RESUMO
The accumulation of senescent cells is a major cause of age-related inflammation and predisposes to a variety of age-related diseases1. However, little is known about the molecular basis underlying this accumulation and its potential as a target to ameliorate the ageing process. Here we show that senescent cells heterogeneously express the immune checkpoint protein programmed death-ligand 1 (PD-L1) and that PD-L1+ senescent cells accumulate with age in vivo. PD-L1- cells are sensitive to T cell surveillance, whereas PD-L1+ cells are resistant, even in the presence of senescence-associated secretory phenotypes (SASP). Single-cell analysis of p16+ cells in vivo revealed that PD-L1 expression correlated with higher levels of SASP. Consistent with this, administration of programmed cell death protein 1 (PD-1) antibody to naturally ageing mice or a mouse model with normal livers or induced nonalcoholic steatohepatitis reduces the total number of p16+ cells in vivo as well as the PD-L1+ population in an activated CD8+ T cell-dependent manner, ameliorating various ageing-related phenotypes. These results suggest that the heterogeneous expression of PD-L1 has an important role in the accumulation of senescent cells and inflammation associated with ageing, and the elimination of PD-L1+ senescent cells by immune checkpoint blockade may be a promising strategy for anti-ageing therapy.
Assuntos
Envelhecimento , Antígeno B7-H1 , Fenótipo , Receptor de Morte Celular Programada 1 , Animais , Camundongos , Envelhecimento/imunologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Inflamação/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Análise de Célula Única , Hepatopatia Gordurosa não Alcoólica , Fígado , RejuvenescimentoRESUMO
Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.
Assuntos
COVID-19 , Proteínas Ativadoras de GTPase , Estudo de Associação Genômica Ampla , Fatores de Troca do Nucleotídeo Guanina , Interações entre Hospedeiro e Microrganismos , SARS-CoV-2 , Alelos , Animais , COVID-19/complicações , COVID-19/genética , COVID-19/imunologia , COVID-19/fisiopatologia , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Predisposição Genética para Doença , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Japão , Pulmão/patologia , Macrófagos , Mesocricetus , Pessoa de Meia-Idade , Pneumonia/complicações , Pirazóis/farmacologia , RNA-Seq , SARS-CoV-2/patogenicidade , Carga Viral , Redução de PesoRESUMO
Histiocytic neoplasms (HNs) in adults have been reported to be associated with a high prevalence of coexisting haematological and solid malignancies. While a proportion of coexisting HNs and haematological malignancies share identical genetic alterations, the genetic association between HNs and solid malignancies has scarcely been reported. We report a case of Rosai-Dorfman disease (RDD) complicated by coexisting clear cell sarcoma (CCS). RDD is a rare HN. CCS is an ultrarare soft tissue sarcoma with a poor prognosis. Mutation analysis with whole-exome sequencing revealed six shared somatic alterations including NRAS p.G12S and TP53 c.559+1G>A in both the RDD and CCS tissue. This is the first evidence of a clonal relationship between RDD and solid malignancies using mutational analysis. We hypothesise that neural crest cells, which originate in CCS, are likely the common cells of origin for RDD and CCS. This case helps to unravel the underlying clinicopathological mechanisms of increased association of solid malignancies in HNs. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
RESUMO
A 64-year-old woman presented with fine motor impairment in both hands. MRI revealed a contrast-enhanced lesion in the medulla oblongata. Lymphoid cells with abnormal blebs were observed and a CD4+/CD8+ double positive (DP) T cell population was detected by flow cytometry (FCM) in the bone marrow (BM) and the peripheral blood (PB). CLEC16A::IL2 fusion gene was identified by whole exome sequencing with DNA prepared from DP T cells. Clonal rearrangement of the T-cell receptor gene and expression of TCL1A protein were detected. This led to a diagnosis of T-cell prolymphocytic leukemia (T-PLL) with central nervous system (CNS) infiltration. Abnormal cells in BM and PB became undetectable on microscopy and FCM, and the CNS lesion disappeared on MRI after second-line therapy with alemtuzumab. Meanwhile, the CLEC16A::IL2 fusion mRNA remained detectable in PB. Allogeneic hematopoietic stem-cell transplantation was performed, and the fusion mRNA has now been undetectable for more than 5 years since transplantation. This is the first report of a T-PLL case with a CLEC16A::IL2 fusion gene.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Prolinfocítica de Células T , Feminino , Humanos , Pessoa de Meia-Idade , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/metabolismo , Leucemia Prolinfocítica de Células T/terapia , Interleucina-2/metabolismo , Alemtuzumab , RNA Mensageiro , Proteínas de Transporte de Monossacarídeos , Lectinas Tipo C/genéticaRESUMO
Langerhans cell histiocytosis (LCH) is a rare disease characterized by clonal expansion of CD1a+ CD207+ myeloid dendritic cells. The features of LCH are mainly described in children and remain poorly defined in adults; therefore, we conducted a nationwide survey to collect clinical data from 148 adult patients with LCH. The median age at diagnosis was 46.5 (range: 20-87) years with male predominance (60.8%). Among the 86 patients with detailed treatment information, 40 (46.5%) had single system LCH, whereas 46 (53.5%) had multisystem LCH. Moreover, 19 patients (22.1%) had an additional malignancy. BRAF V600E in plasma cell-free DNA was associated with a low overall survival (OS) rate and the risk of the pituitary gland and central nervous system involvement. At a median follow-up of 55 months from diagnosis, six patients (7.0%) had died, and the four patients with LCH-related death did not respond to initial chemotherapy. The OS probability at 5 years post-diagnosis was 90.6% (95% confidence interval: 79.8-95.8). Multivariate analysis showed that patients aged ≥60 years at diagnosis had a relatively poor prognosis. The probability of event-free survival at 5 years was 52.1% (95% confidence interval: 36.6-65.5), with 57 patients requiring chemotherapy. In this study, we first revealed the high rate of relapse after chemotherapy and mortality of poor responders in adults as well as children. Therefore, prospective therapeutic studies of adults with LCH using targeted therapies are needed to improve outcomes in adults with LCH.
Assuntos
Histiocitose de Células de Langerhans , Neoplasias , Criança , Humanos , Masculino , Adulto , Adulto Jovem , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Feminino , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Histiocitose de Células de Langerhans/diagnóstico , Histiocitose de Células de Langerhans/terapia , Intervalo Livre de Progressão , MutaçãoRESUMO
PURPOSE: Low-grade adenosquamous carcinoma (LGASC) is a rare type of metaplastic carcinoma of the breast (MBC) with an indolent clinical course. A few LGASC cases with high-grade transformation have been reported; however, the genetics underlying malignant progression of LGASC remain unclear. METHODS: We performed whole-genome sequencing analysis on five MBCs from four patients, including one case with matching primary LGASC and a lymph node metastatic tumor consisting of high-grade MBC with a predominant metaplastic squamous cell carcinoma component (MSC) that progressed from LGASC and three cases of independent de novo MSC. RESULTS: Unlike de novo MSC, LGASC and its associated MSC showed no TP53 mutation and tended to contain fewer structural variants than de novo MSC. Both LGASC and its associated MSC harbored the common GNAS c.C2530T:p.Arg844Cys mutation, which was more frequently detected in the cancer cell fraction of MSC. MSC associated with LGASC showed additional pathogenic deletions of multiple tumor-suppressor genes, such as KMT2D and BTG1. Copy number analysis revealed potential 18q loss of heterozygosity in both LGASC and associated MSC. The frequency of SMAD4::DCC fusion due to deletions increased with progression to MSC; however, chimeric proteins were not detected. SMAD4 protein expression was already decreased at the LGASC stage due to unknown mechanisms. CONCLUSION: Not only LGASC but also its associated high-grade MBC may be genetically different from de novo high-grade MBC. Progression from LGASC to high-grade MBC may involve the concentration of driver mutations caused by clonal selection and inactivation of tumor-suppressor genes.
Assuntos
Neoplasias da Mama , Carcinoma Adenoescamoso , Carcinoma , Humanos , Feminino , Carcinoma Adenoescamoso/genética , Carcinoma Adenoescamoso/química , Carcinoma Adenoescamoso/patologia , Neoplasias da Mama/patologia , Mama/patologiaRESUMO
This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.
Assuntos
Herpesvirus Humano 1 , Proteínas de Membrana Transportadoras , Liberação de Vírus , Animais , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares , Proteômica , Células Vero , Proteínas Virais/metabolismoRESUMO
SUMMARY: It is known that some mutant peptides, such as those resulting from missense mutations and frameshift insertions, can bind to the major histocompatibility complex and be presented to antitumor T cells on the surface of a tumor cell. These peptides are termed neoantigen, and it is important to understand this process for cancer immunotherapy. Here, we introduce an R package termed Neoantimon that can predict a list of potential neoantigens from a variety of mutations, which include not only somatic point mutations but insertions, deletions and structural variants. Beyond the existing applications, Neoantimon is capable of attaching and reflecting several additional information, e.g. wild-type binding capability, allele specific RNA expression levels, single nucleotide polymorphism information and combinations of mutations to filter out infeasible peptides as neoantigen. AVAILABILITY AND IMPLEMENTATION: The R package is available at http://github/hase62/Neoantimon.
Assuntos
Antígenos de Neoplasias , Neoplasias , Antígenos de Neoplasias/genética , Humanos , Imunoterapia , Mutação , Neoplasias/genética , Neoplasias/terapia , Linfócitos TRESUMO
Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos/normas , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Programas de Rastreamento , Proteína 1 Homóloga a MutL/ultraestrutura , Proteína 2 Homóloga a MutS/ultraestrutura , Sequenciamento por Nanoporos , Polimorfismo de Nucleotídeo Único/genética , Conformação ProteicaRESUMO
This study was performed to assess the utility of tumor-derived fragmentary DNA, or circulating tumor DNA (ctDNA), for identifying high-risk patients for relapse of acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after undergoing myeloablative allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively collected tumor and available matched serum samples at diagnosis and 1 and 3 months post-alloSCT from 53 patients with AML/MDS. After identifying driver mutations in 51 patients using next-generation sequencing, we designed at least 1 personalized digital polymerase chain reaction assay per case. Diagnostic ctDNA and matched tumor DNA exhibited excellent correlations with variant allele frequencies. Sixteen patients relapsed after a median of 7 months post-alloSCT. Both mutation persistence (MP) in bone marrow (BM) at 1 and 3 months post-alloSCT and corresponding ctDNA persistence (CP) in the matched serum (MP1 and MP3; CP1 and CP3, respectively) were comparably associated with higher 3-year cumulative incidence of relapse (CIR) rates (MP1 vs non-MP1, 72.9% vs 13.8% [P = .0012]; CP1 vs non-CP1, 65.6% vs 9.0% [P = .0002]; MP3 vs non-MP3, 80% vs 11.6% [P = .0002]; CP3 vs non-CP3, 71.4% vs 8.4% [P < .0001]). We subsequently evaluated whether subset analysis of patients with 3 genes associated with clonal hematopoiesis, DNMT3A, TET2, and ASXL1 (DTA), could also be helpful in relapse prediction. As a result, CP based on DTA gene mutations also had the prognostic effect on CIR. These results, for the first time, support the utility of ctDNA as a noninvasive prognostic biomarker in patients with AML/MDS undergoing alloSCT.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/análise , Leucemia Mieloide Aguda/sangue , Síndromes Mielodisplásicas/sangue , Adolescente , Adulto , Idoso , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/terapia , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Adulto JovemRESUMO
Polymerase proofreading-associated polyposis (PPAP) is a disease caused by germline variations in the POLE and POLD1 genes that encode catalytic subunits of DNA polymerases. Studies of cancer genomes have identified somatic mutations in these genes, suggesting the importance of polymerase proofreading of DNA replication in suppressing tumorigenesis. Here, we identified a germline frameshift variation in the POLE gene (c.4191_4192delCT, p.Tyr1398*) in a case with multiple adenomatous polyps and three synchronous colon cancers. Interestingly, one of the colon cancers showed microsatellite instability-high (MSI-H) and another microsatellite stable. Immunohistochemical staining revealed that the MSI-H tumor cells lost the expression of MLH1 protein. Whole genome sequencing of the MSI-H tumor did not find pathogenic somatic mutations in mismatch repair genes but found frameshift mutations in the TET genes that catalyze 5-methylcytosine hydroxylation. Bisulfite sequencing of the tumor corroborated an increase in the number of hypermethylated regions including the MLH1 promoter. These data indicate that PPAP patients might develop MSI-positive tumors through epigenetic silencing of MLH1. These findings will contribute to comprehensive understanding of the molecular basis of tumors that involve deficiency of proofreading activity of DNA polymerases.
Assuntos
Neoplasias do Colo/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Metilação de DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Instabilidade de Microssatélites , Idoso , Alelos , Neoplasias do Colo/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Análise Mutacional de DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Feminino , Mutação da Fase de Leitura , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genótipo , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Masculino , Estadiamento de Neoplasias , Linhagem , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Repressoras/genética , Sequenciamento Completo do GenomaRESUMO
The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.
Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Retículo Endoplasmático/metabolismo , Hexosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptor 5 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Linhagem Celular , Endonucleases/genética , Genes Reporter/genética , Glicosilação , Proteínas de Fluorescência Verde/genética , Camundongos , Mutagênese , NF-kappa B/genética , Transdução de SinaisRESUMO
The prognosis of multiple myeloma (MM) has been improved due to the introduction of novel agents like proteasome inhibitors and immunomodulatory drugs (IMiDs). However, some cases are refractory to the use of novel agents, and the prognosis of such cases is poor. A 53-year-old male was diagnosed with MM and categorized as follows: Bence-Jones protein lambda type MM, Durie-Salmon IIIA, international staging system (ISS) stage II, and revised ISS stage II. Mutations in K-RAS and IGH/FGFR3 translocation were detected at diagnosis. His tumor was refractory to seven therapeutic regimens including bortezomib, IMiDs (lenalidomide, thalidomide, pomalidomide), conventional chemotherapy, and radiation therapy. N-RAS mutations, CKS1B gains, and C-MYC split signals were detected after treatment. We performed high-dose melphalan/autologous stem cell transplantation (HD-MEL/ASCT) as a salvage therapy and achieved very good partial response. The correlation between K-RAS mutations and poor prognosis or between N-RAS mutations and reduced sensitivity to bortezomib is reported. However, RAS mutations are reported as a favorable factor for HD-MEL/ASCT. In general, mutations of both the K-RAS and N-RAS are known to be mutually exclusive. This rare MM case has mutations in both K-RAS and N-RAS, and the possible relevance of these mutations to both the refractoriness to novel therapies and sensitivity to HD-MEL/ASCT is suggested.
Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/genética , Mieloma Múltiplo/terapia , Mutação , Transplante de Células-Tronco de Sangue Periférico , Proteínas Proto-Oncogênicas p21(ras)/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Recidiva , Transplante AutólogoRESUMO
Familial adenomatous polyposis (FAP) of the colon is characterized by multiple polyps in the intestine and extra-colonic manifestations. Most FAP cases are caused by a germline mutation in the tumor-suppressor gene APC, but some cases of adenomatous polyposis result from germline mutations in MUTYH, POLD1 or POLE. Although sequence analysis of APC by the Sanger method is routinely performed for genetic testing, there remain cases whose mutations are not detected by the analysis. Next-generation sequencing has enabled us to analyze the comprehensive human genome, improving the chance of identifying disease causative variants. In this study, we conducted whole-genome sequencing of a sporadic FAP patient in which we did not find any pathogenic APC mutations by the conventional Sanger sequencing. Whole-genome sequencing and subsequent deep sequencing identified a mosaic mutation of c.3175G>T, p.E1059X in ~12% of his peripheral leukocytes. Additional deep sequencing of his buccal mucosa, hair follicles, non-cancerous mucosa of the stomach and colon disclosed that these tissues harbored the APC mutation at different frequencies. Our data implied that genetic analysis by next-generation sequencing is an effective strategy to identify genetic mosaicism in hereditary diseases.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Mosaicismo , Adulto , Sequência de Bases , Análise Mutacional de DNA , Frequência do Gene , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MasculinoRESUMO
OBJECTIVES: Adipose tissue is an endocrine and energy storage organ composed of several different cell types, including mature adipocytes, stromal cells, endothelial cells, and a variety of immune cells. Adipose tissue aging contributes to the pathogenesis of metabolic dysfunction and is likely induced by crosstalk between adipose progenitor cells (APCs) and immune cells, but the underlying molecular mechanisms remain largely unknown. In this study, we revealed the biological role of p16high senescent APCs, and investigated the crosstalk between each cell type in the aged white adipose tissue. METHODS: We performed the single-cell RNA sequencing (scRNA-seq) analysis on the p16high adipose cells sorted from aged p16-CreERT2/Rosa26-LSL-tdTomato mice. We also performed the time serial analysis on the age-dependent bulk RNA-seq datasets of human and mouse white adipose tissues to infer the transcriptome alteration of adipogenic potential within aging. RESULTS: We show that M2 macrophage-derived TGF-ß induces APCs senescence which impairs adipogenesis in vivo. p16high senescent APCs increase with age and show loss of adipogenic potential. The ligand-receptor interaction analysis reveals that M2 macrophages are the donors for TGF-ß and the senescent APCs are the recipients. Indeed, treatment of APCs with TGF-ß1 induces senescent phenotypes through mitochondrial ROS-mediated DNA damage in vitro. TGF-ß1 injection into gonadal white adipose tissue (gWAT) suppresses adipogenic potential and induces fibrotic genes as well as p16 in APCs. A gWAT atrophy is observed in cancer cachexia by APCs senescence, whose induction appeared to be independent of TGF-ß induction. CONCLUSIONS: Our results suggest that M2 macrophage-derived TGF-ß induces age-related lipodystrophy by APCs senescence. The TGF-ß treatment induced DNA damage, mitochondrial ROS, and finally cellular senescence in APCs.
Assuntos
Adipogenia , Senescência Celular , Macrófagos , Células-Tronco , Fator de Crescimento Transformador beta , Animais , Camundongos , Macrófagos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células-Tronco/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Envelhecimento/metabolismo , Masculino , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Crise Blástica , Neoplasias da Medula Óssea/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Dasatinibe/administração & dosagem , Esquema de Medicação , Substituição de Medicamentos , Evolução Fatal , Feminino , Humanos , Mesilato de Imatinib/uso terapêutico , Imidazóis/administração & dosagem , Pessoa de Meia-Idade , Piridazinas/administração & dosagem , Pirimidinas/administração & dosagem , RecidivaAssuntos
Substituição de Aminoácidos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Neutropenia/congênito , Mutação Puntual , Proteína da Síndrome de Wiskott-Aldrich/genética , Sítios de Ligação , Criança , Feminino , GTP Fosfo-Hidrolases/metabolismo , Redes Reguladoras de Genes , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Genótipo , Humanos , Masculino , Neutropenia/diagnóstico , Neutropenia/genética , FenótipoRESUMO
Most of the intracellular endogenous microRNAs (endo-miRNAs) are considered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells, endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, and endo-miRNAs might also compete for the position in the newly synthesized RISC with each other. This would lead to the fluctuation of global gene expression not only by repression of exo-miRNA target gene expression, but also by the increase of the endo-miRNA target gene expression. In the present study, we quantified the changes in the expression levels of target genes of exo-miRNA and endo-miRNA in the cells transfected with fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs increased ratios of expression levels of target genes of a given endo-miRNA to different extents, suggesting that the replacement efficiencies might differ according to the exo-miRNA types. However, the increased ratios in the expression levels of each endo-miRNA target genes by the transfection of any particular exo-miRNA were mostly equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced with excessive exo-miRNAs at similar levels, probably because they exist in single-stranded forms in the RISC.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Regulação para Baixo/genética , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , MicroRNAs/metabolismo , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Inativação Induzido por RNA , Transfecção , Regulação para Cima/genéticaRESUMO
Background: High-grade fetal adenocarcinoma of the lung (H-FLAC) is a rare variant of pulmonary adenocarcinoma. Our previous study showed a high frequency of KMT2C mutations in lung cancers with an H-FLAC component, showing that KMT2C dysfunction may be associated with the biological features of H-FLACs. Methods: In this study, we performed RNA sequencing and immunohistochemical analysis to identify the differentially expressed genes and corresponding pathways associated with H-FLACs, compared with common adenocarcinomas. Results: Ingenuity pathway analysis based on RNA sequencing data revealed that DNA homologous recombination repair (HRR) pathways were significantly inactivated in H-FLAC. Expression of KMT2C, ATM, ATR, and BRCA2 was significantly lower in H-FLACs than in common adenocarcinomas, and BRCA1 expression showed a decreasing trend. Pearson correlation analyses for all cases revealed that KMT2C expression showed a strong positive correlation (R>0.7) with the expression of ATR, BRCA1, and BRCA2 genes and a moderately positive correlation with ATM expression (R=0.47). Immunohistochemical analysis showed significantly lower levels of KMT2C, ATM, ATR, and BRCA2 expression in H-FLACs than in common adenocarcinomas, and a trend of lower BRCA1 levels. Additionally, KMT2C expression showed a weak to moderate correlation with that of ATM, ATR, BRCA1, and BRCA2. Conclusions: Cancers containing H-FLAC components showed lower levels of KMT2C and HRR factors than common lung adenocarcinomas, and their levels exhibited a positive correlation. These results support the hypothesis that loss of KMT2C function decreases the expression of the HRR factors in H-FLACs. H-FLACs with low KMT2C expression may be a good indication for poly (ADP-ribose) polymerase (PARP) inhibitor-based therapy.
RESUMO
Myxofibrosarcoma (MFS) and undifferentiated sarcoma (US) have been considered as tumors of the same lineage based on genetic/epigenetic profiling. Although MFS shows a notably better prognosis than US, there are no clear criteria for distinguishing between them. Here, we examined 85 patients with MFS/US and found that tumors with infiltrative growth patterns tended to have more myxoid areas and higher local recurrence rates but fewer distant metastases and better overall survival. Morphologically characteristic sickle-shaped blood vessels, which tended to have fewer αSMA-positive cells, were also observed in these tumors, compared with normal vessels. Based on the incidence of these sickle-shaped blood vessels, we subdivided conventionally diagnosed US into two groups. This stratification was significantly correlated with metastasis and prognosis. RNA sequencing of 24 tumors (9 MFS and 15 US tumors) demonstrated that the proteasome, NF-kB, and VEGF pathways were differentially regulated among these tumors. Expression levels of KDR and NFATC4, which encode a transcription factor responsible for the neuritin-insulin receptor angiogenic signaling, were elevated in the sickle-shaped blood vessel-rich US tumors. These findings indicate that further analyses may help elucidate the malignant potential of MFS/US tumors as well as the development of therapeutic strategies for such tumors.