Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks. Asymmetric replication of DNA would lead inevitably to a loss of genetic material, and the telomere repair molecular machinery (a reverse transcriptase, RNA template, and associated proteins) 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, occasionally 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. In the clinic, we routinely now measure telomere length commercially by a CLIA flow-FISH method, and in our research laboratory by gene amplification qpcr. 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 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 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. 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. 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. In some patients, whose blood counts fell (and telomeres shortened rapidly) after danazol was discontinued per protocol, resumption of danazol off protocol and at lower doses restored hematologic improvement. We have developed a protocol to test lower, better tolerated doses of danazol: we will randomize eligible patients to danazol at 400 or 200 mg/day for treatment for 6 months, then cross over to the alternative dose for a further 6 months, with both leadup and washout periods for telomere measurements. This design should allow us to determine efficacy of lower doses at increasing blood counts and reversing rapid telomere attrition. Further, because the protocol duration is shorter and fewer toxic effects are anticipated, we have expanded inclusion criteria so that lung and liver disease can be assessed. 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. 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 shelter in complex. Loss of the 3-prime overhang would place patients at risk of chromosome instability, and indeed in several of our patients, the RTEL1. We are now working to optimize functional assays for assessment of the role of novel variants in telomere biology and telomere homeostasis,: 3 overhang measurement, telomerase activity measurement, telomere length measurement by qPCR and Southern blot, rolling amplification for t-circles detection, protein co-localization by immunofluorescence, and creation of stable cell lines expressing wild type and mutant RTEL1 and POT1 proteins. In a separate project, we are sequencing the TERT promoter in telomeropathy patients to determine if acquired mutations are responsible for modification of clinical phenotype. We also have commenced feasibility experiments to determine whether single cell RNA sequencing can be applied to the limited numbers of cells that can be obtained from bone marrow failure patients. We have had success in obtaining adequate CD34+ cells from bone marrows of GATA2 patients and age-matched healthy controls; cells were harvested using FACS sorting, followed by single cell RNA capture and subsequent cDNA library construction using 10XGenomics technology; libraries were sequenced using Hiseq3000. This method will be applied to marrow cells from patients with telomere gene mutations in order to study both baseline transcriptional patterns and changes with induced hematopoietic stress. For more general purposes, we are standardizing and validating three different NGS protocols for genetic screening of BMF patients: 1. a comprehensive capture-based targeting panel with 165 genes related to BMF and hematologic diseases for screening of germline and somatic variants; 2. an amplicon-based targeting sequencing protocol for the detection of two common variants of the TERT promoter region known to increase this gene expression; 3. error-correcting sequencing standardization using a modified Illumina Trusight Myeloid panel for the detection of somatic clones at very low allele frequency. With the recent availability of very large databases of sequence of tens of thousands of apparently healthy individuals, as well as accumulation of clinical data in our own patients, it is likely that pathogenic mutations may be difficult to discriminate from polymorphisms and private variants, especially absent functional assays. From preliminary and ongoing analyses, it is likely that only a small minority of patients with severe aplastic anemia have an underlying constitutional defect, and conversely a family history and/or clinical features of an inherited syndrome remain important in discriminating acquired from constitutional disease.
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