Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex

Application in a biomonitoring context of three-spined stickleback immunomarker reference ranges

International audienceThe relevance of a biomarker for biomonitoring programs was influenced both by the knowledge on biomarker natural inter-individual and site variabilities and by the sensitivity of the biomarker towards environmental perturbations. To minimize data misinterpretation, robustness reference values for biomarkers were important in biomonitoring programs. Specific three-spined stickleback, Gasterosteus aculeatus, immune reference ranges for field studies had been determined based on laboratory data and one reference station (Contentieuse river at Houdancourt). In this study, data obtained in one uncontaminated and three contaminated sites were compared to these reference ranges as a validation step before considering them for larger scale biomonitoring programs. When the field reference range were compared to data from the uncontaminated station (Béronelle), only few deviations were shown. In this way, data coming from uncontaminated station (Béronelle) was integrated in the field reference ranges to improve the evaluation of site variability. The new field reference ranges provided better discrimination of sites and spanned a larger range of fish lengths than the initial reference ranges. Furthermore, the results suggest lysosomal presence during several months and phagocytosis capacity in autumn may be the most relevant immunomarkers towards identifying contaminated sites. In the future, combining this reference value approach with active biomonitoring could facilitate the obtention of data in multiple stream conditions

Biomarqueurs de génotoxicité chez l’épinoche à trois épines pour une application en biosurveillance de la qualité des milieux aquatiques

International audienc

Reliability evaluation of biomarker reference ranges for mesocosm and field conditions: Cellular innate immunomarkers in Gasterosteus aculeatus

Due to their sensitivity to environmental contamination and their link with fish health status, innate immunomarkers are of great interest for environmental risk assessment studies. Nevertheless, the lack of knowledge about the effect of confounding factors can lead to data misinterpretation and false diagnostics. So, the determination of reference values was of huge interest for the integration of biomarkers in biomonitoring programs. Laboratory immunomarker reference ranges (including cellular mortality, leucocyte distribution, phagocytosis activity, respiratory burst and lysosomal presence) that consider three confounding factors (season, sex and body size) were previously developed in three-spined stickleback, Gasterosteus aculeatus, from our husbandry. Usefulness of these reference ranges in biomonitoring programs depends on how they can be transposed to various experimental levels, such as mesocosm (outdoor artificial pond) and field conditions. Immunomarkers were therefore measured every 2 months over 1 year in one mesocosm and in one site assumed to uncontaminated (Houdancourt, field). Differences between immunomarker seasonal variations in mesocosm and field fish on one side and laboratory fish on the other side were quantified: in some cases, seasonal trends were not significant or did not differ between mesocosm and laboratory conditions, but overall, models developed based on data obtained in laboratory conditions were poorly predictive of data obtained in mesocosm or field conditions. To propose valuable field reference ranges, mesocosm and field data were integrated in innate immunomarker modelling in order to strengthen the knowledge on the effect of confounding factors. As in laboratory conditions, sex was overall a confounding factor only for necrotic cell percentage and granulocyte-macrophage distribution and size was a confounding factor only for cellular mortality, leucocyte distribution and phagocytosis activity. Confounding factors explained a large proportion of immunomarker variability in particular for phagocytosis activity and lysosomal presence. Further research is needed to test the field models in a biomonitoring program to compare the sensitivity of immunomarkers to the confounding factors identified in this study and the sensitivity to various levels of pollution

Optimization of an active biomonitoring approach using three-spined sticklebacks

Active biomonitoring involves exposure of calibrate and well-known organisms to environmental pollution and is increasingly used in ecological studies. Despite of the advantages of caging (standardization of some biotic and abiotic parameters), caged organisms are subject to some constraints which can influence the organisms’ responses to pollution and therefore can be considered as confounding factors. In the way to make biomarkers efficient tools in active biomonitoring, it is necessary to precisely characterize the possible effects of constraints linked to caging on organism physiology. For this purpose, several studies were conducted on threeBspined sticklebacks (Gasterosteus aculeatus) to specify the impacts of i) fish-density inside cages, ii) short-transport after caging, iii) movement limitation and iv) food access restriction induced by caging. The optimization of caging conditions has been validated by assessment of several biomarkers including innate immune responses, antioxidant systems, metabolic detoxification, synaptic transmission and energy metabolism. Results have shown that a high density of fish (> 2.31 kg/m3) and a short transport after caging have induced an oxidative stress manifested by an overproduction of reactive oxygen species and a stimulation of the antioxidant system. The confinement stress had small impact on sticklebacks’ biomarkers. However, this stress seemed to accentuate the negative effects of food restriction on these biomarkers, particularly during their breeding period. Out of breeding time, most of investigate biomarkers were not impacted by food access restriction. These works have allowed to specify the conditions of application and interpretation of biomarkers during active monitoring using stickleback to ensure a good diagnosis of the quality of water bodies with this species

Development of a multi-biomarker approach of genotoxicity on the three spined stickleback for aquatic biomonitoring application

International audienceThe aquatic environment is considered as the receptacle of anthropogenicpressures, including chemical contamination as genotoxic compounds, which canimpact the health of aquatic species. Genotoxic substances may directly or indirectlyaffect the integrity of cell’s genetic material, which can lead to long-term mutagenic,carcinogenic or teratogenic effects. In this context, the measure of damages at differentgenomic scales may be relevant and provide an integrated view of the genotoxic risk ofexposed organism. The finality of our work is to propose their use as early-warningsignals of potential long-term alterations to population and ecosystem health.Genotoxicity assessment was performed on erythrocytes of the three- spinedstickleback by combining the measure of DNA integrity by the alkaline and Fpg(formamidopyrimidines DNA glycosylase)-modified comet assays and the measure ofvariations in nuclear DNA content by flow cytometry (FCM). In this sense, blood cells offish offer many advantages as cellular models as their ease of collection, gives a highdensity of erythrocytes already dissociated allowing to obtain multiple assays on thesame sample. The alkaline comet assay has been largely used with erythrocytes of fishto detect alkali-labile sites, DNA-double- and single- strand breaks. The Fpg-modifiedcomet assay has been optimized in the present study, which expand the comet assaysensitivity to oxidized pyrimidines. Furthermore, FCM may be applied to assess thevariations in the nuclear DNA content of a large population of cells and has alreadydemonstrated its relevance for detecting chromosomal damages in blood cells of fish.The presentation will detail results regarding the definition of baseline levels ofgenotoxicity biomarkers (DNA strand breaks, Fpg-sensitive sites, DNA content,erythrocyte mortality), which are useful in an ecotoxicological context. These biomarkershave been measured in various field context (active biomonitoring) and integrated in theexisting multi-biomarker approach of the three-spined stickleback (Gasterosteusaculeatus L., 1758)

Optimization of an active biomonitoring approach using three-spined sticklebacks

Active biomonitoring involves exposure of calibrate and well-known organisms to environmental pollution and is increasingly used in ecological studies. Despite of the advantages of caging (standardization of some biotic and abiotic parameters), caged organisms are subject to some constraints which can influence the organisms’ responses to pollution and therefore can be considered as confounding factors. In the way to make biomarkers efficient tools in active biomonitoring, it is necessary to precisely characterize the possible effects of constraints linked to caging on organism physiology. For this purpose, several studies were conducted on threeBspined sticklebacks (Gasterosteus aculeatus) to specify the impacts of i) fish-density inside cages, ii) short-transport after caging, iii) movement limitation and iv) food access restriction induced by caging. The optimization of caging conditions has been validated by assessment of several biomarkers including innate immune responses, antioxidant systems, metabolic detoxification, synaptic transmission and energy metabolism. Results have shown that a high density of fish (> 2.31 kg/m3) and a short transport after caging have induced an oxidative stress manifested by an overproduction of reactive oxygen species and a stimulation of the antioxidant system. The confinement stress had small impact on sticklebacks’ biomarkers. However, this stress seemed to accentuate the negative effects of food restriction on these biomarkers, particularly during their breeding period. Out of breeding time, most of investigate biomarkers were not impacted by food access restriction. These works have allowed to specify the conditions of application and interpretation of biomarkers during active monitoring using stickleback to ensure a good diagnosis of the quality of water bodies with this species

Application of the Fpg-modified comet assay on three-spined stickleback in freshwater biomonitoring: toward a multi-biomarker approach of genotoxicity

International audienceAquatic species are exposed to a wide spectrum of substances, which can compromise their genomic integrity by inducing DNA damage or oxidative stress. Genotoxicity biomarkers as DNA strand breaks and chromosomal damages developed on sentinel species have already proved to be relevant in aquatic biomonitoring. However, these biomarkers do not reflect DNA oxidative lesions, i.e., the 8-oxodG, recognized as pre-mutagenic lesion if not or mis-repaired in human biomonitoring. The relevance to include the measure of these lesions by using the Fpg-modified comet assay on erythrocytes of the three-spined stickleback was investigated. An optimization step of the Fpg-modified comet assay considering enzyme buffer impact, Fpg concentration, and incubation time has been performed. Then, this measure was integrated in a battery of genotoxicity and cytotoxicity biomarkers (considering DNA strand breaks, DNA content variation, and cell apoptosis/necrosis and density) and applied in a freshwater monitoring program on six stations of the Artois Picardie watershed (3-week caging of control fish). These biomarkers allowed to discriminate the stations regarding the genotoxic potential of water bodies and specifically by the measure of oxidative DNA lesions, which seem to be a promising tool in environmental genotoxicity risk assessment

An active biomonitoring approach using three-spined stickleback (Gasterosteus aculeatus, L.) to assess the efficiency of a constructed wetland as tertiary treatment of wastewater

The present work aimed to assess the efficiency of a constructed wetland as tertiary treatment on urban wastewater with a multibiomarker approach using caged three-spined sticklebacks (Gasterosteus aculeatus L.). Fish were caged on three sites: at the entrance of the Constructed Wetland (CW), directly inside the wastewater effluent, at the exit of the CW, and in a weakly impacted site considered as a reference. After 21 days of caging, sticklebacks state of health was assessed using several biomarkers representing some biological functions such as innate immune and antioxidant systems, biotransformation enzymes, reproduction parameters and synaptic transmission. A strong inhibition of the innate immune system, an induction of EROD activity and an alteration of the hepatosomatic index were observed in fish caged at the entrance of the CW compared to those caged in the reference site. In addition, wastewater effluent induced a decrease of antioxidant system without induced oxidative damage on cell membranes. No improvement of these biomarkers was observed for antioxidant parameters at the exit of the CW. However, in fish caged at the exit of the CW, the EROD induction observed at the entrance was reduced and the innate immune system presented the same level compared to fish caged in the reference site, underlying the beneficial effect of the CW for these parameters. Integrated Biomarker response (IBR) was equal to 25.2 at the entrance of the CW and 17.4 at the exit of the CW which highlighted the global positive effect of the CW on water quality based on fish biomarker measurement