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1.
J Clin Immunol ; 39(1): 81-89, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30607663

RESUMO

The association of immunodeficiency-related vaccine-derived rubella virus (iVDRV) with cutaneous and visceral granulomatous disease has been reported in patients with primary immunodeficiency disorders (PIDs). The majority of these PID patients with rubella-positive granulomas had DNA repair disorders. To support this line of inquiry, we provide additional descriptive data on seven previously reported patients with Nijmegen breakage syndrome (NBS) (n = 3) and ataxia telangiectasia (AT) (n = 4) as well as eight previously unreported patients with iVDRV-induced cutaneous granulomas and DNA repair disorders including NBS (n = 1), AT (n = 5), DNA ligase 4 deficiency (n = 1), and Artemis deficiency (n = 1). We also provide descriptive data on several previously unreported PID patients with iVDRV-induced cutaneous granulomas including cartilage hair hypoplasia (n = 1), warts, hypogammaglobulinemia, immunodeficiency, myelokathexis (WHIM) syndrome (n = 1), MHC class II deficiency (n = 1), Coronin-1A deficiency (n = 1), X-linked severe combined immunodeficiency (X-SCID) (n = 1), and combined immunodeficiency without a molecular diagnosis (n = 1). At the time of this report, the median age of the patients with skin granulomas and DNA repair disorders was 9 years (range 3-18). Cutaneous granulomas have been documented in all, while visceral granulomas were observed in six cases (40%). All patients had received rubella virus vaccine. The median duration of time elapsed from vaccination to the development of cutaneous granulomas was 48 months (range 2-152). Hematopoietic cell transplantation was reported to result in scarring resolution of cutaneous granulomas in two patients with NBS, one patient with AT, one patient with Artemis deficiency, one patient with DNA Ligase 4 deficiency, one patient with MHC class II deficiency, and one patient with combined immunodeficiency without a known molecular etiology. Of the previously reported and unreported cases, the majority share the diagnosis of a DNA repair disorder. Analysis of additional patients with this complication may clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection, and may lead to defect-specific therapies.


Assuntos
Reparo do DNA/genética , Granuloma/complicações , Granuloma/virologia , Síndromes de Imunodeficiência/complicações , Vírus da Rubéola/patogenicidade , Dermatopatias/etiologia , Dermatopatias/virologia , Adolescente , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/virologia , Criança , Pré-Escolar , Feminino , Granuloma/genética , Cabelo/anormalidades , Cabelo/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Doença de Hirschsprung/genética , Doença de Hirschsprung/virologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/virologia , Masculino , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/virologia , Osteocondrodisplasias/congênito , Osteocondrodisplasias/genética , Osteocondrodisplasias/virologia , Rubéola (Sarampo Alemão)/genética , Rubéola (Sarampo Alemão)/virologia , Pele/virologia , Dermatopatias/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/virologia
2.
Sci Rep ; 7(1): 7516, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28790359

RESUMO

Nijmegen Breakage Syndrome (NBS) is associated with cancer predisposition, premature aging, immune deficiency, microcephaly and is caused by mutations in the gene coding for NIBRIN (NBN) which is involved in DNA damage repair. Dermal-derived fibroblasts from NBS patients were reprogrammed into induced pluripotent stem cells (iPSCs) in order to bypass premature senescence. The influence of antioxidants on intracellular levels of ROS and DNA damage were screened and it was found that EDHB-an activator of the hypoxia pathway, decreased DNA damage in the presence of high oxidative stress. Furthermore, NBS fibroblasts but not NBS-iPSCs were found to be more susceptible to the induction of DNA damage than their healthy counterparts. Global transcriptome analysis comparing NBS to healthy fibroblasts and NBS-iPSCs to embryonic stem cells revealed regulation of P53 in NBS fibroblasts and NBS-iPSCs. Cell cycle related genes were down-regulated in NBS fibroblasts. Furthermore, oxidative phosphorylation was down-regulated and glycolysis up-regulated specifically in NBS-iPSCs compared to embryonic stem cells. Our study demonstrates the utility of NBS-iPSCs as a screening platform for anti-oxidants capable of suppressing DNA damage and a cellular model for studying NBN de-regulation in cancer and microcephaly.


Assuntos
Antioxidantes/farmacologia , Fibroblastos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome de Quebra de Nijmegen/genética , Transdução de Sinais/genética , Transcriptoma , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reprogramação Celular , Dano ao DNA , Derme/efeitos dos fármacos , Derme/metabolismo , Derme/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicólise/genética , Ensaios de Triagem em Larga Escala , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
FEBS J ; 284(15): 2378-2395, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28631426

RESUMO

The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Benzoquinonas/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Transformada , Células Cultivadas , Quinase 1 do Ponto de Checagem/química , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA/efeitos dos fármacos , Deleção de Genes , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Humanos , Lactamas Macrocíclicas/farmacologia , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação/efeitos dos fármacos , Mutação Puntual , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Interferência de RNA , Ubiquitinação/efeitos dos fármacos
4.
Redox Biol ; 11: 375-383, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28063379

RESUMO

Rare pleiotropic genetic disorders, Ataxia-telangiectasia (A-T), Bloom syndrome (BS) and Nijmegen breakage syndrome (NBS) are characterised by immunodeficiency, extreme radiosensitivity, higher cancer susceptibility, premature aging, neurodegeneration and insulin resistance. Some of these functional abnormalities can be explained by aberrant DNA damage response and chromosomal instability. It has been suggested that one possible common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Recent studies indicate new, alternative sources of oxidative stress in A-T, BS and NBS cells, including NADPH oxidase 4 (NOX4), oxidised low-density lipoprotein (ox-LDL) or Poly (ADP-ribose) polymerases (PARP). Mitochondrial abnormalities such as changes in the ultrastructure and function of mitochondria, excess mROS production as well as mitochondrial damage have also been reported in A-T, BS and NBS cells. A-T, BS and NBS cells are inextricably linked to high levels of reactive oxygen species (ROS), and thereby, chronic oxidative stress may be a major phenotypic hallmark in these diseases. Due to the presence of mitochondrial disturbances, A-T, BS and NBS may be considered mitochondrial diseases. Excess activity of antioxidant enzymes and an insufficient amount of low molecular weight antioxidants indicate new pharmacological strategies for patients suffering from the aforementioned diseases. However, at the current stage of research we are unable to ascertain if antioxidants and free radical scavengers can improve the condition or prolong the survival time of A-T, BS and NBS patients. Therefore, it is necessary to conduct experimental studies in a human model.


Assuntos
Ataxia Telangiectasia/genética , Síndrome de Bloom/genética , Reparo do DNA , Mitocôndrias/metabolismo , Síndrome de Quebra de Nijmegen/genética , Estresse Oxidativo/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/patologia , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Mitocôndrias/patologia , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
5.
Oxid Med Cell Longev ; 2017: 6745840, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29456787

RESUMO

This study compared the antioxidant status and major lipophilic antioxidants in patients with ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS). Total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), and concentrations of coenzyme Q10 (CoQ10) and vitamins A and E were estimated in the plasma of 22 patients with AT, 12 children with NBS, and the healthy controls. In AT patients, TAS (median 261.7 µmol/L) was statistically lower but TOS (496.8 µmol/L) was significantly elevated in comparison with the healthy group (312.7 µmol/L and 311.2 µmol/L, resp.). Tocopherol (0.8 µg/mL) and CoQ10 (0.1 µg/mL) were reduced in AT patients versus control (1.4 µg/mL and 0.3 µg/mL, resp.). NBS patients also displayed statistically lower TAS levels (290.3 µmol/L), while TOS (404.8 µmol/L) was comparable to the controls. We found that in NBS patients retinol concentration (0.1 µg/mL) was highly elevated and CoQ10 (0.1 µg/mL) was significantly lower in comparison with those in the healthy group. Our study confirms disturbances in redox homeostasis in AT and NBS patients and indicates a need for diagnosing oxidative stress in those cases as a potential disease biomarker. Decreased CoQ10 concentration found in NBS and AT indicates a need for possible supplementation.


Assuntos
Ataxia Telangiectasia/metabolismo , Síndrome de Quebra de Nijmegen/metabolismo , Oxirredução , Adolescente , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Criança , Feminino , Homeostase , Humanos , Masculino , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/genética , Estresse Oxidativo , Ubiquinona/análogos & derivados , Ubiquinona/sangue , Vitamina A/sangue , Vitamina E/sangue
6.
PLoS One ; 11(12): e0167984, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936167

RESUMO

The vast majority of patients with Nijmegen Breakage Syndrome (NBS) are of Slavic origin and carry a deleterious deletion (c.657del5; rs587776650) in the NBN gene on chromosome 8q21. This mutation is essentially confined to Slavic populations and may thus be considered a Slavic founder mutation. Notably, not a single parenthood of a homozygous c.657del5 carrier has been reported to date, while heterozygous carriers do reproduce but have an increased cancer risk. These observations seem to conflict with the considerable carrier frequency of c.657del5 of 0.5% to 1% as observed in different Slavic populations because deleterious mutations would be eliminated quite rapidly by purifying selection. Therefore, we propose that heterozygous c.657del5 carriers have increased reproductive success, i.e., that the mutation confers heterozygote advantage. In fact, in our cohort study of the reproductive history of 24 NBS pedigrees from the Czech Republic, we observed that female carriers gave birth to more children on average than female non-carriers, while no such reproductive differences were observed for males. We also estimate that c.657del5 likely occurred less than 300 generations ago, thus supporting the view that the original mutation predated the historic split and subsequent spread of the 'Slavic people'. We surmise that the higher fertility of female c.657del5 carriers reflects a lower miscarriage rate in these women, thereby reflecting the role of the NBN gene product, nibrin, in the repair of DNA double strand breaks and their processing in immune gene rearrangements, telomere maintenance, and meiotic recombination, akin to the previously described role of the DNA repair genes BRCA1 and BRCA2.


Assuntos
Proteínas de Ciclo Celular/genética , Efeito Fundador , Mutação , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/genética , Reprodução/genética , Adulto , Estudos de Coortes , República Tcheca , Dano ao DNA , Reparo do DNA , Feminino , Triagem de Portadores Genéticos , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Quebra de Nijmegen/etnologia , Eslováquia
7.
Cell Rep ; 16(9): 2499-511, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545893

RESUMO

Nijmegen breakage syndrome (NBS) results from the absence of the NBS1 protein, responsible for detection of DNA double-strand breaks (DSBs). NBS is characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Here, we show successful reprogramming of NBS fibroblasts into induced pluripotent stem cells (NBS-iPSCs). Our data suggest a strong selection for karyotypically normal fibroblasts to go through the reprogramming process. NBS-iPSCs then acquire numerous chromosomal aberrations and show a delayed response to DSB induction. Furthermore, NBS-iPSCs display slower growth, mitotic inhibition, a reduced apoptotic response to stress, and abnormal cell-cycle-related gene expression. Importantly, NBS neural progenitor cells (NBS-NPCs) show downregulation of neural developmental genes, which seems to be mediated by P53. Our results demonstrate the importance of NBS1 in early human development, shed light on the molecular mechanisms underlying this severe syndrome, and further expand our knowledge of the genomic stress cells experience during the reprogramming process.


Assuntos
Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/genética , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Reprogramação Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Cariotipagem , Proteína Homóloga a MRE11 , Mitose , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/patologia , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
PLoS Genet ; 12(3): e1005942, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26990569

RESUMO

Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Recombinação Homóloga/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/patologia , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Eur J Med Genet ; 59(3): 126-32, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26826318

RESUMO

Nijmegen breakage syndrome (NBS, MIM #251260) is an autosomal recessive chromosomal instability disorder. Majority of patients affected are of Slavic origin and share the same founder mutation of 657del5 within the NBN gene encoding protein involved in DNA double-strand breaks repair. Clinically, this is characterized by a microcephaly, immunodeficiency and a high incidence of pediatric malignancies, mostly lymphomas and leukemias. Anticancer treatment among patients with NBS is challenging because of a high risk of life threatening therapy-related toxicity including severe infections, bone marrow failure, cardio- and nephrotoxicity and occurrence of secondary cancer. Based on systemic review of available literature and the Polish acute lymphoblastic leukemia database we concluded that among patients with NBS, these who suffered from clinically proven severe immunodeficiency are at risk of the complications associated with oncological treatment. Thus, in this group it reasonable to reduce chemotherapy up to 50% especially concerning anthracyclines methotrexate, alkylating agents and epipodophyllotoxines, bleomycin and radiotherapy should be omitted. Moreover, infection prophylaxis using intravenous immunoglobulin supplementation together with antifungal and antibacterial agent is recommended. To replace radiotherapy or some toxic anticancer agents targeted therapy using monoclonal antibodies and kinase inhibitors or bone marrow transplantation with reduced-intensity conditioning should be considered in some cases, however, this statement needs further studies.


Assuntos
Leucemia Linfoide/diagnóstico , Leucemia Linfoide/terapia , Linfoma/diagnóstico , Linfoma/terapia , Síndrome de Quebra de Nijmegen/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transplante de Medula Óssea , Predisposição Genética para Doença , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Leucemia Linfoide/epidemiologia , Leucemia Linfoide/etiologia , Linfoma/epidemiologia , Linfoma/etiologia , Segunda Neoplasia Primária/epidemiologia , Segunda Neoplasia Primária/etiologia , Síndrome de Quebra de Nijmegen/complicações , Síndrome de Quebra de Nijmegen/diagnóstico , Síndrome de Quebra de Nijmegen/terapia , Fenótipo , Radioterapia/efeitos adversos , Radioterapia/métodos , Risco , Resultado do Tratamento
10.
Mol Ther ; 24(1): 117-24, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26265251

RESUMO

Over 90% of patients with Nijmegen breakage syndrome (NBS), a hereditary cancer disorder, are homoallelic for a 5 bp deletion in the NBN gene involved in the cellular response to DNA damage. This hypomorphic mutation leads to a carboxy-terminal protein fragment, p70-nibrin, with some residual function. Average age at malignancy, typically lymphoma, is 9.7 years. NBS patients are hypersensitive to chemotherapeutic and radiotherapeutic treatments, thus prevention of cancer development is of particular importance. Expression of an internally deleted NBN protein, p80-nibrin, has been previously shown to be associated with a milder cellular phenotype and absence of cancer in a 62-year-old NBS patient. Here we show that cells from this patient, unlike other NBS patients, have DNA replication and origin firing rates comparable to control cells. We used here antisense oligonucleotides to enforce alternative splicing in NBS patient cells and efficiently generate the same internally deleted p80-nibrin protein. Injecting the same antisense sequences as morpholino oligomers (VivoMorpholinos) into the tail vein of a humanized NBS murine mouse model also led to efficient alternative splicing in vivo. Thus, proof of principle for the use of antisense oligonucleotides as a potential cancer prophylaxis has been demonstrated.


Assuntos
Processamento Alternativo , Proteínas de Ciclo Celular/genética , Síndrome de Quebra de Nijmegen/terapia , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/administração & dosagem , Deleção de Sequência , Processamento Alternativo/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Criança , Replicação do DNA , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia
11.
J Clin Immunol ; 35(6): 538-49, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26271390

RESUMO

PURPOSE: Nijmegen Breakage Syndrome (NBS) is a rare inherited condition, characterized by microcephaly, chromosomal instability, immunodeficiency, and predisposition to malignancy. This retrospective study, characterizing the clinical and immunological status of patients with NBS at time of diagnosis, was designed to assess whether any parameters were useful in disease prognosis, and could help determine patients qualified for hematopoietic stem cell transplantation. METHODS: The clinical and immunological characteristics of 149 NBS patients registered in the online database of the European Society for Immune Deficiencies were analyzed. RESULTS: Of the 149 NBS patients, 91 (61%), of median age 14.3 years, remained alive at the time of analysis. These patients were clinically heterogeneous, with variable immune defects, ranging from negligible to severe dysfunction. Humoral deficiencies predisposed NBS patients to recurrent/chronic respiratory tract infections and worsened long-term clinical prognosis. Eighty malignancies, most of lymphoid origin (especially non-Hodgkin's lymphomas), were diagnosed in 42% of patients, with malignancy being the leading cause of death in this cohort. Survival probabilities at 5, 10, 20 and 30 years of age were 95, 85, 50 and 35%, respectively, and were significantly lower in patients with than without malignancies. CONCLUSIONS: The extremely high incidence of malignancies, mostly non-Hodgkin's lymphomas, was the main risk factor affecting survival probability in NBS patients. Because treatment of NBS is very difficult and frequently unsuccessful, the search for an alternative medical intervention such as hematopoietic stem cell transplantation is of great clinical importance.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Síndrome de Quebra de Nijmegen/diagnóstico , Fatores de Tempo , Adolescente , Adulto , Criança , Pré-Escolar , Instabilidade Cromossômica , Feminino , Humanos , Síndromes de Imunodeficiência , Lactente , Linfoma não Hodgkin , Masculino , Microcefalia , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/terapia , Prognóstico , Estudos Retrospectivos , Adulto Jovem
12.
J Clin Immunol ; 35(2): 227-33, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25677497

RESUMO

PURPOSE: Severe combined immunodeficiency (SCID) encompasses a group of disorders characterized by reduced or absent T-cell number and function and identified by newborn screening utilizing T-cell receptor excision circles (TRECs). This screening has also identified infants with T lymphopenia who lack mutations in typical SCID genes. We report an infant with low TRECs and non-SCID T lymphopenia, who proved upon whole exome sequencing to have Nijmegen breakage syndrome (NBS). METHODS: Exome sequencing of DNA from the infant and his parents was performed. Genomic analysis revealed deleterious variants in the NBN gene. Confirmatory testing included Sanger sequencing and immunoblotting and radiosensitivity testing of patient lymphocytes. RESULTS: Two novel nonsense mutations in NBN were identified in genomic DNA from the family. Immunoblotting showed absence of nibrin protein. A colony survival assay demonstrated radiosensitivity comparable to patients with ataxia telangiectasia. CONCLUSIONS: Although TREC screening was developed to identify newborns with SCID, it has also identified T lymphopenic disorders that may not otherwise be diagnosed until later in life. Timely identification of an infant with T lymphopenia allowed for prompt pursuit of underlying etiology, making possible a diagnosis of NBS, genetic counseling, and early intervention to minimize complications.


Assuntos
Triagem Neonatal , Síndrome de Quebra de Nijmegen/diagnóstico , Síndrome de Quebra de Nijmegen/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Circular , Exoma , Rearranjo Gênico do Linfócito T , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Síndrome de Quebra de Nijmegen/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Linfócitos T/imunologia
13.
PLoS One ; 9(12): e114651, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25485873

RESUMO

Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Mutação/genética , Síndrome de Quebra de Nijmegen/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Western Blotting , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Redes Reguladoras de Genes , Células HEK293 , Heterozigoto , Homozigoto , Humanos , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Raios X
15.
PLoS One ; 9(8): e104964, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25119968

RESUMO

Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Síndrome de Quebra de Nijmegen/tratamento farmacológico , Proteínas Nucleares/metabolismo , Linfócitos T/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Reparo do DNA/efeitos dos fármacos , Regulação para Baixo , Humanos , Mutação , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/metabolismo , Síndrome de Quebra de Nijmegen/patologia , Proteínas Nucleares/genética , Linfócitos T/metabolismo , Linfócitos T/patologia
16.
Pediatr Blood Cancer ; 61(8): 1469-71, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24619942

RESUMO

Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability disorder characterized by a high incidence of pediatric hematologic malignancies. Majority of patients affected are of Slavic origin and share the same founder mutation of 657del5 within the NBN gene encoding protein involved in DNA double-strand breaks (DSB) repair. We report a case of a pediatric patient with NBS, who developed t(9;11)/AF9-MLL-positive AML as a second malignancy after successful treatment of T-NHL. The coexistence of NBN and MLL mutations suggests that the profound dysfunction of NBN may promote alterations of MLL that is mediated by error-prone non-homologous end joining pathway particularly in patients treated with DNA topoisomerase II inhibitors.


Assuntos
Cromossomos Humanos Par 11/genética , Leucemia Monocítica Aguda/etiologia , Síndrome de Quebra de Nijmegen , Translocação Genética , Adolescente , Proteínas de Ciclo Celular/genética , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Monocítica Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Síndrome de Quebra de Nijmegen/complicações , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/genética
17.
J Neurosci Res ; 92(2): 254-66, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24272991

RESUMO

Nijmegen breakage syndrome (NBS), caused by mutation of the Nbn gene, is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. To explore the underlying molecular mechanisms of NBS microcephaly, Frappart et al. previously inactivated Nbn gene in the central nervous system (CNS) of mice by the nestin-Cre targeting gene system and generated Nbn(CNS-del) mice. Here we first report that Nbn gene inactivation induces the defective proliferation and enhanced apoptosis of the oligodendrocyte precursor cells (OPCs), contributing to the severe hypomyelination of the nerve fibers of the corpus callosum. Under conditions of DNA damage and oxidative stress, the distinct regulatory roles of ATM-Chk2 signaling and AKT/mTOR signaling are responsible for the defective proliferation and enhanced apoptosis of the Nbn-deficient OPCs. In addition, specific HDAC isoforms may play distinctive roles in regulating the myelination of the Nbn-deficient OPCs. However, brain-derived neurotrophic factor and nerve growth factor stimulation attenuates the oxidative stress and thereby increases the proliferation of the Nbn-deficient OPCs, which is accompanied by upregulation of the AKT/mTOR/P70S6K signaling pathway. Taken together, these findings demonstrate that DNA damage and oxidative stress resulting from Nbn gene inactivation are associated with hypomyelination of the nerve fibers of corpus callosum.


Assuntos
Corpo Caloso/patologia , Dano ao DNA/fisiologia , Bainha de Mielina/patologia , Síndrome de Quebra de Nijmegen/patologia , Estresse Oxidativo/fisiologia , Animais , Animais Recém-Nascidos , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , Corpo Caloso/fisiopatologia , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/fisiologia , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/fisiopatologia , Proteínas Nucleares/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Reação em Cadeia da Polimerase em Tempo Real
18.
Hum Mutat ; 35(1): 76-85, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24123394

RESUMO

Ligase IV syndrome is a rare differential diagnosis for Nijmegen breakage syndrome owing to a shared predisposition to lympho-reticular malignancies, significant microcephaly, and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying from malignancy in developmentally normal individuals, to severe combined immunodeficiency and early mortality. Here, we report the identification of biallelic truncating LIG4 mutations in 11 patients with microcephalic primordial dwarfism presenting with restricted prenatal growth and extreme postnatal global growth failure (average OFC -10.1 s.d., height -5.1 s.d.). Subsequently, most patients developed thrombocytopenia and leucopenia later in childhood and many were found to have previously unrecognized immunodeficiency following molecular diagnosis. None have yet developed malignancy, though all patients tested had cellular radiosensitivity. A genotype-phenotype correlation was also noted with position of truncating mutations corresponding to disease severity. This work extends the phenotypic spectrum associated with LIG4 mutations, establishing that extreme growth retardation with microcephaly is a common presentation of bilallelic truncating mutations. Such growth failure is therefore sufficient to consider a diagnosis of LIG4 deficiency and early recognition of such cases is important as bone marrow failure, immunodeficiency, and sometimes malignancy are long term sequelae of this disorder.


Assuntos
DNA Ligases/deficiência , DNA Ligases/genética , Nanismo/genética , Retardo do Crescimento Fetal/genética , Leucopenia/genética , Trombocitopenia/genética , Anormalidades Múltiplas/genética , Imunidade Adaptativa , Adolescente , Linhagem Celular , Criança , Pré-Escolar , DNA Ligase Dependente de ATP , Exoma , Feminino , Retardo do Crescimento Fetal/etiologia , Variação Genética , Genótipo , Heterozigoto , Humanos , Lactente , Masculino , Microcefalia/genética , Neoplasias/genética , Síndrome de Quebra de Nijmegen/genética , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Síndrome
19.
Ital J Pediatr ; 39: 59, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24044622

RESUMO

We report on pediatric patient with Nijmegen breakage syndrome (NBS), a rare DNA repair disorder characterized by microcephaly, immunodeficiency and predisposition to malignant lymphomas, who developed juvenile idiopathic arthritis (JIA)-like polyarthritis. In patients with primary immunodeficiencies (PID), septic arthritis due to pyogenic bacteria or mycoplasmal arthritis are the most common osteoarticular manifestations. In certain PID, chronic, non-infectious arthritis resembling rheumatoid arthritis may occur. In our patient microbiologic cultures of synovial fluid including Mycoplasma spp. were negative. At first, because of suspected mycoplasmal arthritis we used macrolides and doxycycline combined with hydroxychloroquine but without therapeutic response. However, the use of rituximab led to remission of her polyarthritis lasting for 9 months. Autoimmune features were rarely reported in NBS. An occurrence of JIA-like, chronic polyarthritis in NBS, a DNA repair disorder characterized by decreased tolerance of immunosuppressive drugs such as methotrexate and a high natural risk for lymphomas, makes therapeutic approach even more complex.


Assuntos
Artrite/tratamento farmacológico , Artrite/etiologia , Síndrome de Quebra de Nijmegen/complicações , Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos Monoclonais Murinos/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite/diagnóstico por imagem , Artrite/genética , Criança , Doença Crônica , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/fisiopatologia , Radiografia , Doenças Raras , Medição de Risco , Rituximab , Índice de Gravidade de Doença , Resultado do Tratamento
20.
DNA Repair (Amst) ; 12(8): 588-99, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23684796

RESUMO

Ataxia-telangiectasia (A-T) has for a long time stood apart from most other human neurodegenerative syndromes by the characteristic failure of cells derived from these patients to properly repair DNA damage-induced by ionizing radiation. The discovery of mutations in the ATM gene as being the underlying cause for A-T and the demonstration that the ATM protein functions as a DNA damage-responsive kinase has defined current research focusing on decoding how the cell responds to genotoxic stress. Yet, despite significant advances in delineating the cellular DNA damage response pathways coordinated by ATM, very little headway has been made toward understanding how loss of ATM leads to progressive cerebellar ataxia and whether this can be attributed to an underlying defect in DNA double strand break repair (DSBR). Since its identification, A-T has been used as the archetypal model for how a deficiency in DNA repair affects both the development and maintenance of the nervous and immune systems in humans as well as contributing to the process of tumourigenesis. However, following the growing availability and cost effectiveness of next generation sequencing technologies, the increasing recognition of novel human disorders associated with abnormal DNA repair has demonstrated that the neuropathology typified by A-T is an 'exception' rather than the 'rule'. As a consequence, this throws into doubt the longstanding hypothesis that the neurodegeneration seen in A-T is due to the progressive loss of damaged neurons that have acquired toxic levels of unrepaired DNA lesions over time. Therefore, this review aims to address the question: Is defective DNA double strand break repair an underlying cause of neurodegeneration?


Assuntos
Quebras de DNA de Cadeia Dupla , Predisposição Genética para Doença , Sistema Nervoso/patologia , Doenças Neurodegenerativas/genética , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Distúrbios no Reparo do DNA/genética , Distúrbios no Reparo do DNA/patologia , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Humanos , Microcefalia/genética , Microcefalia/patologia , Mutação , Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/citologia , Neurônios/patologia , Síndrome de Quebra de Nijmegen/genética , Síndrome de Quebra de Nijmegen/patologia , Radiação Ionizante
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