Telomere Diseases and Other Constitutional and Acquired Genetic Disorders of Hematopoiesis

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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. Telomere attrition creates chromosome instability and is a mechanism of oncogenesis. Telomere attrition also a reflects generalized DNA damage, and this report includes studies directed at acquisition of somatic mutations, specific genes, and relevant etiologic and functional considerations. In the clinic, 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 are now accruing cases to a new, low dose danazol trial. In the current study, 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 manifestations of telomeropathy. Relevant to the larger clinical issue of distinction between constitutional and acquired etiologies for bone marrow failure, we systematically screen by genomics patients presenting to our clinic with a wide variety of bone marrow failure syndromes. 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. We also have data from collaborators in Sao Paolo, Brazil. We are developing a machine learning approach to identify BMF patients with increased risk of having inherited BMF. As inherited BMF are marked by an overlap of clinical presentations and are very heterogenous, supervised learning may be particularly useful to obtain a model that incorporates the complex clinical and molecular patterns in a predictive model. In collaboration with intra- and extramural colleagues, we have gathered clinical and molecular data from more than 600 patients In early iterations our model displays an accuracy of 90% but moderate specificity (80%). Clustering prior to machine learning should further improve specificity by detecting constitutional syndromes and associated variables. We are exploring scRNAseq in constitutional marrow failure syndromes. In the telomeropathies and related diseases, numbers of CD34 marrow cells are inadequate. We have been successful in defining the hematopoietic defect in GATA2 deficiency and have utilized thid approach now in DADA2 deficiency, in which monocytes from patients were studied. The striking inflammatory clinical phenotype of DADA2 deficiency has not been linked other than generally to innate immunity. We found large differences in classical, non-classical, and transitional subsets between patients and healthy controls, consistent with the abnormal cytokine profile of plasma and a pro-inflammatory clinical phenotype. We performed a comparative analysis to assess the clinical utility of cfDNA for detection and quantification of clonal hematopoiesis. In a comparison of the clinical utility of cfDNA for detection and quantification of clonal hematopoiesis. Compared to matched blood cells, cfDNA sensitivity and specific was high to detect variants only when their VAF was higher than 12%. Also, agreement between cfDNA and blood cells in clones quantification was very poor, suggesting that screening in cfDNA is not interchangeable to bone marrow or peripheral blood cells. We have collaborated with Dr. Mehta from NHLBI and Dr. Grayson from NIAMS in projects that aim to identify mutations associated with clonal hematopoiesis in patients with chronic inflammation, such as psoriasis and ANCA-associated vasculitis. In parallel, we are screening patients with telomere diseases for somatic mutations in genes associated with clonal hematopoiesis and telomere maintenance. POT1 mutations were found exclusively mutated in telomere diseases and are being studied as potential clonal marker of genetic rescuing in these patients. Also, recurrent mutations in U2AF1 were found in patients that presented with MDS and are likely to be a clonal marker of clonal evolution in telomere diseases. In collaboration with other NIH investigators, we helped characterize a novel syndrome in which a somatic mutation in an hematopoietic stem cell that alters an early stage of ubiquitinylation results in a severe multisystem disease, manifesting as polychondritis and vasculitis in adult men; one component is a dysplastic bone marrow featuring vacuolization and variable profound cytopenias. In other related work at the population level, we were successful in obtaining a large research grant from the Department of Defense to establish an international consortium which will undertake the largest multi-ethnic genome wide study of risk genetic risk factors in AA, aimed especially to uncover novel variants associated with the increased frequency of AA in Asian countries