The detection of a strong episignature for Chung–Jansen syndrome, partially overlapping with Börjeson–Forssman–Lehmann and White–Kernohan syndromes

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung–Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung–Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White–Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson–Forssman–Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung–Jansen, Börjeson–Forssman–Lehmann and White–Kernohan syndromes.</p

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Reciprocal Regulation of Shh Trafficking and H₂O₂ Levels via a Noncanonical BOC-Rac1 Pathway

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability

A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

International audienceFar-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially nonfluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far-red fluorescence, frFAST allowed the design of a farred chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors

Reciprocal Regulation of Shh Trafficking and H2O2 Levels via a Noncanonical BOC-Rac1 Pathway

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H2O2) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H2O2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H2O2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H2O2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability

H2O2 and Engrailed 2 paracrine activity synergize to shape the zebrafish optic tectum

International audienceAlthough a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H 2 O 2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H 2 O 2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H 2 O 2 could participate in the patterning of the embryo. We find that perturbations of endogenous H 2 O 2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H 2 O 2 levels and taken up by cells with low H 2 O 2 levels where it leads to increased H 2 O 2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events

A di-Copper Peptidyl Complex Mimics the Activity of Catalase, a Key Antioxidant Metalloenzyme

International audienceCatalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H 2 O 2 concentration by accelerating its dismutation. Many physio-pathological situations are associated with oxidative stress resulting from H 2 O 2 overproduction during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solution and in cells using different analytical methods. The complex exhibited CAT activity in solution and, more interestingly, on HyPer HeLa cells which possess a genetically encoded ratiometric fluorescent sensors of H 2 O 2. These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity

Evaluation of the compounds commonly known as superoxide dismutase and catalase mimics in cellular models

International audienceOxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) mesotetrakis(N-(2′-nbutoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP 5+) and Mn(II) dichloro[(4aR,13aR,17aR,21aR

A Di Copper Peptidyl Complex Mimics Catalase Activity in HeLa HyPer Cells

In this manuscript, we describe the implementation of a combinatorial apporach to synthesize a library of copper complexes, associated with an activity-based screening to discover the first peptidyl di copper complex mimicking CAT redox chemistry. The selected dinuclear complex was studied in detail and characterized for its CAT activity in vitro and in cells. Very interestingly, despite moderate intrinsic catalysis constant, this complex was efficacious in a cellular assay.<br /