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1.
Mol Cell ; 80(6): 1013-1024.e6, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33338401

RESUMO

Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Anemia de Fanconi/genética , Formaldeído/toxicidade , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Anemia de Fanconi/sangue , Anemia de Fanconi/patologia , Formaldeído/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Instabilidade Genômica/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células K562 , Transcrição Genética
3.
Proc Natl Acad Sci U S A ; 117(30): 17785-17795, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651270

RESUMO

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.


Assuntos
Recombinação Homóloga/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Biologia Computacional , Metilases de Modificação do DNA/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Fator de Necrose Tumoral alfa/metabolismo
4.
BMC Med Genet ; 21(1): 118, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487094

RESUMO

BACKGROUND: Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome with 22 related genes identified. The ALDH2 rs671variant has been proved related to accelerate the progression of BMF in FA patients. The phenotype and genetic basis of Chinese FA patients have not been investigated yet. METHODS: We analyzed the 22 FA-related genes of 63 BMF patients suspected to be FA. Clinical manifestations, morphological and cytogenetic feathers, ALDH2 genotypes, treatment, and outcomes of the definite cases were retrospectively studied. RESULTS: A total of 21 patients were confirmed the diagnosis of FA with the median age of BMF onset was 4-year-old. The number of patients manifested as congenital malformations and growth retardation were 20/21 and 14/21, respectively. BM dysplasia and cytogenetic abnormalities were found in 13/20 and 8/19 patients. All the patients with abnormal karyotypes also manifested as BM dysplasia or had evident blasts. Thirty-five different mutations were identified involving six genes and including twenty novel mutations. FANCA mutations contributed to 66.67% of cases. Eight patients harboring ALDH2-G/A genotype have a significantly younger age of BMF onset (p = 0.025). Within the 19 patients adhering to continuous follow-up, 15 patients underwent hematopoietic stem cell transplantations (HSCTs). During the 29 months of follow-up, 8/19 patients died, seven of which were HSCT-related, and one patient who did not receive HSCT died from severe infection. CONCLUSIONS: The phenotypic and genetic spectrum of Chinese FA patients is broad. Bone marrow dysplasia and cytogenetic abnormalities are prevalent and highly consistent. The overall outcome of HSCTs is disappointing. Nationwide multicenter studies are needed for the rarity and adverse outcome of this disease.


Assuntos
Aldeído-Desidrogenase Mitocondrial/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Estudos de Associação Genética , Genótipo , Mutação , Fenótipo , Adolescente , Medula Óssea/patologia , Criança , Pré-Escolar , Quebra Cromossômica , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Cariótipo , Masculino
5.
Genes Dev ; 34(11-12): 832-846, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32354836

RESUMO

DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2-WRN nuclease-helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.


Assuntos
Proteína BRCA2/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Proteína BRCA2/genética , Linhagem Celular , DNA/química , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Hidroxiureia/farmacologia , Mutação , Domínios Proteicos/genética , Rad51 Recombinase/metabolismo
6.
Mol Cell ; 78(5): 951-959.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32359443

RESUMO

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.


Assuntos
Proteína BRCA1/genética , Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Éxons , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
8.
Nature ; 580(7802): 278-282, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269332

RESUMO

The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions1. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL2; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown1,3. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.


Assuntos
Microscopia Crioeletrônica , DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , DNA/química , Anemia de Fanconi/genética , Humanos , Modelos Moleculares , Conformação Proteica , Ubiquitina/química
9.
Nat Commun ; 11(1): 1120, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111838

RESUMO

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Anemia de Fanconi/enzimologia , Anemia de Fanconi/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genética
10.
Nat Struct Mol Biol ; 27(2): 179-191, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32042152

RESUMO

Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.


Assuntos
Proteína BRCA1/metabolismo , Reparo do DNA , Elementos Nucleotídeos Longos e Dispersos , Fase S , Proteína BRCA1/genética , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Deleção de Genes , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Microscopia
11.
Nucleic Acids Res ; 48(6): 3328-3342, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32002546

RESUMO

Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA-FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/ultraestrutura , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/ultraestrutura , Proteína do Grupo de Complementação G da Anemia de Fanconi/ultraestrutura , Anemia de Fanconi/genética , Animais , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/química , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Ligação Proteica/genética , Conformação Proteica , Xenopus laevis/genética
12.
Mol Cells ; 43(2): 99-106, 2020 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-32024352

RESUMO

Cells are constantly exposed to endogenous and exogenous stresses that can result in DNA damage. In response, they have evolved complex pathways to maintain genomic integrity. RUNX family transcription factors (RUNX1, RUNX2, and RUNX3 in mammals) are master regulators of development and differentiation, and are frequently dysregulated in cancer. A growing body of research also implicates RUNX proteins as regulators of the DNA damage response, often acting in conjunction with the p53 and Fanconi anemia pathways. In this review, we discuss the functional role and mechanisms involved in RUNX factor mediated response to DNA damage and other cellular stresses. We highlight the impact of these new findings on our understanding of cancer predisposition associated with RUNX factor dysregulation and their implications for designing novel approaches to prevent cancer formation in affected individuals.


Assuntos
Subunidades alfa de Fatores de Ligação ao Core/genética , Dano ao DNA/genética , Anemia de Fanconi/genética , Genômica/métodos , Fatores de Transcrição/genética , Anemia de Fanconi/patologia , Humanos
13.
Ann Hematol ; 99(5): 913-924, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32065290

RESUMO

Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1-6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.


Assuntos
Células da Medula Óssea/metabolismo , Anemia de Fanconi , Neoplasias Hematológicas , Células-Tronco Hematopoéticas/metabolismo , Mosaicismo , Células da Medula Óssea/patologia , Anemia de Fanconi/sangue , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos
14.
Nucleic Acids Res ; 48(5): 2442-2456, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31960047

RESUMO

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Proteínas de Choque Térmico/metabolismo , Recombinação Homóloga , Animais , Proteína BRCA2/metabolismo , Linhagem Celular , Dano ao DNA , Anemia de Fanconi/genética , Humanos , Camundongos , Ligação Proteica , Proteólise , Rad51 Recombinase/metabolismo , Xenopus
15.
Cell Rep ; 30(4): 1235-1245.e4, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995761

RESUMO

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Camptotecina/farmacologia , Reparo do DNA/genética , Etoposídeo/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Técnicas Genéticas , Células HeLa , Humanos , Inibidores da Topoisomerase I/farmacologia
16.
PLoS One ; 15(1): e0227603, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31940411

RESUMO

In-vitro studies with different Fanconi anemia (FA) cell lines and FANC gene silenced cell lines indicating involvement of mitochondria function in pathogenesis of FA have been reported. However, in-vivo studies have not been studied so far to understand the role of mitochondrial markers in pathogenesis of FA. We have carried out a systematic set of biomarker studies for elucidating involvement of mitochondrial dysfunction in disease pathogenesis for Indian FA patients. We report changes in the mtDNA number in 59% of FA patients studied, a high frequency of mtDNA variations (37.5% of non-synonymous variations and 62.5% synonymous variations) and downregulation of mtDNA complex-I and complex-III encoding genes of OXPHOS (p<0.05) as strong biomarkers for impairment of mitochondrial functions in FA. Deregulation of expression of mitophagy genes (ATG; p>0.05, Beclin-1; p>0.05, and MAP1-LC3, p<0.05) has also been observed, suggesting inability of FA cells to clear off impaired mitochondria. We hypothesize that accumulation of such impaired mitochondria in FA cells therefore may be the principal cause for bone marrow failure (BMF) and a plausible effect of inefficient clearance of impaired mitochondria in FA.


Assuntos
DNA Mitocondrial/genética , Anemia de Fanconi/genética , Adolescente , Adulto , Proteína 12 Relacionada à Autofagia/genética , Proteína 12 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Criança , DNA Mitocondrial/metabolismo , Anemia de Fanconi/metabolismo , Feminino , Variação Genética/genética , Humanos , Índia , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitofagia , Espécies Reativas de Oxigênio/metabolismo
17.
Congenit Anom (Kyoto) ; 60(1): 32-39, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30809872

RESUMO

Fanconi anemia (FA) is a recessive disorder that predispose to bone marrow failure and multiple congenital anomalies in affected individuals worldwide. To date, 22 FA genes are known to harbor sequence variations in disease phenotype. Among these, mutations in the FANCA gene are associated with 60% to 70% of FA cases. The aim of the present study was to screen FA cases belonging to consanguineous Pakistani families for selected exons of FANCA gene which are known mutational hotspots for Asian populations. Blood samples were collected from 20 FA cases and 20 controls. RNA was extracted and cDNA was synthesized from blood samples of cases. DNA was extracted from blood samples of cases and ethnically matched healthy controls. Sanger's sequencing of the nine selected exons of FANCA gene in FA cases revealed 19 genetic alterations of which 15 were single nucleotide variants, three were insertions and one was microdeletion. Of the total 19 sequence changes, 13 were novel and six were previously reported. All identified variants were evaluated by computational programs including SIFT, PolyPhen-2 and Mutation taster. Seven out of 20 analyzed patients were carrying homozygous novel sequence variations, predicted to be associated with FA. These disease associated novel variants were not detected in ethnically matched controls and depict genetic heterogeneity of disease.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação/genética , Adolescente , Criança , Análise Mutacional de DNA , Anemia de Fanconi/epidemiologia , Anemia de Fanconi/patologia , Feminino , Genótipo , Homozigoto , Humanos , Íntrons/genética , Masculino , Paquistão/epidemiologia
18.
In Vivo ; 34(1): 33-38, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31882460

RESUMO

BACKGROUND/AIM: Radiation mitigator, GS-nitroxide, JP4-039, was evaluated for mitigation of total body irradiation (TBI) in Fanconi anemia (FA) Fancd2-/- (129/Sv), Fancg-/- (B6), and Fanca-/- (129/Sv) mice. MATERIALS AND METHODS: JP4-039 dissolved in 30% 2-hydroxypropyl-ß-cyclodextrin was injected intramuscularly 24 h after total body irradiation (9.25 Gy) into Fanca-/-, Fancd2-/- and Fancg-/- mice. Irradiation survival curves were performed in vitro using bone marrow stromal cell lines derived from Fanca-/-, Fancd2-/- and Fancg-/- mice. RESULTS: FA mice demonstrate genotype specific differences in TBI mitigation by JP4-039. Radiation effects in derived bone marrow stromal cell lines in vitro were mitigated by drugs that block apoptosis, but not necroptosis or ferroptosis. CONCLUSION: FA mouse models are valuable for elucidating DNA repair pathways in cell and tissue responses to TBI, and the role of drugs that target distinct cell death pathways.


Assuntos
Anemia de Fanconi/genética , Óxidos de Nitrogênio/farmacologia , Protetores contra Radiação/farmacologia , Irradiação Corporal Total/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Linhagem Celular , Modelos Animais de Doenças , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Genótipo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/tratamento farmacológico , Lesões Experimentais por Radiação/genética , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética
19.
Cancer Lett ; 472: 1-7, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830560

RESUMO

Fanconi anemia (FA) is a rare genome instability syndrome characterized by progressive bone marrow failure and predisposition to cancer, especially head and neck squamous cell carcinoma. Surgical resection is the standard of care for solid tumors, as patients with FA do not tolerate genotoxic chemotherapies or radiation, leading to poor prognosis. It is therefore imperative to develop chemoprevention strategies such as the identification of novel biomarkers to detect the formation of the tumor before its emergence and to use them in clinical trials aimed to counteract genome instability of patients with FA in tissues at risk. Micronuclei (MN) are chromosome fragments that are left behind in anaphase and appear in daughter cells as small additional nuclei. In this work, we analyzed MN frequencies in exfoliated buccal cells from 40 patients with FA and 24 controls. We found that MN frequency was significantly increased in the FA cohort indicating that we can detect chromosome fragility in patients with FA in basal conditions and in a tissue that is divided in vivo. Consequently, the MN assay in exfoliated buccal cells of patients with FA could be used in cancer risk studies and clinical trials aimed to identify cancer chemopreventive drugs.


Assuntos
Fragilidade Cromossômica/genética , Anemia de Fanconi/genética , Micronúcleos com Defeito Cromossômico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adolescente , Adulto , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Dano ao DNA/genética , Epitélio/metabolismo , Epitélio/patologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Feminino , Instabilidade Genômica/genética , Humanos , Masculino , Testes para Micronúcleos/métodos , Pessoa de Meia-Idade , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto Jovem
20.
J Clin Invest ; 130(3): 1377-1391, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31877112

RESUMO

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.


Assuntos
Transtornos da Insuficiência da Medula Óssea/metabolismo , Dano ao DNA , Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição Associado à Microftalmia/metabolismo , Animais , Ácido Ascórbico , Transtornos da Insuficiência da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea/patologia , Linhagem Celular , Colecalciferol , Desidroepiandrosterona/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Knockout , Fator de Transcrição Associado à Microftalmia/genética , Ácidos Nicotínicos , Extratos Vegetais , Proteínas Smad/genética , Proteínas Smad/metabolismo
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