RESUMO
Dyskeratosis congenita (DC) is a genetic syndrome with progressive multisystem involvement classically characterized by the clinical triad of oral leukoplakia, nail dystrophy, and reticular hyperpigmentation. Frequent complications are bone marrow failure, increased rate of malignancy, lung and liver diseases. DC results from an anomalous progressive shortening of telomeres resulting in DNA replication problems inducing replicative senescence. We report a death due to DC in a 16-year-old male with bone marrow failure and multiple organ dysfunction. At autopsy, nail dystrophy and skin hypopigmentation were observed. Gross and microscopic examinations of the internal organs showed cardiac hypertrophy, multiple lung consolidations and prominent interstitial fibrosis, liver cirrhosis, and fibrosis. Multiple foci of extramedullary hematopoiesis were identified, including on the epidural surface of the dura, that is an infrequent location, mimicking a focal area of epidural hemorrhage. Only a few autopsy studies about DC are reported in the literature. Further research should be done to understand the pathophysiology of the disease and its complications.
Assuntos
Humanos , Masculino , Adolescente , Disceratose Congênita/patologia , Autopsia , Hematopoese Extramedular , Evolução Fatal , Encurtamento do TelômeroRESUMO
Telomeropathies are a group of phenotypically heterogeneous diseases molecularly unified by pathogenic mutations in telomere-maintenance genes causing critically short telomeres. X-linked dyskeratosis congenita (DC), the prototypical telomere disease, manifested with ectodermal dysplasia, cancer predisposition, and severe bone marrow failure, is caused by mutations in DKC1, encoding a protein responsible for telomerase holoenzyme complex stability. To investigate the effects of pathogenic DKC1 mutations on telomere repair and hematopoietic development, we derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DC patient carrying the most frequent mutation: DKC1 p.A353V. Telomeres eroded immediately after reprogramming in DKC1-mutant iPSCs but stabilized in later passages. The telomerase activity of mutant iPSCs was comparable to that observed in human embryonic stem cells, and no evidence of alternative lengthening of telomere pathways was detected. Hematopoietic differentiation was carried out in DKC1-mutant iPSC clones that resulted in increased capacity to generate hematopoietic colony-forming units compared to controls. Our study indicates that telomerase-dependent telomere maintenance is defective in pluripotent stem cells harboring DKC1 mutation and unable to elongate telomeres, but sufficient to maintain cell proliferation and self-renewal, as well as to support the primitive hematopoiesis, the program that is recapitulated with our differentiation protocol.
Assuntos
Proteínas de Ciclo Celular/genética , Diferenciação Celular , Hematopoese , Proteínas Nucleares/genética , Telômero/metabolismo , Células Cultivadas , Reprogramação Celular , Disceratose Congênita/genética , Disceratose Congênita/patologia , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Mutação , Telomerase/genética , Telomerase/metabolismo , Encurtamento do TelômeroRESUMO
Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8 ± 7.1%) and qPCR (9.5 ± 7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6 ± 7.6% vs. 16 ± 19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes.
Assuntos
Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Encurtamento do Telômero/fisiologia , Telômero/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/metabolismo , Anemia Aplástica/patologia , Criança , Pré-Escolar , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Lactente , Recém-Nascido , Leucócitos/citologia , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. OBJECTIVES: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. METHODS: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. RESULTS: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. CONCLUSIONS: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence.
Assuntos
Dano ao DNA/genética , Disceratose Congênita/genética , Fibroblastos/metabolismo , Ribossomos/fisiologia , Telômero/genética , Proteína Supressora de Tumor p53/genética , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Senescência Celular/fisiologia , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Fibroblastos/citologia , Perfilação da Expressão Gênica , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.
Assuntos
Proteínas de Ciclo Celular/genética , Disceratose Congênita/genética , Disceratose Congênita/terapia , Mutação de Sentido Incorreto/fisiologia , Proteínas Nucleares/genética , Estabilidade de RNA/genética , RNA/metabolismo , Telomerase/metabolismo , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/fisiologia , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/uso terapêutico , Células Cultivadas , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Ativação Enzimática/genética , Terapia Genética , Células HeLa , Humanos , Transferases Intramoleculares/química , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Proteínas Nucleares/uso terapêutico , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , RNA/química , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/fisiologia , Telomerase/química , Valina/genéticaRESUMO
Hereditary benign intraepithelial dyskeratosis (HBID) is a rare disorder first described in 1960. To date, all but one published case trace their ancestry back to an Indian tribe in North Carolina. Affected patients usually develop asymptomatic ocular and oral lesions. The latter may resemble other dermatologic conditions that affect the oral mucosa, such as white sponge nevus. This report describes a case of a Brazilian patient who showed clinical and histological features of HBID, which appears to be the first reported case in South America. Although genetic analysis could not be carried out, the family history suggests a genetic etiology.