Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect

Anoctamins are a family of Ca$^{2+}$-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca$^{2+}$ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patchclamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca$^{2+}$-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca$^{2+}$-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease

Etudes des altérations épigénétiques promues par l’exposition au chlordécone au cours du développement chez l’homme et la souris

Epigenetic mechanisms regulate many cellular processes and they are critical for establishing cell identity. The work performed during my Ph.D. aimed to determine whether exposure to an environmental pollutant, chlordecone (CD), could affect the major epigenetic marks in the reproductive system in directly-exposed mice and whether these changes could be inherited by subsequent generations through epigenetic transgenerational inheritance. Our work revealed that direct developmental exposure to CD leads to impairment of meiosis in male and female mice, causes a reduction in viable reproductive cells, i.e., both spermatogonia and spermatozoid, and oocytes in females. The altered morphological changes were accompanied by altered histone H3K4me3 occupancy in the genes related to pluripotency regulation. We also performed studies in the umbilical cord blood of males from the TIMOUN cohort with the known internal concentration of CD. We found that exposure to CD causes epigenetic changes in major histone trimethylation marks important for the maintenance of genetic stability. Using whole exome-sequencing, we detected de novo mutations in genes encoding proteins with important functions, such as DNA repair and metabolic genes. Comparative analysis of mice and human exposures revealed some common genes to be altered in both organisms, notably genes related to chromosome organization, transcription factors, and metabolic functions. In summary, our studies brought some evidence for the ability of CD to induce epigenetic and genetic alterations. Identified genes and molecular mechanisms affected by CD will be further investigated using in vivo and in vitro model systems.Les mécanismes épigénétiques régulent de nombreux processus cellulaires et sont essentiels pour en établir l'identité. Le travail effectué pendant mon doctorat visait à déterminer si l'exposition à un polluant environnemental, le chlordécone (CD), pouvait affecter les principales marques épigénétiques du système reproducteur chez la souris et si ces changements pouvaient être hérités par les générations suivantes par héritage épigénétique transgénérationnel. Les travaux réalisés dans le cadre de cette thèse ont révélé qu'une exposition développementale au CD conduit à une altération de la méiose chez les souris mâles et femelles, entraîne une réduction des cellules reproductrices viables, à la fois la spermatogonie et les spermatozoïdes chez les mâles, et les ovocytes chez les femelles. Les modifications morphologiques sont accompagnées d'une modification de l'occupation de l'histone H3K4me3 dans les gènes liés à la régulation de la pluripotence. Par ailleurs des études ont été menées sur le sang du cordon ombilical des garçons de la cohorte TIMOUN dont la concentration interne en CD est connue. Ces travaux ont permis de constater que l'exposition au CD provoque des changements de tri-méthylation des principales marques histones impliquées dans le maintien de la stabilité génétique. Le séquençage de l’exome complet a permis la détection des mutations de novo dans des gènes codant pour des protéines ayant des fonctions importantes, telles que la réparation de l'ADN et le métabolisme. L'analyse comparative des données d’exposition des souris et des humains a révélé que certains gènes étaient modifiés dans les deux organismes, notamment les gènes liés à l'organisation des chromosomes, aux facteurs de transcription et aux fonctions métaboliques. En résumé, nos études ont apporté des preuves de la capacité du CD à induire des altérations épigénétiques et génétiques. Il reste nécessaire d’étudier plus en détail les gènes identifiés et les mécanismes moléculaires affectés par le CD à l'aide de modèles in vivo et in vitro

Transgenerational Inheritance of Environmentally Induced Epigenetic Alterations during Mammalian Development

Genetic studies traditionally focus on DNA as the molecule that passes information on from parents to their offspring. Changes in the DNA code alter heritable information and can more or less severely affect the progeny’s phenotype. While the idea that information can be inherited between generations independently of the DNA’s nucleotide sequence is not new, the outcome of recent studies provides a mechanistic foundation for the concept. In this review, we attempt to summarize our current knowledge about the transgenerational inheritance of environmentally induced epigenetic changes. We focus primarily on studies using mice but refer to other species to illustrate salient points. Some studies support the notion that there is a somatic component within the phenomenon of epigenetic inheritance. However, here, we will mostly focus on gamete-based processes and the primary molecular mechanisms that are thought to contribute to epigenetic inheritance: DNA methylation, histone modifications, and non-coding RNAs. Most of the rodent studies published in the literature suggest that transgenerational epigenetic inheritance through gametes can be modulated by environmental factors. Modification and redistribution of chromatin proteins in gametes is one of the major routes for transmitting epigenetic information from parents to the offspring. Our recent studies provide additional specific cues for this concept and help better understand environmental exposure influences fitness and fidelity in the germline. In summary, environmental cues can induce parental alterations and affect the phenotypes of offspring through gametic epigenetic inheritance. Consequently, epigenetic factors and their heritability should be considered during disease risk assessment

Transgenerational Inheritance of Environmentally Induced Epigenetic Alterations during Mammalian Development

International audienceGenetic studies traditionally focus on DNA as the molecule that passes information on from parents to their offspring. Changes in the DNA code alter heritable information and can more or less severely affect the progeny's phenotype. While the idea that information can be inherited between generations independently of the DNA's nucleotide sequence is not new, the outcome of recent studies provides a mechanistic foundation for the concept. In this review, we attempt to summarize our current knowledge about the transgenerational inheritance of environmentally induced epigenetic changes. We focus primarily on studies using mice but refer to other species to illustrate salient points. Some studies support the notion that there is a somatic component within the phenomenon of epigenetic inheritance. However, here, we will mostly focus on gamete-based processes and the primary molecular mechanisms that are thought to contribute to epigenetic inheritance: DNA methylation, histone modifications, and non-coding RNAs. Most of the rodent studies published in the literature suggest that transgenerational epigenetic inheritance through gametes can be modulated by environmental factors. Modification and redistribution of chromatin proteins in gametes is one of the major routes for transmitting epigenetic information from parents to the offspring. Our recent studies provide additional specific cues for this concept and help better understand environmental exposure influences fitness and fidelity in the germline. In summary, environmental cues can induce parental alterations and affect the phenotypes of offspring through gametic epigenetic inheritance. Consequently, epigenetic factors and their heritability should be considered during disease risk assessment

Design of epidemiological studies measuring genital and plasma HIV-1 outcomes: lessons from a randomised controlled trial.

OBJECTIVE: HIV-1 genital viral loads have not been extensively used as markers of HIV transmissibility. We set out to determine whether the variability of genital HIV-1 RNA over time necessitates design adjustments in studies measuring genital shedding to account for this variability. METHODS: We used data from a completed trial of HSV suppressive therapy to estimate the correlation of plasma and genital HIV-1 RNA quantities sampled at different times. These correlation estimates were used to estimate the relative sample sizes needed to detect an impact on HIV-1 genital and plasma quantities assuming a variable number of pre- and post-randomisation repeated measurements. The treatment effect on quantities of genital and plasma HIV-1 RNA were analysed using random effects linear regression. RESULTS: Post-randomisation plasma HIV-1 RNA was highly correlated within-women, while genital HIV-1 RNA was less strongly correlated. Related to this, the sample size required to detect a treatment effect on genital HIV-1 RNA decreased with increasing numbers of post-randomisation measurements up to 6-7 measurements, but varied less for plasma HIV-1 RNA. In contrast, repeated pre-randomisation measurements of plasma HIV-1 RNA increased study power more than genital HIV-1 RNA because of the high correlation of plasma HIV-1 RNA measurements between the pre- and post-randomisation samples. Re-analysis of the trial data illustrated the increased precision of the treatment effect on genital HIV-1 with increasing post-randomisation measurements. CONCLUSIONS: Designs allowing for repeated post-randomisation measures should be used to increase the precision in estimates of genital HIV-1 RNA. Repeated post-randomisation measurements of plasma HIV-1 RNA are of limited benefit

In utero exposure to chlordecone affects histone modifications and activates LINE-1 in cord blood

International audienceEnvironmental factors can induce detrimental consequences into adulthood life. In this study, we examined the epigenetic effects induced by in utero chlordecone (CD) exposure on human male cord blood as well as in blood-derived Ke-37 cell line. Genome-wide analysis of histone H3K4me3 distribution revealed that genes related to chromosome segregation, chromatin organization, and cell cycle have altered occupancy in their promoters. The affected regions were enriched in ESR1, SP family, and IKZF1 binding motifs. We also observed a global reduction in H3K9me3, markedly in repeated sequences of the genome. Decrease in H3K9me3 after CD exposure correlates with decreased methylation in LINE-1 promoters and telomere length extension. These observations on human cord blood were assessed in the Ke-37 human cell line. H3K4me3 and the expression of genes related to immune response, DNA repair, and chromatin organization, which were affected in human cord blood were also altered in CD-exposed Ke-37 cells. Our data suggest that developmental exposure to CD leads to profound changes in histone modification patterns and affects the processes controlled by them in human cord blood

Histone deacetylase inhibition leads to regulatory histone mark alterations and impairs meiosis in oocytes

International audienceBackground Panobinostat (PB), a histone deacetylase (HDAC) inhibitor drug, is clinically used in the treatment of cancers. We investigated the effects of PB on murine ovarian functions in embryos and adult animals. Methods C57BL/6J mice were treated with 5 mg/kg PB on alternate days from embryonic day (E) 6.5 to E15.5. We analysed the effects of PB on the ovaries by using immunofluorescence, gene expression analysis and DNA methylation analysis techniques. Results At E15.5, we observed increases in histone H3K9Ac, H4Ac and H3K4me3 marks, while the level of the silencing H3K9me3 mark decreased. Synaptonemal complex examination at E15.5, E17.5 and E18.5 showed a delay in meiotic progression characterized by the absence of synaptonemal complexes at E15.5 and the persistence of double-strand breaks (DSBs) at E17.5 and E18.5 in PB-exposed oocytes. We found that exposure to PB led to changes in the expression of 1169 transcripts at E15.5. Genes regulated by the male-specific factors SRY-Box Transcription Factor 9 (SOX9) and Doublesex and Mab-3-related Transcription factor 1 (DMRT1) were among the most upregulated genes in the ovaries of PB-exposed mice. In contrast, PB treatment led to decreases in the expression of genes regulated by the WNT4 pathway. Notably, we observed 119 deregulated genes encoding Zn-finger proteins. The observed alterations in epigenetic marks and gene expression correlated with decreases in the numbers of germ cells at E15.5. After birth, PB-exposed ovaries showed increased proliferation of primary and secondary follicles. We also observed decreases in the numbers of primordial, primary and secondary follicles in adult ovaries from mice that were exposed to PB in utero. Finally, epigenetic alterations such as decreased H3K4me3 and increased H4 acetylation levels were also detected in somatic cells surrounding fully grown oocytes. Conclusion Our data suggest that inhibition of histone deacetylase by PB during a critical developmental window affects reprogramming and germ cell specification via alteration of epigenetic marks

Exploring the relationship between metal exposure, BDNF, and behavior in adolescent males

This research would not have been achieved without the selfless collaboration of the INMA-Granada adolescents and families who took part in the study. The authors also acknowledge the Human Genotyping Laboratory at the Spanish National Cancer Research Center, CeGen-PRB3, which is supported by grant no. PT17/0019, of the PE I+D+i 2013-2016, funded by the Instituto de Salud Carlos III (ISCIII) and ERDF. They thank the European Union's Horizon 2020 research and innovation program HBM4EU for financial support under Grant Agree-ment No. 733032. The study was also supported by the ISCIII with grant no. CP16/00085. The authors also acknowledge the funding received from the University of Granada for the open access publishing costs and the support of the Biomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP) , and the ISCIII (FIS 17/01526, FIS-PI16/01820 and FIS-PI16/01858) . Vicente Mustieles and Shereen Cynthia D'Cruz were under contract with the HBM4EU project. A. Rodriguez-Carrillo received a predoctoral fellowship (FPU 16/03011) from the Spanish Ministry of Education and C. Freire (grant no. MS16/00085) received a grant under the Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) . This article forms part of the doctoral thesis developed by Andrea Rodriguez-Carrillo in the context of the "Clinical Medicine and Public Health Program" of the University of Granada (Spain) .Background: Brain-derived neurotrophic factor (BDNF) plays an important role in brain development by regulating multiple pathways within the central nervous system. In the Human Biomonitoring for Europe Project (HBM4EU), this neurotrophin is being implemented as a novel effect biomarker to evaluate the potential threats of environmental chemicals on neurodevelopment. Objectives: To explore the relationships among exposure to environmental metals, BDNF biomarkers at two levels of biological complexity, and behavioral function in adolescent males. Methods: Data were gathered from 125 adolescents on: spot urine sample total concentrations of the neurotoxic metal(oid)s arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb); serum BDNF protein concentrations; and concurrent behavioral functioning according to the Child Behavior Check List (CBCL/6–18). In 113 of the participants, information was also collected on blood BDNF DNA methylation at six CpGs. Associations were evaluated by multivariate linear regression analysis adjusted for confounders. Results: As, Cd, Hg, and Pb were detected in 100%, 98.5%, 97.0%, and 89.5% of urine samples, respectively. Median serum BDNF concentration was 32.6 ng/mL, and total percentage of BDNF gene methylation was 3.8%. In the adjusted models, urinary As was non-linearly associated with more internalizing problems and Cd with more externalizing behaviors. The percentage BDNF DNA methylation at CPGs #5 and the mean percentage CpG methylation increased across As tertiles (p-trend = 0.04 and 0.03, respectively), while 2nd tertile and 3rd tertile of Cd concentrations were associated with lower serum BDNF and higher CpG3 methylation percentage. Additionally, when BDNF was categorized in tertiles, serum BDNF at the 3rd tertile was associated with fewer behavioral problems, particularly withdrawn (p-trend = 0.04), social problems (p-trend = 0.12), and thought problems (p-trend = 0.04). Conclusion: Exposure to As and Cd was associated with BDNF gene DNA methylation BDNF gene and serum BDNF, respectively. Associations with DNA methylation may be attributable to a higher variability over time in circulating BDNF concentrations than in the methylation status of this gene. Caution should be taken when interpreting the results relating postnatal Pb and Hg to behavioral functioning. Further studies are needed to verify these findings.Instituto de Salud Carlos III European Commission PT17/0019European CommissionEuropean Union's Horizon 2020 research and innovation program HBM4EU 733032Instituto de Salud Carlos III CP16/00085 FIS 17/01526 FIS-PI16/01820 FIS-PI16/01858University of GranadaBiomedical Research Networking Center-CIBER de Epidemiologia y Salud Publica (CIBERESP)Spanish Government FPU 16/03011Miguel Servet Type I program of the ISCIII "Fondo Europeo de Desarrollo Regional" (ISCIII/FEDER) MS16/0008

Epigenetic Effects Promoted by Neonicotinoid Thiacloprid Exposure

International audienceBackground Neonicotinoids, a widely used class of insecticide, have attracted much attention because of their widespread use that has resulted in the decline of the bee population. Accumulating evidence suggests potential animal and human exposure to neonicotinoids, which is a cause of public concern. Objectives In this study, we examined the effects of a neonicotinoid, thiacloprid (thia), on the male reproductive system. Methods The pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using "0," "0.06," "0.6," and "6" mg/kg/day doses. Adult male progeny was analyzed for morphological and cytological defects in the testes using hematoxylin and eosin (HandE) staining. We also used immunofluorescence, Western blotting, RT-qPCR and RNA-seq techniques for the analyses of the effects of thia on testis. Results We found that exposure to thia causes a decrease in spermatozoa at doses "0.6" and "6" and leads to telomere defects at all tested doses. At doses "0.6" and "6," thia exposure leads to an increase in meiotic pachytene cells and a decrease in lumen size, these changes were accompanied by increased testis-to-body weight ratios at high dose. By using RNA-seq approach we found that genes encoding translation, ATP production, ATP-dependent proteins and chromatin-modifying enzymes were deregulated in testes. In addition, we found that exposure to thia results in a decrease in H3K9me3 levels in spermatocytes. The changes in H3K9me3 were associated with a dramatic increase in activity of retroelements. Conclusion Our study suggests that gestational exposure to thia affects epigenetic mechanisms controlling meiosis which could lead to deleterious effects on male spermatogenesis

Gestational exposure to chlordecone promotes transgenerational changes in the murine reproductive system of males

International audienceEnvironmental factors can affect epigenetic events during germline reprogramming and impose distinctive transgenerational consequences onto the offspring. In this study, we examined the transgenerational effects of chlordecone (CD), an organochlorine insecticide with well-known estrogenic properties. We exposed pregnant mice to CD from embryonic day 6.5 to 15.5 and observed a reduction in spermatogonia (SG) numbers in F3, meiotic defects in spermatocytes and decrease in spermatozoa number in the first and third generation of male progeny. The RNA qRT-PCR expression analysis in F1 and transcriptomics analysis in F3 males using the whole testes revealed changes in the expression of genes associated with chromosome segregation, cell division and DNA repair. The expression of the master regulator of pluripotency, Pou5f1, decreased in foetal and increased in adult F1, but not in F3 adult testes. Analysis of histone H3K4me3 distribution revealed widespread changes in its occupancy in the genome of F1 and F3 generations. We established that 7.1% of altered epigenetic marks were conserved between F1 and F3 generations. The overlapping changes common to F1 and F3 include genes implicated in cell adhesion and transcription factor activities functions. Differential peaks observed in F1 males are significantly enriched in predicted ESR1 binding sites, some of which we confirmed to be functional. Our data demonstrate that CD-mediated impairment of reproductive functions could be transmitted to subsequent generations