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1.
Nat Commun ; 11(1): 3883, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753598

RESUMO

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Regiões Promotoras Genéticas/genética , RNA-Seq , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
2.
Nucleic Acids Res ; 48(12): 6672-6684, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32504494

RESUMO

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Enzimas Reparadoras do DNA/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Apraxias/genética , Apraxias/patologia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Reparo do DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Microcefalia/genética , Microcefalia/patologia , Mutação/genética , Convulsões/genética , Convulsões/patologia
3.
PLoS One ; 15(6): e0233900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520976

RESUMO

OBJECTIVES: To identify differences in the mutational profile of endometrial tumours between British White (BW) and South Asian (BSA) women. METHODS: We analysed primary tumours from matched cohorts of British White (BW) and British South Asian (BSA) women resident in Leicestershire diagnosed with EC. Next Generation Sequencing was performed to investigate mutational differences in a panel of 10 genes previously identified as being commonly mutated in EC. The presence of somatic Mismatch Repair (MMR) gene deficiencies was determined by immunohistochemistry. RESULTS: In total, 57 tumours (27 BSA and 30 BW) were sequenced. There was no significant difference in the overall mutation frequency of the 10 genes analysed; however, numerous differences were observed between the groups. There was a positive association between PIK3CA and PTEN mutations in the BSA group, with 78% of PIK3CA-mutant tumours harbouring a PTEN mutation, whereas only 11% of PIK3CA wild-type (wt) tumours were PTEN mutant positive (p = 0.0012). In BW women, 90% of ARID1A mutant tumours had co-existent PI3K pathway mutations versus 50% of wild-type (wt) ARID1A patients (p = 0.0485). This trend was not significant in the BSA group (p = 0.66). The age at diagnosis was significantly higher in the BW group with a somatic MMR gene deficiency compared to those with no deficiency (72.8 years versus 59.6 years, p = 0.007), whereas this difference was not seen in the BSA group (64 years versus 60 years, p = 0.37). CONCLUSION: We have identified differences in the mutational profile of primary EC tumours from BW and BSA women. Further research is needed to confirm these findings and to explore their potential implications for early detection, treatment response and prognosis.


Assuntos
Carcinoma Endometrioide/genética , Enzimas Reparadoras do DNA/genética , DNA de Neoplasias/genética , Neoplasias do Endométrio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Estudos de Coortes , Análise Mutacional de DNA/métodos , Grupo com Ancestrais do Continente Europeu , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Mutação , Reino Unido
4.
Nat Commun ; 11(1): 3088, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555206

RESUMO

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.


Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Brain Tumor Pathol ; 37(2): 50-59, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32361941

RESUMO

Aging is a known negative prognostic factor in glioblastomas (GBM). Whether particular genetic backgrounds are a factor in poor outcomes of elderly patients with GBM warrants investigation. We aim to elucidate any differences between older and younger adult patients with IDH-wildtype GBM regarding both molecular characteristics and clinical outcomes. We collected adult cases diagnosed with IDH-wildtype GBM from the Kansai Network. Clinical and pathological characteristics were analyzed retrospectively and compared between older (≥ 70 years) and younger (≤ 50 years) cases. Included were 92 older vs. 33 younger cases. The older group included more patients with preoperative Karnofsky performance status score < 70 and had a shorter survival time than the younger group. MGMT promoter was methylated more frequently in the older group. TERT promoter mutation was more common in the older group. There were significant differences in DNA copy-number alteration profiles between age groups in PTEN deletion and CDK4 amplification/gain. In the older group, no molecular markers were identified, but surgical resection was an independent prognostic factor. Age-specific survival difference was significant in the MGMT methylated and TERT wildtype subgroup. Elderly patients have several potential factors in poor prognosis of glioblastomas. Varying molecular profiles may explain differing rates of survival between generations.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Isocitrato Desidrogenase/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Estudos de Coortes , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Glioblastoma/mortalidade , Humanos , Japão , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
J Clin Neurosci ; 75: 45-51, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32229069

RESUMO

Due to the varied overall survival (OS), limited studies focus on the factors that affect the prognosis for lower grade glioma patients (LGGs) with MGMT promoter methylated. A total of 579 samples (TCGA LGGs 456; CGGA LGGs 123) were included to identify potential genes for LGGs with MGMT promoter methylated. All bioinformatics analyses were conducted using SPSS software and GraphPad Prism 6. Based on COX regression analysis, we established a four-gene signature (ALDOC, APOBEC3C, ANXA1 and ARPP21) and divided LGGs into two groups based on median risk score. The OS of LGGs in high risk group was shorter than low risk group (P < 0.0001). Furthermore, the OS in high risk group were shorter than low risk group in Grade II and III, respectively (P = 0.0003; P = 0.0104). It showed that the signature was an independent prognosis factor on multivariate Cox regression analysis (P = 0.033). Patients in high group tended to displayed high grade (GIII), IDH1 wild type and mesenchymal subtype preference. Four-gene signature was discovered for LGGs with MGMT promoter methylated. Our findings suggested that the four genes could serve as prognostic biomarkers.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , RNA Mensageiro/genética , Transcriptoma/genética , Adulto , Neoplasias Encefálicas/mortalidade , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Glioma/mortalidade , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Fatores de Risco , Proteínas Supressoras de Tumor/genética
7.
Nucleic Acids Res ; 48(9): 4960-4975, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232411

RESUMO

G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.


Assuntos
Enzimas Reparadoras do DNA/fisiologia , Replicação do DNA , Exodesoxirribonucleases/fisiologia , Quadruplex G , Telômero/química , Aminoquinolinas/farmacologia , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Quadruplex G/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidade , Ácidos Picolínicos/farmacologia , Prognóstico
8.
Am J Surg Pathol ; 44(5): 649-656, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32294063

RESUMO

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Neoplasias Ovarianas/genética , Fatores Etários , Biomarcadores Tumorais/deficiência , Carcinoma/mortalidade , Carcinoma/patologia , Carcinoma/terapia , Neoplasias Colorretais Hereditárias sem Polipose/mortalidade , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Neoplasias Colorretais Hereditárias sem Polipose/terapia , Metilação de DNA , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/deficiência , Progressão da Doença , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/patologia , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Fenótipo , Intervalo Livre de Progressão , Estudos Prospectivos , Sistema de Registros , Fatores de Risco , Espanha , Fatores de Tempo
9.
PLoS One ; 15(4): e0231932, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324779

RESUMO

BACKGROUND: Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) DNA repair enzyme via promoter hypermethylation (hmMGMT) may increase mutations in the TP53 oncosuppressor gene and contribute to carcinogenesis. The effects of smoking, which is a risk factor for head and neck squamous cell carcinoma (HNSCC), were investigated to determine whether they up- or down-regulate hmMGMT. Additionally, the impact of hmMGMT and disruptive TP53-mutations on relapse was investigated in patients with HNSCC. METHODS: This study included 164 patients with HNSCC who were negative for both p16 protein expression and human papilloma virus infection. The association of smoking and hmMGMT was investigated using multiple logistic regression analysis. Competing risk regression was used to evaluate the effects of hmMGMT and TP53-mutations in exon 2 to 11 on relapse of HNSCC. RESULTS: hmMGMT was observed in 84% of the 164 patients. TP53-mutations, specifically, G:C>A:T transition, were more frequent in patients with hmMGMT (32%) than in those without hmMGMT (8%). The frequency of disruptive TP53-mutations was not significantly different between groups. Compared with nonsmoking, heavy smoking of 20 pack-years or more was significantly associated with decreased hmMGMT (adjusted odds ratio, 0.08; 95% CI, 0.01 to 0.56; P = 0.01). Patients who had both hmMGMT and disruptive TP53-mutations showed a significantly higher relapse rate than all other patients (subdistribution hazard ratio, 1.77; 95% CI, 1.07 to 2.92; P = 0.026). CONCLUSIONS: It was found that hmMGMT was suppressed by heavy smoking, and hmMGMT combined with disruptive TP53-mutations may indicate a poor prognosis in patients with HNSCC.


Assuntos
Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Mutação , Fumar , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Recidiva
10.
PLoS Genet ; 16(3): e1008422, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32187176

RESUMO

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinogênese/genética , Saccharomyces cerevisiae/genética , Animais , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Genômica/métodos , Proteína Homóloga a MRE11/genética , Mutação/genética , Células Sf9 , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética
11.
World Neurosurg ; 137: e213-e220, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32001415

RESUMO

BACKGROUND: Patients of lower socioeconomic status (SES) may experience barriers to their oncologic care, but current data conflict over whether SES affects the prognosis of patients with glioblastoma (GB). OBJECTIVE: We sought to determine whether SES disparities impaired delivery of neuro-oncologic care and affected the prognosis of GB patients. METHODS: The records of GB patients treated from 2010 to 2014 at a safety-net hospital (SNH) or private hospital (PH), both served by 1 academic medical institution, were retrospectively reviewed and compared. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. RESULTS: A total of 55 SNH and 39 PH GB patients were analyzed with median 11-month follow-up. SNH patients were predominantly Hispanic, low income, enrolled in Medicaid, were less likely to receive radiation (89% vs. 100%), took longer to start radiation (41 vs. 29 days), and were less likely to complete radiation treatment (80% vs. 95%). Concurrent and adjuvant temozolomide use were also lower (85% vs. 94% and 60% vs. 71%, respectively). OS and PFS were not significantly different (15 vs. 16 months and 8 vs. 11 months, respectively). On multivariate analysis, adjuvant chemotherapy and RT completion predicted for better OS, whereas hospital type, income, and insurance did not. CONCLUSION: Although GB patients at our SNH received less adjuvant treatment compared with PH, outcomes were similar. Access to multidisciplinary care staffed by academic physicians may play an important role in overcoming socioeconomic barriers to treatment availability and quality at SNHs.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Disparidades em Assistência à Saúde/estatística & dados numéricos , Hospitais Privados , Procedimentos Neurocirúrgicos , Provedores de Redes de Segurança , Temozolomida/uso terapêutico , Tempo para o Tratamento/estatística & dados numéricos , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Quimiorradioterapia Adjuvante/estatística & dados numéricos , Quimioterapia Adjuvante/estatística & dados numéricos , Estudos de Coortes , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Grupos Étnicos/estatística & dados numéricos , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Masculino , Medicaid , Pessoa de Meia-Idade , Pobreza/estatística & dados numéricos , Intervalo Livre de Progressão , Radioterapia Adjuvante/estatística & dados numéricos , Estudos Retrospectivos , Classe Social , Padrão de Cuidado , Taxa de Sobrevida , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Estados Unidos
12.
Doc Ophthalmol ; 141(1): 89-97, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32048102

RESUMO

BACKGROUND: Cockayne syndrome is a rare autosomal recessive neurodegenerative disorder caused by mutations of either the ERCC6/CSB or ERCC8/CSA genes. Here, we describe two sisters with Cockayne syndrome caused by compound heterozygous mutations in the ERCC8 gene using multimodal imaging. Significant ophthalmic and systemic phenotypic variability is discussed. MATERIALS AND METHODS: Multimodal imaging was performed in two affected sisters and included electroretinography, optical coherence tomography, ultra-wide-field confocal scanning laser ophthalmoscopy, fundus autofluorescence and fluorescein angiography, and magnetic resonance imaging. Genetic analyses were performed on the affected sisters, both parents, and three unaffected siblings. RESULTS: The older sister (Patient 1) had mental retardation, bilateral hearing loss, ataxia, and decreased visual acuity with retinal dystrophy. Radiographic studies revealed microcephaly, cerebral and cerebellar atrophy, ventriculomegaly, and a diffusely thickened skull. Full-field electroretinography waveforms were severely diminished with attenuation of cone and rod responses. The younger sister (Patient 2) had similar clinical features, including ataxia, bilateral hearing loss, and decreased visual acuity with retinal dystrophy. She also had paranoid schizophrenia. Wide-field fundus autofluorescence showed scattered areas of retinal pigment epithelium atrophy, which was different from her sister. Genetic analysis revealed two mutations in the ERCC8 gene shared by the sisters. These include an unreported missense point mutation: p.Thr328Ser:c.983C > G, and another previously reported pathogenic missense mutation: p.Ala205Pro:c.613G > C. Familial testing showed in trans segregation of these mutations with unaffected siblings inheriting one or neither mutation, but not both. CONCLUSION: The clinical presentation and genetic studies confirmed a diagnosis of Cockayne syndrome in both sisters caused by compound heterozygous mutations in the ERCC8 gene on chromosome 10. Multimodal ocular imaging and systemic findings revealed wide phenotypic variability between the affected siblings.


Assuntos
Síndrome de Cockayne/genética , Enzimas Reparadoras do DNA/genética , Mutação de Sentido Incorreto/genética , Fatores de Transcrição/genética , Adulto , Variação Biológica da População , Síndrome de Cockayne/diagnóstico , Eletrorretinografia , Feminino , Angiofluoresceinografia , Humanos , Imagem por Ressonância Magnética , Imagem Multimodal , Células Fotorreceptoras Retinianas Cones/patologia , Distrofias Retinianas/genética , Irmãos , Tomografia de Coerência Óptica
13.
BMC Cancer ; 20(1): 79, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005184

RESUMO

BACKGROUND: Gliomas consist of a heterogeneous group of tumors. This study aimed to report the incidences of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, 1p19q co-deletion, isocitrate dehydrogenase (IDH) gene mutations, and inactivating mutations of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) in high-grade gliomas in an ethnically diverse population. METHODS: Records of patients who underwent surgery for high-grade gliomas from January 2013 to March 2017 at our institution were obtained. The patients' age, gender, ethnicity, Karnofsky Performance Scale (KPS) score, ability to perform activities of daily living (ADLs), tumor location and biomarkers status were recorded. Data were analyzed using chi-square and Mann-Whitney U tests, Kaplan-Meier estimates and log-rank test. RESULTS: 181 patients were selected (56 with grade III gliomas, 125 with grade IV gliomas). In the grade III group, 55% had MGMT promoter methylation, 41% had 1p19q co-deletion, 35% had IDH1 mutation and none had ATRX loss. In the grade IV group, 30% had MGMT promoter methylation, 2% had 1p19q co-deletion, 15% had IDH1 mutation and 8% had ATRX loss. After adjusting for effects of age, surgery and pre-operative ADL statuses, only MGMT promoter methylation was found to be significantly associated with longer overall survival time in grade III (p = 0.024) and IV patients (p = 0.006). CONCLUSIONS: The incidences of MGMT promoter methylation and IDH1 mutation were found to be comparable to globally reported rates, but those of 1p19q co-deletion and ATRX loss seemed to be lower in our cohort. MGMT promoter methylation was associated with increased overall survival in our cohort and might serve as favorable prognostic factor.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioma/cirurgia , Isocitrato Desidrogenase/genética , Proteínas Supressoras de Tumor/genética , Proteína Nuclear Ligada ao X/genética , Atividades Cotidianas , Adulto , Ásia Sudeste/etnologia , Cromossomos Humanos Par 1/genética , Epigênese Genética , Feminino , Glioma/genética , Glioma/mortalidade , Glioma/patologia , Humanos , Incidência , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Prognóstico , Regiões Promotoras Genéticas , Estudos Retrospectivos , Deleção de Sequência , Análise de Sobrevida
14.
Nat Commun ; 11(1): 857, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051414

RESUMO

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.


Assuntos
Endodesoxirribonucleases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Espermatócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Feminino , Histona-Lisina N-Metiltransferase/genética , Proteína Homóloga a MRE11/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
15.
Gynecol Oncol ; 157(1): 245-251, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31980219

RESUMO

OBJECTIVES: To apply the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to a consecutive series of endometrial cancer (EC) patients diagnosed at a tertiary referral center and assign EC specimens to one of four molecular subgroups using immunohistochemistry (IHC) for p53/mismatch repair protein expression and sequencing for Polymerase Epsilon Exonuclease Domain Mutations (POLE-EDM). Mismatch Repair Deficient (MMR-D) cases were more thoroughly investigated to identify underlying somatic or germline genetic defects. METHODS: Hundred-and eight consecutive endometrial cancer patients, diagnosed between March 2017 and April 2019, were subjected to immunohistochemical and molecular analysis, according to ProMisE. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM by Sanger sequencing. In addition, tumor and corresponding normal tissue of cases with abnormal MMR IHC were tested by PCR for microsatellite instability (MSI) (MSI analysis system, Promega). Hypermethylation of MLH1 promotor was tested with (methylation specific) multiplex ligation dependent probe amplification. MMR-D cases were subjected to germline mutation analysis of the mismatch repair genes, using next generation sequencing on MiSeq (Illumina) with the BRCA Hereditary Cancer MASTR Plus, (Multiplicom/Agilent), RNA mutation analysis and MLPA. RESULTS: FIGO classification was stage IA (n = 54), IB (n = 22) II(n = 8), III(n = 18) and IV(n = 6). Of the 33 patients with MMR-D on IHC (31%), 26 showed MLH1 promotor hypermethylation as the probable cause of MMR-D. The remaining 7 patients without MLH1 promotor hypermethylation were referred for germline analysis of Lynch syndrome. Six patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n = 3), PMS2(n = 1), MLH1(n = 1) and MSH2 (n = 1). Pathogenic POLE-EDM were identified in 7 (6%) patients. Multiple molecular features (POLE-EDM + MMR-D or POLE-EDM + p53 abnormal) were observed in 4 patients (4%). A high concordance between MMR-D and microsatellite instability was observed in our cohort. In cases of a genetic defect in the MMR genes, we do note a large proportion of cases exhibiting microsatellite instability. On the contrary a hypermutation state, as seen in POLE EDM, does not result in accompanied phenotypic changes in MSI status. CONCLUSION: The ProMisE classification proved to be an efficient and easily implementable system. Future research should elucidate the precise biological and prognostic meaning of the cases with multiple molecular markers.


Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Neoplasias do Endométrio/classificação , Idoso , Idoso de 80 Anos ou mais , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Estadiamento de Neoplasias , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteína Supressora de Tumor p53/genética
16.
Nat Commun ; 11(1): 236, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932649

RESUMO

Alternative DNA structure-forming sequences can stimulate mutagenesis and are enriched at mutation hotspots in human cancer genomes, implicating them in disease etiology. However, the mechanisms involved are not well characterized. Here, we discover that Z-DNA is mutagenic in yeast as well as human cells, and that the nucleotide excision repair complex, Rad10-Rad1(ERCC1-XPF), and the mismatch repair complex, Msh2-Msh3, are required for Z-DNA-induced genetic instability in yeast and human cells. Both ERCC1-XPF and MSH2-MSH3 bind to Z-DNA-forming sequences, though ERCC1-XPF recruitment to Z-DNA is dependent on MSH2-MSH3. Moreover, ERCC1-XPF-dependent DNA strand-breaks occur near the Z-DNA-forming region in human cell extracts, and we model these interactions at the sub-molecular level. We propose a relationship in which these complexes recognize and process Z-DNA in eukaryotes, representing a mechanism of Z-DNA-induced genomic instability.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/química , Instabilidade Genômica , Linhagem Celular , Simulação por Computador , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Genéticos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética
17.
Gynecol Oncol ; 156(3): 669-675, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924330

RESUMO

OBJECTIVE: Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. METHODS: N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. RESULTS: MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. CONCLUSIONS: Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.


Assuntos
Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/deficiência , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Endometrioide/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/deficiência , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/deficiência , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/deficiência , Proteína 2 Homóloga a MutS/genética , Análise Serial de Tecidos , Adulto Jovem
18.
Neurology ; 94(5): e529-e537, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31831598

RESUMO

OBJECTIVE: To determine the role of olfactory function in patients with glioblastoma multiforme (GBM) as a prognostic clinical measure. METHODS: In a prospective case-control study, olfactory testing was performed in 73 patients with primary GBM at baseline during first-line treatment and at later follow-ups. An age-matched control cohort consisted of 49 patients with neurologic diseases, excluding those known to affect olfactory function per se. Depending on the olfactory testing score, patients were allotted to a hyposmia group (HG) or normosmia group (NG). MRI analysis was performed to assess whether tumor location affects olfactory pathways. RESULTS: Patients with GBM had olfactory dysfunction significantly more often compared to the control cohort (p = 0.003). Tumor location could not explain this finding since no relevant difference in MRI-based olfactory pathway involvement was found between HG and NG (p = 0.131). Patients with olfactory dysfunction had significantly worse overall survival (OS) and progression-free survival (PFS) compared to those without dysfunction (median OS 20.9 vs 40.6 months, p = 0.035; median PFS, 9 vs 19 months, p = 0.022). Multivariate analysis in patients without MRI-based involvement of olfactory pathways confirmed olfaction is an independent prognostic factor for OS (hazard ratio [HR] 0.43; p = 0.042) and PFS (HR 0.51; p = 0.049). CONCLUSION: This pilot study provides the first indication that olfactory dysfunction is frequently observed in GBM and may be associated with worse survival outcome in GBM. However, validation of these results in an independent cohort is needed.


Assuntos
Neoplasias Encefálicas/mortalidade , Glioblastoma/mortalidade , Transtornos do Olfato/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/epidemiologia , Neoplasias Encefálicas/terapia , Estudos de Casos e Controles , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/epidemiologia , Glioblastoma/terapia , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Projetos Piloto , Prognóstico , Intervalo Livre de Progressão , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Estudos Prospectivos , Radioterapia , Olfato , Taxa de Sobrevida , Proteínas Supressoras de Tumor/genética , Adulto Jovem
19.
J Clin Pathol ; 73(2): 112-115, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31422371

RESUMO

AIMS: O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation is a high predictive factor for therapy results of temozolomide in patients with glioma. The objective of this work was to analyse the impact of MGMT promoter methylation in patients with primary diagnosed glioblastoma (GBM) relating to survival using a quantitative method (methylation quantification of endonuclease-resistant DNA, MethyQESD) by verifying a cut-off point for MGMT methylation provided by the literature (

Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioblastoma/genética , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/mortalidade , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Temozolomida/uso terapêutico , Fatores de Tempo
20.
Nucleic Acids Res ; 48(1): 231-248, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31722399

RESUMO

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição/genética , Transcrição Genética , Ubiquitina/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Raios Ultravioleta
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