Fine Mapping of the NRG1 Hirschsprung's Disease Locus

The primary pathology of Hirschsprung's disease (HSCR, colon aganglionosis) is the absence of ganglia in variable lengths of the hindgut, resulting in functional obstruction. HSCR is attributed to a failure of migration of the enteric ganglion precursors along the developing gut. RET is a key regulator of the development of the enteric nervous system (ENS) and the major HSCR-causing gene. Yet the reduced penetrance of RET DNA HSCR-associated variants together with the phenotypic variability suggest the involvement of additional genes in the disease. Through a genome-wide association study, we uncovered a ∼350 kb HSCR-associated region encompassing part of the neuregulin-1 gene (NRG1). To identify the causal NRG1 variants contributing to HSCR, we genotyped 243 SNPs variants on 343 ethnic Chinese HSCR patients and 359 controls. Genotype analysis coupled with imputation narrowed down the HSCR-associated region to 21 kb, with four of the most associated SNPs (rs10088313, rs10094655, rs4624987, and rs3884552) mapping to the NRG1 promoter. We investigated whether there was correlation between the genotype at the rs10088313 locus and the amount of NRG1 expressed in human gut tissues (40 patients and 21 controls) and found differences in expression as a function of genotype. We also found significant differences in NRG1 expression levels between diseased and control individuals bearing the same rs10088313 risk genotype. This indicates that the effects of NRG1 common variants are likely to depend on other alleles or epigenetic factors present in the patients and would account for the variability in the genetic predisposition to HSCR

Trial Protocol: The use of mindfulness-based intervention for improving bracing compliance for adolescent idiopathic scoliosis patients: protocol for a randomised, controlled trial

Introduction: Adolescent idiopathic scoliosis (AIS) is the most prevalent deforming orthopaedic condition; it causes significant disability when spinal curves progress beyond 45 deg. Bracing is the primary treatment prescribed for adolescents with an immature skeleton who have spinal curves between 25 and 45 deg. New evidence suggests that compliance with bracing significantly decreases the progression of high-risk curves to the threshold for surgery. Nonetheless, bracing is a stressful experience. Therefore, interventions that mediate health-related quality of life for AIS patients are of great interest. In the past few decades, numerous studies have documented the benefits of mindfulness training on chronic pain, stress management, anxiety and emotional disorders. Mindfulness might additionally provide AIS patients with psychosocial support. Research questions: This study will investigate the effects of a mindfulness-based intervention on bracing compliance and quality of life among AIS patients with poor bracing compliance. The study also plans to evaluate if the mindfulness-based intervention effect is sustained after the intervention period. The potential mechanism by which mindfulness affects bracing compliance will be explored. Design: Single-blind, two-arm, randomised, controlled trial. Participants and setting: The study will recruit 120 AIS patients aged between 10 and 15 years with non-satisfactory bracing compliance. Patients who have previously practised or are currently practising meditation or mindful yoga or who cannot finish the whole intervention will be excluded. The study will take place at the Jockey Club School of Public Health and Primary Care building. Intervention: Patients in the mindfulness-based intervention group will join weekly sessions for 8 weeks. This program is a short version of a mindfulness-based stress relaxation program to address the specific issues of AIS patients. Two to three experienced instructors will deliver the program. Control: Control group patients will participate in an 8-week physiotherapy exercise program as recommended in the International Scientific Society on Scoliosis Orthopaedic and Rehabilitation Treatment (SOSORT) 2011 guideline. Measurements: The primary outcome is the 6-month post-intervention total score of bracing compliance. Secondary measures are non-bracing-specific quality of life, bracing-specific quality of life, self-compassion, emotional regulation, mindful awareness and acceptance, self-efficacy, perception of stress, and general measure of health outcome. Procedure: 120 participants will be assigned to either an intervention or control arm by simple randomisation, and the randomisation result will only be revealed once participants have confirmed availability to attend intervention classes. Clinicians of the scoliosis clinic and research staff will be blinded to the treatment allocation. Analysis: ANCOVA will be conducted to compare the effect of mindfulness-based intervention versus physiotherapy exercise on the outcome measures. To investigate significant change over time, linear mixed models analyses will be conducted following the intention-to-treat principle. The R-package lavaan will be used to conduct structural equation modelling to study the potential mechanism of mindfulness. Discussion/significance: This will be the first psychosocial intervention study conducted on braced AIS patients with the aim of improving patients’ bracing compliance and quality of life. The results from this study will potentially carry significant impact on future AIS treatment by emphasising psychosocial care for braced AIS patients

Docosahexaenoic acid reduces microglia phagocytic activity via miR-124 and induces neuroprotection in rodent models of spinal cord contusion injury

Microglia are activated after spinal cord injury (SCI), but their phagocytic mechanisms and link to neuroprotection remain incompletely characterized. Docosahexaenoic acid (DHA) has been shown to have significant neuroprotective effects after hemisection and compression SCI and can directly affect microglia in these injury models. In rodent contusion SCI, we demonstrate that DHA (500 nmol/kg) administered acutely post-injury confers neuroprotection and enhances locomotor recovery, and also exerts a complex modulation of the microglial response to injury. In rodents, at 7 days after SCI, the level of phagocytosed myelin within Iba1-positive or P2Y12-positive cells was significantly lower after DHA treatment, and this occurred in parallel with an increase in intracellular miR-124 expression. Furthermore, intraspinal administration of a miR-124 inhibitor significantly reduced the DHA-induced decrease in myelin phagocytosis in mice at 7 days post-SCI. In rat spinal primary microglia cultures, DHA reduced the phagocytic response to myelin, which was associated with an increase in miR-124, but not miR-155. A similar response was observed in a microglia cell line (BV2) treated with DHA, and the effect was blocked by a miR-124 inhibitor. Furthermore, the phagocytic response of BV2 cells to stressed neurones was also reduced in the presence of DHA. In peripheral monocyte-derived macrophages, the expression of the M1, but not the M0 or M2 phenotype, was reduced by DHA, but the phagocytic activation was not altered. These findings show that DHA induces neuroprotection in contusion injury. Furthermore, the improved outcome is via a miR-124-dependent reduction in the phagocytic response of microglia. © 2019 The Author(s).Department of Defence. Estados Unidos W81XWH-10-1-1040Chang Gung Memorial Hospital. Taiwán CMRPG3A1051–1054Ministerio de Economía y Competitividad (MINECO). España RYC-2017-2180

TET2 Regulates the Neuroinflammatory Response in Microglia.

Epigenomic mechanisms regulate distinct aspects of the inflammatory response in immune cells. Despite the central role for microglia in neuroinflammation and neurodegeneration, little is known about their epigenomic regulation of the inflammatory response. Here, we show that Ten-eleven translocation 2 (TET2) methylcytosine dioxygenase expression is increased in microglia upon stimulation with various inflammogens through a NF-κB-dependent pathway. We found that TET2 regulates early gene transcriptional changes, leading to early metabolic alterations, as well as a later inflammatory response independently of its enzymatic activity. We further show that TET2 regulates the proinflammatory response in microglia of mice intraperitoneally injected with LPS. We observed that microglia associated with amyloid β plaques expressed TET2 in brain tissue from individuals with Alzheimer's disease (AD) and in 5xFAD mice. Collectively, our findings show that TET2 plays an important role in the microglial inflammatory response and suggest TET2 as a potential target to combat neurodegenerative brain disorders

Genome-Wide Copy Number Analysis Uncovers a New HSCR Gene: <em>NRG3</em>

<div><p>Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified <em>NRG1</em> as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (<em>p</em> = 1.50×10⁻⁵), particularly for those encompassing genes (<em>p</em> = 5.00×10⁻⁶). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a <em>NRG3</em> deletion (<em>p</em> = 1.64×10⁻³). Subsequent follow-up (96 additional patients and 220 controls) on <em>NRG3</em> revealed 9 deletions (combined <em>p</em> = 3.36×10⁻⁵) and 2 <em>de novo</em> duplications among patients and two deletions among controls. Importantly, <em>NRG3</em> is a paralog of <em>NRG1</em>. Stratification of patients by presence/absence of HSCR–associated syndromes showed that while syndromic–HSCR patients carried significantly longer CNVs than the non-syndromic or controls (<em>p</em> = 1.50×10⁻⁵), non-syndromic patients were enriched in CNV number when compared to controls (<em>p</em> = 4.00×10⁻⁶) or the syndromic counterpart. Our results suggest a role for <em>NRG3</em> in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.</p> </div

Global CNV burden in HSCR patients.

a<p>Rate: number of CNVs per individual.</p>b<p>Baseline: size, rate or gene count of controls.</p>c<p>Ratio: Case/control ratio.</p>d<p><i>P</i>-value for conditional permutation based on score of LRR_SD, BAF_drift and median absolute deviation.</p>e<p>Del+Dup of all rare CNVs was specified as above.</p>*<p>0.01<<i>p</i><0.05;</p>**<p>10⁻⁴<<i>p</i><0.01;</p>***<p><i>p</i><10⁻⁴.</p

CNV burden for syndromic (Syn) and non-syndromic (Non-syn) HSCR patients relative to controls (Ctrl).

<p>The burden was measured with reference to the (A) size, (B) rate and (C) gene count of CNVs. Red and blue bars denote the mean value of the corresponding test for deletion and duplication respectively. Summary statistics as well as conditional permutation <i>p</i>-value was shown in (D).</p

<i>NRG3</i> deletions identified in HSCR patients.

<p>(A) Intensity signals of 5 HSCR patients (CN = 1; red) with <i>NRG3</i> deletions together with other samples of normal copy number (CN = 2; grey). Deleted regions are shown by the dark red bar and are highlighted in pink. (B) Consensus CNV segments of the 5 <i>NRG3</i> deletions (red) and the overlapping DGV segments (blue; with DGV ID). (C, D and E) Box plot of <i>NRG3</i> copy number estimates by real-time PCR. Samples were grouped according to the called copy number states (CN = 1, red; CN = 2, white; CN = 3, blue); (C) Validation of 5 deletions (CN = 1) and 24 copy-neutral (CN = 2) HSCR patients in the discovery phase; (D) Follow-up analysis on independent case-controls set and (E) Transmission analysis for probands with <i>NRG3</i> deletions (child CN = 1) or duplications (child CN = 3). (F) Sequence of the <i>NRG3</i> deletion boundary region showing the breakpoint (upstream boundary chr10: 84032610; downstream boundary chr10: 84052262).</p

Large (>1 Mb), rare CNVs identified in HSCR patients.

a<p>S: Syndromic patient; NS: non-syndromic patient.</p>b<p>Genes overlapped by recurrent CNVs unique to patients.</p>c<p>S27 have 2 overlapping duplications within the 16p11-12 locus.</p