Summary Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow failure and cancer susceptibility syndrome. Decreased accumulation of telomerase RNA resulting in excessive telomere shortening and premature cellular senescence is thought to be the primary cause of disease in X-linked DC. Affected tissues are those that require constant renewal by stem cell activity. We previously showed that in Dkc1Δ15 mice, which contain a mutation that is a copy of a human mutation causing DC, mutant cells have a telomerase dependent proliferative defect and increased accumulation of DNA damage in the first generation before the telomeres are short. We now demonstrate the presence of the growth defect in Dkc1Δ15 mouse embryonic fibroblasts in vitro and show that accumulation of DNA damage and levels of reactive oxygen species increase with increasing population doublings. Treatment with the antioxidant, N-acetyl cysteine, partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive bone marrow repopulation experiments showed that the Dkc1Δ15 mutation is associated with a functional stem cell defect that becomes more severe with increasing age, consistent with accelerated senescence, a hallmark of DC hematopoiesis. This stem cell phenotype was partially corrected by N-acetyl cysteine treatment. These results suggest that a pathogenic Dkc1 mutation, accelerates stem cell aging, that increased oxidative stress might play a role in the pathogenesis of X-linked DC, and that some manifestations of DC may be prevented or delayed by antioxidant treatment.

Key Points Success of hematopoietic stem cell transplantation for MIRAGE syndrome may be limited by syndrome-specific comorbidities. SAMD9 mutations associated with MIRAGE syndrome are a newly described cause of congenital amegakaryocytic thrombocytopenia.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed “corrected” lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human patients have so far not shown defects in pseudouridylation or ribosomal RNA processing. None of the mutant iPS cells presented here show decreased pseudouridine levels in rRNA or defective rRNA processing suggesting telomere maintenance defects account for most of the phenotype of X-linked DC. Finally gene expression analysis of the iPS cells shows that WNT signaling is significantly decreased in all mutant cells, raising the possibility that defective WNT signaling may contribute to disease pathogenesis.

Key Points Acquired aplastic anemia is a T-cell–mediated autoimmune bone marrow aplasia, without a known etiologic trigger. Clonal expansion of CD8+ effector T lymphocytes can occur following vaccination and accompany graft dysfunction or aplastic anemia relapse.