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. Having successfully completed our clinical protocol testing danazol at high doses and prolonged administration for clinical efficacy and telomere effects in patients with telomere disease, we have initiated a new, low dose danazol trial. In the current, patients are randomized to initially receive either half or quarter doses (400 mg or 200 mg daily) compared to the original protocol, for 6 months, and then crossed over the other dose for a further 6 months. These regimens should avoid the toxicities of high dose sex hormones. The design of the current protocol also addresses deficiencies of the original study: 1. We will utilize both q-pcr and also flow-FISH for telomere length and 2. 6 month observation and wash-out periods precede and follow drug administration in order to provide baseline telomere attrition information. Additionally, because of the suggestion of stabilization and possibly improvement in pulmonary function in the earlier protocol, the inclusion criteria have been expanded to recruit patients with mainly lung manifestions of telomeropathy. Accrual is proceeding as anticipated to the low dose danazol protocol. Also, in the clinic, relating to telomere disease especially but relevant to the larger clinical issue of distinction between constitutional and acquired etiologies for bone marrow failure, we are systematically screening by genomics, in collaboration with the University of Chicago, patients presenting to our clinic with a wide variety of manifestations of bone marrow failure. We assess for mutations and polymorphisms in >50 genes etiologic in inherited marrow failure syndromes for both research purposes and clinical reporting to the patient. For comparative purposes, we also have data from collaborators at a marrow failure center in Sao Paolo, Brazil. To date, our results indicate: first, patients with acute onset of severe aplastic anemia, defined by convention based on peripheral blood counts, very rarely have mutations in constitutional marrow failure genes, and second, that clinical correlates such as family history and multi-organ involvement are highly predictive of a germline etiology and positive genomic testing. Obvious but important, frequencies of genomic diagnoses are highly dependent on clinic demographics and referral patterns. RTEL1 is a germline mutation that affects telomere repair and in which we have special interest, as mutations have been present in several patients on other of our protocols who progressed from marrow failure to myeloid malignancy. Due to its function as helicase, RTEL1 deficiency may be subtle, and not cause accelerated telomere shortening. We continue to pursue screening for RTEL1 by next generation sequencing and optimization of functional assays. We have screened telomeropathy patients for TERT promoter acquired mutations, and they appear to be strongly associated in this population with older age and pulmonary disease, and milder hematologic manifestations. We are also investigating novel genes in pedigrees in which marrow failure is variably clinical severe. As a component of our single cell effort, we are exploring scRNAseq of DADA2 T cells and GATA2 CD34 obtained from patients with these specific deficiency syndromes, who suffer marrow failure but have adequate numbers of CD34 marrow cells for such efforts, as well as the occasional telomeropathy patient with relatively well preserved hematopoiesis. Because telomeropathy is associated with chromosome instability and myeloid neoplasm, we are testing new methods for sensitive detection of single copy number variation as a harbinger of gross aneuploidy and transformation.

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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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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

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