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The choroid plexus (ChP) is a vital brain barrier and source of cerebrospinal fluid (CSF). Here, we use longitudinal two-photon imaging in awake mice and single-cell transcriptomics to elucidate the mechanisms of ChP regulation of brain inflammation. We used intracerebroventricular injections of lipopolysaccharides (LPS) to model meningitis in mice and observed that neutrophils and monocytes accumulated in the ChP stroma and surged across the epithelial barrier into the CSF. Bi-directional recruitment of monocytes from the periphery and, unexpectedly, macrophages from the CSF to the ChP helped eliminate neutrophils and repair the barrier. Transcriptomic analyses detailed the molecular steps accompanying this process and revealed that ChP epithelial cells transiently specialize to nurture immune cells, coordinating their recruitment, survival, and differentiation as well as regulation of the tight junctions that control the permeability of the ChP brain barrier. Collectively, we provide a mechanistic understanding and a comprehensive roadmap of neuroinflammation at the ChP brain barrier.
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We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).
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Distrofina , Terapia Genética , Distrofia Muscular de Duchenne , Síndrome do Desconforto Respiratório , Transgenes , Adulto , Humanos , Anticorpos , Distrofina/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/imunologia , Transgenes/genética , Transgenes/imunologia , Evolução Fatal , Imunidade Inata/genética , Imunidade Inata/imunologiaRESUMO
INTRODUCTION/AIMS: Heterogeneous nuclear ribonucleoprotein A1 is involved in nucleic acid homeostatic functions. The encoding gene HNRNPA1 has been associated with several neuromuscular disorders including an amyotrophic lateral sclerosis-like phenotype, distal hereditary motor neuropathy, multisystem proteinopathy, and various myopathies. We report two unrelated individuals with monoallelic stop loss variants affecting the same codon of HNRNPA1. METHODS: Two individuals with unsolved juvenile-onset myopathy were enrolled under approved institutional protocols. Phenotype data were collected and genetic analyses were performed, including whole-exome sequencing (WES). RESULTS: The two probands (MNOT002-01 and K1440-01) showed a similar onset of slowly progressive extremity and facial weakness in early adolescence. K1440-01 presented with facial weakness, winged scapula, elevated serum creatine kinase (CK) levels, and mild neck weakness. MNOT002-01 also exhibited elevated CK levels along with facial weakness, cardiomyopathy, respiratory dysfunction, pectus excavatum, a mildly rigid spine, and loss of ambulation. On quadriceps muscle biopsy, K1440-01 displayed rounded myofibers, mild variation in fiber diameter, and type 2 fiber hypertrophy, while MNOT002-01 displayed rimmed vacuoles. Monoallelic stop-loss variants in HNRNPA1 were identified for both probands: c.1119A>C p.*373Tyrext*6 (K1440-01) and c.1118A>C p.*373Serext*6 (MNOT002-01) affect the same codon and are both predicted to lead to the addition of six amino acids before termination at an alternative stop codon. DISCUSSION: Both stop-loss variants in our probands are likely pathogenic. Our findings contribute to the disease characterization of pathogenic variants in HNRNPA1. This gene should be screened in clinical diagnostic testing of unsolved cases of sporadic or dominant juvenile-onset myopathy.
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PHACE (posterior fossa malformations, hemangiomas, arterial anomalies, cardiac anomalies, eye anomalies) association has many recognized clinical features. A link between PHACE and non-vascular intracranial lesions has not been well-described. We report three pediatric patients with PHACE and non-vascular intracranial lesions.
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Anormalidades Múltiplas , Coartação Aórtica , Anormalidades do Olho , Síndromes Neurocutâneas , Humanos , Criança , Lactente , Síndromes Neurocutâneas/diagnóstico , Síndromes Neurocutâneas/patologia , Coartação Aórtica/complicações , Coartação Aórtica/diagnóstico , Coartação Aórtica/patologia , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/patologiaRESUMO
DTNA encodes α-dystrobrevin, a component of the macromolecular dystrophin-glycoprotein complex (DGC) that binds to dystrophin/utrophin and α-syntrophin. Mice lacking α-dystrobrevin have a muscular dystrophy phenotype, but variants in DTNA have not previously been associated with human skeletal muscle disease. We present 12 individuals from four unrelated families with two different monoallelic DTNA variants affecting the coiled-coil domain of α-dystrobrevin. The five affected individuals from family A harbor a c.1585G > A; p.Glu529Lys variant, while the recurrent c.1567_1587del; p.Gln523_Glu529del DTNA variant was identified in the other three families (family B: four affected individuals, family C: one affected individual, and family D: two affected individuals). Myalgia and exercise intolerance, with variable ages of onset, were reported in 10 of 12 affected individuals. Proximal lower limb weakness with onset in the first decade of life was noted in three individuals. Persistent elevations of serum creatine kinase (CK) levels were detected in 11 of 12 affected individuals, 1 of whom had an episode of rhabdomyolysis at 20 years of age. Autism spectrum disorder or learning disabilities were reported in four individuals with the c.1567_1587 deletion. Muscle biopsies in eight affected individuals showed mixed myopathic and dystrophic findings, characterized by fiber size variability, internalized nuclei, and slightly increased extracellular connective tissue and inflammation. Immunofluorescence analysis of biopsies from five affected individuals showed reduced α-dystrobrevin immunoreactivity and variably reduced immunoreactivity of other DGC proteins: dystrophin, α, ß, δ and γ-sarcoglycans, and α and ß-dystroglycans. The DTNA deletion disrupted an interaction between α-dystrobrevin and syntrophin. Specific variants in the coiled-coil domain of DTNA cause skeletal muscle disease with variable penetrance. Affected individuals show a spectrum of clinical manifestations, with severity ranging from hyperCKemia, myalgias, and exercise intolerance to childhood-onset proximal muscle weakness. Our findings expand the molecular etiologies of both muscular dystrophy and paucisymptomatic hyperCKemia, to now include monoallelic DTNA variants as a novel cause of skeletal muscle disease in humans.
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Transtorno do Espectro Autista , Distrofias Musculares , Neuropeptídeos , Camundongos , Humanos , Animais , Criança , Distrofina/genética , Distrofina/metabolismo , Transtorno do Espectro Autista/metabolismo , Distrofias Musculares/metabolismo , Distroglicanas/metabolismo , Processamento Alternativo , Músculo Esquelético/patologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismoRESUMO
Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.
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Epilepsia , Hemimegalencefalia , Malformações do Desenvolvimento Cortical , Caderinas , Proteínas de Ciclo Celular , Feminino , Humanos , Malformações do Desenvolvimento Cortical do Grupo I , Mutação , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Protocaderinas , Serina-Treonina Quinases TORRESUMO
Central nervous system (CNS) germ cell tumors (GCTs) represent 2-3% of all primary CNS tumors. The majority are germinomas, which are radiosensitive and have an excellent prognosis. Contrarily, CNS non-germinomatous GCTs (NGGCTs) have less favorable prognosis and require more aggressive treatment. The expression of checkpoint/immune markers in CNS GCTs, particularly NGGCTs, is unknown. We previously reported a case of a patient whose intracranial NGGCT (predominantly choriocarcinoma) responded to immune checkpoint inhibition therapy. This case led us to evaluate our archive of intracranial GCTs for expression of PD-L1 and PD-1. With IRB approval, we searched the pathology archives at our institution for CNS GCTs. Demographic, radiologic, clinical, and histologic information was extracted from the medical records. Immunohistochemistry for lymphocytic markers (CD4, CD8, CD20), PD-1, and PD-L1 was performed. PD-L1 was considered positive if greater than 1% of tumor cells were positive and PD-1 was reported as a percentage of positive inflammatory cells. Fifty cases were identified, including 28 germinomas (mean age at diagnosis: 15.5 years; 17 males, 11 females), and 22 NGGCTs (mean age at diagnosis: 12.0 years, 21 males, 1 female). Germinomas were mostly suprasellar (17/28) and NGGCTs were predominantly pineal (17/22). Twenty-two germinomas (79%) were positive for PD-L1 expression, and 13 NGGCTs (57%) were positive for PD-L1. Cases of choriocarcinoma showed the most diffuse PD-L1 expression. PD-1 expression was seen in lymphocytes among 27/28 of the germinomas and 20/23 of the NGGCTs (ranging from 1-40% of lymphocytes). As expected, larger quantities of inflammatory cells were present in cases of germinoma. We demonstrate immune activity in CNS GCTs, and our results suggest that immune checkpoint inhibitors may be efficacious in the treatment of intracranial GCTs. Among NGGCTs, cases of choriocarcinoma showed the highest expression of PD-L1 in tumor cells, suggesting that this subtype may have the greatest benefit from checkpoint blockade.
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Neoplasias Encefálicas , Neoplasias do Sistema Nervoso Central , Coriocarcinoma , Germinoma , Neoplasias Embrionárias de Células Germinativas , Criança , Masculino , Humanos , Feminino , Adolescente , Receptor de Morte Celular Programada 1 , Antígeno B7-H1 , Germinoma/patologia , Neoplasias Encefálicas/patologia , Neoplasias do Sistema Nervoso Central/patologia , Sistema Nervoso Central/patologiaRESUMO
Gain of function PIK3CA pathogenic variants have been identified in overgrowth syndromes collectively termed "PIK3CA-related overgrowth spectrum" (PROS). There are no previously reported cases of cerebrovascular venous malformations in PROS syndromes, though somatic activating PIK3CA variants have been identified in extracranial venous malformation. This study was approved by the Institutional Review Boar at Boston Children's Hospital. A 14-year-old female mosaic for the de novo p.R108H pathogenic variant in the PIK3CA gene was found to have a large tumor involving the superior sagittal sinus with mass effect on the motor cortex most consistent with a parafalcine meningioma. She underwent surgical resection with pathology demonstrating a venous malformation. PIK3CA pathogenic variants have been identified in nonsyndromic extracranial venous and lymphatic malformations as well in brain tumors, including glioma and meningioma. However, PIK3CA variants have not previously been identified in purely intracranial venous malformations. This distinction is relevant to treatment decisions, given that mTOR inhibitors may provide an alternative option for noninvasive therapy in cases of suspected venous malformation.
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Neoplasias Meníngeas , Meningioma , Malformações Vasculares , Adolescente , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Humanos , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/genética , Meningioma/diagnóstico , Meningioma/genética , Mutação , Síndrome , Fatores de Transcrição/genética , Malformações Vasculares/diagnóstico , Malformações Vasculares/genéticaRESUMO
Population averaged diffusion atlases can be utilized to characterize complex microstructural changes with less bias than data from individual subjects. In this study, a fetal diffusion tensor imaging (DTI) atlas was used to investigate tract-based changes in anisotropy and diffusivity in vivo from 23 to 38 weeks of gestational age (GA). Healthy pregnant volunteers with typically developing fetuses were imaged at 3 T. Acquisition included structural images processed with a super-resolution algorithm and DTI images processed with a motion-tracked slice-to-volume registration algorithm. The DTI from individual subjects were used to generate 16 templates, each specific to a week of GA; this was accomplished by means of a tensor-to-tensor diffeomorphic deformable registration method integrated with kernel regression in age. Deterministic tractography was performed to outline the forceps major, forceps minor, bilateral corticospinal tracts (CST), bilateral inferior fronto-occipital fasciculus (IFOF), bilateral inferior longitudinal fasciculus (ILF), and bilateral uncinate fasciculus (UF). The mean fractional anisotropy (FA) and mean diffusivity (MD) was recorded for all tracts. For a subset of tracts (forceps major, CST, and IFOF) we manually divided the tractograms into anatomy conforming segments to evaluate within-tract changes. We found tract-specific, nonlinear, age related changes in FA and MD. Early in gestation, these trends appear to be dominated by cytoarchitectonic changes in the transient white matter fetal zones while later in gestation, trends conforming to the progression of myelination were observed. We also observed significant (local) heterogeneity in within-tract developmental trajectories for the CST, IFOF, and forceps major.
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Imagem de Tensor de Difusão , Feto/diagnóstico por imagem , Diagnóstico Pré-Natal , Substância Branca/diagnóstico por imagem , Anisotropia , Atlas como Assunto , Feminino , Idade Gestacional , Humanos , Masculino , GravidezRESUMO
Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.
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Astrocitoma/genética , Neoplasias Encefálicas/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Adolescente , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Transcriptoma , Adulto JovemRESUMO
AIMS: Primary intracranial sarcoma, DICER1-mutant is a recently described central nervous system tumour with specific genomic and DNA-methylation profiles. Although some of its histological features (focal spindle-cell morphology, intracytoplasmic eosinophilic granules, and focal heterologous differentiation) are common across most reported cases, the presence of significant histological variability and the lack of differentiation pose diagnostic challenges. We aim to further define the immunoprofile of this tumor. METHODS AND RESULTS: We reviewed the clinical history and performed immunohistochemistry for glial fibrillary acidic protein, oligodendrocyte transcription factor 2, SOX2, SOX10, S100, histone H3 trimethylated on lysine 27 (H3K27me3), desmin, myogenin, CD99, epithelial membrane antigen (EMA) and transducin-like enhancer of split 1 (TLE1) on six primary intracranial sarcomas, DICER1-mutant, with appropriate controls. Targeted exome sequencing was performed on all cases. The sarcomas showed diffuse (n = 4), mosaic (n = 1) or minimal (≤5%, n = 1) loss of H3K27 trimethylation and nuclear TLE1 expression (n = 6). Four had immunohistochemical evidence of myogenic differentiation. SOX2, SOX10, S100 and EMA were negative; CD99 expression ranged from focal cytoplasmic (n = 4) to crisp diffuse membranous (n = 2). One tumour had focal cartilaginous differentiation. Similar immunohistochemical findings were observed in a pleuropulmonary blastoma (albeit with focal TLE1 expression), a DICER1-related pineoblastoma, and an embryonal tumour with a multilayered rosette-like DICER1-related cerebellar tumour. Targeted exome sequencing confirmed the presence of pathogenic biallelic DICER1 mutations in all tumours included in this study. CONCLUSION: We conclude that H3K27me3 and TLE1 immunostains, when utilised in combination, can be helpful diagnostic markers for primary intracranial sarcoma, DICER1-mutant.
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Neoplasias Encefálicas , RNA Helicases DEAD-box/genética , Histonas/metabolismo , Ribonuclease III/genética , Sarcoma , Transducina , Adolescente , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica/métodos , Lactente , Lisina/metabolismo , Masculino , Metilação , Mutação , Sarcoma/genética , Sarcoma/patologia , Transducina/genética , Transducina/metabolismoRESUMO
Choroid plexus tumors and ciliary body medulloepithelioma are predominantly pediatric neoplasms. Progress in understanding the pathogenesis of these tumors has been hindered by their rarity and lack of models that faithfully recapitulate the disease. Here, we find that endogenous Myc proto-oncogene protein is down-regulated in the forebrain neuroepithelium, whose neural plate border domains give rise to the anterior choroid plexus and ciliary body. To uncover the consequences of persistent Myc expression, MYC expression was forced in multipotent neural precursors (nestin-Cre:Myc), which produced fully penetrant models of choroid plexus carcinoma and ciliary body medulloepithelioma. Nestin-mediated MYC expression in the epithelial cells of choroid plexus leads to the regionalized formation of choroid plexus carcinoma in the posterior domain of the lateral ventricle choroid plexus and the fourth ventricle choroid plexus that is accompanied by loss of multiple cilia, up-regulation of protein biosynthetic machinery, and hydrocephalus. Parallel MYC expression in the ciliary body leads also to up-regulation of protein biosynthetic machinery. Additionally, Myc expression in human choroid plexus tumors increases with aggressiveness of disease. Collectively, our findings expose a select vulnerability of the neuroepithelial lineage to postnatal tumorigenesis and provide a new mouse model for investigating the pathogenesis of these rare pediatric neoplasms.
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Carcinogênese/patologia , Neoplasias do Plexo Corióideo/patologia , Corpo Ciliar/patologia , Modelos Animais de Doenças , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adolescente , Adulto , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Criança , Pré-Escolar , Neoplasias do Plexo Corióideo/genética , Neoplasias do Plexo Corióideo/metabolismo , Corpo Ciliar/metabolismo , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Adulto JovemRESUMO
OBJECTIVE: Somatic variants are a recognized cause of epilepsy-associated focal malformations of cortical development (MCD). We hypothesized that somatic variants may underlie a wider range of focal epilepsy, including nonlesional focal epilepsy (NLFE). Through genetic analysis of brain tissue, we evaluated the role of somatic variation in focal epilepsy with and without MCD. METHODS: We identified somatic variants through high-depth exome and ultra-high-depth candidate gene sequencing of DNA from epilepsy surgery specimens and leukocytes from 18 individuals with NLFE and 38 with focal MCD. RESULTS: We observed somatic variants in 5 cases in SLC35A2, a gene associated with glycosylation defects and rare X-linked epileptic encephalopathies. Nonsynonymous variants in SLC35A2 were detected in resected brain, and absent from leukocytes, in 3 of 18 individuals (17%) with NLFE, 1 female and 2 males, with variant allele frequencies (VAFs) in brain-derived DNA of 2 to 14%. Pathologic evaluation revealed focal cortical dysplasia type Ia (FCD1a) in 2 of the 3 NLFE cases. In the MCD cohort, nonsynonymous variants in SCL35A2 were detected in the brains of 2 males with intractable epilepsy, developmental delay, and magnetic resonance imaging suggesting FCD, with VAFs of 19 to 53%; Evidence for FCD was not observed in either brain tissue specimen. INTERPRETATION: We report somatic variants in SLC35A2 as an explanation for a substantial fraction of NLFE, a largely unexplained condition, as well as focal MCD, previously shown to result from somatic mutation but until now only in PI3K-AKT-mTOR pathway genes. Collectively, our findings suggest a larger role than previously recognized for glycosylation defects in the intractable epilepsies. Ann Neurol 2018.
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Encéfalo/patologia , Epilepsia Resistente a Medicamentos/genética , Proteínas de Transporte de Monossacarídeos/genética , Neocórtex/patologia , Adolescente , Criança , Exoma/genética , Feminino , Humanos , Masculino , Malformações do Desenvolvimento Cortical/genética , Mutação/genética , Neurônios/patologia , Fosfatidilinositol 3-Quinases/genética , Serina-Treonina Quinases TOR/genética , Adulto JovemRESUMO
BACKGROUND: Pediatric brain tumors are associated with high morbidity and mortality, in part due to insufficient understanding of tumor biology. With limited tissue allocation for research from surgical specimens, a key barrier to improving biological understanding, brain tumor autopsies have become an increasingly valuable resource. This study reviews the brain tumor autopsy practice at our institution and describes specific emerging research utilization patterns beyond the clinical autopsy report. METHODS: We performed a retrospective analysis of brain tumor autopsies at Boston Children's Hospital (BCH) between 2007 and 2017 and reviewed their consents, neuropathology reports and final diagnoses. We reviewed the method of tissue triaging for research consented autopsies (bioregistry, frozen and fresh tissue) and documented their specific uses. RESULTS: Ninety-six deaths at BCH were due to brain tumors; 56 autopsies were performed (58.3%), of which 49 (87.5%) were consented for research. Tumor mapping was performed on all cases and tissue was allocated for DNA- and RNA-based sequencing studies (published and ongoing). Three tissue allocations with a postmortem interval of 8 h or less resulted in successful cell lines. Tissue from 14 autopsies was contributed to the National DIPG Registry. CONCLUSION: Our institutional pediatric brain tumor autopsy clinical experience demonstrates the increased utility and wide utilization of autopsy-derived tissue for multiple types of research. These results support the increased efforts to obtain research consent for brain tumor autopsy and active collection of unfixed autopsy material in the molecular era.
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Autopsia/métodos , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Oncologia/métodos , Pesquisa Biomédica , Criança , Humanos , Estudos RetrospectivosRESUMO
AIMS: The PHOX2B gene regulates neuronal maturation in the brain stem nuclei associated with cardiorespiratory function and in the autonomic sympathetic and enteric nervous system. PHOX2B expression is a reliable immunomarker for peripheral neuroblastic tumours; however, no systematic evaluation of central nervous system (CNS) embryonal tumours was included in the studies. We encountered two cases in which the differential diagnosis included neuroblastoma and CNS embryonal tumour, and we hypothesised that PHOX2B immunostain would be helpful in establishing the diagnosis. METHODS AND RESULTS: PHOX2B immunostain was performed on 29 paediatric cases, with adequate controls: one retroperitoneal embryonal tumour in a child with retinoblastoma (index 1), one posterior fossa embryonal tumour in a child with a neuroblastoma (index 2), seven medulloblastomas, four atypical teratoid/rhabdoid tumours (ATRT), four retinoblastomas, six pineoblastomas, four embryonal tumours with multilayered rosettes (ETMR) and two CNS embryonal tumours, not elsewhere classified. Cell lineage immunomarkers (GFAP, OLIG2, synaptophysin, NeuN, CRX, PGP 9.5), immunosurrogates for molecular alterations (beta-catenin, INI1, Lin-28), array CGH and OncoPanel were performed as needed. Medulloblastomas, ATRTs, ETMRs, retinoblastomas and CNS embryonal tumours not elsewhere classified were essentially negative for PHOX2B. Two of six pineoblastomas had significant PHOX2B expression, while the rest were negative. Index 1 was negative for PHOX2B and PGP 9.5 and positive for CRX, consistent with retinoblastoma. Index 2 had diffuse PHOX2B expression, MYCN amplification and no copy number changes of medulloblastoma, in keeping with neuroblastoma. CONCLUSION: PHOX2B antibody is helpful in distinguishing between peripheral neuroblastic and CNS embryonal tumours, which are immunonegative, with the caveat that a subset of pineoblastomas has significant expression.
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Biomarcadores Tumorais/análise , Proteínas de Homeodomínio/biossíntese , Neoplasias Infratentoriais/diagnóstico , Metástase Neoplásica/diagnóstico , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Neuroblastoma/diagnóstico , Retinoblastoma/diagnóstico , Fatores de Transcrição/biossíntese , Pré-Escolar , Diagnóstico Diferencial , Proteínas de Homeodomínio/análise , Humanos , Lactente , Neoplasias Infratentoriais/secundário , Masculino , Metástase Neoplásica/patologia , Neoplasias Embrionárias de Células Germinativas/patologia , Neuroblastoma/secundário , Retinoblastoma/secundário , Neoplasias Retroperitoneais/diagnóstico , Neoplasias Retroperitoneais/patologia , Fatores de Transcrição/análiseRESUMO
The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.
Assuntos
Exoma/genética , Testes Genéticos/métodos , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Sequência de Bases , Miopatias Distais/diagnóstico , Miopatias Distais/genética , Feminino , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/genética , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular Facioescapuloumeral/diagnóstico , Distrofia Muscular Facioescapuloumeral/genética , Mutação/genética , Miopatias Congênitas Estruturais/diagnóstico , Miopatias Congênitas Estruturais/genética , Análise de Sequência de DNA/métodos , Estados UnidosRESUMO
Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in Drosophila melanogaster. We identified 39 different EPG5 mutations, most of them truncating and predicted to result in reduced EPG5 protein. Most mutations were private, but three recurrent mutations (p.Met2242Cysfs*5, p.Arg417*, and p.Gln336Arg) indicated possible founder effects. Presentation was mainly neonatal, with marked hypotonia and feeding difficulties. In addition to the five principal features (callosal agenesis, cataracts, hypopigmentation, cardiomyopathy, and immune dysfunction), we identified three equally consistent features (profound developmental delay, progressive microcephaly, and failure to thrive). The manifestation of all eight of these features has a specificity of 97%, and a sensitivity of 89% for the presence of an EPG5 mutation and will allow informed decisions about genetic testing. Clinical progression was relentless and many children died in infancy. Survival analysis demonstrated a median survival time of 24 months (95% confidence interval 0-49 months), with only a 10th of patients surviving to 5 years of age. Survival outcomes were significantly better in patients with compound heterozygous mutations (P = 0.046), as well as in patients with the recurrent p.Gln336Arg mutation. Acquired microcephaly and regression of skills in long-term survivors suggests a neurodegenerative component superimposed on the principal neurodevelopmental defect. Two-thirds of patients had a severe seizure disorder, placing EPG5 within the rapidly expanding group of genes associated with early-onset epileptic encephalopathies. Consistent neuroradiological features comprised structural abnormalities, in particular callosal agenesis and pontine hypoplasia, delayed myelination and, less frequently, thalamic signal intensity changes evolving over time. Typical muscle biopsy features included fibre size variability, central/internal nuclei, abnormal glycogen storage, presence of autophagic vacuoles and secondary mitochondrial abnormalities. Nerve biopsy performed in one case revealed subtotal absence of myelinated axons. Post-mortem examinations in three patients confirmed neurodevelopmental and neurodegenerative features and multisystem involvement. Finally, downregulation of epg5 (CG14299) in Drosophila resulted in autophagic abnormalities and progressive neurodegeneration. We conclude that EPG5-related Vici syndrome defines a novel group of neurodevelopmental disorders that should be considered in patients with suggestive features in whom mitochondrial, glycogen, or lysosomal storage disorders have been excluded. Neurological progression over time indicates an intriguing link between neurodevelopment and neurodegeneration, also supported by neurodegenerative features in epg5-deficient Drosophila, and recent implication of other autophagy regulators in late-onset neurodegenerative disease.
Assuntos
Agenesia do Corpo Caloso/diagnóstico , Agenesia do Corpo Caloso/genética , Autofagia/genética , Catarata/diagnóstico , Catarata/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Proteínas/genética , Agenesia do Corpo Caloso/complicações , Animais , Proteínas Relacionadas à Autofagia , Catarata/complicações , Pré-Escolar , Estudos Transversais , Drosophila melanogaster , Feminino , Hipocampo/patologia , Humanos , Proteínas de Membrana Lisossomal , Masculino , Mutação/genética , Transtornos do Neurodesenvolvimento/complicações , Estudos Retrospectivos , Proteínas de Transporte VesicularRESUMO
A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.
Assuntos
Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Quarto Ventrículo/metabolismo , Ventrículos Laterais/metabolismo , Transcriptoma , Envelhecimento/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Humanos , Macaca mulatta , Masculino , CamundongosAssuntos
Ciclina D2/genética , Predisposição Genética para Doença , Hidrocefalia/genética , Malformações do Desenvolvimento Cortical/genética , Meduloblastoma/genética , Polidactilia/genética , Feminino , Humanos , Hidrocefalia/complicações , Hidrocefalia/patologia , Lactente , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/patologia , Meduloblastoma/complicações , Meduloblastoma/patologia , Polidactilia/complicações , Polidactilia/patologiaRESUMO
Background: Choroid plexus tumors (CPTs) are rare, potentially aggressive CNS tumors with defined histologic criteria for grading. In recent years, several patients within our practice have demonstrated discordance between the histologic diagnosis and clinical behavior. DNA methylation profiling has emerged as a potential diagnostic adjunct for aiding the clinical approach. Methods: We reviewed the clinical and pathologic data of all CPTs diagnosed at Boston Children's Hospital from 1995 to 2023. All cases with available material (38/48) underwent DNA methylation profiling at NIH/NCI, and the classifier results were correlated with the WHO histologic grade and patient outcomes. Survival information was analyzed using Kaplan-Meier curves. Results: There was good correlation (11/12, 92%) between methylation class and WHO histologic grade for choroid plexus carcinomas (CPC); one histologic CPC grouped with choroid plexus papilloma (CPP) group pediatric (P). Five CPPs grouped with methylation class CPC (5/17, 29%). In the group of atypical CPPs (nâ =â 9), there were two that grouped with methylation class CPC. Survival analysis showed utility of methylation classes in the prediction of biologic behavior. Conclusions: Results indicated that methylation profiling may serve as a valuable tool in the clinical decision-making process for patients with CPTs, providing additional prognostic information compared to WHO histologic grade alone. The value of methylation array analysis is particularly important given the lack of consensus on treatment regimens for CPTs.