RESUMEN
BACKGROUND: The retinoic acid (RA) pathway plays a crucial role in both eye morphogenesis and the visual cycle. Individuals with monoallelic and biallelic pathogenic variants in retinol-binding protein 4 (RBP4), encoding a serum retinol-specific transporter, display variable ocular phenotypes. Although few families have been reported worldwide, recessive inherited variants appear to be associated with retinal degeneration, while individuals with dominantly inherited variants manifest ocular development anomalies, mainly microphthalmia, anophthalmia and coloboma (MAC). METHODS: We report here seven new families (13 patients) with isolated and syndromic MAC harbouring heterozygous RBP4 variants, of whom we performed biochemical analyses. RESULTS: For the first time, malformations that overlap the clinical spectrum of vitamin A deficiency are reported, providing a link with other RA disorders. Our data support two distinct phenotypes, depending on the nature and mode of inheritance of the variants: dominantly inherited, almost exclusively missense, associated with ocular malformations, in contrast to recessive, mainly truncating, associated with retinal degeneration. Moreover, we also confirm the skewed inheritance and impact of maternal RBP4 genotypes on phenotypical expression in dominant forms, suggesting that maternal RBP4 genetic status and content of diet during pregnancy may modify MAC occurrence and severity. Furthermore, we demonstrate that retinol-binding protein blood dosage in patients could provide a biological signature crucial for classifying RBP4 variants. Finally, we propose a novel hypothesis to explain the mechanisms underlying the observed genotype-phenotype correlations in RBP4 mutational spectrum. CONCLUSION: Dominant missense variants in RBP4 are associated with MAC of incomplete penetrance with maternal inheritance through a likely dominant-negative mechanism.
Asunto(s)
Anoftalmos , Microftalmía , Degeneración Retiniana , Embarazo , Femenino , Humanos , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Microftalmía/genética , Anoftalmos/genética , Tretinoina/metabolismo , Proteínas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol/química , Proteínas Plasmáticas de Unión al Retinol/metabolismoRESUMEN
The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.
Asunto(s)
Encéfalo/anomalías , Anomalías del Ojo/genética , Dedos/anomalías , Mutación Missense , Fenotipo , Ubiquitina-Proteína Ligasas/genética , Proteínas con Repetición de beta-Transducina/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , MasculinoRESUMEN
Two distinct syndromes arise from pathogenic variants in the X-linked gene BCOR (BCL-6 corepressor): oculofaciocardiodental (OFCD) syndrome, which affects females, and a severe microphthalmia ('Lenz'-type) syndrome affecting males. OFCD is an X-linked dominant syndrome caused by a variety of BCOR null mutations. As it manifests only in females, it is presumed to be lethal in males. The severe male X-linked recessive microphthalmia syndrome ('Lenz') usually includes developmental delay in addition to the eye findings and is caused by hypomorphic BCOR variants, mainly by a specific missense variant c.254C > T, p.(Pro85Leu). Here, we detail 16 new cases (11 females with 4 additional, genetically confirmed, affected female relatives; 5 male cases each with unaffected carrier mothers). We describe new variants and broaden the phenotypic description for OFCD to include neuropathy, muscle hypotonia, pituitary underdevelopment, brain atrophy, lipoma and the first description of childhood lymphoma in an OFCD case. Our male X-linked recessive cases show significant new phenotypes: developmental delay (without eye anomalies) in two affected half-brothers with a novel BCOR variant, and one male with high myopia, megalophthalmos, posterior embryotoxon, developmental delay, and heart and bony anomalies with a previously undescribed BCOR splice site variant. Our female OFCD cases and their affected female relatives showed variable features, but consistently had early onset cataracts. We show that a mosaic carrier mother manifested early cataract and dental anomalies. All female carriers of the male X-linked recessive cases for whom genetic confirmation was available showed skewed X-inactivation and were unaffected. In view of the extended phenotype, we suggest a new term of X-linked BCOR-related syndrome.
Asunto(s)
Anomalías Múltiples/genética , Catarata/congénito , Cromosomas Humanos X/genética , Genes Ligados a X/genética , Defectos de los Tabiques Cardíacos/genética , Microftalmía/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Adolescente , Adulto , Catarata/genética , Preescolar , Anomalías del Ojo/genética , Femenino , Variación Genética/genética , Heterocigoto , Humanos , Lactante , Masculino , Fenotipo , Síndrome , Inactivación del Cromosoma X/genética , Adulto JovenRESUMEN
GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.
Asunto(s)
Conexinas/genética , Anomalías del Ojo/genética , Mutación Missense/genética , Catarata/genética , Estudios de Cohortes , Proteínas del Ojo/genética , Femenino , Uniones Comunicantes/genética , Estudios de Asociación Genética/métodos , Heterocigoto , Humanos , Cristalino/patología , Masculino , Linaje , FenotipoRESUMEN
Nystagmus (involuntary, rhythmical eye movements) can arise due to sensory eye defects, in association with neurological disorders or as an isolated condition. We identified a family with early onset nystagmus and additional neurological features carrying a partial duplication of FGF14, a gene associated with spinocerebellar ataxia type 27 (SCA27) and episodic ataxia. Detailed eye movement analysis revealed oculomotor anomalies strikingly similar to those reported in a previously described four-generation family with early onset nystagmus and linkage to a region on chromosome 13q31.3-q33.1 (NYS4). Since FGF14 lies within NYS4, we revisited the original pedigree using whole genome sequencing, identifying a 161 kb heterozygous deletion disrupting FGF14 and ITGBL1 in the affected individuals, suggesting an FGF14-related condition. Therefore, our study reveals the genetic variant underlying NYS4, expands the spectrum of pathogenic FGF14 variants, and highlights the importance of screening FGF14 in apparently isolated early onset nystagmus.
Asunto(s)
Nistagmo Patológico , Degeneraciones Espinocerebelosas , Humanos , Ataxia/genética , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Integrina beta1/genética , Linaje , Degeneraciones Espinocerebelosas/genéticaRESUMEN
Biallelic pathogenic variants in ALDH1A3 are responsible for approximately 11% of recessively inherited cases of severe developmental eye anomalies. Some individuals can display variable neurodevelopmental features, but the relationship to the ALDH1A3 variants remains unclear. Here, we describe seven unrelated families with biallelic pathogenic ALDH1A3 variants: four compound heterozygous and three homozygous. All affected individuals had bilateral anophthalmia/microphthalmia (A/M), three with additional intellectual or developmental delay, one with autism and seizures and three with facial dysmorphic features. This study confirms that individuals with biallelic pathogenic ALDH1A3 variants consistently manifest A/M, but additionally display neurodevelopmental features with significant intra- and interfamilial variability. Furthermore, we describe the first case with cataract and highlight the importance of screening ALDH1A3 variants in nonconsanguineous families with A/M.
Asunto(s)
Anoftalmos , Anomalías del Ojo , Microftalmía , Humanos , Microftalmía/genética , Anoftalmos/genética , Mutación , Aldehído Oxidorreductasas/genética , FenotipoRESUMEN
BACKGROUND: Anophthalmia, microphthalmia and coloboma are a genetically heterogenous spectrum of developmental eye disorders. Recently, variants in the Wnt-pathway gene Frizzled Class Receptor 5 (FZD5) have been identified in individuals with coloboma and rarely microphthalmia, sometimes with additional phenotypes and variable penetrance. MATERIALS AND METHODS: We identified variants in FZD5 in individuals with developmental eye disorders from the UK (including the DDD Study [www.ddduk.org/access.html]), France and Spain using whole genome/exome sequencing or customized NGS panels of ocular development genes. RESULTS: We report eight new families with FZD5 variants and ocular coloboma. Three individuals presented with additional syndromic features, two explicable by additional variants in other genes (SLC12A2 and DDX3X). In two families initially showing incomplete penetrance, re-examination of apparently unaffected carrier individuals revealed subtle ocular colobomatous phenotypes. Finally, we report two families with microphthalmia in addition to coloboma, representing the second and third reported cases of this phenotype in conjunction with FZD5 variants. CONCLUSIONS: Our findings indicate FZD5 variants are typically associated with isolated ocular coloboma, occasionally microphthalmia, and that extraocular phenotypes are likely to be explained by other gene alterations.
Asunto(s)
Anoftalmos , Coloboma , Microftalmía , Humanos , Microftalmía/genética , Coloboma/diagnóstico , Coloboma/genética , Ojo , Anoftalmos/genética , Fenotipo , Receptores Frizzled/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
PURPOSE: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase overexpressed in a variety of human malignancies, against which targeted therapies have shown efficacy in lung and brain tumors. Clinical responses to EGFR inhibitors have been found to be highly dependent on the presence of activating mutations, whereas gene amplification, downstream activation of Akt, and abnormalities in PTEN are also reported predictive factors. We sought to evaluate these variables in pediatric renal tumors. EXPERIMENTAL DESIGN: We screened a series of 307 pediatric renal tumors for EGFR expression by immunohistochemistry and gene amplification by chromogenic in situ hybridization. In identifying a striking predilection for certain tumor types, we further analyzed the clear cell sarcomas of the kidney (CCSK) for mutations in EGFR and PTEN. RESULTS: Although only 23 of 177 (13.0%) nonanaplastic Wilms' tumors were EGFR positive, 4 of 11 (36.4%) anaplastic tumors showed receptor overexpression. In addition, 5 of 9 (55.6%) mesoblastic nephromas and 12 of 12 (100%) CCSKs were strongly immunoreactive for EGFR. In studying the CCSKs in more detail, we identified gene amplification in 1 of 12 (8.3%) cases and a somatic T790M EGFR mutation in a further case. These two samples additionally harbored mutations in PTEN. Downstream pathway activation, as assayed by phosphorylated Akt expression, was observed in 8 of 12 (66.7%) cases. CONCLUSIONS: Together, these data show dysregulation of the EGFR pathway at multiple levels in CCSKs. Identification of factors predictive of poor response to targeted therapy, including the drug resistance T790M mutation, may provide a rationale for upfront trials with irreversible inhibitors of EGFR in children with these tumors.
Asunto(s)
Receptores ErbB/metabolismo , Neoplasias Renales/metabolismo , Fosfohidrolasa PTEN/metabolismo , Sarcoma de Células Claras/metabolismo , Transducción de Señal , Preescolar , Amplificación de Genes , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Lactante , Neoplasias Renales/patología , Mutación , Nefroma Mesoblástico/metabolismo , Nefroma Mesoblástico/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tumor Rabdoide/metabolismo , Tumor Rabdoide/patología , Sarcoma de Células Claras/patología , Análisis de Matrices Tisulares , Tumor de Wilms/metabolismo , Tumor de Wilms/patologíaRESUMEN
Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.
Asunto(s)
Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , FMN Reductasa/genética , Helicobacter pylori/efectos de los fármacos , Metronidazol/farmacología , Nitrorreductasas/genética , Amoxicilina/farmacología , Anaerobiosis , Antiinfecciosos/farmacología , Proteínas Bacterianas/metabolismo , Cloranfenicol/farmacología , Claritromicina/farmacología , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Farmacorresistencia Bacteriana/fisiología , FMN Reductasa/metabolismo , Eliminación de Gen , Genes Bacterianos , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Nitrorreductasas/metabolismo , Análisis de Secuencia de ADN , Tetraciclina/farmacologíaRESUMEN
The outcome for children with high-grade gliomas (HGG) remains dismal, with a 2-year survival rate of only 10% to 30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in the brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet-derived growth factor receptor α (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic-activating mutations were identified in 14.4% (13 of 90) of nonbrainstem pediatric HGGs and 4.7% (2 of 43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. Forty percent of tumors with mutation showed concurrent amplification, whereas 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 nonbrainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.
Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , Mutación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Adolescente , Animales , Neoplasias Encefálicas/patología , Niño , Preescolar , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Glioma/patología , Humanos , Ratones , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
UNLABELLED: Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcriptionK27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein. SIGNIFICANCE: We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Histonas/genética , Mutación , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Adolescente , Línea Celular Tumoral , Niño , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Biología Computacional , Análisis Mutacional de ADN , Bases de Datos de Ácidos Nucleicos , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Genotipo , Glioblastoma/metabolismo , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Humanos , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Unión Proteica , Transcripción Genética , Regulación hacia ArribaRESUMEN
Intratumoral heterogeneity in human solid tumors represents a major barrier for the development of effective molecular treatment strategies, as treatment efficacies will reflect the molecular variegation in individual tumors. In glioblastoma, the generation of composite genomic profiles from bulk tumor samples has allowed one to map the genomic amplifications of putative genetic drivers and to prioritize therapeutic targeting strategies aimed at eradicating the tumor burden. Notably, amplification of multiple receptor tyrosine kinases (RTK) within a single tumor specimen obtained from patients is frequently observed. In this study, use of a detailed multicolor FISH mapping procedure in pathologic specimens revealed a mutual exclusivity of gene amplification in the majority of glioblastoma tumors examined. In particular, the two most commonly amplified RTK genes, EGFR and PDGFRA, were found to be present in variable proportions across the tumors, with one or the other gene predominating in certain areas of the same specimen. Our findings have profound implications for designing efficacious therapeutic regimens, as it remains unclear that how the cells with different gene amplification events contribute to disease propagation or the response to molecular targeted therapies.
Asunto(s)
Neoplasias Encefálicas/genética , Receptores ErbB/genética , Amplificación de Genes , Glioblastoma/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Dosificación de Gen , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is one of the most frequent malignant pediatric brain tumor and its prognosis is universaly fatal. No significant improvement has been made in last thirty years over the standard treatment with radiotherapy. To address the paucity of understanding of DIPGs, we have carried out integrated molecular profiling of a large series of samples obtained with stereotactic biopsy at diagnosis. While chromosomal imbalances did not distinguish DIPG and supratentorial tumors on CGHarrays, gene expression profiling revealed clear differences between them, with brainstem gliomas resembling midline/thalamic tumours, indicating a closely-related origin. Two distinct subgroups of DIPG were identified. The first subgroup displayed mesenchymal and pro-angiogenic characteristics, with stem cell markers enrichment consistent with the possibility to grow tumor stem cells from these biopsies. The other subgroup displayed oligodendroglial features, and appeared largely driven by PDGFRA, in particular through amplification and/or novel missense mutations in the extracellular domain. Patients in this later group had a significantly worse outcome with an hazard ratio for early deaths, ie before 10 months, 8 fold greater that the ones in the other subgroup (p = 0.041, Cox regression model). The worse outcome of patients with the oligodendroglial type of tumors was confirmed on a series of 55 paraffin-embedded biopsy samples at diagnosis (median OS of 7.73 versus 12.37 months, p = 0.045, log-rank test). Two distinct transcriptional subclasses of DIPG with specific genomic alterations can be defined at diagnosis by oligodendroglial differentiation or mesenchymal transition, respectively. Classifying these tumors by signal transduction pathway activation and by mutation in pathway member genes may be particularily valuable for the development of targeted therapies.
Asunto(s)
Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Transición Epitelial-Mesenquimal/genética , Perfilación de la Expresión Génica/métodos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Neoplasias del Tronco Encefálico/patología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Mutación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
High grade gliomas (HGG) are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI) in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults) using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7%) pediatric cases, significantly more than observed in adults (5/73, 6.8%; pâ=â0.02, Chi-square test). MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI.
Asunto(s)
Silenciador del Gen , Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Glioma/genética , Inestabilidad de Microsatélites , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Roturas del ADN de Doble Cadena , Reparación de la Incompatibilidad de ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Mutación del Sistema de Lectura/genética , Humanos , Lactante , Proteína Homóloga de MRE11 , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo Genético , Adulto JovenRESUMEN
PURPOSE: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. EXPERIMENTAL DESIGN: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (<23 years). RESULTS: The genomic profiles of these tumors could be subclassified into four categories: those with stable genomes, which were associated with a better prognosis; those with aneuploid and those with highly rearranged genomes; and those with an amplifier genotype, which had a significantly worse clinical outcome. Independent of this was a clear segregation of cases with 1q gain (more common in children) from those with concurrent 7 gain/10q loss (a defining feature of adults). Detailed mapping of all the amplification and deletion events revealed numerous low-frequency amplifications, including IGF1R, PDGFRB, PIK3CA, CDK6, CCND1, and CCNE1, and novel homozygous deletions encompassing unknown genes, including those at 5q35, 10q25, and 22q13. Despite this, aberrations targeting the "core signaling pathways" in adult glioblastomas are significantly underrepresented in the pediatric setting. CONCLUSIONS: These data highlight that although there are overlaps in the genomic events driving gliomagenesis of all ages, the pediatric disease harbors a distinct spectrum of copy number aberrations compared with adults.
Asunto(s)
Neoplasias Encefálicas/genética , Variaciones en el Número de Copia de ADN , Glioma/genética , Adolescente , Adulto , Niño , Preescolar , Hibridación Genómica Comparativa , Amplificación de Genes , Eliminación de Gen , Glioblastoma/genética , HumanosRESUMEN
Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.
Asunto(s)
Dacarbazina/análogos & derivados , Proteínas de Homeodominio/genética , O(6)-Metilguanina-ADN Metiltransferasa/genética , Fosfatidilinositol 3-Quinasas/genética , Antineoplásicos Alquilantes/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Niño , Análisis por Conglomerados , Metilación de ADN/efectos de los fármacos , Dacarbazina/farmacología , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Proteínas Homeobox A10 , Proteínas de Homeodominio/metabolismo , Humanos , Concentración 50 Inhibidora , Células Madre Neoplásicas/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas/genética , TemozolomidaRESUMEN
PURPOSE: To define copy number alterations and gene expression signatures underlying pediatric high-grade glioma (HGG). PATIENTS AND METHODS: We conducted a high-resolution analysis of genomic imbalances in 78 de novo pediatric HGGs, including seven diffuse intrinsic pontine gliomas, and 10 HGGs arising in children who received cranial irradiation for a previous cancer using single nucleotide polymorphism microarray analysis. Gene expression was analyzed with gene expression microarrays for 53 tumors. Results were compared with publicly available data from adult tumors. RESULTS: Significant differences in copy number alterations distinguish childhood and adult glioblastoma. PDGFRA was the predominant target of focal amplification in childhood HGG, including diffuse intrinsic pontine gliomas, and gene expression analyses supported an important role for deregulated PDGFRalpha signaling in pediatric HGG. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Pediatric and adult glioblastomas were clearly distinguished by frequent gain of chromosome 1q (30% v 9%, respectively) and lower frequency of chromosome 7 gain (13% v 74%, respectively) and 10q loss (35% v 80%, respectively). PDGFRA amplification and 1q gain occurred at significantly higher frequency in irradiation-induced tumors, suggesting that these are initiating events in childhood gliomagenesis. A subset of pediatric HGGs showed minimal copy number changes. CONCLUSION: Integrated molecular profiling showed substantial differences in the molecular features underlying pediatric and adult HGG, indicating that findings in adult tumors cannot be simply extrapolated to younger patients. PDGFRalpha may be a useful target for pediatric HGG, including diffuse pontine gliomas.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Perfilación de la Expresión Génica , Glioma/genética , Polimorfismo de Nucleótido Simple/genética , Adolescente , Adulto , Neoplasias Encefálicas/patología , Niño , Preescolar , Cromosomas Humanos Par 1/genética , Irradiación Craneana , Glioma/patología , Humanos , Lactante , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Adulto JovenRESUMEN
BACKGROUND: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines. PRINCIPAL FINDINGS: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response. SIGNIFICANCE: These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.
Asunto(s)
Línea Celular Tumoral , Diseño de Fármacos , Glioma , Fenotipo , Adulto , Astrocitos/metabolismo , Biomarcadores/metabolismo , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/patología , Niño , Aberraciones Cromosómicas , Activación Enzimática , Epigénesis Genética , Perfilación de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Inmunofenotipificación , Pérdida de Heterocigocidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
PURPOSE: The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG). EXPERIMENTAL DESIGN: We retrospectively studied a total of 90 archival pediatric HGG specimens for EGFR protein overexpression, gene amplification, and mutation and assessed the in vitro sensitivity of pediatric glioma cell line models to the small-molecule EGFR inhibitor erlotinib. RESULTS: Amplification was detected in 11% of cases, with corresponding overexpression of the receptor. No kinase or extracellular domain mutations were observed; however, 6 of 35 (17%) cases harbored the EGFRvIII deletion, including two anaplastic oligodendrogliomas and a gliosarcoma overexpressing EGFRvIII in the absence of gene amplification and coexpressing platelet-derived growth factor receptor alpha. Pediatric glioblastoma cells transduced with wild-type or deletion mutant EGFRvIII were not rendered more sensitive to erlotinib despite expressing wild-type PTEN. Phosphorylated receptor tyrosine kinase profiling showed a specific activation of platelet-derived growth factor receptor alpha/beta in EGFRvIII-transduced pediatric glioblastoma cells, and targeted coinhibition with erlotinib and imatinib leads to enhanced efficacy in this model. CONCLUSIONS: These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric HGG than previously recognized and show the likely necessity of targeting multiple genetic alterations in the tumors of these children.