RESUMO
Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.
Assuntos
Neoplasias Encefálicas , Glioma Pontino Intrínseco Difuso , Glioma , Humanos , Criança , Histonas/genética , Adenosina Trifosfatases/metabolismo , Lisina/genética , Cromatina , Glioma/genética , Neoplasias Encefálicas/genética , Mutação , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Since the 1990s, the sheer number of defined central nervous system (CNS) embryonal tumor entities has continuously increased, with the trend accelerating in the most recent editions of the World Health Organization (WHO) Classification of Tumours of the CNS. The introduction of increasingly specific tumor groups is an effort to create more internally homogeneous categories, to allow more precise prognostication, and potentially to develop targeted therapies. However, these ever-smaller categories within an already rare group of tumors pose a challenge for pediatric pathologists. In this article we review the current categorization of non-medulloblastoma CNS embryonal tumors (including atypical teratoid/rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, FOXR2-activated, and CNS tumor with BCOR internal tandem duplication) and provide an overview of available ancillary techniques to characterize these tumors. We provide a practical approach to workup and development of an integrated diagnosis for CNS embryonal tumors.
Assuntos
Neoplasias do Sistema Nervoso Central , Neoplasias Cerebelares , Meduloblastoma , Neoplasias Embrionárias de Células Germinativas , Sistema Nervoso Central , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Fatores de Transcrição Forkhead , Humanos , Meduloblastoma/diagnóstico , Meduloblastoma/terapia , Neoplasias Embrionárias de Células Germinativas/diagnósticoRESUMO
Tumor classification in neuropathology is a dynamic and complex topic, with many changes emerging in the past 5 years, up to and including the 2021 publication of the 5th edition of the World Health Organization Classification of Tumours of the Central Nervous System (CNS). For pediatric pathologists who will encounter brain tumors with varying frequency, it is important to understand the principles of these classification updates, particularly the inclusion of molecular genetic features and development of a layered, or integrated, diagnosis. This issue of Perspectives in Pediatric Pathology is dedicated to the examination of pediatric brain tumors, and features articles on intraoperative diagnosis and updated information on molecular-based classification for pediatric glial, glioneuronal, ependymal, and embryonal tumors of the CNS.
Assuntos
Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Neoplasias Embrionárias de Células Germinativas , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Sistema Nervoso Central , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Humanos , Organização Mundial da SaúdeRESUMO
Central nervous system (CNS) tumors are now the most common type of solid tumor in individuals aged 0-19 years, with an incidence rate in the United States around 5 per 100,000, accounting for about 1 out of 4 childhood cancers. Pediatric pathologists encounter brain tumor cases with varying frequency, but many of these encounters begin in the context of intraoperative consultation or "frozen section." This review provides an overview of the technical aspects of intraoperative consultation specific to, or more helpful in, CNS tumors, emphasizing helpful cytologic and histologic features of the more commonly encountered pediatric CNS tumors, and illustrating some common diagnostic pitfalls and how these may be avoided.
Assuntos
Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Adolescente , Adulto , Neoplasias Encefálicas/diagnóstico , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Pré-Escolar , Secções Congeladas , Humanos , Lactente , Recém-Nascido , Encaminhamento e Consulta , Adulto JovemRESUMO
Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed, and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.
Assuntos
Apoptose/genética , Asparagina/metabolismo , Aspartato-Amônia Ligase/antagonistas & inibidores , Citrato (si)-Sintase/genética , Glutamina/deficiência , Fator 4 Ativador da Transcrição/metabolismo , Asparagina/biossíntese , Asparagina/química , Aspartato-Amônia Ligase/biossíntese , Ácido Aspártico/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ciclo do Ácido Cítrico , Humanos , Ácido Oxaloacético/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Atypical teratoid rhabdoid tumor (ATRT) is a fatal pediatric malignancy of the central neural system lacking effective treatment options. It belongs to the rhabdoid tumor family and is usually caused by biallelic inactivation of SMARCB1, encoding a key subunit of SWI/SNF chromatin remodeling complexes. Previous studies proposed that SMARCB1 loss drives rhabdoid tumor by promoting cell cycle through activating transcription of cyclin D1 while suppressing p16. However, low cyclin D1 protein expression is observed in most ATRT patient tumors. The underlying mechanism and therapeutic implication of this molecular trait remain unknown. Here, we show that SMARCB1 loss in ATRT leads to the reduction of cyclin D1 expression by upregulating MIR17HG, a microRNA (miRNA) cluster known to generate multiple miRNAs targeting CCND1. Furthermore, we find that this cyclin D1 deficiency in ATRT results in marked in vitro and in vivo sensitivity to the CDK4/6 inhibitor palbociclib as a single agent. Our study identifies a novel genetic interaction between SMARCB1 and MIR17HG in regulating cyclin D1 in ATRT and suggests a rationale to treat ATRT patients with FDA-approved CDK4/6 inhibitors. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Proteínas/genética , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Teratoma/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclina D1/metabolismo , Humanos , Proteínas/metabolismo , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patologia , Proteína SMARCB1/metabolismo , Teratoma/metabolismo , Teratoma/patologia , Regulação para CimaRESUMO
BACKGROUND: The contribution of neonatal cyanosis, inherent to cyanotic congenital heart disease, to the magnitude of neurologic injury during deep hypothermic circulatory arrest has not been fully delineated. This study investigates the impact of cyanosis and deep hypothermic circulatory arrest on brain injury. METHODS: Neonatal piglets were randomised to placement of a pulmonary artery to left atrium shunt to create cyanosis or sham thoracotomy. At day 7, animals were randomised to undergo deep hypothermic circulatory arrest or sham. Arterial oxygen tension and haematocrit were obtained. Neurobehavioural performance was serially assessed. The animals were sacrificed on day 14. Brain tissue was assessed for neuronal necrosis using a 5-point histopathologic score. RESULTS: Four experimental groups were analysed (sham, n = 10; sham + deep hypothermic circulatory arrest, n = 8; shunt, n = 9; shunt + deep hypothermic circulatory arrest, n = 7). Cyanotic piglets had significantly higher haematocrit and lower partial pressure of oxygen at day 14 than non-cyanotic piglets. There were no statistically significant differences in neurobehavioural scores at day 1. However, shunt + deep hypothermic circulatory arrest piglets had evidence of greater neuronal injury than sham animals (median (range): 2 (0-4) versus 0 (0-0), p = 0.02). DISCUSSION: Cyanotic piglets undergoing deep hypothermic circulatory arrest had increased neuronal injury compared to sham animals. Significant injury was not seen for either cyanosis or deep hypothermic circulatory arrest alone relative to shams. These findings suggest an interaction between cyanosis and deep hypothermic circulatory arrest and may partially explain the suboptimal neurologic outcomes seen in children with cyanotic heart disease who undergo deep hypothermic circulatory arrest.
Assuntos
Parada Circulatória Induzida por Hipotermia Profunda , Hipotermia Induzida , Animais , Animais Recém-Nascidos , Encéfalo , Ponte Cardiopulmonar , Parada Circulatória Induzida por Hipotermia Profunda/efeitos adversos , Cianose/etiologia , Parada Cardíaca Induzida/efeitos adversos , Necrose , SuínosRESUMO
Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.
Assuntos
Ependimoma/metabolismo , Neoplasias Infratentoriais/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Criança , Pré-Escolar , Intervalo Livre de Doença , Ependimoma/mortalidade , Ependimoma/patologia , Feminino , Humanos , Lactente , Neoplasias Infratentoriais/mortalidade , Neoplasias Infratentoriais/patologia , Masculino , Prognóstico , Sistema de Registros , Taxa de SobrevidaRESUMO
Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM.
Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Glioblastoma/diagnóstico , Glioblastoma/genética , Histonas/genética , Mutação/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Humanos , Recém-Nascido , Lisina/genética , Masculino , Metionina/genética , Pediatria , Modelos de Riscos Proporcionais , Sensibilidade e Especificidade , Adulto JovemRESUMO
The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of mtDNA to nuclear DNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify â¼200â bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress.
RESUMO
SMARCB1 gene alterations were first described in highly malignant rhabdoid tumors of the kidney, brain (atypical teratoid/rhabdoid tumor) and soft tissue. An increasing number of tumors have now shown loss of SMARCB1 protein expression by immunohistochemistry, including the majority of epithelioid sarcomas. However, investigations of SMARCB1 gene alterations in epithelioid sarcoma have produced conflicting results. The aim of this study was to evaluate SMARCB1 status using Sanger sequencing of the coding region and multiplex ligation-dependent probe amplification, a rapid and sensitive method for detecting intragenic deletions and duplications, which has not been used in previous studies. Twenty-one epithelioid sarcomas of both classical and proximal type were selected for SMARCB1 gene testing and SMARCB1 immunohistochemistry. Nineteen of 21 (90%) epithelioid sarcomas were SMARCB1 negative by immunohistochemistry. Twelve of the 19 (63%) had adequate DNA recovery for evaluation. Ten of 12 (83%) tumors showed homozygous deletions of the gene. Two cases showed heterozygous deletions and polymorphisms, but no sequence mutations. These results confirm the high frequency of SMARCB1 deletions in epithelioid sarcoma and show that multiplex ligation-dependent probe amplification is a reliable method for detection of deletions in these cases, which can be performed on formalin-fixed, paraffin-embedded tissue. Given the high percentage of SMARCB1 alterations in epithelioid sarcoma, these findings argue against using SMARCB1 gene deletion as a tool in distinguishing them from malignant rhabdoid tumors.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Proteínas Cromossômicas não Histona/análise , Análise Mutacional de DNA , Proteínas de Ligação a DNA/análise , Diagnóstico Diferencial , Éxons , Feminino , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Minnesota , Reação em Cadeia da Polimerase Multiplex , Fenótipo , Philadelphia , Polimorfismo Genético , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Proteína SMARCB1 , Sarcoma/química , Sarcoma/patologia , Neoplasias de Tecidos Moles/química , Neoplasias de Tecidos Moles/patologia , Fatores de Transcrição/análise , Adulto JovemRESUMO
Loss-of-function of alpha thalassemia/mental retardation syndrome X-linked (ATRX) protein leads to a phenotype called alternative lengthening of telomeres (ALT) in some tumors. High-grade astrocytomas comprise a heterogeneous group of central nervous system tumors. We examined a large cohort of adult (91) and pediatric (n=88) high-grade astrocytomas as well as lower grade forms (n=35) for immunohistochemical loss of ATRX protein expression and the presence of ALT using telomere-specific fluorescence in situ hybridization, with further correlation to other known genetic alterations. We found that in pediatric high-grade astrocytomas, 29.6% of tumors were positive for ALT and 24.5% were immunonegative for the ATRX protein, these two alterations being highly associated with one another (P<0.0001). In adult high-grade astrocytomas, 26.4% of tumors were similarly positive for ALT, including 80% of ATRX protein immunonegative cases (P<0.0001). Similar frequencies were found in 11 adult low-grade astrocytomas, whereas all 24 pilocytic astrocytomas were negative for ALT. We did not find any significant correlations between isocitrate dehydrogenase status and either ALT positivity or ATRX protein expression in our adult high-grade astrocytomas. In both cohorts, however, the ALT positive high-grade astrocytomas showed more frequent amplification of the platelet-derived growth factor receptor alpha gene (PDGFRA; 45% and 50%, respectively) than the ALT negative counterparts (18% and 26%; P=0.03 for each). In summary, our data show that the ALT and ATRX protein alterations are common in both pediatric and adult high-grade astrocytomas, often with associated PDGFRA gene amplification.
Assuntos
Astrocitoma/química , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , DNA Helicases/análise , Proteínas Nucleares/análise , Homeostase do Telômero , Telômero/genética , Adulto , Fatores Etários , Astrocitoma/mortalidade , Astrocitoma/patologia , Astrocitoma/cirurgia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Criança , Feminino , Amplificação de Genes , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Isocitrato Desidrogenase/análise , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Masculino , Mutação , Gradação de Tumores , América do Norte , Modelos de Riscos Proporcionais , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Proteína Nuclear Ligada ao XRESUMO
Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography-tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle.
Assuntos
Corpos de Inclusão/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Musculares/genética , Doenças Musculares/genética , Mutação , Proteômica/métodos , Sequência de Aminoácidos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas com Domínio LIM , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/análise , Proteínas Musculares/química , Doenças Musculares/metabolismo , TransfecçãoRESUMO
BACKGROUND: Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15-20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications. PROCEDURE: One hundred matched tumor and blood samples from patients with rhabdoid tumors of the brain, kidney, or soft tissues were analyzed for mutations and deletions of SMARCB1 by FISH, multiplex ligation-dependent probe amplification (MLPA), sequence analysis and high resolution Illumina 610K SNP-based oligonucleotide array studies. RESULTS: Thirty-five of 100 patients were found to have a germline SMARCB1 abnormality. These abnormalities included point and frameshift mutations, intragenic deletions and duplications, and larger deletions including regions both proximal and distal to SMARCB1. There were nine cases that demonstrated parent to child transmission of a mutated copy of SMARCB1. In eight of the nine cases, one or more family members were also diagnosed with rhabdoid tumor or schwannoma, and two of the eight families presented with multiple affected children in a manner consistent with gonadal mosaicism. CONCLUSIONS: Approximately one-third of newly diagnosed patients with rhabdoid tumor have an underlying genetic predisposition to tumors due to a germline SMARCB1 alteration. Families may demonstrate incomplete penetrance and gonadal mosaicism, which must be considered when counseling families of patients with rhabdoid tumor.
Assuntos
Proteínas Cromossômicas não Histona/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Tumor Rabdoide/genética , Fatores de Transcrição/genética , Pré-Escolar , Cromossomos Humanos Par 22 , Família , Feminino , Deleção de Genes , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Mosaicismo , Penetrância , Tumor Rabdoide/etiologia , Proteína SMARCB1RESUMO
Mutations in the SARS-CoV-2 Membrane (M) gene are relatively uncommon. The M gene encodes the most abundant viral structural protein, and is implicated in multiple viral functions, including initial attachment to the host cell via heparin sulphate proteoglycan, viral protein assembly in conjunction with the N and E genes, and enhanced glucose transport. We have identified a recent spike in the frequency of reported SARS-CoV-2 genomes carrying M gene mutations. This is associated with emergence of a new sub-B.1 clade, B.1.I82T, defined by the previously unreported M:I82T mutation within TM3, the third of three membrane spanning helices implicated in glucose transport. The frequency of this mutation increased in the USA from 0.014% in October 2020 to 1.62% in February 2021, a 116-fold change. While constituting 0.7% of the isolates overall, M:I82T sub-B.1 lineage accounted for 14.4% of B.1 lineage isolates in February 2021, similar to the rapid initial increase previously seen with the B.1.1.7 and B.1.429 lineages, which quickly became the dominant lineages in Europe and California over a period of several months. A similar increase in incidence was also noted in another related mutation, V70L, also within the TM2 transmembrane helix. These M mutations are associated with younger patient age (4.6 to 6.3 years). The rapid emergence of this B.1.I82T clade, recently named Pangolin B.1.575 lineage, suggests that this M gene mutation is more biologically fit, perhaps related to glucose uptake during viral replication, and should be included in ongoing genomic surveillance efforts and warrants further evaluation for potentially increased pathogenic and therapeutic implications.
Assuntos
COVID-19/virologia , Mutação , SARS-CoV-2/genética , Proteínas da Matriz Viral/genética , Adulto , Linhagem da Célula , Criança , Pré-Escolar , Humanos , FilogeniaRESUMO
The SARS-CoV-2 B.1.1.7 lineage is highly infectious and as of April 2021 accounted for 92% of COVID-19 cases in Europe and 59% of COVID-19 cases in the U.S. It is defined by the N501Y mutation in the receptor-binding domain (RBD) of the Spike (S) protein, and a few other mutations. These include two mutations in the N terminal domain (NTD) of the S protein, HV69-70del and Y144del (also known as Y145del due to the presence of tyrosine at both positions). We recently identified several emerging SARS-CoV-2 variants of concerns, characterized by Membrane (M) protein mutations, including I82T and V70L. We now identify a sub-lineage of B.1.1.7 that emerged through sequential acquisitions of M:V70L in November 2020 followed by a novel S:D178H mutation first observed in early February 2021. The percentage of B.1.1.7 isolates in the US that belong to this sub-lineage increased from 0.15% in February 2021 to 1.8% in April 2021. To date, this sub-lineage appears to be U.S.-specific with reported cases in 31 states, including Hawaii. As of April 2021, it constituted 36.8% of all B.1.1.7 isolates in Washington. Phylogenetic analysis and transmission inference with Nextstrain suggest this sub-lineage likely originated in either California or Washington. Structural analysis revealed that the S:D178H mutation is in the NTD of the S protein and close to two other signature mutations of B.1.1.7, HV69-70del and Y144del. It is surface exposed and may alter NTD tertiary configuration or accessibility, and thus has the potential to affect neutralization by NTD directed antibodies.
Assuntos
Mutação , SARS-CoV-2/classificação , Glicoproteína da Espícula de Coronavírus/genética , Proteínas da Matriz Viral/genética , Sequenciamento Completo do Genoma/métodos , Sítios de Ligação , Humanos , Modelos Moleculares , Filogenia , Domínios Proteicos , Estrutura Terciária de Proteína , SARS-CoV-2/genética , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Estados UnidosRESUMO
BACKGROUND: The full spectrum of the disease phenotype and viral genotype of coronavirus disease 2019 (COVID-19) have yet to be thoroughly explored in children. Here, we analyze the relationships between viral genetic variants and clinical characteristics in children. METHODS: Whole-genome sequencing was performed on respiratory specimens collected for all SARS-CoV-2-positive children (n = 141) between March 13 and June 16, 2020. Viral genetic variations across the SARS-CoV-2 genome were identified and investigated to evaluate genomic correlates of disease severity. RESULTS: Higher viral load was detected in symptomatic patients (P = .0007) and in children <5 years old (P = .0004). Genomic analysis revealed a mean pairwise difference of 10.8 single nucleotide variants (SNVs), and the majority (55.4%) of SNVs led to an amino acid change in the viral proteins. The D614G mutation in the spike protein was present in 99.3% of the isolates. The calculated viral mutational rate of 22.2 substitutions/year contrasts the 13.5 substitutions/year observed in California isolates without the D614G mutation. Phylogenetic clade 20C was associated with severe cases of COVID-19 (odds ratio, 6.95; P = .0467). Epidemiological investigation revealed major representation of 3 of 5 major Nextstrain clades (20A, 20B, and 20C) consistent with multiple introductions of SARS-CoV-2 in Southern California. CONCLUSIONS: Genomic evaluation demonstrated greater than expected genetic diversity, presence of the D614G mutation, increased mutation rate, and evidence of multiple introductions of SARS-CoV-2 into Southern California. Our findings suggest a possible association of phylogenetic clade 20C with severe disease, but small sample size precludes a definitive conclusion. Our study warrants larger and multi-institutional genomic evaluation and has implications for infection control practices.
RESUMO
BACKGROUND: Recent large-scale genomic studies have revealed a spectrum of genetic variants associated with specific subtypes of central nervous system (CNS) tumors. The aim of this study was to determine the clinical utility of comprehensive genomic profiling of pediatric, adolescent and young adult (AYA) CNS tumors in a prospective setting, including detection of DNA sequence variants, gene fusions, copy number alterations (CNAs), and loss of heterozygosity. METHODS: OncoKids, a comprehensive DNA- and RNA-based next-generation sequencing (NGS) panel, in conjunction with chromosomal microarray analysis (CMA) was employed to detect diagnostic, prognostic, and therapeutic markers. NGS was performed on 222 specimens from 212 patients. Clinical CMA data were analyzed in parallel for 66% (146/222) of cases. RESULTS: NGS demonstrated clinically significant alterations in 66% (147/222) of cases. Diagnostic markers were identified in 62% (138/222) of cases. Prognostic information and targetable genomic alterations were identified in 22% (49/222) and 18% (41/222) of cases, respectively. Diagnostic or prognostic CNAs were revealed by CMA in 69% (101/146) of cases. Importantly, clinically significant CNAs were detected in 57% (34/60) of cases with noncontributory NGS results. Germline cancer predisposition testing was indicated for 27% (57/212) of patients. Follow-up germline testing was performed for 20 patients which confirmed a germline pathogenic/likely pathogenic variant in 9 cases: TP53 (2), NF1 (2), SMARCB1 (1), NF2 (1), MSH6 (1), PMS2 (1), and a patient with 47,XXY Klinefelter syndrome. CONCLUSIONS: Our results demonstrate the significant clinical utility of integrating genomic profiling into routine clinical testing for pediatric and AYA patients with CNS tumors.
RESUMO
High-grade gliomas with arginine or valine substitutions of the histone H3.3 glycine-34 residue (H3.3G34R/V) carry a dismal prognosis, and current treatments, including radiotherapy and chemotherapy, are not curative. Because H3.3G34R/V mutations reprogram epigenetic modifications, we undertook a comprehensive epigenetic approach using ChIP sequencing and ChromHMM computational analysis to define therapeutic dependencies in H3.3G34R/V gliomas. Our analyses revealed a convergence of epigenetic alterations, including (i) activating epigenetic modifications on histone H3 lysine (K) residues such as H3K36 trimethylation (H3K36me3), H3K27 acetylation (H3K27ac), and H3K4 trimethylation (H3K4me3); (ii) DNA promoter hypomethylation; and (iii) redistribution of repressive histone H3K27 trimethylation (H3K27me3) to intergenic regions at the leukemia inhibitory factor (LIF) locus to drive increased LIF abundance and secretion by H3.3G34R/V cells. LIF activated signal transducer and activator of transcription 3 (STAT3) signaling in an autocrine/paracrine manner to promote survival of H3.3G34R/V glioma cells. Moreover, immunohistochemistry and single-cell RNA sequencing from H3.3G34R/V patient tumors revealed high STAT3 protein and RNA expression, respectively, in tumor cells with both inter- and intratumor heterogeneity. We targeted STAT3 using a blood-brain barrierpenetrable small-molecule inhibitor, WP1066, currently in clinical trials for adult gliomas. WP1066 treatment resulted in H3.3G34R/V tumor cell toxicity in vitro and tumor suppression in preclinical mouse models established with KNS42 cells, SJ-HGGx42-c cells, or in utero electroporation techniques. Our studies identify the LIF/STAT3 pathway as a key epigenetically driven and druggable vulnerability in H3.3G34R/V gliomas. This finding could inform development of targeted, combination therapies for these lethal brain tumors.