Fatality assessment and variant risk monitoring for COVID-19 using three new hospital occupancy related metrics.

BACKGROUND: Though case fatality rate (CFR) is widely used to reflect COVID-19 fatality risk, its use is limited by large temporal and spatial variation. Hospital mortality rate (HMR) is also used to assess the severity of COVID-19, but HMR data is not directly available globally. Alternative metrics are needed for COVID-19 severity and fatality assessment. METHODS: We introduce new metrics for COVID-19 fatality risk measurements/monitoring and a new mathematical model to estimate average hospital length of stay for deaths (Ldead) and discharges (Ldis). Multiple data sources were used for our analyses. FINDINGS: We propose three, new metrics: hospital occupancy mortality rate (HOMR), ratio of total deaths to hospital occupancy (TDHOR), and ratio of hospital occupancy to cases (HOCR), for dynamic assessment of COVID-19 fatality risk. Estimated Ldead and Ldis for 501,079 COVID-19 hospitalizations in 34 US states between 7 August 2020 and 1 March 2021 were 18·2(95%CI:17·9-18·5) and 14·0(95%CI:13·9-14·0) days, respectively. We found the dramatic changes in COVID-19 CFR observed in 27 countries during early stages of the pandemic were mostly caused by undiagnosed cases. Compared to the first week of November 2021, the week mean HOCRs (mimics hospitalization-to-case ratio) for Omicron variant (58·6% of US new cases as of 25 December 2021) decreased 65·16% in the US as of 16 January 2022. INTERPRETATION: The new and reliable measurements described here could be useful for COVID-19 fatality risk and variant-associated risk monitoring. FUNDING: No specific funding was associated with the present study.

Thyroid hormone signaling specifies cone subtypes in human retinal organoids.

The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone-degrading and -activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.

Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons.

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Astroglial transcriptome dysregulation in early disease of an ALS mutant SOD1 mouse model.

Astroglia are a morphologically diverse and highly abundant cell type in the CNS. Despite these obvious observations, astroglia still remain largely uncharacterized at the cellular and molecular level. In disease contexts such as amyotrophic lateral sclerosis (ALS), it has been widely shown that astroglia downregulate crucial physiological functions, become hypertrophied, reactive, and toxic to motor neurons. However, little is known about the astroglia-specific transcriptomic changes that occur during ALS disease progression, especially early in disease. To address this, we FACS-isolated pure astroglia from early and mid-symptomatic superoxide dismutase 1 (SOD1) G93A spinal cord and performed microarray sequencing, in hopes to uncover markers and pathways driving astroglia dysfunction in ALS. After extensive analyses, we uncovered genes selectively enriched and downregulated in both control and SOD1 astroglia at both disease points. In addition, we were able to identify genes and pathways differentially expressed that may have relevance with other neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, suggesting a common theme among astroglial dysfunction in neurodegenerative disease. In aggregate, this study sheds light on the common and unique themes of dysfunction that astroglia undergo during neurodegenerative disease progression and provides candidate targets for therapeutic approaches.

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100ß, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

A comprehensive library of familial human amyotrophic lateral sclerosis induced pluripotent stem cells.

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons, leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS), among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations, usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies, however due to the differences between rodents and humans, it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore, we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations, sod1 mutations, FUS, ANG and FIG4 mutations. Certain mutations are represented with more than one line, which allows for studies of variable genetic backgrounds. In addition, these iPSCs can be successfully differentiated to astroglia, a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics.

RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

A prolyl-isomerase mediates dopamine-dependent plasticity and cocaine motor sensitization.

Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction.

Homers at the Interface between Reward and Pain.

Pain alters opioid reinforcement, presumably via neuroadaptations within ascending pain pathways interacting with the limbic system. Nerve injury increases expression of glutamate receptors and their associated Homer scaffolding proteins throughout the pain processing pathway. Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs. Thus, we investigated a potential role for Homers in the interactions between pain and drug reward in mice. Chronic constriction injury (CCI) of the sciatic nerve elevated Homer1b/c and/or Homer2a/b expression within all mesolimbic structures examined and for the most part, the Homer increases coincided with elevated mGluR5, GluN2A/B, and the activational state of various down-stream kinases. Behaviorally, CCI mice showed pain hypersensitivity and a conditioned place-aversion (CPA) at a low heroin dose that supported conditioned place-preference (CPP) in naïve controls. Null mutations of Homer1a, Homer1, and Homer2, as well as transgenic disruption of mGluR5-Homer interactions, either attenuated or completely blocked low-dose heroin CPP, and none of the CCI mutant strains exhibited heroin-induced CPA. However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI's effects) were sufficient to blunt heroin CPP in uninjured mice. However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect (intrathecal) or attenuated (NAC) heroin CPA. Together, these data implicate increases in glutamate receptor/Homer/kinase activity within limbic structures, perhaps outside the NAC, as possibly critical for switching the incentive motivational properties of heroin following nerve injury, which has relevance for opioid psychopharmacology in individuals suffering from neuropathic pain.

Oligodendroglia metabolically support axons and contribute to neurodegeneration.

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Preso1 dynamically regulates group I metabotropic glutamate receptors.

Group I metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, are G protein­coupled receptors (GPCRs) that are expressed at excitatory synapses in brain and spinal cord. GPCRs are often negatively regulated by specific G protein­coupled receptor kinases and subsequent binding of arrestin-like molecules. Here we demonstrate an alternative mechanism in which group I mGluRs are negatively regulated by proline-directed kinases that phosphorylate the binding site for the adaptor protein Homer, and thereby enhance mGluR­Homer binding to reduce signaling. This mechanism is dependent on a multidomain scaffolding protein, Preso1, that binds mGluR, Homer and proline-directed kinases and that is required for their phosphorylation of mGluR at the Homer binding site. Genetic ablation of Preso1 prevents dynamic phosphorylation of mGluR5, and Preso1(−/−) mice exhibit sustained, mGluR5-dependent inflammatory pain that is linked to enhanced mGluR signaling. Preso1 creates a microdomain for proline-directed kinases with broad substrate specificity to phosphorylate mGluR and to mediate negative regulation.

Nucleus accumbens mGluR5-associated signaling regulates binge alcohol drinking under drinking-in-the-dark procedures.

BACKGROUND: Alcohol increases the expression of Group 1 metabotropic glutamate receptors (mGluRs) and their associated scaffolding protein Homer2 and stimulates phosphatidylinositol 3-kinase (PI3K) within the nucleus accumbens (NAC). Moreover, functional studies suggest that NAC Group 1 mGluR/Homer2/PI3K signaling may be a potential target for pharmacotherapeutic intervention in alcoholism. METHODS: Immunoblotting was conducted to examine the effects of alcohol consumption under drinking-in-the-dark (DID) procedures on Group 1 mGluR-associated proteins in C57BL/6J (B6) mice. Follow-up behavioral studies examined the importance of Group 1 mGluR/Homer2/PI3K signaling within the NAC shell for limited-access alcohol drinking. Finally, immunoblotting examined whether the NAC expression of Group 1 mGluR-associated proteins is a genetic correlate of high alcohol drinking using a selectively bred high DID (HDID-1) mouse line. RESULTS: Limited-access alcohol drinking under DID procedures up-regulated NAC shell Homer2 levels, concomitant with increases in mGluR5 and NR2B. Intra-NAC shell blockade of mGluR5, Homer2, or PI3K signaling, as well as transgenic disruption of the Homer binding site on mGluR5, decreased alcohol consumption in B6 mice. Moreover, transgenic disruption of the Homer binding site on mGluR5 and Homer2 deletion both prevented the attenuating effect of mGluR5 and PI3K blockade upon intake. Finally, the basal NAC shell protein expression of mGluR1 and Homer2 was increased in offspring of HDID-1 animals. CONCLUSIONS: Taken together, these data further implicate Group 1 mGluR signaling through Homer2 within the NAC in excessive alcohol consumption.

Meta-analysis and genome-wide interpretation of genetic susceptibility to drug addiction.

BACKGROUND: Classical genetic studies provide strong evidence for heritable contributions to susceptibility to developing dependence on addictive substances. Candidate gene and genome-wide association studies (GWAS) have sought genes, chromosomal regions and allelic variants likely to contribute to susceptibility to drug addiction. RESULTS: Here, we performed a meta-analysis of addiction candidate gene association studies and GWAS to investigate possible functional mechanisms associated with addiction susceptibility. From meta-data retrieved from 212 publications on candidate gene association studies and 5 GWAS reports, we linked a total of 843 haplotypes to addiction susceptibility. We mapped the SNPs in these haplotypes to functional and regulatory elements in the genome and estimated the magnitude of the contributions of different molecular mechanisms to their effects on addiction susceptibility. In addition to SNPs in coding regions, these data suggest that haplotypes in gene regulatory regions may also contribute to addiction susceptibility. When we compared the lists of genes identified by association studies and those identified by molecular biological studies of drug-regulated genes, we observed significantly higher participation in the same gene interaction networks than expected by chance, despite little overlap between the two gene lists. CONCLUSIONS: These results appear to offer new insights into the genetic factors underlying drug addiction.

Harmine, a natural beta-carboline alkaloid, upregulates astroglial glutamate transporter expression.

Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2/GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2/GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

A human-specific de novo protein-coding gene associated with human brain functions.

To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203). Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

Genome wide association for addiction: replicated results and comparisons of two analytic approaches.

BACKGROUND: Vulnerabilities to dependence on addictive substances are substantially heritable complex disorders whose underlying genetic architecture is likely to be polygenic, with modest contributions from variants in many individual genes. "Nontemplate" genome wide association (GWA) approaches can identity groups of chromosomal regions and genes that, taken together, are much more likely to contain allelic variants that alter vulnerability to substance dependence than expected by chance. METHODOLOGY/PRINCIPAL FINDINGS: We report pooled "nontemplate" genome-wide association studies of two independent samples of substance dependent vs control research volunteers (n = 1620), one European-American and the other African-American using 1 million SNP (single nucleotide polymorphism) Affymetrix genotyping arrays. We assess convergence between results from these two samples using two related methods that seek clustering of nominally-positive results and assess significance levels with Monte Carlo and permutation approaches. Both "converge then cluster" and "cluster then converge" analyses document convergence between the results obtained from these two independent datasets in ways that are virtually never found by chance. The genes identified in this fashion are also identified by individually-genotyped dbGAP data that compare allele frequencies in cocaine dependent vs control individuals. CONCLUSIONS/SIGNIFICANCE: These overlapping results identify small chromosomal regions that are also identified by genome wide data from studies of other relevant samples to extents much greater than chance. These chromosomal regions contain more genes related to "cell adhesion" processes than expected by chance. They also contain a number of genes that encode potential targets for anti-addiction pharmacotherapeutics. "Nontemplate" GWA approaches that seek chromosomal regions in which nominally-positive associations are found in multiple independent samples are likely to complement classical, "template" GWA approaches in which "genome wide" levels of significance are sought for SNP data from single case vs control comparisons.

Binge drinking upregulates accumbens mGluR5-Homer2-PI3K signaling: functional implications for alcoholism.

The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC metabotropic glutamate receptor (mGluR)-Homer2-phosphatidylinositol 3-kinase (PI3K) signaling in regulating excessive alcohol consumption within the context of the scheduled high alcohol consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking ( approximately 1.5 g/kg per 30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knockdown of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride] (0.1-1 microg/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared with wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5-Homer-PI3K signaling may be a mechanism governing excessive alcohol intake, the "anti-binge" effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5(F1128R) transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2, and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5-Homer2-PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5-Homer2-PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.

OKCAM: an ontology-based, human-centered knowledgebase for cell adhesion molecules.

'Cell adhesion molecules' (CAMs) are essential elements of cell/cell communication that are important for proper development and plasticity of a variety of organs and tissues. In the brain, appropriate assembly and tuning of neuronal connections is likely to require appropriate function of many cell adhesion processes. Genetic studies have linked and/or associated CAM variants with psychiatric, neurologic, neoplastic, immunologic and developmental phenotypes. However, despite increasing recognition of their functional and pathological significance, no systematic study has enumerated CAMs or documented their global features. We now report compilation of 496 human CAM genes in six gene families based on manual curation of protein domain structures, Gene Ontology annotations, and 1487 NCBI Entrez annotations. We map these genes onto a cell adhesion molecule ontology that contains 850 terms, up to seven levels of depth and provides a hierarchical description of these molecules and their functions. We develop OKCAM, a CAM knowledgebase that provides ready access to these data and ontologic system at http://okcam.cbi.pku.edu.cn. We identify global CAM properties that include: (i) functional enrichment, (ii) over-represented regulation modes and expression patterns and (iii) relationships to human Mendelian and complex diseases, and discuss the strengths and limitations of these data.

Genome wide association for substance dependence: convergent results from epidemiologic and research volunteer samples.

BACKGROUND: Dependences on addictive substances are substantially-heritable complex disorders whose molecular genetic bases have been partially elucidated by studies that have largely focused on research volunteers, including those recruited in Baltimore. Maryland. Subjects recruited from the Baltimore site of the Epidemiological Catchment Area (ECA) study provide a potentially-useful comparison group for possible confounding features that might arise from selecting research volunteer samples of substance dependent and control individuals. We now report novel SNP (single nucleotide polymorphism) genome wide association (GWA) results for vulnerability to substance dependence in ECA participants, who were initially ascertained as members of a probability sample from Baltimore, and compare the results to those from ethnically-matched Baltimore research volunteers. RESULTS: We identify substantial overlap between the home address zip codes reported by members of these two samples. We find overlapping clusters of SNPs whose allele frequencies differ with nominal significance between substance dependent vs control individuals in both samples. These overlapping clusters of nominally-positive SNPs identify 172 genes in ways that are never found by chance in Monte Carlo simulation studies. Comparison with data from human expressed sequence tags suggests that these genes are expressed in brain, especially in hippocampus and amygdala, to extents that are greater than chance. CONCLUSION: The convergent results from these probability sample and research volunteer sample datasets support prior genome wide association results. They fail to support the idea that large portions of the molecular genetic results for vulnerability to substance dependence derive from factors that are limited to research volunteers.

Molecular neurobiological methods in marijuana-cannabinoid research.

Recent aggregation of evidence for the roles of endogenous agonist and receptor systems that are mimicked or activated by cannabanoid ligands has provided a focus for work that has elucidated details of some of the multiple physiological roles and pharmacological functions that these systems play in brain and peripheral tissues. This chapter reviews some of the approaches to improved elucidation of these systems, with special focus on the human genes that encode cannabanoid receptors and the variants in these receptors that appear likely to contribute to human addiction vulnerabilities.