Telomere Diseases and Other Constitutional and Acquired Genetic Disorders of Hematopoiesis

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

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.Telomere attrition creates chromosome instability and is a mechanism of oncogenesis. Telomere biology diseases are among an increasing number of constitutional marrow failure syndromes which can manifest initially in adults, and without physical anomalies or suggestive pedigrees. In the clinic, following on our successful clinical protocol testing danazol at high doses, we are now accruing cases to a new danazol trial, at low doses that should avoid toxicities. The design of the current protocol also addresses deficiencies of the original study in utilizing flow-FISH for telomere length. And both extended observation and wash-out periods that will provide baseline telomere length 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 telomere biology disorders. 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 for both research purposes and clinical reporting to the patient. We also have data from collaborators in Brazil. We have completed application of artificial intelligence to the difficult problem of distinguishing acquired immune aplastic anemia from constitutional bone marrow failure syndromes. A machine learning approach has required broad collaborations with multiple other NIH clinical groups, as well as extensive systems biology expertise in AI. Our aims include development of algorithms based on available clinical testing, to allow more accurate diagnosis in low resource medical setting, and to expedite assignment of appropriate therapies without delays in genomic laboratories. We have clinical and molecular data from more almost 500 patients from the NIH and Brazil. Our model was based on a two-step data-driven clustering and classification process based on 27 clinical and laboratory variables recorded at initial clinical encounter. K-means clustering was first applied to resolve highly dimensional data into two main clusters, and an optimized bootstrap aggregation ensemble was trained with cases from the largest cluster of the training cohort ; the model was then validated with an external cohort. Unsupervised clustering separately grouped datasets into Cluster A, the largest group mostly represented by immune or inherited aplastic anemia (AA), and Cluster B, those underrepresented in our cohort and including some classical IBMFS. The ensemble model Cluster A-specific was accurate (88%) to predict the BMF etiology, correctly predicting inherited and likely immune BMF in 72% and 92% of cases, respectively. Telomere length, age, mucocutaneos and physical findings, longstanding cytopenias and macrocytosis and multi-organ disease, and blood counts were most important for the models predictive accuracy, highlighting their importance as part of the initial patient history and evaluation. It is of interest and reassuring that machine-learning reproduced the clinical knowledge used by clinicians specialized in BMF, with the addition of a relatively simple assay, telomere length, of particular importance. Further, the model was especially effective in classifying immune aplastic anemia, a diagnosis of exclusion and also the syndrome likely to present acutely and in urgent need to immediate definitive therapies. A public web-based algorithm will be made available as a service to the marrow failure community and ultimately to increase the models accuracy by accruing more well curated constitutional syndromes to the database. Single cell RNA sequencing (scRNAseq) is a major technical focus of our laboratory (see Aplastic Anemia annual report). In constitutional marrow failure syndromes. We first studied hematopoiesis in GATA2 deficiency. GATA2 CD34 cells displayed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. Patients cells exhibited distinct patterns of gene expression and coexpression compared with healthy donors. Distinct lineages showed differently altered transcriptional profiles, dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. Deficient lympho-myeloid lineage commitment was associated with aberrant gene programs in stem cells prior to lineage commitment. In DADA2 deficiency, molecular immune mechanisms responsible for diverse clinical phenotypes are poorly understood. To perform scRNAseq, purified monocytes of patients and then subpopulations were imputed from transcriptomes. Monocytes could be classified as classical, intermediate, and nonclassical, and we used gene pathway analytics to interpret patterns of gene regulation. In DADA2, the frequency of nonclassical monocytes was higher compared with that of healthy donors, and M1 macrophage markers were up-regulated. We identified upregulated immune response for IFN and IFN signaling in all monocyte subtypes, but the TNFR2 noncanonical NF-B pathway was up-regulated only in nonclassical monocytes. Elevated IFN activates cell signaling, leading to differentiation into M1 macrophages from monocytes and release of TNF. Immune responses and more general response to stimuli pathways were up-regulated in DADA2 monocytes, and protein synthesis pathways were down-regulated. In other work, T cells of DADA2 patients have been related by novel algorithms to innate immune responses, implicating specific lymphocyte subsets and immune pathways in the pathogenesis of the disease. In collaboration with M.D. Anderson, we applied single cell methods to telomere disease. Based on experiments utilizing cells from human patients and also genetically deficient mice, telomere attrition was implicated in maintenenance of HSCs in a state of persistent metabolic activation and differentiation towards the megakaryocytic lineage, via cell-intrinsic upregulation of the innate immune signaling response; these effects directly compromised self-renewal and lead to stem cell exhaustion.