Telomere Diseases and Other Constitutional and Acquired Genetic Disorders of Hematopoiesis

National Heart, Lung, And Blood Institute

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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 continue to accrue cases to a new danazol trial, using low doses that should avoid common 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 will provide firmer 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. In a collaborative and parallel study conducted in Brazil, decadurabolin, another, more potent synthetic androgen, was shown to lengthen telomeres, as measured by flow-FISH, to an extent comparable to improvement we observed with danazol. Further, there was evidence for stabilization and even improvement in a few patients suffering predominantly pulmomary fibrosis as a consequence of their telomere disorder. We have completed our (relatively) large, inter-Institute and interinstitutional machine learning systems approach to distinguish constitutional from immune aplastic anemia. We show that about 20 clinical laboratory testsplus telomere length of leukocyteshas high sensitivity and specificity for this purpose. Development of algorithms based only on readily available clinical testing allows more accurate diagnosis in low resource medical setting, and to expedite assignment of appropriate therapies without delays in genomic laboratories. Our results should be useful in 1. Strengthening and focusing dependence expert clinicians knowledge; 2. Encourage performance of telomere length measurement; 3. Expedite treatment of immune AA; and 4. Provide an interactive web-based program for physicians wordwide, both to guide practice and care of individual patients and potentially increase the size and variety of the database. We have examined clonal hematopoiesis in recurrently mutated myeloid malignancy genes in patients with telomere disease and other syndromes. In the telomeropathies, as in immune AA, clones stereotypically involve limited numbers of genes. With a single exception, the presence of clonal hematopoiesis does not correlate with either clinical manifestations, outcomes, or survival, perhaps due to the time required for clones to expand in vivo. Second mutations in POT1 (which associate with germline TINF2 mutations) and in the promoter region of TERT appear to rescue the telomere deficient phenotype. However, clones mutated in U2AF1, a spliceasome gene, appear as drivers of malignancy and were strongly associated with a poor prognosis, a finding of clinical importance in decision-making for stem cell transplant. Our clinic, in coordination with colleagues in NIAMS, has become a major referral center for the newly described syndrome VEXAS (vacuoles, E1 enzyme, X linked, autoinflammatory, somatic), in which an acquired mutation in UBA1, a key gene in ubiquitinylation, leads to severe multisystem disease, predominantly rheumatologic and hematologic in its manifestations (see also AR VEXAS is not a rare disease and is now recognized by knowledgeable rheumatologists, dermatologists, and hematologists. We have contributed to characterizing VEXAS clinically and pathologically and investigating its complex and uncertain pathophysiology as a multisystem syndrome arising through an acquired mutation in the UBA1 gene of an hematopoietic cell. Among our prominent findings have been stereotypical patterns of clonal hematopoiesis in patients and the detection of a an unusual, strikingly broad pattern of activation of immune and inflammatory markers in the earliest affected human stem cells. Clonal hematopoiesis also is a feature of VEXAS. Secondary clones are frequent in VEXAS patients, especially mutated in DNMT3A, a prominent component of clonal hematopoiesis of indeterminate prognosis (CHIP). Such clones are markedly more frequent in general in autoinflammatory diseases, when adjusted for age, from young patients with Kawasaki arteritis to older patients with vasculitic syndromes. We have used single cell DNA sequencing with error correction to demonstrate variable clonal architectures in VEXAS cases; in almost all instances studied, the dominant clone arose from the underlying UBA1 mutated stem cell. Paradoxically, VEXAS bone marrow cells are physically fragile and difficult to manipulate in the laboratory, and knock down or gene edited cells do not show a proliferative advantage in vitro or in animal models to date.