Marie-Sylvie Gross

Sex- and diet-specific changes of imprinted gene expression and DNA methylation in mouse placenta under a high-fat diet.

Background Changes in imprinted gene dosage in the placenta may compromise the prenatal control of nutritional resources. Indeed monoallelic behaviour and sensitivity to changes in regional epigenetic state render imprinted genes both vulnerable and adaptable. Methods and Findings We investigated whether a high-fat diet (HFD) during pregnancy modified the expression of imprinted genes and local and global DNA methylation patterns in the placenta. Pregnant mice were fed a HFD or a control diet (CD) during the first 15 days of gestation. We compared gene expression patterns in total placenta homogenates, for male and female offspring, by the RT-qPCR analysis of 20 imprinted genes. Sexual dimorphism and sensitivity to diet were observed for nine genes from four clusters on chromosomes 6, 7, 12 and 17. As assessed by in situ hybridization, these changes were not due to variation in the proportions of the placental layers. Bisulphite-sequencing analysis of 30 CpGs within the differentially methylated region (DMR) of the chromosome 17 cluster revealed sex- and diet-specific differential methylation of individual CpGs in two conspicuous subregions. Bioinformatic analysis suggested that these differentially methylated CpGs might lie within recognition elements or binding sites for transcription factors or factors involved in chromatin remodelling. Placental global DNA methylation, as assessed by the LUMA technique, was also sexually dimorphic on the CD, with lower methylation levels in male than in female placentae. The HFD led to global DNA hypomethylation only in female placenta. Bisulphite pyrosequencing showed that neither B1 nor LINE repetitive elements could account for these differences in DNA methylation. Conclusions A HFD during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes important in the control of many cellular, metabolic and physiological functions potentially involved in adaptation and/or evolution. These findings highlight the importance of studying both sexes in epidemiological protocols and dietary interventions.

Maternal Diets Trigger Sex-Specific Divergent Trajectories of Gene Expression and Epigenetic Systems in Mouse Placenta

Males and females responses to gestational overnutrition set the stage for subsequent sex-specific differences in adult onset non communicable diseases. Placenta, as a widely recognized programming agent, contibutes to the underlying processes. According to our previous findings, a high-fat diet during gestation triggers sex-specific epigenetic alterations within CpG and throughout the genome, together with the deregulation of clusters of imprinted genes. We further investigated the impact of diet and sex on placental histology, transcriptomic and epigenetic signatures in mice. Both basal gene expression and response to maternal high-fat diet were sexually dimorphic in whole placentas. Numerous genes showed sexually dimorphic expression, but only 11 genes regardless of the diet. In line with the key role of genes belonging to the sex chromosomes, 3 of these genes were Y-specific and 3 were X-specific. Amongst all the genes that were differentially expressed under a high-fat diet, only 16 genes were consistently affected in both males and females. The differences were not only quantitative but remarkably qualitative. The biological functions and networks of genes dysregulated differed markedly between the sexes. Seven genes of the epigenetic machinery were dysregulated, due to effects of diet, sex or both, including the Y- and X-linked histone demethylase paralogues Kdm5c and Kdm5d, which could mark differently male and female epigenomes. The DNA methyltransferase cofactor Dnmt3l gene expression was affected, reminiscent of our previous observation of changes in global DNA methylation. Overall, this striking sexual dimorphism of programming trajectories impose a considerable revision of the current dietary interventions protocols.

Nutri-epigenomics: lifelong remodelling of our epigenomes by nutritional and metabolic factors and beyond.

Abstract The phenotype of an individual is the result of complex interactions between genotype, epigenome and current, past and ancestral environment, leading to lifelong remodelling of our epigenomes. Various replication-dependent and -independent epigenetic mechanisms are involved in developmental programming, lifelong stochastic and environmental deteriorations, circadian deteriorations, and transgenerational effects. Several types of sequences can be targets of a host of environmental factors and can be associated with specific epigenetic signatures and patterns of gene expression. Depending on the nature and intensity of the insult, the critical spatiotemporal windows and developmental or lifelong processes involved, these epigenetic alterations can lead to permanent changes in tissue and organ structure and function, or to reversible changes using appropriate epigenetic tools. Given several encouraging trials, prevention and therapy of age- and lifestyle-related diseases by individualised tailoring of optimal epigenetic diets or drugs are conceivable. However, these interventions will require intense efforts to unravel the complexity of these epigenetic, genetic and environment interactions and to evaluate their potential reversibility with minimal side effects. Clin Chem Lab Med 2007;45:321–7.

ABCC8 (SUR1) and KCNJ11 (KIR6.2) Mutations in Persistent Hyperinsulinemic Hypoglycemia of Infancy and Evaluation of Different Therapeutic Measures

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) can occur as a result of mutations in the subunits that form the ATP-sensitive potassium channel (K+ATP) in pancreatic beta-cells which play a major role in modulating insulin secretion from the beta-cells. Mutations have been shown in the genes for these subunits, namely for the plasma membrane sulfonylurea receptor (SUR1), ABCC8, and its associated inwardly rectifying potassium channel (KIR6.2) KCNJ11. Drugs which act on K+ATP channels, such as diazoxide, seem to need intact ABCC8 to be able to show their effects. Thus, it would be desirable to know the exact locus of the abnormality in the beta-cell to be able to choose the right therapeutic agent or to perform early pancreatectomy. The aim of this study was to search for the correlation between the mutations of the K+ATP channel and the outcome of therapeutic measures in patients with PHHI followed for a duration of 4 months to 7.3 years. Thirteen patients (5 F, 8 M) with PHHI with a median age of 2.5 months (8 days-12.1 years) were included in the study. Therapy for PHHI was initiated either with diazoxide (n = 9) or with calcium channel blocker (n = 4) as the agent of first choice. Three patients unresponsive to drugs underwent 95% pancreatectomy. Mutation analysis was performed by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) in DNA samples extracted from patients peripheral leukocytes. The PCR products were directly sequenced. Screening of ABCC8 and KCNJ11 for mutations revealed abnormalities in the ABCC8 gene in three patients out of 13: homozygosity for the 155del1 mutation, compound heterozygosity for T267-->G/A4612-2-->G, and compound heterozygosity for G4310-->A/ R1494Q. No mutations in the KCNJ11 gene were identified. Of the three patients who underwent pancreatectomy, two had identified mutations and one did not have any known mutation. In two patients in whom hyperinsulinism recurred after surgery and in the rest of the children, therapy with either diazoxide or calcium channel blocker proved to be effective in controlling hypoglycemia over the follow-up period. Thus it may be concluded that mutations in the ABCC8 gene were not predictive of the response to drugs. Unidentified mutations in the K+ATP channels other than those screened or other functional abnormalities in these channels may account for the different therapeutic responses.

Epigénomique nutritionnelle : impact de régimes alimentaires déséquilibrés sur les processus épigénétiques de programmation au cours de la vie et transgénérationnels

Les modifications epigenetiques - associant une methylation de l’ADN et des modifications des histones - assurent par un remodelage adequat de la chromatine la modulation de l’expression des genes. Ces modifications sous-tendent la programmation, au cours du developpement fœtal et postnatal, aboutissant au faconnage des tissus et des organes. Outre l’heritage d’un genotype d’epargne les individus avec les desordres metaboliques conduisant au syndrome metabolique, a l’obesite, au diabete de type 2 et aux maladies cardiovasculaires ont subi au cours de fenetres critiques du developpement une « programmation epigenetique » anormale en liaison avec les desequilibres nutritionnels et les defauts metaboliques de la mere pendant la gestation et la lactation. Une programmation erronee peut avoir des effets sur la sante de l’enfant et surtout plus tard a l’age adulte et pourrait meme etre transmise a la generation suivante. Les marques et processus epigenetiques et en particulier celles portees par les transposons et les genes soumis a empreinte sont, du fait de leur labilite, hautement sensibles aux agents environnementaux et en particulier a l’alimentation et, en principe, reversibles. Elles jouent donc un role crucial au cours du developpement et de l’evolution. Dans cette revue, nous decrivons brievement les consequences physiopathologiques, a l’age adulte, des conditions nutritionnelles influencant les principales etapes de la programmation epigenetique au cours du developpement fœto-placentaire et post-natal, les coadaptations evolutives des generations et les effets trans-generationnels, ainsi que les principaux objectifs actuels de recherche.

An oligonucleotide microarray for mouse imprinted genes profiling

Genomic imprinting is an epigenetic phenomenon unique to mammals that causes some genes to be expressed according to their parental origin. It results in developmental asymmetry in the function of the parental genomes. We describe here a method for the profiling of imprinted genes based on the development of a mouse imprinting microchip containing oligonucleotides corresponding to 493 genes, including most of the known imprinted genes (IG = 63), genes involved in epigenetic processes (EPI = 15), in metabolism (= 147), in obesity (= 10) and in neurotransmission (= 256) and housekeeping reference genes (= 2). This custom oligonucleotide microarray has been constructed to make data analysis and handling more manageable than pangenomic microarrays. As a proof of concept we present the differential expression of these 493 genes in different tissues (liver, placenta, embryo) of C57BL6/J mice fed different diets. Appropriate experimental strategies and statistical tools were defined at each step of the data analysis process with regard to the different sources of constraints. Data were confirmed by expression analyses based on quantitative real-time PCR. These oligochips should make it possible to increase our understanding of the involvement of imprinted genes in the timing of expression programs, tissue by tissue, stage by stage, in response to nutrients, lifestyles and other as yet unknown critical environmental factors in a variety of physiopathological situations, and in animals of different strains, ages and sexes. The use of oligonucleotides makes it possible to expand this microchip to include the increasing number of imprinted genes discovered.

Chromosomal assignment of 14 genomic probes for highly polymorphic loci

Over 500 probes revealing restriction fragment length polymorphisms (RFLPs) have been isolated by Schumm et al. (1988). We describe here the chromosomal assignment of 14 of the most highly polymorphic markers in that set of probes, with polymorphism information content values of up to 0.98. The probes were mapped using a panel of human x rodent somatic cell hybrids and were found to be distributed among nine different autosomes. Chromosome localization of such highly polymorphic markers has been an important step in the construction of the human genetic map, as a large number of RFLP probes has now been localized by genetic linkage studies to these loci.

Association of modifiers and other genetic factors explain Marfan syndrome clinical variability

Marfan syndrome (MFS) is a rare autosomal dominant connective tissue disorder related to variants in the FBN1 gene. Prognosis is related to aortic risk of dissection following aneurysm. MFS clinical variability is notable, for age of onset as well as severity and number of clinical manifestations. To identify genetic modifiers, we combined genome-wide approaches in 1070 clinically well-characterized FBN1 disease-causing variant carriers: (1) an FBN1 eQTL analysis in 80 fibroblasts of FBN1 stop variant carriers, (2) a linkage analysis, (3) a kinship matrix association study in 14 clinically concordant and discordant sib-pairs, (4) a genome-wide association study and (5) a whole exome sequencing in 98 extreme phenotype samples.Three genetic mechanisms of variability were found. A new genotype/phenotype correlation with an excess of loss-of-cysteine variants (Pu2009=u20090.004) in severely affected subjects. A second pathogenic event in another thoracic aortic aneurysm gene or the COL4A1 gene (known to be involved in cerebral aneurysm) was found in nine individuals. A polygenic model involving at least nine modifier loci (named gMod-M1-9) was observed through cross-mapping of results. Notably, gMod-M2 which co-localizes with PRKG1, in which activating variants have already been described in thoracic aortic aneurysm, and gMod-M3 co-localized with a metalloprotease (proteins of extra-cellular matrix regulation) cluster. Our results represent a major advance in understanding the complex genetic architecture of MFS and provide the first steps toward prediction of clinical evolution.

Marfan Syndrome Variability: Investigation of the Roles of Sarcolipin and Calcium as Potential Transregulator of FBN1 Expression

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder that displays a great clinical variability. Previous work in our laboratory showed that fibrillin-1 (FBN1) messenger RNA (mRNA) expression is a surrogate endpoint for MFS severity. Therefore, an expression quantitative trait loci (eQTL) analysis was performed to identify trans-acting regulators of FBN1 expression, and a significant signal reached genome-wide significant threshold on chromosome 11. This signal delineated a region comprising one expressed gene, SLN (encoding sarcolipin), and a single pseudogene, SNX7-ps1 (CTD-2651C21.3). We first investigated the region and then looked for association between the genes in the region and FBN1 expression. For the first time, we showed that the SLN gene is weakly expressed in skin fibroblasts. There is no direct correlation between SLN and FBN1 gene expression. We showed that calcium influx modulates FBN1 gene expression. Finally, SLN gene expression is highly correlated to that of the neighboring SNX7-ps1. We were able to confirm the impact of calcium influx on FBN1 gene expression but we could not conclude regarding the role of sarcolipin and/or the eQTL locus in this regulation.