Serum cytokines and inflammatory proteins in individuals with heroin use disorder: potential mechanistically based biomarkers for diagnosis.

Opioid use disorders cause major morbidity and mortality, and there is a pressing need for novel mechanistic targets and biomarkers for diagnosis and prognosis. Exposure to mu-opioid receptor (MOR) agonists causes changes in cytokine and inflammatory protein networks in peripheral blood, and also in brain glia and neurons. Individuals with heroin use disorder (iHUD) show dysregulated levels of several cytokines in the blood. However, there is limited data on a comprehensive panel of such markers in iHUD versus healthy controls (HC), especially considered as a multi-target biomarker. We used a validated proximity extension assay for the relative quantification of 92 cytokines and inflammatory proteins in the serum of iHUD on medication-assisted therapy (MAT; n = 21), compared to HC (n = 24). Twenty-nine targets showed significant group differences (primarily iHUD>HC), surviving multiple comparison corrections (p = 0.05). These targets included 19 members of canonical cytokine families, including specific chemokines, interleukins, growth factors, and tumor necrosis factor (TNF)-related proteins. For dimensionality reduction, data from these 19 cytokines were entered into a principal component (PC) analysis, with PC1 scores showing significant group differences (iHUD > HC; p < 0.0001). A receiver-operating characteristic (ROC) curve analysis yielded an AUROC = 91.7% (p < 0.0001). This PC1 score remained a positive predictor of being in the HUD group in a multivariable logistic regression, that included select demographic/clinical variables. Overall, this study shows a panel of cytokines that differ significantly between iHUD and HC, providing a multi-target "cytokine biomarker score" for potential diagnostic purposes, and future examination of disease severity.

Divergent landscapes of A-to-I editing in postmortem and living human brain.

Adenosine-to-inosine (A-to-I) editing is a prevalent post-transcriptional RNA modification within the brain. Yet, most research has relied on postmortem samples, assuming it is an accurate representation of RNA biology in the living brain. We challenge this assumption by comparing A-to-I editing between postmortem and living prefrontal cortical tissues. Major differences were found, with over 70,000 A-to-I sites showing higher editing levels in postmortem tissues. Increased A-to-I editing in postmortem tissues is linked to higher ADAR and ADARB1 expression, is more pronounced in non-neuronal cells, and indicative of postmortem activation of inflammation and hypoxia. Higher A-to-I editing in living tissues marks sites that are evolutionarily preserved, synaptic, developmentally timed, and disrupted in neurological conditions. Common genetic variants were also found to differentially affect A-to-I editing levels in living versus postmortem tissues. Collectively, these discoveries offer more nuanced and accurate insights into the regulatory mechanisms of RNA editing in the human brain.

Genome-Wide Association Study of Treatment-Resistant Depression: Shared Biology With Metabolic Traits.

OBJECTIVE: Treatment-resistant depression (TRD) occurs in roughly one-third of all individuals with major depressive disorder (MDD). Although research has suggested a significant common variant genetic component of liability to TRD, with heritability estimated at 8% when compared with non-treatment-resistant MDD, no replicated genetic loci have been identified, and the genetic architecture of TRD remains unclear. A key barrier to this work has been the paucity of adequately powered cohorts for investigation, largely because of the challenge in prospectively investigating this phenotype. The objective of this study was to perform a well-powered genetic study of TRD. METHODS: Using receipt of electroconvulsive therapy (ECT) as a surrogate for TRD, the authors applied standard machine learning methods to electronic health record data to derive predicted probabilities of receiving ECT. These probabilities were then applied as a quantitative trait in a genome-wide association study of 154,433 genotyped patients across four large biobanks. RESULTS: Heritability estimates ranged from 2% to 4.2%, and significant genetic overlap was observed with cognition, attention deficit hyperactivity disorder, schizophrenia, alcohol and smoking traits, and body mass index. Two genome-wide significant loci were identified, both previously implicated in metabolic traits, suggesting shared biology and potential pharmacological implications. CONCLUSIONS: This work provides support for the utility of estimation of disease probability for genomic investigation and provides insights into the genetic architecture and biology of TRD.

Serum cytokine and inflammatory markers in individuals with heroin use disorder: potential biomarkers for diagnosis and disease severity.

Opioid use disorders cause major morbidity and mortality, and there is a pressing need for novel mechanistic targets and biomarkers for diagnosis and prognosis. Exposure to mu-opioid receptor (MOR) agonists causes changes in cytokine and inflammatory protein networks in peripheral blood, and also in brain glia and neurons. Individuals with heroin use disorder (iHUD) show dysregulated levels of several cytokines in blood. However, there is limited data on a comprehensive panel of such markers in iHUD versus healthy controls (HC), especially as a multi-target biomarker. We used a validated proximity extension assay for relative quantification of 92 cytokines and inflammatory proteins in serum of iHUD on medication assisted therapy (MAT; n=21), versus HC (n=24). Twenty-nine targets showed significant group differences (primarily iHUD>HC), surviving multiple comparison correction (p=0.05). This included 19 members of canonical cytokine families, including specific chemokines, interleukins, growth factors, and tumor necrosis factor (TNF)-related proteins. For dimensionality reduction, data from these 19 cytokines were entered into a principal component (PC) analysis, and PC1 scores were iHUD>HC (p<0.0001). A receiver-operating characteristic (ROC) curve analysis yielded an AUROC=91.7% (p<0.0001). This PC1 score remained a positive predictor of being in the HUD group in a multivariable logistic regression, which included demographic/clinical variables. Overall, this study shows a panel of cytokines that differ significantly between iHUD and HC, and provides a multi-target "cytokine biomarker score" for potential diagnostic purposes, and examination of disease severity.

Divergent landscapes of A-to-I editing in postmortem and living human brain.

Adenosine-to-inosine (A-to-I) editing is a prevalent post-transcriptional RNA modification within the brain. Yet, most research has relied on postmortem samples, assuming it is an accurate representation of RNA biology in the living brain. We challenge this assumption by comparing A-to-I editing between postmortem and living prefrontal cortical tissues. Major differences were found, with over 70,000 A-to-I sites showing higher editing levels in postmortem tissues. Increased A-to-I editing in postmortem tissues is linked to higher ADAR1 and ADARB1 expression, is more pronounced in non-neuronal cells, and indicative of postmortem activation of inflammation and hypoxia. Higher A-to-I editing in living tissues marks sites that are evolutionarily preserved, synaptic, developmentally timed, and disrupted in neurological conditions. Common genetic variants were also found to differentially affect A-to-I editing levels in living versus postmortem tissues. Collectively, these discoveries illuminate the nuanced functions and intricate regulatory mechanisms of RNA editing within the human brain.

Genome-wide analysis of a model-derived binge eating disorder phenotype identifies risk loci and implicates iron metabolism.

Binge eating disorder (BED) is the most common eating disorder, yet its genetic architecture remains largely unknown. Studying BED is challenging because it is often comorbid with obesity, a common and highly polygenic trait, and it is underdiagnosed in biobank data sets. To address this limitation, we apply a supervised machine-learning approach (using 822 cases of individuals diagnosed with BED) to estimate the probability of each individual having BED based on electronic medical records from the Million Veteran Program. We perform a genome-wide association study of individuals of African (n = 77,574) and European (n = 285,138) ancestry while controlling for body mass index to identify three independent loci near the HFE, MCHR2 and LRP11 genes and suggest APOE as a risk gene for BED. We identify shared heritability between BED and several neuropsychiatric traits, and implicate iron metabolism in the pathophysiology of BED. Overall, our findings provide insights into the genetics underlying BED and suggest directions for future translational research.

Integration of Alzheimer's disease genetics and myeloid genomics identifies disease risk regulatory elements and genes.

Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer's disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.

Chromatin accessibility mapping of the striatum identifies tyrosine kinase FYN as a therapeutic target for heroin use disorder.

The current opioid epidemic necessitates a better understanding of human addiction neurobiology to develop efficacious treatment approaches. Here, we perform genome-wide assessment of chromatin accessibility of the human striatum in heroin users and matched controls. Our study reveals distinct neuronal and non-neuronal epigenetic signatures, and identifies a locus in the proximity of the gene encoding tyrosine kinase FYN as the most affected region in neurons. FYN expression, kinase activity and the phosphorylation of its target Tau are increased by heroin use in the post-mortem human striatum, as well as in rats trained to self-administer heroin and primary striatal neurons treated with chronic morphine in vitro. Pharmacological or genetic manipulation of FYN activity significantly attenuates heroin self-administration and responding for drug-paired cues in rodents. Our findings suggest that striatal FYN is an important driver of heroin-related neurodegenerative-like pathology and drug-taking behavior, making FYN a promising therapeutic target for heroin use disorder.

Genetic Variation in Long-Range Enhancers.

Cis-regulatory elements (CREs), including insulators, promoters, and enhancers, play critical roles in the establishment and maintenance of normal cellular function. Within each cell, the 3D structure of chromatin is arranged in specific patterns to expose the CREs required for optimal spatiotemporal regulation of gene expression. CREs can act over large distances along the linear genome, facilitated by looping of the intervening chromatin to allow direct interaction between distal regulatory elements and their target genes. A number of pathologies are associated with dysregulation of CRE function, including developmental disorders, cancers, and neuropsychiatric disease. A majority of known neuropsychiatric disease risk loci are noncoding, and increasing evidence suggests that they contribute to disease through disruption of CREs. As such, rather than directly altering the amino acid content of proteins, these variants are instead thought to affect where, when, and to what extent a given gene is expressed. The distances over which CREs can operate often render their target genes difficult to identify. Furthermore, as many risk loci contain multiple variants in high linkage disequilibrium, identification of the causative single nucleotide polymorphism(s) therein is not straightforward. Thus, deciphering the genetic etiology of complex neuropsychiatric disorders presents a significant challenge.

Biopsy During Minimally Invasive Intracerebral Hemorrhage Clot Evacuation.

BACKGROUND: The safety and efficacy of brain parenchyma biopsy during minimally invasive (MIS) intracerebral hemorrhage (ICH) clot evacuation has not been previously reported. The objective of this study was to establish the safety and diagnostic efficacy of brain biopsy during MIS ICH clot evacuation and to validate the modified Boston criteria as a predictor of cerebral amyloid angiopathy (CAA) in this cohort. METHODS: From October 2016 to March 2018, superficial and perihematomal biopsies were collected for 40 patients undergoing MIS ICH clot evacuation and analyzed by the pathology department to assess for various ICH etiologies. Additionally, the admission magnetic resonance imaging or computed tomography scan of each patient was analyzed and evaluated for the likelihood of a CAA etiology based on the modified Boston criteria. Student t test was used to analyze intergroup differences in continuous variables, and a 2-tailed Fisher exact test was used to determine intergroup differences of categorical variables, with significance set at P < 0.05. RESULTS: Two of the 40 patients (5%) experienced postoperative rebleed. Four of the 40 patients (10%) had evidence of CAA on biopsy. Patients with CAA on biopsy were older (P = 0.005) and had a higher prevalence of parietal lobe (P = 0.02) and occipital lobe (P = 0.001) hemorrhage. The modified Boston criteria had a sensitivity of 100% (95% confidence interval [CI], 39.6%-100%) and a specificity of 72.2% (95% CI, 54.6%-84.2%) for predicting CAA on biopsy. CONCLUSIONS: Brain biopsy in MIS ICH clot evacuation is safe and allows for the diagnosis of various ICH etiologies.

A functional genomics predictive network model identifies regulators of inflammatory bowel disease.

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

Striatal H3K27 Acetylation Linked to Glutamatergic Gene Dysregulation in Human Heroin Abusers Holds Promise as Therapeutic Target.

BACKGROUND: Opiate abuse and overdose reached epidemic levels in the United States. However, despite significant advances in animal and in vitro models, little knowledge has been directly accrued regarding the neurobiology of the opiate-addicted human brain. METHODS: We used postmortem human brain specimens from a homogeneous European Caucasian population of heroin users for transcriptional and epigenetic profiling, as well as direct assessment of chromatin accessibility in the striatum, a brain region central to reward and emotion. A rat heroin self-administration model was used to obtain translational molecular and behavioral insights. RESULTS: Our transcriptome approach revealed marked impairments related to glutamatergic neurotransmission and chromatin remodeling in the human striatum. A series of biochemical experiments tracked the specific location of the epigenetic disturbances to hyperacetylation of lysine 27 of histone H3, showing dynamic correlations with heroin use history and acute opiate toxicology. Targeted investigation of GRIA1, a glutamatergic gene implicated in drug-seeking behavior, verified the increased enrichment of lysine-27 acetylated histone H3 at discrete loci, accompanied by enhanced chromatin accessibility at hyperacetylated regions in the gene body. Analogous epigenetic impairments were detected in the striatum of heroin self-administering rats. Using this translational model, we showed that bromodomain inhibitor JQ1, which blocks the functional readout of acetylated lysines, reduced heroin self-administration and cue-induced drug-seeking behavior. CONCLUSIONS: Overall, our data suggest that heroin-related histone H3 hyperacetylation contributes to glutamatergic transcriptional changes that underlie addiction behavior and identify JQ1 as a promising candidate for targeted clinical interventions in heroin use disorder.

The Role of H3K4me3 in Transcriptional Regulation Is Altered in Huntington's Disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion of CAG repeats in the Huntingtin (HTT) gene. Previous studies have shown mutant HTT can alter expression of genes associated with dysregulated epigenetic modifications. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls, and its association with gene expression measured by RNA-sequencing. We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down-regulated in HD. Although H3K4me3 signals are expected to be associated with mRNA levels, we found an unexpected discordance between altered H3K4me3 peaks and mRNA levels. Gene ontology (GO) term enrichment analysis of the genes with differential H3K4me3 peaks, revealed statistically significantly enriched GO terms only in the genes with down-regulated signals in HD. The most frequently implicated biological process terms are organ morphogenesis and positive regulation of gene expression. More than 9,000 H3K4me3 peaks were located not near any recognized transcription start sites and approximately 36% of these "distal" peaks co-localized to known enhancer sites. Six transcription factors and chromatin remodelers are differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, PRC2 repressive state was significantly depleted in HD-enriched peaks, suggesting the epigenetic role of PRC2 inhibition associated with up-regulated H3K4me3 in Huntington's disease. In summary, our study provides new insights into transcriptional dysregulation of Huntington's disease by analyzing the differentiation of H3K4me3 enrichment.

The relationship between dopamine receptor D1 and cognitive performance.

BACKGROUND: Cognitive impairment cuts across traditional diagnostic boundaries and is one of the most typical symptoms in various psychiatric and neurobiological disorders. AIMS: The objective of this study was to examine the genetic association between 94 candidate genes, including receptors and enzymes that participate in neurotransmission, with measures of cognition. METHODS: The Clinical Dementia Rating (CDR), a global measure of cognition, and genotypes derived from a custom array of 1,536 single-nucleotide polymorphisms (SNPs) in 94 genes were available for a large postmortem cohort of Caucasian cases with Alzheimer's disease (AD), schizophrenia and controls (n=727). A cohort of healthy young males (n=1,493) originating from the LOGOS project (Learning On Genetics Of Schizophrenia Spectrum) profiled across multiple cognitive domains was available for targeted SNP genotyping. Gene expression was quantified in the superior temporal gyrus of control samples (n=109). The regulatory effect on transcriptional activity was assessed using the luciferase reporter system. RESULTS: The rs5326-A allele at the promoter region of dopamine receptor D1 (DRD1) locus was associated with: (i) poorer cognition (higher CDR) in the postmortem cohort (P=9.325×10(-4)); (ii) worse cognitive performance relevant to strategic planning in the LOGOS cohort (P=0.008); (iii) lower DRD1 gene expression in the superior temporal gyrus of controls (P=0.038); and (iv) decreased transcriptional activity in human neuroblastoma (SH-SY5Y) cells (P=0.026). CONCLUSIONS: An interdisciplinary approach combining genetics with cognitive and molecular neuroscience provided a possible mechanistic link among DRD1 and alterations in cognitive performance.

A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide.

Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide.

The CSMD1 genome-wide associated schizophrenia risk variant rs10503253 affects general cognitive ability and executive function in healthy males.

BACKGROUND: The single-nucleotide polymorphism (SNP) rs10503253, located within the CUB and Sushi multiple domains-1 (CSMD1) gene on 8p23.2, has reached genome-wide support as a risk factor for schizophrenia. There is initial but inconclusive evidence for a role of this variant in aspects of cognition. METHODS: We investigated the neurocognitive effects of the CSMD1 rs10503253 (C/A) polymorphism in a large, demographically homogeneous sample of young, healthy Greek Caucasian males (n=1149) phenotyped for a wide range of neuropsychological measures, most of which have been shown to be reliable endophenotypes for schizophrenia. RESULTS: The risk 'A' allele was associated with poorer performance on measures of general cognitive ability, strategy formation, spatial and visual working memory, set shifting, target detection and planning for problem solving but not for emotional decision making. Most of these effects were dependent on risk "A" allele dose, with AA and CC homozygotes being the worse and the best respectively, while CA individuals were intermediate. Potential genotype effects in Stroop and verbal memory performance were also suggested by our dataset. DISCUSSION: These results underline the relevance of the risk "A" allele to neurocognitive functioning and suggest that its detrimental effects on cognition, may be part of the mechanism by which the CSMD1 mediates risk for schizophrenia.

Cannabis-dependence risk relates to synergism between neuroticism and proenkephalin SNPs associated with amygdala gene expression: case-control study.

BACKGROUND: Many young people experiment with cannabis, yet only a subgroup progress to dependence suggesting individual differences that could relate to factors such as genetics and behavioral traits. Dopamine receptor D2 (DRD2) and proenkephalin (PENK) genes have been implicated in animal studies with cannabis exposure. Whether polymorphisms of these genes are associated with cannabis dependence and related behavioral traits is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Healthy young adults (18-27 years) with cannabis dependence and without a dependence diagnosis were studied (N = 50/group) in relation to a priori-determined single nucleotide polymorphisms (SNPs) of the DRD2 and PENK genes. Negative affect, Impulsive Risk Taking and Neuroticism-Anxiety temperamental traits, positive and negative reward-learning performance and stop-signal reaction times were examined. The findings replicated the known association between the rs6277 DRD2 SNP and decisions associated with negative reinforcement outcomes. Moreover, PENK variants (rs2576573 and rs2609997) significantly related to Neuroticism and cannabis dependence. Cigarette smoking is common in cannabis users, but it was not associated to PENK SNPs as also validated in another cohort (N = 247 smokers, N = 312 non-smokers). Neuroticism mediated (15.3%-19.5%) the genetic risk to cannabis dependence and interacted with risk SNPs, resulting in a 9-fold increase risk for cannabis dependence. Molecular characterization of the postmortem human brain in a different population revealed an association between PENK SNPs and PENK mRNA expression in the central amygdala nucleus emphasizing the functional relevance of the SNPs in a brain region strongly linked to negative affect. CONCLUSIONS/SIGNIFICANCE: Overall, the findings suggest an important role for Neuroticism as an endophenotype linking PENK polymorphisms to cannabis-dependence vulnerability synergistically amplifying the apparent genetic risk.

The 5-min pupillary alertness test is sensitive to modafinil: a placebo controlled study in patients with sleep apnea.

RATIONALE: The extent of pupillary miosis during 5 min in darkness is a simple, recently introduced alertness test which may become useful in the clinical assessment of normal and pathological sleepiness. OBJECTIVES: In this study, we further validated this test by testing its sensitivity to the effects of modafinil, a non-stimulant, alertness-promoting drug. METHODS: Twelve unmedicated patients recently diagnosed with obstructive sleep apnea (OSA) after polysomnography, received placebo or modafinil (200 mg), according to a double-blind, cross-over design. The patients' resting pupil diameter (RPD) was sampled over 5 min in darkness before (10:00 A.M.) and after treatment (2:00 P.M.), and their light reflexes were elicited and recorded in darkness with an infrared video pupillometer. RESULTS: We found a circadian miosis at 2:00 P.M. in the placebo treatment condition, which was reversed by modafinil. This effect correlated with modafinil-induced increase in subjective alertness, and it was greater in the most severely affected patients in terms of lowest oxygen saturation, independently of body mass index, age, or apneic episodes during sleep. Modafinil reduced the light reflex amplitude, suggesting an increase in the inhibitory input at the pupilloconstrictor Edinger-Westphal nucleus. CONCLUSIONS: These effects of modafinil are best explained via an activation of the hypoxia-sensitive nucleus locus coeruleus. The 5-min pupillary alertness test has promising predictive validity, and it holds promise as a fast and sensitive method for the objective assessment of excessive daytime sleepiness, monitoring of disease progression, and response to treatment.