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1.
J Clin Invest ; 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33290277

RESUMO

Inborn errors of immunity cause monogenic immune dysregulatory conditions such as severe and recurrent pathogen infection, inflammation, allergy and malignancy. Somatic reversion refers to the spontaneous repair of a pathogenic germline genetic variant and has been reported to occur in a number of inborn errors of immunity with a range of impacts on clinical outcomes of these conditions. DOCK8 deficiency due to bi-allelic inactivating mutations in DOCK8 causes a combined immunodeficiency characterised by severe bacterial, viral and fungal infections, as well as allergic disease and some cancers. Here, we describe the clinical, genetic and cellular features of three patients with bi-allelic DOCK8 variants who, following somatic reversion in multiple lymphocyte subsets, exhibited improved clinical features, including complete resolution of infection and allergic disease, cure over time. Acquisition of DOCK8 expression restored defective lymphocyte signalling, survival and proliferation, as well as CD8+ T cell cytotoxicity, CD4+ T cell cytokine production, and memory B cell generation compared to typical DOCK8-deficient patients. Our temporal analysis of DOCK8-revertant and DOCK8-deficient cells within the same individual established mechanisms of clinical improvement in these patients following somatic reversion and revealed further non-redundant functions of DOCK8 in human lymphocyte biology. Lastly, our findings have significant implications for future therapeutic options for the treatment of DOCK8 deficiency.

2.
Cell Rep ; 33(13): 108559, 2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33378670

RESUMO

The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50E1035Δ) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain. This mutation represents a human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins indicate that RAD50E1035Δ impairs MRE11 nuclease activity. The corresponding mutation in Saccharomyces cerevisiae causes severe thermosensitive defects in both DNA repair and Tel1ATM-dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled coil suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize the importance of the RAD50 coiled coil to regulate MRE11-dependent DNA end resection in humans.

4.
JCI Insight ; 5(5)2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32161190

RESUMO

Cytidine triphosphate (CTP) synthetase 1 (CTPS1) deficiency is caused by a unique homozygous frameshift splice mutation (c.1692-1G>C, p.T566Dfs26X). CTPS1-deficient patients display severe bacterial and viral infections. CTPS1 is responsible for CTP nucleotide de novo production involved in DNA/RNA synthesis. Herein, we characterized in depth lymphocyte defects associated with CTPS1 deficiency. Immune phenotyping performed in 7 patients showed absence or low numbers of mucosal-associated T cells, invariant NKT cells, memory B cells, and NK cells, whereas other subsets were normal. Proliferation and IL-2 secretion by T cells in response to TCR activation were markedly decreased in all patients, while other T cell effector functions were preserved. The CTPS1T566Dfs26X mutant protein was found to be hypomorphic, resulting in 80%-90% reduction of protein expression and CTPS activity in cells of patients. Inactivation of CTPS1 in a T cell leukemia fully abolished cell proliferation. Expression of CTPS1T566Dfs26X failed to restore proliferation of CTPS1-deficient leukemia cells to normal, except when forcing its expression to a level comparable to that of WT CTPS1. This indicates that CTPS1T566Dfs26X retained normal CTPS activity, and thus the loss of function of CTPS1T566Dfs26X is completely attributable to protein instability. This study supports that CTPS1 represents an attractive therapeutic target to selectively inhibit pathological T cell proliferation, including lymphoma.

5.
DNA Repair (Amst) ; 88: 102801, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32032862

RESUMO

High fidelity of genetic transmission in neural stem and progenitor cells (NSPCs) has been long time considered to be crucial for brain development and homeostasis. However, recent studies have identified recurrent DSB clusters in dividing NSPCs, which may underlie the diversity of neuronal cell types. This raised the interest in understanding how NSPCs sense and repair DSBs and how this mechanism could be altered by environmental genotoxic stress caused by pollutants or ionizing radiation. Here, we show that embryonic mouse neural stem and progenitor cells (NSPCs) have significantly higher capacity than mouse embryonic fibroblasts (MEFs) to maintain their chromosome stability in response to acute (γ-radiation) and chronic (tritiated thymidine -3H-T- incorporation into DNA) genotoxic stress. Cells deficient for XLF/Cernunnos, which is involved in non-homologous end joining DNA (NHEJ) repair, highlighted important variations in fidelity of DNA repair pathways between the two cell types. Strikingly, a progressive and generalized chromosome instability was observed in MEFs cultured with 3H-T at long-term, whereas NSPCs cultured in the same conditions, preserved their chromosome stability thanks to higher DNA repair activity further enhanced by an adaptive response and also to the elimination of damaged cells by apoptosis. This specific DNA damage response of NSPCs may rely on the necessity for preservation of their genome stability together with their possible function in creating neuronal genetic diversity.


Assuntos
Instabilidade Cromossômica/genética , Dano ao DNA , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Células-Tronco Neurais/metabolismo , Animais , Reparo do DNA/genética , Camundongos , Fatores de Tempo
6.
J Biol Chem ; 295(8): 2398-2406, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31915249

RESUMO

Repair of DNA double-strand breaks by the nonhomologous end joining pathway is central for proper development of the adaptive immune system. This repair pathway involves eight factors, including XRCC4-like factor (XLF)/Cernunnos and the paralog of XRCC4 and XLF, PAXX nonhomologous end joining factor (PAXX). Xlf-/- and Paxx-/- mice are viable and exhibit only a mild immunophenotype. However, mice lacking both PAXX and XLF are embryonic lethal because postmitotic neurons undergo massive apoptosis in embryos. To decipher the roles of PAXX and XLF in both variable, diversity, and joining recombination and immunoglobulin class switch recombination, here, using Cre/lox-specific deletion to prevent double-KO embryonic lethality, we developed two mouse models of a conditional Xlf KO in a Paxx-/- background. Cre expressed under control of the iVav or CD21 promoter enabled Xlf deletion in early hematopoietic progenitors and splenic mature B cells, respectively. We demonstrate the XLF and PAXX interplay during variable, diversity, and joining recombination in vivo but not during class switch recombination, for which PAXX appeared to be fully dispensable. Xlf/Paxx double KO in hematopoietic progenitors resulted in a shorter lifespan associated with onset of thymic lymphomas, revealing a genome caretaking function of XLF/PAXX.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Linfócitos/metabolismo , Animais , Medula Óssea/metabolismo , Proteínas de Ligação a DNA/deficiência , Switching de Imunoglobulina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sobrevida , Recombinação V(D)J/genética
7.
Hum Mol Genet ; 29(6): 907-922, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31985013

RESUMO

Telomeres are nucleoprotein structures at the end of chromosomes. The telomerase complex, constituted of the catalytic subunit TERT, the RNA matrix hTR and several cofactors, including the H/ACA box ribonucleoproteins Dyskerin, NOP10, GAR1, NAF1 and NHP2, regulates telomere length. In humans, inherited defects in telomere length maintenance are responsible for a wide spectrum of clinical premature aging manifestations including pulmonary fibrosis (PF), dyskeratosis congenita (DC), bone marrow failure and predisposition to cancer. NHP2 mutations have been so far reported only in two patients with DC. Here, we report the first case of Høyeraal-Hreidarsson syndrome, the severe form of DC, caused by biallelic missense mutations in NHP2. Additionally, we identified three unrelated patients with PF carrying NHP2 heterozygous mutations. Strikingly, one of these patients acquired a somatic mutation in the promoter of TERT that likely conferred a selective advantage in a subset of blood cells. Finally, we demonstrate that a functional deficit of human NHP2 affects ribosomal RNA biogenesis. Together, our results broaden the functional consequences and clinical spectrum of NHP2 deficiency.

8.
Trends Cell Biol ; 30(2): 87-96, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31818700

RESUMO

DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the 'price to pay' to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.

9.
Mol Ther Methods Clin Dev ; 15: 232-245, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31720302

RESUMO

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID.

10.
EMBO Mol Med ; 11(7): e10201, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273937

RESUMO

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.


Assuntos
Disceratose Congênita/metabolismo , Exorribonucleases/deficiência , Retardo do Crescimento Fetal/metabolismo , Deficiência Intelectual/metabolismo , Microcefalia/metabolismo , RNA Ribossômico/biossíntese , Homeostase do Telômero , Telômero/metabolismo , Animais , Pré-Escolar , Modelos Animais de Doenças , Disceratose Congênita/genética , Disceratose Congênita/patologia , Exorribonucleases/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/patologia , RNA Ribossômico/genética , Telômero/genética , Telômero/patologia
11.
Blood ; 134(3): 277-290, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31151987

RESUMO

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.


Assuntos
Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Iniciação de Peptídeos/biossíntese , Ribonucleoproteína Nuclear Pequena U5/genética , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/metabolismo , Adolescente , Animais , Células Cultivadas , Análise Mutacional de DNA , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Estudo de Associação Genômica Ampla , Humanos , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Linhagem , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Fenótipo , Conformação Proteica , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Síndrome de Shwachman-Diamond/diagnóstico , Relação Estrutura-Atividade , Sequenciamento Completo do Genoma
12.
Front Immunol ; 10: 443, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30923523

RESUMO

Xlf/Cernunnos is unique among the core factors of the non-homologous end joining (NHEJ) DNA double strand breaks (DSBs) repair pathway, in the sense that it is not essential for V(D)J recombination in vivo and in vitro. Unlike other NHEJ deficient mice showing a SCID phenotype, Xlf -/- mice present a unique immune phenotype with a moderate B- and T-cell lymphopenia, a decreased cellularity in the thymus, and a characteristic TCRα repertoire bias associated with the P53-dependent apoptosis of CD4+CD8+ DP thymocytes. Here, we thoroughly analyzed Xlf -/- mice immune phenotype and showed that it is specifically related to the DP stage but independent of the MHC-driven antigen presentation and T-cell activation during positive selection. Instead, we show that V(D)J recombination is subefficient in Xlf -/- mice in vivo, exemplified by the presence of unrepaired DSBs in the thymus. This results in a moderate developmental delay of both B- and T-lymphocytes at key V(D)J recombination dependent stages. Furthermore, subefficient V(D)J recombination waves are accumulating during TCRα rearrangement, causing the typical TCRα repertoire bias with loss of distal Vα and Jα rearrangements.


Assuntos
Linfócitos B/imunologia , Proteínas de Ligação a DNA/deficiência , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Recombinação V(D)J/imunologia , Animais , Linfócitos B/citologia , Proteínas de Ligação a DNA/imunologia , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
13.
J Allergy Clin Immunol ; 143(1): 325-334.e2, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29906526

RESUMO

BACKGROUND: V(D)J recombination ensures the diversity of the adaptive immune system. Although its complete defect causes severe combined immunodeficiency (ie, T-B- severe combined immunodeficiency), its suboptimal activity is associated with a broad spectrum of immune manifestations, such as late-onset combined immunodeficiency and autoimmunity. The earliest molecular diagnosis of these patients is required to adopt the best therapy strategy, particularly when it involves a myeloablative conditioning regimen for hematopoietic stem cell transplantation. OBJECTIVE: We aimed at developing biomarkers based on analysis of the T-cell receptor (TCR) α repertoire to assist in the diagnosis of patients with primary immunodeficiencies with V(D)J recombination and DNA repair deficiencies. METHODS: We used flow cytometric (fluorescence-activated cell sorting) analysis to quantify TCR-Vα7.2-expressing T lymphocytes in peripheral blood and developed PROMIDISα, a multiplex RT-PCR/next-generation sequencing assay, to evaluate a subset of the TCRα repertoire in T lymphocytes. RESULTS: The combined fluorescence-activated cell sorting and PROMIDISα analyses revealed specific signatures in patients with V(D)J recombination-defective primary immunodeficiencies or ataxia telangiectasia/Nijmegen breakage syndromes. CONCLUSION: Analysis of the TCRα repertoire is particularly appropriate in a prospective way to identify patients with partial immune defects caused by suboptimal V(D)J recombination activity, a DNA repair defect, or both. It also constitutes a valuable tool for the retrospective in vivo functional validation of variants identified through exome or panel sequencing. Its broader implementation might be of interest to assist early diagnosis of patients presenting with hypomorphic DNA repair defects inclined to experience acute toxicity during prehematopoietic stem cell transplantation conditioning.


Assuntos
Síndromes de Imunodeficiência , Receptores de Antígenos de Linfócitos T alfa-beta , Recombinação V(D)J/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Estudos Retrospectivos
14.
Cell Discov ; 4: 61, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30455981

RESUMO

A loss-of-function mutation in tetratricopeptide repeat domain 7A (TTC7A) is a recently identified cause of human intestinal and immune disorders. However, clues to related underlying molecular dysfunctions remain elusive. It is now shown based on the study of TTC7A-deficient and wild-type cells that TTC7A is an essential nuclear protein. It binds to chromatin, preferentially at actively transcribed regions. Its depletion results in broad range of epigenomic changes at proximal and distal transcriptional regulatory elements and in altered control of the transcriptional program. Loss of WT_TTC7A induces general decrease in chromatin compaction, unbalanced cellular distribution of histones, higher nucleosome accessibility to nuclease digestion along with genome instability, and reduced cell viability. Our observations characterize for the first time unreported functions for TTC7A in the nucleus that exert a critical role in chromatin organization and gene regulation to safeguard healthy immune and intestinal status.

15.
PLoS Genet ; 14(7): e1007541, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30059501

RESUMO

DNA replication stress (DRS) leads to the accumulation of stalled DNA replication forks leaving a fraction of genomic loci incompletely replicated, a source of chromosomal rearrangements during their partition in mitosis. MUS81 is known to limit the occurrence of chromosomal instability by processing these unresolved loci during mitosis. Here, we unveil that the endonucleases ARTEMIS and XPF-ERCC1 can also induce stalled DNA replication forks cleavage through non-epistatic pathways all along S and G2 phases of the cell cycle. We also showed that both nucleases are recruited to chromatin to promote replication fork restart. Finally, we found that rapid chromosomal breakage controlled by ARTEMIS and XPF is important to prevent mitotic segregation defects. Collectively, these results reveal that Rapid Replication Fork Breakage (RRFB) mediated by ARTEMIS and XPF in response to DRS contributes to DNA replication efficiency and limit chromosomal instability.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Fase G2/genética , Proteínas Nucleares/metabolismo , Fase S/genética , Linhagem Celular Tumoral , Segregação de Cromossomos/fisiologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Fibroblastos , Instabilidade Genômica/fisiologia , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Proteínas Nucleares/genética , RNA Interferente Pequeno/metabolismo
16.
Oncotarget ; 9(3): 3779-3793, 2018 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-29423082

RESUMO

The Fanconi anemia (FA) pathway is implicated in the repair of DNA interstrand crosslinks (ICL). In this process, it has been shown that FA factors regulate the choice for DNA double strand break repair towards homologous recombination (HR). As this mechanism is impaired in FA deficient cells exposed to crosslinking agents, an inappropriate usage of non-homologous end joining (NHEJ) leads to the accumulation of toxic chromosomal abnormalities. We studied a family with two FANCG patients and found a genetically inherited attenuation of mitomycin C sensitivity resulting in-vitro in an attenuated phenotype for one patient or in increased resistance for two healthy relatives. A heterozygous mutation in ATM was identified in these 3 subjects but was not directly linked to the observed phenotype. However, the attenuation of ICL sensitivity was associated with a reduced recruitment of 53BP1 during the course of ICL repair, and increased HR levels. These results further demonstrate the importance of favoring HR over NHEJ for the survival of cells challenged with ICLs.

18.
Cell Death Differ ; 25(2): 444-452, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29077092

RESUMO

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf-/-PAXX-/- E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCRß gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Síndromes de Imunodeficiência/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Cimentos de Resina/metabolismo
19.
Nat Genet ; 49(10): 1539-1545, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28846100

RESUMO

Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/fisiologia , Osteogênese/genética , Animais , Sequência de Bases , Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Deformidades Congênitas do Pé/genética , Deleção de Genes , Dosagem de Genes , Duplicação Gênica , Técnicas de Inativação de Genes , Genes Reporter , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Camundongos , Camundongos Endogâmicos C57BL , Polidactilia/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Crânio/anormalidades , Transcrição Genética
20.
Hum Mol Genet ; 26(10): 1900-1914, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28369633

RESUMO

NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.


Assuntos
Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Telomerase/genética , Linhagem Celular , Criança , Enzimas Reparadoras do DNA/sangue , Proteínas de Ligação a DNA/sangue , Regulação para Baixo , Expressão Gênica , Humanos , Masculino , Mutação/genética , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero , Encurtamento do Telômero/genética
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