ALLELE-SPECIFIC TRANSCRIPTIONAL ACTIVITY OF THE VARIABLE NUMBER OF TANDEM REPEATS OF THE INDUCIBLE NITRIC OXIDE SYNTHASE GENE IS ASSOCIATED WITH IDIOPATHIC ACHALASIA

Background: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved in both immune function and inhibitory neurotransmission. Objective: to assess the association and the functional relevance of the CCTTT inducible Nitric Oxide Synthase (NOS2) gene promoter polymorphism in achalasia. Methods: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. Results: The alleles’ distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3-0.5 and OR 1.6, 95% CI 1-2.4, all p<0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13-2.53, p=0.01). Transfection experiments’ revealed a similar allele-specific iNOS transcriptional activity. Conclusion: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production

Candidate biomarkers from the integration of methylation and gene expression in discordant autistic sibling pairs

While the genetics of autism spectrum disorders (ASD) has been intensively studied, resulting in the identification of over 100 putative risk genes, the epigenetics of ASD has received less attention, and results have been inconsistent across studies. We aimed to investigate the contribution of DNA methylation (DNAm) to the risk of ASD and identify candidate biomarkers arising from the interaction of epigenetic mechanisms with genotype, gene expression, and cellular proportions. We performed DNAm differential analysis using whole blood samples from 75 discordant sibling pairs of the Italian Autism Network collection and estimated their cellular composition. We studied the correlation between DNAm and gene expression accounting for the potential effects of different genotypes on DNAm. We showed that the proportion of NK cells was significantly reduced in ASD siblings suggesting an imbalance in their immune system. We identified differentially methylated regions (DMRs) involved in neurogenesis and synaptic organization. Among candidate loci for ASD, we detected a DMR mapping to CLEC11A (neighboring SHANK1) where DNAm and gene expression were significantly and negatively correlated, independently from genotype effects. As reported in previous studies, we confirmed the involvement of immune functions in the pathophysiology of ASD. Notwithstanding the complexity of the disorder, suitable biomarkers such as CLEC11A and its neighbor SHANK1 can be discovered using integrative analyses even with peripheral tissues

Demographic, clinical, and service-use characteristics related to the clinician’s recommendation to transition from child to adult mental health services

Purpose: The service configuration with distinct child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) may be a barrier to continuity of care. Because of a lack of transition policy, CAMHS clinicians have to decide whether and when a young person should transition to AMHS. This study describes which characteristics are associated with the clinicians’ advice to continue treatment at AMHS. Methods: Demographic, family, clinical, treatment, and service-use characteristics of the MILESTONE cohort of 763 young people from 39 CAMHS in Europe were assessed using multi-informant and standardized assessment tools. Logistic mixed models were fitted to assess the relationship between these characteristics and clinicians’ transition recommendations. Results: Young people with higher clinician-rated severity of psychopathology scores, with self- and parent-reported need for ongoing treatment, with lower everyday functional skills and without self-reported psychotic experiences were more likely to be recommended to continue treatment. Among those who had been recommended to continue treatment, young people who used psychotropic medication, who had been in CAMHS for more than a year, and for whom appropriate AMHS were available were more likely to be recommended to continue treatment at AMHS. Young people whose parents indicated a need for ongoing treatment were more likely to be recommended to stay in CAMHS. Conclusion: Although the decision regarding continuity of treatment was mostly determined by a small set of clinical characteristics, the recommendation to continue treatment at AMHS was mostly affected by service-use related characteristics, such as the availability of appropriate services

Cohort profile : demographic and clinical characteristics of the MILESTONE longitudinal cohort of young people approaching the upper age limit of their child mental health care service in Europe

Purpose: The presence of distinct child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) impacts continuity of mental health treatment for young people. However, we do not know the extent of discontinuity of care in Europe nor the effects of discontinuity on the mental health of young people. Current research is limited, as the majority of existing studies are retrospective, based on small samples or used non-standardised information from medical records. The MILESTONE prospective cohort study aims to examine associations between service use, mental health and other outcomes over 24 months, using information from self, parent and clinician reports. Participants: Seven hundred sixty-three young people from 39 CAMHS in 8 European countries, their parents and CAMHS clinicians who completed interviews and online questionnaires and were followed up for 2 years after reaching the upper age limit of the CAMHS they receive treatment at. Findings to date: This cohort profile describes the baseline characteristics of the MILESTONE cohort. The mental health of young people reaching the upper age limit of their CAMHS varied greatly in type and severity: 32.8% of young people reported clinical levels of self-reported problems and 18.6% were rated to be ‘markedly ill’, ‘severely ill’ or ‘among the most extremely ill’ by their clinician. Fifty-seven per cent of young people reported psychotropic medication use in the previous half year. Future plans: Analysis of longitudinal data from the MILESTONE cohort will be used to assess relationships between the demographic and clinical characteristics of young people reaching the upper age limit of their CAMHS and the type of care the young person uses over the next 2 years, such as whether the young person transitions to AMHS. At 2 years follow-up, the mental health outcomes of young people following different care pathways will be compared. Trial registration number: NCT03013595

Role of environmental oxygen in the regulation of human embryonic stem cells

Human embryonic stem (hES) cells derived from the inner cell mass of the blastocyst propagate by self-renewal and can give rise to all cells of the body. However, they have a tendency to spontaneously differentiate in vitro, an effect which can be abrogated by culture at low, 5% oxygen tensions. This response is mediated by hypoxia inducible factors (HIFs) and in particular HIF-2α. However, the mechanism of this regulation is still unknown. Chromatin immunoprecipitation (ChIP) analysis showed that HIF-2α directly binds to a predicted hypoxia response element (HRE) in the proximal promoter of OCT4, NANOG, SOX2, GLUT1 and eNOS under hypoxic conditions. An increased level of enrichment (P&lt;0.01) was observed in hES cells cultured at 5% oxygen whereas no significant binding was observed in cells maintained at 20% oxygen. Interestingly, HIF-2α induced an array of histone modifications that are associated with gene transcription within the predicted HRE site for all the genes analysed. ChIP assays showed that the chromatin state is more accessible and transcriptionally active in hES cells cultured under hypoxic conditions. In contrast, a heterochromatin state exists in the HRE of OCT4, SOX2, NANOG, GLUT1 and eNOS in hES cells cultured under 20% oxygen tension. This was also confirmed using pyrosequence analysis which revealed a significant hypomethylation pattern (P&lt;0.01) in the HRE site within the OCT4 proximal promoter in hES cells cultured at 5% oxygen compared to those maintained at 20% oxygen. hES cells were then used as a model to investigate the mechanisms acquired by resident stem cell populations to maintain an undifferentiate and proliferative state at the site of injury which are characterized by hypoxia and oxidative stress. Interestingly, an enhanced euchromatic state was found when hES cells were exposed to hypoxia/reoxygenation for 72 hours. Surprisingly, this was sustained by HIF-2α which was found significantly enriched within the HRE of all core pluripotency genes but particularly within the NANOG gene promoter. Furthermore, HIF-2α was found responsible of the establishment of a multiprotein complex thereby allowing interaction with an oct-sox cis-regulatory element in the NANOG promoter and sustaining selfrenewal. Thus, these data have uncovered a novel role of HIF-2α as a direct regulator of key transcription factors controlling self-renewal and epigenetic modifications which enhances the regenerative potential of hES cells exposed to hypoxia and reoxygenation

Environmental oxygen tension regulates the energy metabolism and self-renewal of human embryonic stem cells

Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2? at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs

HIF-2α regulates NANOG expression in human embryonic stem cells following hypoxia and reoxygenation through the interaction with an Oct-Sox Cis regulatory element

Low O2 tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is unknown. HIF-2? was found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O2). This binding induced an array of histone modifications associated with gene transcription while a heterochromatic state existed at atmospheric O2. Interestingly, an enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This was sustained by HIF-2? which enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG promoter. Thus, these data have uncovered a novel role of HIF-2? as a direct regulator of key transcription factors controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation which enhances the regenerative potential of these cells

The Liver-Gut axis in colorectal cancer development: a single center 8 years prospective study in 1145 metabolic subjects

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