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Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma.Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity.Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity.Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

Supplementary Figures S1-S6; Supplementary Table S1.

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in theCrb1gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-β and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia.

International audience; There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

Item does not contain fulltext The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.

Background Data on patients affected by chronic mucocutaneous candidiasis underscore the preponderant role of IL-17 receptor A (IL-17RA) in preserving mucocutaneous immunity. Little is known about the role of adenosine deaminase (ADA) 2 in regulation of immune responses, although recent reports linked ADA2 deficiency with inflammation and vasculitis. Objective We sought to investigate the mechanisms of chronic inflammation and vasculitis in a child lacking IL-17RA and ADA2 to identify therapeutic targets. Methods We report a family with 2 siblings who have had recurrent mucocutaneous infections with Candida albicans and Staphylococcus aureus and chronic inflammatory disease and vasculitis since early childhood, which were refractory to classical treatments. Array-based comparative genomic hybridization analysis showed that both siblings are homozygous for a 770-kb deletion on chr22q11.1 encompassing both IL17RA and cat eye critical region 1 ( CECR1 ). Immunologic studies were carried out by means of flow cytometry, ELISA, and RIA. Results As expected, in the affected child we found a lack of IL-17RA expression, which implies a severe malfunction in the IL-17 signaling pathway, conferring susceptibility to recurrent mucocutaneous infections. Surprisingly, we detected an in vitro and in vivo upregulation of proinflammatory cytokines, notably IL-1β and TNF-α, which is consistent with the persistent systemic inflammation. Conclusions This work emphasizes the utility of whole-genome analyses combined with immunologic investigation in patients with unresolved immunodeficiency. This approach is likely to provide an insight into immunologic pathways and mechanisms of disease. It also provides molecular evidence for more targeted therapies. In addition, our report further corroborates a potential role of ADA2 in modulating immunity and inflammation.