RESUMO
The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Glioma/patologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Glioblastoma/metabolismo , Glioma/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/metabolismo , Pediatria , Tecnologia/métodosRESUMO
Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K(+) channels.
Assuntos
Anormalidades Múltiplas/genética , Deficiências do Desenvolvimento/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Deficiência Intelectual/genética , Modelos Moleculares , Anormalidades Múltiplas/patologia , Sequência de Bases , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Primers do DNA/genética , Deficiências do Desenvolvimento/patologia , Exoma/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Humanos , Deficiência Intelectual/patologia , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Análise de Sequência de DNA , Deleção de Sequência/genética , SíndromeRESUMO
Exome sequencing has led to the comprehension of the molecular bases of several forms of neurodevelopmental disorders, a clinically heterogeneous group of diseases characterized by intellectual disability (ID) and autism spectrum disorder (ASD). De novo mutations in POGZ has been causally linked to isolated ASD and syndromic ID, only recently. Here we report on a 15 year-old girl in whom exome sequencing allowed to identify a de novo POGZ truncating mutation as the molecular cause underlying a complex phenotype apparently not fitting any recognized syndrome. We describe the evolution of her clinical features with age, and review published clinical data of patients with POGZ mutations to systematically analyze the clinical spectrum associated with mutations. Our finding expands the clinical and molecular spectrum of POGZ mutations. Revision of the literature indicate that moderate to severe ID, microcephaly, variable CNS malformations, reduced growth, brachytelephalangy, and facial dysmorphism represent recurrent features associated with POGZ mutations.
RESUMO
BACKGROUND: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. METHODS: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. RESULTS: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. CONCLUSION: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.
Assuntos
Neoplasias do Tronco Encefálico , Glioma , Imunoterapia Adotiva , Receptor IGF Tipo 1 , Receptor de Insulina , Neoplasias do Tronco Encefálico/patologia , Criança , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Humanos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Linfócitos T/metabolismoRESUMO
Nephropathic cystinosis (NC) is an autosomal recessive disorder caused by mutations of the CTNS gene that encodes for a cystine transmembrane transporter. Several mutations have been described in the coding and promoter regions of the CTNS gene in affected individuals. We selected three patients with NC from two unrelated families, in whom sequence analysis of the CTNS gene detected only one or no mutations. Total RNA was isolated from peripheral blood mononuclear cells or fibroblasts and CTNS transcripts were analyzed. We observed a skipping of exon 5 (85 bp) in two siblings and an intron 9 retention of 75 bp associated with partial replication of exon 9 in the third patient. Genomic DNA analysis of intron regions surrounding exon 5 showed a point mutation in the hypothetical lariat branch site of intron 4 at position -24 (c.141-24 T > C) in the first two patients and a duplication of 266 bp including a part of exon and intron 9 in the third patient. Analysis of CTNS gene transcripts allowed identification of mutations in patients in whom CTNS mutations could not be detected by traditional DNA sequencing. These results support the hypothesis that cystinosis is a monogenic disorder.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinose/genética , Rim/patologia , Mutação Puntual , Processamento Alternativo , Pré-Escolar , Cistinose/patologia , DNA/análise , Análise Mutacional de DNA , Feminino , Fibroblastos/química , Fibroblastos/patologia , Duplicação Gênica , Humanos , Lactente , Leucócitos Mononucleares/química , Leucócitos Mononucleares/patologia , MasculinoRESUMO
Classic nephropathic or infantile cystinosis (NC) is an autosomal recessive disorder; the gene coding for the integral membrane protein cystinosin, which is responsible for membrane transport of cystine (CTNS), was cloned. Mutation analysis of the CTNS gene of Caucasian patients revealed a common 57-kb deletion, and several other mutations spread throughout the entire gene. In the present study, we report the CTNS mutations identified in 42 of 46 Italian families with NC. The percentage of mutations characterized in this study is 86%. The mutational spectrum of the Italian population is different from that of populations of North European origin: the 57-kb deletion is present in a lower percentage, while the splicing mutations represent 30% of mutation detected in our sample. In all, six novel mutations have been identified, and the origin of one recurrent mutation has been traced.
Assuntos
Cistinose/genética , Glicoproteínas , Proteínas de Membrana/genética , Adolescente , Adulto , Sistemas de Transporte de Aminoácidos Neutros , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Itália , Masculino , Proteínas de Membrana Transportadoras , Polimorfismo Conformacional de Fita SimplesRESUMO
Aims: To screen cases of infantile cystinosis among different forms of proximal renal tubular acidosis (RTA). Study Design: Cross sectional. Place and Duration of Study: From a total of 25 families of RTA followed up in Nephrology unit of Mansoura University Children's Hospital (MUCH), Egypt, two unrelated families were diagnosed as infantile nephropathic cystinosis using clinical suspicion plus mutation analysis of CTNS gene in the period between January 2008 and November 2012. Methodology: Two families with multiple cases of infantile nephropathic cystinosis have been diagnosed. In absence of high-performance liquid chromatography and tandem mass spectrometry used for measuring intraleucocyte cystine, diagnostic tools for cystinosis used in the current work were clinical and laboratory evidences of PRTA, slit lamp detection of corneal cystine crystals and finally identification of CTNS gene mutations. All patients were subjected to routine echocardiography because of accidental discovery of heart malformation in one case. Rare mutant variant of the first family was subjected to RNA analysis which unfortunately failed, alternatively an in silico study was used to predict splice site. Results: All patients with cystinosis manifested a severe clinical course. Proband of family 1 showed two known mutations; deletion in the exon 3 (c.18_21 del GACT) and substitution in acceptor splice site of intron 11 (c.971 -12G>A). In silico study predicted an anticipated splice site that modified the open reading frame in carboxy-terminal region. Probands of family 2 were affected by ventricular and atrial septal defects in younger, and mild mitral and aortic incompetence in older patient; their DNA analysis revealed a novel nonsense mutation (c.734 G>A) which caused a premature stop codon in position 245 of protein. Conclusion: Nephropathic cystinosis has been diagnosed with ease in Egyptian population without need of sophisticated investigations. A novel mutation had been added to the list of CTNS gene variants.
RESUMO
Bethlem myopathy is an early-onset benign myopathy characterized by proximal muscular weakness and multiple flexion contractures. It is a dominantly inherited disorder associated with mutations in the three COL6 genes encoding type VI collagen. We detected a g-->a substitution at +1 position of COL6A1 intron 3 in a four-generation Italian family affected by a mild form of Bethlem myopathy. The mutation results in the activation of a cryptic splice donor site at the 3' end of exon 3, leading to the loss of 66 nucleotides and an "in-frame" deletion of 22 amino acids in the NH2-domain. Molecular analysis on fibroblasts of the propositus showed that the mutated mRNA was present and stable, but the mutated protein could not be detected. Western blot and immunofluorescence analyses showed a decreased level of collagen VI synthesis and deposition in fibroblasts of the propositus. Together, the results suggest that the mutated protein was highly unstable and rapidly degraded, and that the mild phenotype was caused by a reduced amount of normal collagen VI microfibrils. In addition, we demonstrated that lymphocytes can be used for the first mutation screening analysis of patients with Bethlem myopathy.