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Background & Aims: Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods: We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results: Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2–4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5–8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2–4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions: Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. Lay summary: MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy.

Background Parkinson’s disease (PD) is a systemic disease clinically defined by the degeneration of dopaminergic neurons in the brain. While alterations in the gut microbiome composition have been reported in PD, their functional consequences remain unclear. Herein, we addressed this question by an analysis of stool samples from the Luxembourg Parkinson’s Study (n = 147 typical PD cases, n = 162 controls). Results All individuals underwent detailed clinical assessment, including neurological examinations and neuropsychological tests followed by self-reporting questionnaires. Stool samples from these individuals were first analysed by 16S rRNA gene sequencing. Second, we predicted the potential secretion for 129 microbial metabolites through personalised metabolic modelling using the microbiome data and genome-scale metabolic reconstructions of human gut microbes. Our key results include the following. Eight genera and seven species changed significantly in their relative abundances between PD patients and healthy controls. PD-associated microbial patterns statistically depended on sex, age, BMI, and constipation. Particularly, the relative abundances of Bilophila and Paraprevotella were significantly associated with the Hoehn and Yahr staging after controlling for the disease duration. Furthermore, personalised metabolic modelling of the gut microbiomes revealed PD-associated metabolic patterns in the predicted secretion potential of nine microbial metabolites in PD, including increased methionine and cysteinylglycine. The predicted microbial pantothenic acid production potential was linked to the presence of specific non-motor symptoms. Conclusion Our results suggest that PD-associated alterations of the gut microbiome can translate into substantial functional differences affecting host metabolism and disease phenotype.

Additional file 1. Extended results on microbial abundance analyses (Fig. S1, S2, and S3) and background information about characteristics of the community models and read counts (Tables S1, S2, S3, and S4).

International audience; “General-purpose cohorts” in epidemiology and public health are designed to cover a broad scope of determinants and outcomes, in order to answer several research questions, including those not defined at study inception. In this context, the general objective of the CONSTANCES project is to set up a large population-based cohort that will contribute to the development of epidemiological research by hosting ancillary projects on a wide range of scientific domains, and to provide public health information. CONSTANCES was designed as a randomly selected sample of French adults aged 18–69 years at study inception; 202,045 subjects were included over an 8-year period. At inclusion, the selected participants are invited to attend one of the 24 participating Health Prevention Centers (HPCs) for a comprehensive health examination. The follow-up includes a yearly self-administered questionnaire, and a periodic visit to an HPC. Procedures have been developed to use the national healthcare databases to allow identification and validation of diseases over the follow-up. The biological collection (serum, lithium heparinized plasma, EDTA plasma, urine and buffy coat) began gradually in June 2018. At the end of the inclusions, specimens from 83,000 donors will have been collected. Specimens are collected according to a standardized protocol, identical in all recruitment centers. All operations relating to bio-banking have been entrusted by Inserm to the Integrated Biobank of Luxembourg (IBBL). A quality management system has been put in place. Particular attention has been paid to the traceability of all operations. The nature of the biological samples stored has been deliberately limited due to the economic and organizational constraints of the inclusion centers. Some research works may require specific collection conditions, and can be developed on request for a limited number of subjects and in specially trained centers. The biological specimens that are collected will allow for a large spectrum of biomarkers studies and genetic and epigenetic markers through candidate or agnostic approaches. By linking the extensive data on personal, lifestyle, environmental, occupational and social factors with the biomarker data, the CONSTANCES cohort offers the opportunity to study the interplays between these factors using an integrative approach and state-of-the-art methods.

Table S1. Commonly reported haplotypes. Table S2. GR SNPs PCR primers and their reaction condition. Table S3. Association of GR gene SNPs and haplotypes with response of blood pressure, heart rate and cortisol response effect by SECPT.

Gene expression is regulated by a wide range of mechanisms including endogenicmechanisms and external environmental factors. Epigenetic factors areconsidered to be the principal interface between the genome and theenvironment (Fraga, M 2005) (Szyf, M 2011a) (Szyf, M 2011b). Among thesemechanisms DNA methylation is probably the best understood. Moreover theDNA methylation pattern is susceptible to modifications induced byenvironmental factorsTaking into account its implication in the regulation of gene transcription andsilencing (Bird, A 2007), DNA methylation may also play a critical role in thedevelopment of various diseases including stress related disorders..The Department of Immunology of the “Centre de Recherche Public de la Santé”(CRP-Santé) in Luxembourg has gained considerable experience in clinicalstudies as well as in epigenetics, especially concerning stress response genes. Ihad the opportunity to pursue my Master internship in this Department workingon two academic exploratory studies projects related to the link between DNAmethylation and stress response.First, I was responsible for the data analysis of the “Cold Pressor Test” study.This study demonstrated the association between the stress response induced bya validated stress test: the Cold Pressor Test, and the methylation of theglucocorticoid receptor (GR) gene.These results, together with previous non clinical studies lead thePsychoImmunology team to design the “Long term epigenetic and physiologicaleffects of early life adversity” (EpiPath) study. EpiPath will examine the impact ofEarly Life Adversity (ELA) on the potential risk factors of major public healthproblems: cardiovascular, mental, immune system diseases and stress responsealterations. These are multifactorial diseases meaning that these conditionsdevelopment depends on the presence of genetics, epigenetics andenvironmental factors. The link between ELA and those pathologies in adulthoodis already settled and the aim of this present study is to provide a betterunderstanding of the impact of ELA on epigenetic methylation and on its clinicconsequences.; L’expression des gènes est régulée en partie par de nombreux mécanismes endogènes, mais également par desfacteurs environnementaux. Les modifications épigénétiques représentent la principale interface entre le génomeet l’environnement[1][2][3]. Parmi les différents mécanismes épigénétiques, la méthylation de l’ADN est sansdoute la mieux connue. De plus le profil de méthylation de l’ADN est susceptible de subir des modificationsinduites par des facteurs environnementaux.Compte tenu de son implication dans la régulation de la transcription et l’inactivation des gènes[4], laméthylation de l’ADN pourrait également jouer un rôle clé dans l’apparition de différentes maladies, incluant lesmaladies liées au stress.Le Département d’Immunologie du Centre de Recherche Public de la Santé (CRP-Santé) de Luxembourg aacquis une expérience considérable dans le domaine de l’épigénétique, en particulier concernant les gènesimpliqués dans la réponse au stress ainsi que dans la conduite d’études cliniques. Mon stage de Master 2 au seinde ce Département, m’a permis de travailler sur deux études portant sur le niveau de méthylation de l’ADN.L’étude “Cold Pressor Test” a montré un lien entre la réponse à un stress induite par un test validé- le ColdPressor Test - et la méthylation du gène du récepteur aux glucocorticoïdes (GR). Durant mon stage, je me suischargée de l’analyse des données de cette étude.Ces résultats ainsi que ceux d’études précédentes menées par le groupe PsychoImmunologie du CRP-Santé ontconduit à l’élaboration du protocole de l’étude “Long term epigenetic and physiological effects of early lifeadversity” (EpiPath). EpiPath analysera l’impact des maltraitances subies durant la petite enfance, ou "Early LifeAdversity" (ELA) sur les facteurs de risque de problèmes de santé publiques majeurs: maladiescardiovasculaires, mentales, du système immunitaire et altération de la réponse au stress. Le lien entre desantécédents d’ELA et la survenue de ces pathologies ayant déjà été mis en évidence, l’objectif de cette étudeest de fournir une meilleure compréhension de l’impact de l’ELA sur la méthylation de l’ADN et lesconséquences cliniques.

Background & Aims Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2–4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5–8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2–4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. Lay summary MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy., Graphical abstract, Highlights • Serum miRNA was sequenced in 183 NAFLD cases of varying severity and 10 population controls. • Plasma levels of miR-193a-5p were significantly increased in patients with advanced fibrosis, high NAS scores, or high SAF scores. • Other miRNAs including miR378d and miR378e were also significantly increased in certain comparisons. • The findings for miR-193a-5p were replicated in a cohort of 372 additional NAFLD cases.