Severe Thrombocytopenia Due to Bone Marrow Failure in Children With Dyskeratosis Congenita Does Not Respond to Eltrombopag Treatment: Case Series.

Dyskeratosis congenita is a rare inherited disease with classic cutaneous symptoms, sometimes accompanied with more severe extracutaneous manifestations such as bone marrow failure, which can be lethal. Eltrombopag is an orally available thrombopoietin receptor agonist in clinical use for increasing platelet levels in patients with immune thrombocytopenia and aplastic anemia. Here, 3 pediatric patients with dyskeratosis congenita are presented with varying disease severity, in which off-label eltrombopag treatment had no clinical effect on bone marrow failure. This, in addition to the negative results in a previous case report, supports the preclusion of eltrombopag use in dyskeratosis congenita.

Bilateral cytomegalovirus retinitis as the presenting feature of Dyskeratosis Congenita.

PURPOSE: To describe a young male with bilateral sequential Cytomegalovirus retinitis (CMVR) as the presenting feature of Dyskeratosis Congenita. CASE REPORT: A 25-year-old human immunodeficiency virus (HIV) negative male developed CMVR in his left eye, while on a three week course of oral valganciclovir therapy for CMV retinitis in his right eye. Systemic examination revealed reticular hypopigmentation of the forearms, dystrophic nails, oral leukoplakia and complete blood counts showed pancytopenia. A diagnosis of Dyskeratosis Congenita was confirmed with genetic testing. CONCLUSION: CMVR in non-HIV individuals should be considered as a harbinger of systemic immunosuppressive conditions. Ophthalmologists may be the first ones to suspect and diagnose congenital immunosuppressive disorders like Dyskeratosis Congenita in these patients.

Lipoprotein particle alterations due to androgen therapy in individuals with dyskeratosis congenita.

BACKGROUND: Dyskeratosis congenita (DC) is a telomere biology disorder associated with high rates of bone marrow failure (BMF) and other medical complications. Oral androgens are successfully used to treat BMF in DC but often have significant side effects, including elevation of serum lipids. This study sought to determine the extent to which oral androgen therapy altered lipid and lipoprotein levels. METHODS: Nuclear magnetic resonance (NMR) was used to evaluate serum lipid profiles, and lipoprotein particle number and size in nine androgen-treated individuals with DC, 45 untreated individuals with DC, 72 unaffected relatives of DC patients, and 19 untreated individuals with a different inherited BMF syndrome, Fanconi anaemia (FA). FINDINGS: Androgen-treated individuals with DC had significantly decreased serum HDL cholesterol, HDL particle number and HDL particle size (p < 0·001, p < 0·001 and p < 0·001, respectively); significantly increased serum LDL cholesterol and LDL particle number (p < 0·001, p < 0·001, respectively), decreased apoA-I and increased apoB (p < 0⋅001, p < 0⋅05 respectively) when compared with untreated individuals with DC. There were no significant lipid profile differences between untreated DC and untreated FA participants; or between untreated DC participants and their unaffected relatives. Branched chain amino acids and lipoprotein insulin resistance were not significantly different with androgen treatment. GlycA, an inflammatory acute phase reactant, was significantly increased with androgen treatment (p < 0⋅001). INTERPRETATION: Androgen treatment in DC creates an atherogenic lipoprotein profile, raising concern for the potential of elevated cardiovascular disease risk. Clinical guidelines for individuals on androgens for DC-related BMF should include cardiovascular disease monitoring. These findings could be relevant in individuals treated with androgen for other indications. FUNDING: Intramural research programs of the Division of Cancer Epidemiology and Genetics of the National Cancer Institute and National Heart, Lung, and Blood Institute.

Androgen derivatives improve blood counts and elongate telomere length in adult cryptic dyskeratosis congenita.

Dyskeratosis Congenita (DKC) is a systemic disorder caused by mutations resulting in impaired telomere maintenance. Clinical features include bone marrow failure and an increased risk of developing hematological malignancies. There are conflicting data whether androgen derivatives (AD) can elongate telomeres in vivo and whether AD treatment enhances the risk of gaining myelodysplastic syndrome-related mutations. Seven TERC or TERT-mutated DKC patients underwent AD treatment. All patients revealed hematological response. Telomere length of lymphocytes and granulocytes increased significantly and no MDS-related mutations were detected. Pending longer follow-up, treatment with AD seems to represent an efficient and safe therapy for DKC patients.

Comparable Effects of the Androgen Derivatives Danazol, Oxymetholone and Nandrolone on Telomerase Activity in Human Primary Hematopoietic Cells from Patients with Dyskeratosis Congenita.

Dyskeratosis congenita (DKC) is a rare inherited disease of impaired telomere maintenance that progressively leads to multi-organ failure, including the bone marrow. By enhancing telomerase activity, androgen derivatives (ADs) are a potential therapeutic option able to re-elongate previously shortened telomeres. Danazol, oxymetholone, and nandrolone are ADs most frequently used to treat DKC. However, no direct in vitro analyses comparing the efficacy of these ADs have been conducted so far. We therefore treated mononuclear cells derived from peripheral blood and bone marrow of four patients with mutations in telomerase reverse transcriptase (TERT, n = 1),in the telomerase RNA component (TERC, n = 2) and in dyskerin pseudouridine synthase 1 (DKC1, n = 1) and found no substantial differences in the activity of these three agents in patients with TERC/TERT mutations. All AD studied produced comparable improvements of proliferation rates as well as degrees of telomere elongation. Increased TERT expression levels were shown with danazol and oxymetholone. The beneficial effects of all ADs on proliferation of bone marrow progenitors could be reversed by tamoxifen, an estrogen antagonist abolishing estrogen receptor-mediated TERT expression, thereby underscoring the involvement of TERT in AD mechanism of action. In conclusion, no significant differences in the ability to functionally enhance telomerase activity could be observed for the three AD studied in vitro. Physicians therefore might choose treatment based on patients' individual co-morbidities, e.g., pre-existing liver disease and expected side-effects.

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration in vitro, in stem cell models, and in vivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

Similar telomere attrition rates in androgen-treated and untreated patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome and the prototypic telomere biology disorder (TBD). Leukocyte telomere length (TL) less than the first percentile for age, measured by flow cytometry with in situ hybridization (flow FISH), is diagnostic of DC. Androgens are a therapeutic option for DC/TBD-associated bone marrow failure (BMF). One report has shown an apparent increase in TL in patients while on treatment with the attenuated androgen danazol. The aim of this study was to compare TL over time in 10 androgen-treated and 16 untreated patients with DC. All subjects were enrolled in institutional review board-approved longitudinal cohort studies of inherited BMF. TL in 6-panel leukocyte subsets was measured by flow FISH. Generalized estimating equations (GEE) methodology was used to compare TL changes over time between groups. Unadjusted analyses showed annual median total lymphocyte TL attrition of -62 base pairs/year (bp/y) in androgen-treated patients with DC compared with -76 bp/y in untreated DC patients (P = .71). Longitudinal analysis using a GEE model, adjusted for age at sample collection, showed no statistically significant difference in TL change over time between treated and untreated patients (P = .24). The results were similar for each individual leukocyte subset evaluated. In summary, our data show the expected age-associated longitudinal telomere shortening in patients with DC, irrespective of androgen therapy. Caution is warranted when recommending androgen therapy for non-BMF manifestations of DC or TBDs until the biological mechanisms are better understood.

Immune status of patients with inherited bone marrow failure syndromes.

Immune function abnormalities have been reported in patients with Fanconi anemia (FA), dyskeratosis congenita (DC) and, rarely, in Shwachman-Diamond syndrome (SDS), and Diamond-Blackfan anemia (DBA), but large systematic studies are lacking. We assessed immunological parameters in 118 patients with these syndromes and 202 unaffected relatives. We compared the results in patients with reference values, and with values in relatives after adjusting for age, sex, corticosteroid treatment, and severe bone marrow failure (BMF). Adult patients (≥18 years) with FA had significantly lower immunoglobulins (IgG, IgA and IgM), total lymphocytes, and CD4 T cells than reference values or adult relatives (P < 0.001); children with FA had normal values. Both children and adults with FA had lower B- and NK cells (P < 0.01) than relatives or reference values. Patients with DC had essentially normal immunoglobulins but lower total lymphocytes than reference values or relatives, and lower T-, B-, and NK-cells; these changes were more marked in children than adults (P < 0.01). Most patients with DBA and SDS had normal immunoglobulins and lymphocytes. Lymphoproliferative responses, serum cytokine levels, including tumor necrosis factor-α and interferon-γ, and cytokine levels in supernatants from phytohemagglutinin-stimulated cultures were similar across patient groups and relatives. Only patients with severe BMF, particularly those with FA and DC, had higher serum G-CSF and Flt3-ligand and lower RANTES levels compared with all other groups or relatives (P < 0.05). Overall, immune function abnormalities were seen mainly in adult patients with FA, which likely reflects their disease-related progression, and in children with DC, which may be a feature of early-onset severe disease phenotype.

Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

Response to androgen therapy in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome and telomere biology disorder characterized by dysplastic nails, reticular skin pigmentation and oral leucoplakia. Androgens are a standard therapeutic option for bone marrow failure in those patients with DC who are unable to undergo haematopoietic stem cell transplantation, but there are no systematic data on its use in those patients. We evaluated haematological response and side effects of androgen therapy in 16 patients with DC in our observational cohort study. Untreated DC patients served as controls. Seventy percent of treated DC patients had a haematological response with red blood cell and/or platelet transfusion independence. The expected age-related decline in telomere length was noted in androgen-treated patients. All treated DC patients had at least one significant lipid abnormality. Additional treatment-related findings included a significant decrease in thyroid binding globulin, accelerated growth in pre-pubertal children and splenic peliosis in two patients. Liver enzymes were elevated in both androgen-treated and untreated patients, suggesting underlying liver involvement in DC. This study suggests that androgen therapy can be effectively used to treat bone marrow failure in DC, but that side effects need to be closely monitored.

Dyskeratosis congenita--two siblings with a new missense mutation in the DKC1 gene.

Dyskeratosis congenital is reported in two siblings. They presented with the classic triad of mucocutaneous features: leukoplakia of the tongue, dystrophic nails, and a widespread reticulate pigmentation on the neck and upper chest. A genetic analysis was performed and a new missense mutation S356P, hemizygous, was identified in the DKC1 gene in both patients. Acitretin was started at a low-dose in both patients, resulting in clinical improvement and important, positive psychosocial effects.

Accelerated hematopoietic stem cell aging in a mouse model of dyskeratosis congenita responds to antioxidant treatment.

Mutations in DKC1, encoding telomerase associated protein dyskerin, cause X-linked dyskeratosis congenita (DC), a bone marrow (BM) 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 Dkc1(Δ15) 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 Dkc1(Δ15) 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 (NAC), partially rescued the growth disadvantage of mutant cells in vitro and in vivo. Competitive BM repopulation experiments showed that the Dkc1(Δ15) 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 NAC 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.

Reduced-intensity conditioning using fludarabine and antithymocyte globulin alone allows stable engraftment in a patient with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by the triad of nail dystrophy, mucosal leukoplakia, and reticular pigmentation. Bone marrow failure is the principal cause of early mortality, and stem cell transplantation is the only cure for these patients. However, the results of conventional hematopoietic stem cell transplantation (HSCT) for patients with DC are poor because of the high incidence of transplant-related complications. We describe the successful treatment of a 21-year-old male with DC by nonmyeloablative HSCT from a matched unrelated donor. The gene responsible for the X-linked form of DC was screened and hemizygosity for the mutation Gln31Lys was found, which is consistent with the diagnosis. The conditioning regimen consisted of only fludarabine and antithymocyte globulin. Additionally, a graft-versus-host disease (GVHD) prophylaxis was administered with cyclosporine A (CSA) and mycophenolate mofetil (MMF). The regimen was well tolerated, no severe posttransplantation complications were observed, and engraftment was rapid and complete (granulocytes on day +11 and platelets on day +13). Seven months after HSCT, the patient developed GVHD of the liver after tapering CSA which was successfully treated with prednisolone, CSA, and MMF. At the time of reporting, 3 years after HSCT, the patient remained in good clinical condition with minimal signs of chronic GVHD of the oral mucosa. Thus, we conclude that a low-intensity conditioning regimen might be sufficient to induce permanent engraftment by using matched unrelated donor HSCT in DC patients and may avoid severe organ toxicity. Although allogeneic HSCT in patients with DC will not cure the underlying genetic defect it may significantly prolong survival through effective therapy for hematologic complications.

A child with severe form of dyskeratosis congenita and TINF2 mutation of shelterin complex.

A 26-month-old male presented with bone marrow failure and dystrophic nail lesions mimicking onychomycosis. There was no skin finding. Treatment with androgen and methylprednisolone was started due to unavailability of a matched-related hematopoietic stem cell donor. After 30 months, transfusion support was required. TINF2 mutation was identified at the age of five and dyskeratosis congenita (DC) was confirmed. TIN2 mutation analysis must be carried out in patients younger than 10 years presenting with bone marrow failure even if characteristic physical anomalies of DC is missing. Genetic confirmation of DC prevents ineffective immunotherapy with misdiagnosis of acquired aplastic anemia.

Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.

Androgens have been used in the treatment of bone marrow failure syndromes without a clear understanding of their mechanism of action. Blood counts of patients with dyskeratosis congenita or aplastic anemia with mutations in telomerase genes can improve with androgen therapy. Here we observed that exposure in vitro of normal peripheral blood lymphocytes and human bone marrow-derived CD34(+) cells to androgens increased telomerase activity, coincident with higher TERT mRNA levels. Cells from patients who were heterozygous for telomerase mutations had low baseline telomerase activity, which was restored to normal levels by exposure to androgens. Estradiol had an effect similar to androgens on TERT gene expression and telomerase enzymatic activity. Tamoxifen abolished the effects of both estradiol and androgens on telomerase function, and letrozole, an aromatase inhibitor, blocked androgen effects on telomerase activity. Conversely, flutamide, an androgen receptor antagonist, did not affect androgen stimulation of telomerase. Down-regulation by siRNA of estrogen receptor-alpha (ER alpha), but not ER beta, inhibited estrogen-stimulated telomerase function. Our results provide a mechanism for androgen therapy in bone marrow failure: androgens appear to regulate telomerase expression and activity mainly by aromatization and through ER alpha. These findings have potential implications for the choice of current androgenic compounds and the development of future agents for clinical use.