Methylation-associated PHOX2B gene silencing is a rare event in human neuroblastoma.

International audienceNeuroblastoma (NB), an embryonic tumour originating from neural crest cells, is one of the most common solid tumours in childhood. Although NB is characterised by numerous recurrent, large-scale chromosome rearrangements, the genes targeted by these imbalances have remained elusive. We recently identified the paired-like homeobox 2B (PHOX2B, MIM 603851) gene as disease-causing in dysautonomic disorders including Congenital Central Hypoventilation Syndrome (CCHS), Hirschsprung disease (HSCR) and NB in various combinations. Most patients with NB due to a germline heterozygous PHOX2B gene mutation are familial and/or syndromic. PHOX2B, at chromosome 4p12, does not lie in a commonly rearranged locus in NB. To evaluate the role of PHOX2B in sporadic, isolated NB, we analysed 13 NB cell lines and 45 tumours for expression, mutations of coding and promoter sequences, loss of heterozygosity (LOH), or aberrant hypermethylation of PHOX2B (13 cell lines and 18 tumours). We didn't identify any mutation but LOH in about 10% of the cases and aberrant CpG dinucleotide methylation of the 500 bp PHOX2B promoter region in 4/31 tumours and cell lines (12.9%). Altogether, both germinal and somatic anomalies at the PHOX2B locus are found in NB

A review of Dynamin 2 involvement in cancers highlights a promising therapeutic target

International audienceDynamin 2 (DNM2) is an ubiquitously expressed large GTPase well known for its role in vesicle formation in endocytosis and intracellular membrane trafficking also acting as a regulator of cytoskeletons. During the last two decades, DNM2 involvement, through mutations or overexpression, emerged in an increasing number of cancers and often associated with poor prognosis. A wide panel of DNM2-dependent processes was described in cancer cells which explains DNM2 contribution to cancer pathomechanisms. First, DNM2 dysfunction may promote cell migration, invasion and metastasis. Second, DNM2 acts on intracellular signaling pathways fostering tumor cell proliferation and survival. Relative to these roles, DNM2 was demonstrated as a therapeutic target able to reduce cell proliferation, induce apoptosis, and reduce the invasive phenotype in a wide range of cancer cells in vitro. Moreover, proofs of concept of therapy by modulation of DNM2 expression was also achieved in vivo in several animal models. Consequently, DNM2 appears as a promising molecular target for the development of anti-invasive agents and the already provided proofs of concept in animal models represent an important step of preclinical development

Etude du gène PHOX2B dans des anomalies de développement du système nerveux autonome

L Hypoventilation Alvéolaire Centrale Congénitale (HVACC, MIM209880) est une maladie du contrôle autonome de la ventilation. Elle s associée à la maladie de Hirschsprung dans 20% des cas et prédispose au neuroblastome (NB). Paired-like Homeobox 2B (PHOX2B) est le gène majeur de l HVACC. J ai réuni une série de plus de 300 patients et identifié une mutation hétérozygote de novo de PHOX2B dans plus de 90% des cas. Les mutations sont de trois types: i) expansion de +5 à +13 alanines, ii) mutations décalage de phase iii) faux sens de l homéodomaine. Des corrélations génotype/phénotype ont pu être établies et le spectre phénotypique élargi aux formes retardées d HVAC et à des NB familiaux et/ou syndromique. Une étude fonctionnelle in vitro de mutants des 3 types, a permis d observer une diminution de la transactivation de promoteurs cibles de PHOX2B corrélée à la taille de l expansion et, une anomalie de conformation des protéines mutées allant de l oligomérisation à l agrégation.Congenital Central Hypoventilation Syndrome (CCHS, Ondine's curse, MIM209880) is a disorder of autonomic respiratory control. The range of dysautonomia is wide and includes Hirschsprung disease (HSCR) in 20% of the cases and predisposition to neuroblastoma (NB). Paired-like Homeobox 2B (PHOX2B) is the major disease-causing gene. We collected 300 patients and identified a de novo heterozygous mutation in 90% of the cases. Mutations are of 3 main types: i) polyalanine expansions ranging from +5 to +13 alanines ii) frameshift mutations and iii) homeodomain missense mutations. Genotype/Phenotype correlations could be drawn and the clinical presentation encompasses late onset CHS and familial and/or syndromic NB. Finally, in vitro functional studies showed a decrease PHOX2B transcriptionnal activity correlated with the size of the alanine expansion, as well as protein misfolding of expansion and frameshift mutations ranging from oligomerization to aggregation.PARIS-BIUP (751062107) / SudocSudocFranceF

Therapy for Dominant Inherited Diseases by Allele-Specific RNA Interference: Successes and Pitfalls

International audienceRNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called “allele-specific-RNA interference” devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in Pachyonychia Congenita, an inherited skin disorder due to dominant mutations in the Keratin 6 gene. Our purpose is to review the successes of this strategy aiming to treat dominant inherited diseases and to highlight the pitfalls to avoid

Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development

Data from: Local predation risk and matrix permeability interact to shape movement strategy

In fragmented landscapes, the reduced connectivity among patches drives the evolution of movement strategies through an increase of transience costs. Reduced movements may further alter heterogeneity in biotic and abiotic conditions experienced by individuals. The joint action of local conditions and matrix permeability may shape emigration decisions. Here, we tested the interactive effects of predation risk and matrix permeability on movement propensity, movement costs and movers’ phenotype in the common toad Bufo bufo. In a full-crossed experimental design, we assessed the movement propensity of juveniles in three connectivity treatments (from poorly to highly permeable matrix), with or without predation risk in their living patch. We also assessed the relationships between movement propensity and morphological traits (i.e. body and leg length) and how it affected the movement cost (i.e. mass loss). Movement propensity increased in presence of predation risk, while matrix permeability had no effect. However, matrix permeability interacted with predation risk to influence movers’ phenotype and the physiological cost they endured while moving. In particular, a well-known movement syndrome in toads (i.e. movement propensity positively related to longer legs) depended on the interaction between matrix permeability and predation risk and resulted in differences in mass loss among matrix types. Movers lost more mass on average than residents except when they also displayed longer legs or when they crossed the most permeable matrix in the presence of predation risk. Our results show that matrix permeability shapes the physiological cost of dispersal by changing the identity of individuals moving away from local conditions. As the movers’ phenotype can importantly alter (meta)population dynamics, context-dependency of dispersal syndromes should be considered in studies predicting the functioning of human-altered natural systems

Local predation risk and matrix permeability interact to shape movement strategy

International audienceIn fragmented landscapes, the reduced connectivity among patches drives the evolution of movement strategies through an increase of transience costs. Reduced movements may further alter heterogeneity in biotic and abiotic conditions experienced by individuals. The joint action of local conditions and matrix permeability may shape emigration decisions. Here, we tested the interactive effects of predation risk and matrix permeability on movement propensity, movement costs and movers’ phenotype in the common toad Bufo bufo. In a full‐crossed experimental design, we assessed the movement propensity of juveniles in three connectivity treatments (from poorly to highly permeable matrix), with or without predation risk in their living patch. We also assessed the relationships between movement propensity and morphological traits (i.e. body and leg length) and how it affected the movement cost (i.e. mass loss). Movement propensity increased in presence of predation risk, while matrix permeability had no effect. However, matrix permeability interacted with predation risk to influence movers’ phenotype and the physiological cost they endured while moving. In particular, a well‐known movement syndrome in toads (i.e. movement propensity positively related to longer legs) depended on the interaction between matrix permeability and predation risk and resulted in differences in mass loss among matrix types. Movers lost more mass on average than residents except when they also displayed longer legs or when they crossed the most permeable matrix in the presence of predation risk. Our results show that matrix permeability shapes the physiological cost of dispersal by changing the identity of individuals moving away from local conditions. As the movers’ phenotype can importantly alter (meta)population dynamics, context‐dependency of dispersal syndromes should be considered in studies predicting the functioning of human‐altered natural systems

Development of versatile allele-specific siRNAs able to silence all the dominant dynamin 2 mutations

International audienceDominant centronuclear myopathy (CNM) is a rare form of congenital myopathy associated with a wide clinical spectrum, from severe neonatal to milder adult forms. There is no available treatment for this disease due to heterozygous mutations in the DNM2 gene encoding Dynamin 2 (DNM2). Dominant DNM2 mutations also cause rare forms of Charcot-Marie-Tooth disease and hereditary spastic paraplegia, and deleterious DNM2 overexpression was noticed in several diseases. The proof of concept for therapy by allele-specific RNA interference devoted to silence the mutated mRNA without affecting the normal allele was previously achieved in a mouse model and patient-derived cells, both expressing the most frequent DNM2 mutation in CNM. In order to have versatile small interfering RNAs (siRNAs) usable regardless of the mutation, we have developed allele-specific siRNAs against two non-pathogenic single-nucleotide polymorphisms (SNPs) frequently heterozygous in the population. In addition, allele-specific siRNAs against the p.S619L DNM2 mutation, a mutation frequently associated with severe neonatal cases, were developed. The beneficial effects of these new siRNAs are reported for a panel of defects occurring in patient-derived cell lines. The development of these new molecules allows targeting the large majority of the patients harboring DNM2 mutations or overexpression by only a few siRNAs

Population sex ratio and dispersal in experimental, two-patch metapopulations of butterflies

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Spatial genetic structure of Lissotriton helveticus L. following the restoration of a forest ponds network

International audiencePreserving amphibian genetic diversity through ecological restoration and conservation actions is a major challenge since their populations are declining worldwide. We studied the genetic diversity and spatial genetic structure of the palmate newt (Lissotriton helveticus) 2 years after the restoration of a pond network in northwestern France with the aim of reconstructing fine-scale genetic structure and patterns of colonization. We sampled newts from 29 forest ponds including both restored and non-degraded reference ponds, and genotyped 391 individuals at 12 microsatellite loci. We used two Bayesian clustering methods to spatially delineate genetic clusters, and we also detected potential recent migrants within the network. All ponds showed low levels of observed heterozygosity (Ho = 0.534) and a mean F IS of 0.251, possibly indicating a Wahlund or bottleneck effect. Pairwise F ST suggested limited evidence of genetic differentiation among ponds. Within the pond network, we identified 3 to 4 genetic clusters. Combined with the detection of migrants, the results suggest an increase in gene flow within the restored pond network and that a high number of migrants came from the reference ponds. Our findings indicate an unexpected high dispersal ability for this small-bodied species. Overall, the absence of population structure represents a positive beginning for the restoration project. It also emphasizes the importance of spatial design in restoring a pond network and that such genetic data and methods should be used to monitor amphibians in restored habitats