone, in patients with telomere disease. The protocol specified two year treatment at very high doses of hormone, with the aim of reducing the accelerated rate of telomere attrition in patients with telomere disease. In 27 cases, we unexpectedly observed elongation of telomere length, which occurred in most patients and during the first six months of therapy. Toxicity was frequent but tolerated by patients; abnormalities of lipids as well as symptoms of bloating and emotional liability, were most frequent. Blood counts improved in almost all patients, most prominently hemoglobin and platelets, and patients who were transfusion-dependent prior to danazol treatment often achieved transfusion-independence. Telomere disease also involves the lung and liver, producing fibrosis and cirrhosis, respectively. Although function in these organ systems were not among primary outcomes, patients showed stability or even improvement in pulmonary functions as measured by CO diffusion, and in the liver, a reduction in fibrosis as determined by imaging utilizing the fibroscan method. From these results, we inferred that pharmacologic modulation of telomere length in humans is feasible. A likely mechanism is increased telomerase transcription, as was observed in vitro previously by us in primary hematopoietic CD34 cells in lymphocytes and by others in tumor cell lines. We have examined patients with bone marrow failure and a history suggestive of familial disease or specific signs and symptoms suggestive of telomeropathy for mutations in RTEL1. RTEL1 encodes a DNA helicase, and RTEL1 mutations have been shown to be etiologic in children with dyskeratosis congenita and the Hoyeraal- Hreidarsson syndrome, a particularly severe variant of dyskeratosis congenita with neurological and ophthalmologic manifestations. In our series of over 60 patients, we detected RTEL1 mutation in 8. In silico, these mutations could be predicted to be deleterious, and they often occurred in well conserved regions of the gene. Of particular note was our observation that in some patients, telomere length was not markedly decreased, but the 3-prime overhang at the chromosome end was diminished, consistent with the action of the helicase. Further, in several patients RTEL1 mutations occurred in combination with other mutations in genes involved in telomere maintenance and repair, as for example TERC and TERT. In contrast to children with dyskeratosis congenita, who have compound heterozygous or homozygous RTEL1 mutations, in adults, RTEL1 mutations occurred in the heterozygous state. In other functional studies, we were able to demonstrate with the specific ring domain variant abnormalities and co-localization of RTEL1 with TRF2, a protein member of the shelterin complex. Our results indicate that RTEL1 should be included in screening in patients with acquired aplastic anemia, some of whom will not have evidence of telomere attrition. In addition, loss of the 3-prime overhang would place patients at risk of chromosome instability, and indeed in several of our patients, the RTEL1 mutation was associated with marked chromosome abnormalities and myeloid malignancy progression. We plan to initiate a new study of danazol in patients with telomeropathy, to replicate our initial findings of telomere elongation and clinical improvement in blood, lung, and liver abnormalities. We will utilize lower doses, of 200 or 400 mg daily in order to reduce toxicity. As we did not observe major abnormalities of liver function, the new protocol will be more inclusive of patients with predominantly pulmonary fibrosis and liver cirrhosis manifestations. Low doses of danazol may be useful in the prevention as well as the treatment of multiple clinical abnormalities in patients who have no other good therapeutic options, given their often multiorgan dysfunction. We are investigating danazols effects on single cells using RNA seq as described in our accompanying annual report. We have observed 2-3x increased TERT transcription in preliminary experiments, and we will specifically interrogate the transcriptome of individual cells for changes in hemapoietic cell self-renewal, differentiation, and response to DNA damage. This method will be used in both normal cells and in cells derived from patients with telomere disease. To investigate the utility of danazol in patients at high risk of chromosome instability, telomerase transcription will be measured in bulk cells from patients who carry mutations for increased telomerase transcription and effects on both telomere attrition and the loss of 3-overhang.

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Marsh, Judith C W; Gutierrez-Rodrigues, Fernanda; Cooper, James et al. (2018) Heterozygous RTEL1 variants in bone marrow failure and myeloid neoplasms. Blood Adv 2:36-48
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