Transcriptome-wide gene-gene interaction associations elucidate pathways and functional enrichment of complex traits.

It remains unknown to what extent gene-gene interactions contribute to complex traits. Here, we introduce a new approach using predicted gene expression to perform exhaustive transcriptome-wide interaction studies (TWISs) for multiple traits across all pairs of genes expressed in several tissue types. Using imputed transcriptomes, we simultaneously reduce the computational challenge and improve interpretability and statistical power. We discover (in the UK Biobank) and replicate (in independent cohorts) several interaction associations, and find several hub genes with numerous interactions. We also demonstrate that TWIS can identify novel associated genes because genes with many or strong interactions have smaller single-locus model effect sizes. Finally, we develop a method to test gene set enrichment of TWIS associations (E-TWIS), finding numerous pathways and networks enriched in interaction associations. Epistasis is may be widespread, and our procedure represents a tractable framework for beginning to explore gene interactions and identify novel genomic targets.

South Asia: The Missing Diverse in Diversity.

South Asia, making up around 25% of the world's population, encompasses a wide range of individuals with tremendous genetic and environmental diversity. This region, which spans eight countries, is home to over 4500 anthropologically defined groups that speak numerous languages and have an array of religious beliefs and cultures, making it one of the most diverse places in the world. Much of the region's rich genetic diversity and structure is the result of a complex combination of population history, migration patterns, and endogamous practices. Despite the overwhelming size and diversity, South Asians have often been underrepresented in genetic research, making up less than 2% of the participants in genetic studies. This has led to a lack of population specific understanding of genetic disease risks. We aim to raise awareness about underlying genetic diversity in this ancestry group, call attention to the lack of representation of the group, and to highlight strategies for future studies in South Asians.

Reinvigoration of a Diversity, Equity, and Inclusion Committee at the Institute for Behavioral Genetics: Student-driven Progress.

Extremist far-right ideologies, including scientifically inaccurate beliefs about race, are on the rise (Mierina and Koroleva 2015; Youngblood 2020); individuals perpetuating such ideologies occasionally cite genetics research, including behavioral genetics research. This highlights the need for behavioral geneticists to actively confront extremist ideology and promote anti-racism. We emphasize the need for Diversity, Equity and Inclusion (DEI) committees within behavioral genetics institutions. DEI committees can lead to: greater awareness of ways in which behavioral genetics has been misused (historically and currently) to harm minoritized communities, increased discussions on conducting ethical behavioral genetics research, and increased collaboration for conducting more diverse behavioral genetics research. We discuss the activities and goals of the student-driven DEI committee at the Institute for Behavior Genetics (IBG). At the same time, we acknowledge we have a long way to go, both as a committee and as a field. Our committee is still in its early stages; we discuss challenges to increasing DEI in the field and present future goals for both IBG and the behavioral genetics community as we explore the process of implementing DEI work.

Testing Association of Previously Implicated Gene Sets and Gene-Networks in Nicotine Exposed Mouse Models with Human Smoking Phenotypes.

INTRODUCTION: Smoking behaviors are partly heritable, yet the genetic and environmental mechanisms underlying smoking phenotypes are not fully understood. Developmental nicotine exposure (DNE) is a significant risk factor for smoking and leads to gene expression changes in mouse models; however, it is unknown whether the same genes whose expression is impacted by DNE are also those underlying smoking genetic liability. We examined whether genes whose expression in D1-type striatal medium spiny neurons due to DNE in the mouse are also associated with human smoking behaviors. METHODS: Specifically, we assessed whether human orthologs of mouse-identified genes, either individually or as a set, were genetically associated with five human smoking traits using MAGMA and S-LDSC while implementing a novel expression-based gene-SNP annotation methodology. RESULTS: We found no strong evidence that these genes sets were more strongly associated with smoking behaviors than the rest of the genome, but ten of these individual genes were significantly associated with three of the five human smoking traits examined (p < 2.5e-6). Three of these genes have not been reported previously and were discovered only when implementing the expression-based annotation. CONCLUSIONS: These results suggest the genes whose expression is impacted by DNE in mice are largely distinct from those contributing to smoking genetic liability in humans. However, examining a single mouse neuronal cell type may be too fine a resolution for comparison, suggesting that experimental manipulation of nicotine consumption, reward, or withdrawal in mice may better capture genes related to the complex genetics of human tobacco use. IMPLICATIONS: Genes whose expression is impacted by DNE in mouse D1-type striatal medium spiny neurons were not found to be, as a whole, more strongly associated with human smoking behaviors than the rest of the genome, though ten individual mouse-identified genes were associated with human smoking traits. This suggests little overlap between the genetic mechanisms impacted by DNE and those influencing heritable liability to smoking phenotypes in humans. Further research is warranted to characterize how developmental nicotine exposure paradigms in mice can be translated to understand nicotine use in humans and their heritable effects on smoking.

Whole-Genome Sequencing of Inbred Mouse Strains Selected for High and Low Open-Field Activity.

We conducted whole-genome sequencing of four inbred mouse strains initially selected for high (H1, H2) or low (L1, L2) open-field activity (OFA), and then examined strain distribution patterns for all DNA variants that differed between their BALB/cJ and C57BL/6J parental strains. Next, we assessed genome-wide sharing (3,678,826 variants) both between and within the High and Low Activity strains. Results suggested that about 10% of these DNA variants may be associated with OFA, and clearly demonstrated its polygenic nature. Finally, we conducted bioinformatic analyses of functional genomics data from mouse, rat, and human to refine previously identified quantitative trait loci (QTL) for anxiety-related measures. This combination of sequence analysis and genomic-data integration facilitated refinement of previously intractable QTL findings, and identified possible genes for functional follow-up studies.

Little Evidence of Modified Genetic Effect of rs16969968 on Heavy Smoking Based on Age of Onset of Smoking.

INTRODUCTION: Tobacco smoking is the leading cause of preventable death globally. Smoking quantity, measured in cigarettes per day, is influenced both by the age of onset of regular smoking (AOS) and by genetic factors, including a strong effect of the nonsynonymous single-nucleotide polymorphism rs16969968. A previous study by Hartz et al. reported an interaction between these two factors, whereby rs16969968 risk allele carriers who started smoking earlier showed increased risk for heavy smoking compared with those who started later. This finding has yet to be replicated in a large, independent sample. METHODS: We performed a preregistered, direct replication attempt of the rs16969968 × AOS interaction on smoking quantity in 128 383 unrelated individuals from the UK Biobank, meta-analyzed across ancestry groups. We fit statistical association models mirroring the original publication as well as formal interaction tests on multiple phenotypic and analytical scales. RESULTS: We replicated the main effects of rs16969968 and AOS on cigarettes per day but failed to replicate the interaction using previous methods. Nominal significance of the rs16969968 × AOS interaction term depended strongly on the scale of analysis and the particular phenotype, as did associations stratified by early/late AOS. No interaction tests passed genome-wide correction (α = 5e-8), and all estimated interaction effect sizes were much smaller in magnitude than previous estimates. CONCLUSIONS: We failed to replicate the strong rs16969968 × AOS interaction effect previously reported. If such gene-moderator interactions influence complex traits, they likely depend on scale of measurement, and current biobanks lack the power to detect significant genome-wide associations given the minute effect sizes expected. IMPLICATIONS: We failed to replicate the strong rs16969968 × AOS interaction effect on smoking quantity previously reported. If such gene-moderator interactions influence complex traits, current biobanks lack the power to detect significant genome-wide associations given the minute effect sizes expected. Furthermore, many potential interaction effects are likely to depend on the scale of measurement employed.

Novel characterization of the multivariate genetic architecture of internalizing psychopathology and alcohol use.

Genetic correlations suggest that the genetic relationship of alcohol use with internalizing psychopathology depends on the measure of alcohol use. Problematic alcohol use (PAU) is positively genetically correlated with internalizing psychopathology, whereas alcohol consumption ranges from not significantly correlated to moderately negatively correlated with internalizing psychopathology. To explore these different genetic relationships of internalizing psychopathology with alcohol use, we performed a multivariate genome-wide association study of four correlated factors (internalizing psychopathology, PAU, quantity of alcohol consumption, and frequency of alcohol consumption) and then assessed genome-wide and local genetic covariance between these factors. We identified 14 significant regions of local, largely positive, genetic covariance between PAU and internalizing psychopathology and 12 regions of significant local genetic covariance (including both positive and negative genetic covariance) between consumption factors and internalizing psychopathology. Partitioned genetic covariance among functional annotations suggested that brain tissues contribute significantly to positive genetic covariance between internalizing psychopathology and PAU but not to the genetic covariance between internalizing psychopathology and quantity or frequency of alcohol consumption. We hypothesize that genome-wide genetic correlations between alcohol use and psychiatric traits may not capture the more complex shared or divergent genetic architectures at the locus or tissue specific level. This study highlights the complexity of genetic architectures of alcohol use and internalizing psychopathology, and the differing shared genetics of internalizing disorders with PAU compared to consumption.

The Role of A Priori-Identified Addiction and Smoking Gene Sets in Smoking Behaviors.

INTRODUCTION: Smoking is a leading cause of death, and genetic variation contributes to smoking behaviors. Identifying genes and sets of genes that contribute to risk for addiction is necessary to prioritize targets for functional characterization and for personalized medicine. METHODS: We performed a gene set-based association and heritable enrichment study of two addiction-related gene sets, those on the Smokescreen Genotyping Array and the nicotinic acetylcholine receptors, using the largest available GWAS summary statistics. We assessed smoking initiation, cigarettes per day, smoking cessation, and age of smoking initiation. RESULTS: Individual genes within each gene set were significantly associated with smoking behaviors. Both sets of genes were significantly associated with cigarettes per day, smoking initiation, and smoking cessation. Age of initiation was only associated with the Smokescreen gene set. Although both sets of genes were enriched for trait heritability, each accounts for only a small proportion of the single nucleotide polymorphism-based heritability (2%-12%). CONCLUSIONS: These two gene sets are associated with smoking behaviors, but collectively account for a limited amount of the genetic and phenotypic variation of these complex traits, consistent with high polygenicity. IMPLICATIONS: We evaluated evidence for the association and heritable contribution of expert-curated and bioinformatically identified sets of genes related to smoking. Although they impact smoking behaviors, these specifically targeted genes do not account for much of the heritability in smoking and will be of limited use for predictive purposes. Advanced genome-wide approaches and integration of other 'omics data will be needed to fully account for the genetic variation in smoking phenotypes.

Common and Rare Variants Genetic Association Analysis of Cigarettes per Day Among Ever-Smokers in Chronic Obstructive Pulmonary Disease Cases and Controls.

INTRODUCTION: Cigarette smoking is a major environmental risk factor for many diseases, including chronic obstructive pulmonary disease (COPD). There are shared genetic influences on cigarette smoking and COPD. Genetic risk factors for cigarette smoking in cohorts enriched for COPD are largely unknown. METHODS: We performed genome-wide association analyses for average cigarettes per day (CPD) across the Genetic Epidemiology of COPD (COPDGene) non-Hispanic white (NHW) (n = 6659) and African American (AA) (n = 3260), GenKOLS (the Genetics of Chronic Obstructive Lung Disease) (n = 1671), and ECLIPSE (the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) (n = 1942) cohorts. In addition, we performed exome array association analyses across the COPDGene NHW and AA cohorts. We considered analyses across the entire cohort and stratified by COPD case-control status. RESULTS: We identified genome-wide significant associations for CPD on chromosome 15q25 across all cohorts (lowest p = 1.78 × 10-15), except in the COPDGene AA cohort alone. Previously reported associations on chromosome 19 had suggestive and directionally consistent associations (RAB4, p = 1.95 × 10-6; CYP2A7, p = 7.50 × 10-5; CYP2B6, p = 4.04 × 10-4). When we stratified by COPD case-control status, single nucleotide polymorphisms on chromosome 15q25 were nominally associated with both NHW COPD cases (ß = 0.11, p = 5.58 × 10-4) and controls (ß = 0.12, p = 3.86 × 10-5) For the gene-based exome array association analysis of rare variants, there were no exome-wide significant associations. For these previously replicated associations, the most significant results were among COPDGene NHW subjects for CYP2A7 (p = 5.2 × 10-4). CONCLUSIONS: In a large genome-wide association study of both common variants and a gene-based association of rare coding variants in ever-smokers, we found genome-wide significant associations on chromosome 15q25 with CPD for common variants, but not for rare coding variants. These results were directionally consistent among COPD cases and controls. IMPLICATIONS: We examined both common and rare coding variants associated with CPD in a large population of heavy smokers with and without COPD of NHW and AA descent. We replicated genome-wide significant associations on chromosome 15q25 with CPD for common variants among NHW subjects, but not for rare variants. We demonstrated for the first time that common variants on chromosome 15q25 associated with CPD are similar among COPD cases and controls. Previously reported associations on chromosome 19 showed suggestive and directionally consistent associations among common variants (RAB4, CYP2A7, and CYP2B6) and for rare variants (CYP2A7) among COPDGene NHW subjects. Although the genetic effect sizes for these single nucleotide polymorphisms on chromosome 15q25 are modest, we show that this creates a substantial smoking burden over the lifetime of a smoker.

Examination of the Involvement of Cholinergic-Associated Genes in Nicotine Behaviors in European and African Americans.

INTRODUCTION: Cigarette smoking is a physiologically harmful habit. Nicotinic acetylcholine receptors (nAChRs) are bound by nicotine and upregulated in response to chronic exposure to nicotine. It is known that upregulation of these receptors is not due to a change in mRNA of these genes, however, more precise details on the process are still uncertain, with several plausible hypotheses describing how nAChRs are upregulated. We have manually curated a set of genes believed to play a role in nicotine-induced nAChR upregulation. Here, we test the hypothesis that these genes are associated with and contribute risk for nicotine dependence (ND) and the number of cigarettes smoked per day (CPD). METHODS: Studies with genotypic data on European and African Americans (EAs and AAs, respectively) were collected and a gene-based test was run to test for an association between each gene and ND and CPD. RESULTS: Although several novel genes were associated with CPD and ND at P < 0.05 in EAs and AAs, these associations did not survive correction for multiple testing. Previous associations between CHRNA3, CHRNA5, CHRNB4 and CPD in EAs were replicated. CONCLUSIONS: Our hypothesis-driven approach avoided many of the limitations inherent in pathway analyses and provided nominal evidence for association between cholinergic-related genes and nicotine behaviors. IMPLICATIONS: We evaluated the evidence for association between a manually curated set of genes and nicotine behaviors in European and African Americans. Although no genes were associated after multiple testing correction, this study has several strengths: by manually curating a set of genes we circumvented the limitations inherent in many pathway analyses and tested several genes that had not yet been examined in a human genetic study; gene-based tests are a useful way to test for association with a set of genes; and these genes were collected based on literature review and conversations with experts, highlighting the importance of scientific collaboration.

Evidence for Association Between Low Frequency Variants in CHRNA6/CHRNB3 and Antisocial Drug Dependence.

Common SNPs in nicotinic acetylcholine receptor genes (CHRN genes) have been associated with drug behaviors and personality traits, but the influence of rare genetic variants is not well characterized. The goal of this project was to identify novel rare variants in CHRN genes in the Center for Antisocial Drug Dependence (CADD) and Genetics of Antisocial Drug Dependence (GADD) samples and to determine if low frequency variants are associated with antisocial drug dependence. Two samples of 114 and 200 individuals were selected using a case/control design including the tails of the phenotypic distribution of antisocial drug dependence. The capture, sequencing, and analysis of all variants in 16 CHRN genes (CHRNA1-7, 9, 10, CHRNB1-4, CHRND, CHRNG, CHRNE) were performed independently for each subject in each sample. Sequencing reads were aligned to the human reference sequence using BWA prior to variant calling with the Genome Analysis ToolKit (GATK). Low frequency variants (minor allele frequency < 0.05) were analyzed using SKAT-O and C-alpha to examine the distribution of rare variants among cases and controls. In our larger sample, the region containing the CHRNA6/CHRNB3 gene cluster was significantly associated with disease status using both SKAT-O and C-alpha (unadjusted p values <0.05). More low frequency variants in the CHRNA6/CHRNB3 gene region were observed in cases compared to controls. These data support a role for genetic variants in CHRN genes and antisocial drug behaviors.

Examination of genetic variation in GABRA2 with conduct disorder and alcohol abuse and dependence in a longitudinal study.

Previous studies have shown associations between single nucleotide polymorphisms (SNPs) in gamma aminobutyric acid receptor alpha 2 (GABRA2) and adolescent conduct disorder (CD) and alcohol dependence in adulthood, but not adolescent alcohol dependence. The present study was intended as a replication and extension of this work, focusing on adolescent CD, adolescent alcohol abuse and dependence (AAD), and adult AAD. Family based association tests were run using Hispanics and non-Hispanic European American subjects from two independent longitudinal samples. Although the analysis provided nominal support for an association with rs9291283 and AAD in adulthood and CD in adolescence, the current study failed to replicate previous associations between two well replicated GABRA2 SNPs and CD and alcohol dependence. Overall, these results emphasize the utility of including an independent replication sample in the study design, so that the results from an individual sample can be weighted in the context of its reproducibility.

Opioid trail: Tracking contributions to opioid use disorder from host genetics to the gut microbiome.

Opioid use disorder (OUD) is a worldwide public health crisis with few effective treatment options. Traditional genetics and neuroscience approaches have provided knowledge about biological mechanisms that contribute to OUD-related phenotypes, but the complexity and magnitude of effects in the brain and body remain poorly understood. The gut-brain axis has emerged as a promising target for future therapeutics for several psychiatric conditions, so characterizing the relationship between host genetics and the gut microbiome in the context of OUD will be essential for development of novel treatments. In this review, we describe evidence that interactions between host genetics, the gut microbiome, and immune signaling likely play a key role in mediating opioid-related phenotypes. Studies in humans and model organisms consistently demonstrated that genetic background is a major determinant of gut microbiome composition. Furthermore, the gut microbiome is susceptible to environmental influences such as opioid exposure. Additional work focused on gene by microbiome interactions will be necessary to gain improved understanding of their effects on OUD-related behaviors.

Loci on chromosome 20 interact with rs16969968 to influence cigarettes per day in European ancestry individuals.

BACKGROUND: The understanding of the molecular genetic contributions to smoking is largely limited to the additive effects of individual single nucleotide polymorphisms (SNPs), but the underlying genetic risk is likely to also include dominance, epistatic, and gene-environment interactions. METHODS: To begin to address this complexity, we attempted to identify genetic interactions between rs16969968, the most replicated SNP associated with smoking quantity, and all SNPs and genes across the genome. RESULTS: Using the UK Biobank European subsample, we found one SNP, rs1892967, and two genes, PCNA and TMEM230, that showed a significant genome-wide interaction with rs16969968 for log10 CPD and raw CPD, respectively, in a sample of 116 442 individuals who self-reported currently or previously smoking. We extended these analyses to individuals of South Asian descent and meta-analyzed the combined sample of 117 212 individuals of European and South Asian ancestry. We replicated the gene findings in a meta-analysis of five Finnish samples (N=40 140): FinHealth, FINRISK, Finnish Twin Cohort, GeneRISK, and Health-2000-2011. CONCLUSIONS: To our knowledge, this represents the first reliable epistatic association between single nucleotide polymorphisms for smoking behaviors and provides a novel direction for possible future functional studies related to this interaction. Furthermore, this work demonstrates the feasibility of these analyses by pooling multiple datasets across various ancestries, which may be applied to other top SNPs for smoking and/or other phenotypes.

Genetic background and sex influence somatosensory sensitivity and oxycodone analgesia in the Hybrid Rat Diversity Panel.

Opioid use disorder (OUD) is an ongoing public health concern in the United States, and relatively little work has addressed how genetic background contributes to OUD. Understanding the genetic contributions to oxycodone-induced analgesia could provide insight into the early stages of OUD development. Here, we present findings from a behavioral phenotyping protocol using several inbred strains from the Hybrid Rat Diversity Panel. Our behavioral protocol included a modified "up-down" von Frey procedure to measure inherent strain differences in the sensitivity to a mechanical stimulus on the hindpaw. We also performed the tail immersion assay, which measures the latency to display tail withdrawal in response to a hot water bath. Initial withdrawal thresholds were taken in drug-naïve animals to record baseline thermal sensitivity across the strains. Oxycodone-induced analgesia was measured after administration of oxycodone over the course of 2 h. Both mechanical and thermal sensitivity are shaped by genetic factors and display moderate heritability (h2 = 0.23-0.40). All strains displayed oxycodone-induced analgesia that peaked at 15-30 min and returned to baseline by 2 h. There were significant differences between the strains in the magnitude and duration of their analgesic response to oxycodone, although the heritability estimates were quite modest (h2 = 0.10-0.15). These data demonstrate that genetic background confers differences in mechanical sensitivity, thermal sensitivity, and oxycodone-induced analgesia.

AKT2 modulates astrocytic nicotine responses in vivo.

A better understanding of nicotine neurobiology is needed to reduce or prevent chronic addiction, ameliorate the detrimental effects of nicotine withdrawal, and increase successful cessation of use. Nicotine binds and activates two astrocyte-expressed nicotinic acetylcholine receptors (nAChRs), α4ß2 and α7. We recently found that Protein kinase B-ß (Pkb-ß or Akt2) expression is restricted to astrocytes in mice and humans. To determine if AKT2 plays a role in astrocytic nicotinic responses, we generated astrocyte-specific Akt2 conditional knockout (cKO) and full Akt2 KO mice for in vivo and in vitro experiments. For in vivo studies, we examined mice exposed to chronic nicotine for two weeks in drinking water (200 µg/mL) and following acute nicotine challenge (0.09, 0.2 mg/kg) after 24 hrs. Our in vitro studies used cultured mouse astrocytes to measure nicotine-dependent astrocytic responses. We validated our approaches using lipopolysaccharide (LPS) exposure inducing astrogliosis. Sholl analysis was used to measure glial fibrillary acidic protein responses in astrocytes. Our data show that wild-type (WT) mice exhibit increased astrocyte morphological complexity during acute nicotine exposure, with decreasing complexity during chronic nicotine use, whereas Akt2 cKO mice showed increased astrocyte morphology complexity. In culture, we found that 100µM nicotine was sufficient for morphological changes and blocking α7 or α4ß2 nAChRs prevented observed morphologic changes. Finally, we performed conditioned place preference (CPP) in Akt2 cKO mice and found that astrocytic AKT2 deficiency reduced nicotine preference compared to controls. These findings show the importance of nAChRs and Akt2 signaling in the astrocytic response to nicotine.

Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD.

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10(-35) and <10(-8) respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10(-6)). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10(-20)) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Collaborative study on the genetics of alcoholism: The strength of collaboration, team science, and longitudinal data.

This issue contains a series of articles describing the various resources, studies, results, and future directions for the collaborative study on the genetics of alcoholism (COGA). The collaborative and integrative approach initiated by this group ~30 years ago serves as an excellent example of the strength of team science. Individually, various aspects of COGA would be limited in their impact toward improved understanding of alcohol use disorder. Collectively, their wholistic approach which spans deep longitudinal phenotypic assessments in families to include the application of large-scale omics technologies and cell-culture based molecular studies has demonstrated the power of working together.

Diazepam effects on anxiety-related defensive behavior of male and female high and low open-field activity inbred mouse strains.

Open-field activity is a commonly used measure of anxiety-related behavior in rodents. The inbred High and Low Activity strains of mice, selected for extreme differences in open-field activity, have been used as a genetic model of anxiety-related behaviors. These selected strains have been thoroughly studied through extensive behavioral testing, quantitative trait locus (QTL) mapping, whole-genome sequencing, and RNA sequencing, to uncover phenotypic and genotypic differences related to anxiety-related behavior. However, the effects of anxiolytic drugs on anxiety-related behavior in these strains have not been studied previously. This study allowed us to expand on previous findings to further characterize the anxiety-related behavior of these unique strains, using an anxiolytic drug. The goal of this study was to determine whether the treatment of adult male and female High Activity (low anxiety) and Low Activity (high anxiety) mice with diazepam, an agonist at the benzodiazepine allosteric site on the GABAA receptor and a drug commonly prescribed to treat anxiety disorders in humans, led to decreases in anxiety-like defensive behavioral responses as assessed in the open-field test (OFT) and elevated plus-maze (EPM). We tested the effects of three doses of diazepam (0, 0.5, 1.0, 3.0 mg/kg, i.p.), given 30 min before behavioral testing to one High Activity strain (H2) and two Low Activity strains (L1 and L2). There was an anxiolytic effect of diazepam observed in the High Activity strain, with more entries into the open arms of the elevated plus-maze, an effect similar to that seen in common mouse strains. However, the only anxiolytic effect of diazepam seen in the Low Activity strains was a reduction in stretch attend posture (SAP). Low Activity strains also displayed freezing behavior in both the OFT and EPM. The combination of the observed freezing behavior, that was not reduced by diazepam, and the reduction in SAP seen with diazepam, suggests a more complex phenotype that includes a component of innate fear in addition to anxiety-related risk assessment behaviors. Since fear and anxiety are distinguishable traits, and both contribute to human anxiety disorders, these results provide novel insight about interpretation of previous genetic and phenotypic differences observed between the High and Low Activity strains.

Hippocampal RNA sequencing in mice selectively bred for high and low activity.

High and Low Activity strains of mice were bidirectionally selected for differences in open-field activity (DeFries et al., 1978, Behavior Genetics, 8: 3-13) and subsequently inbred to use as a genetic model for studying anxiety-like behaviors (Booher et al., 2021, Genes, Brain and Behavior, 20: e12730). Hippocampal RNA-sequencing of the High and Low Activity mice identified 3901 differentially expressed protein-coding genes, with both sex-dependent and sex-independent effects. Functional enrichment analysis (PANTHER) highlighted 15 gene ontology terms, which allowed us to create a narrow list of 264 top candidate genes. Of the top candidate genes, 46 encoded four Complexes (I, II, IV and V) and two electron carriers (cytochrome c and ubiquinone) of the mitochondrial oxidative phosphorylation process. The most striking results were in the female high anxiety, Low Activity mice, where 39/46 genes relating to oxidative phosphorylation were upregulated. In addition, comparison of our top candidate genes with two previously curated High and Low Activity gene lists highlight 24 overlapping genes, where Ndufa13, which encodes the supernumerary subunit A13 of complex I, was the only gene to be included in all three lists. Mitochondrial dysfunction has recently been implicated as both a cause and effect of anxiety-related disorders and thus should be further explored as a possible novel pharmaceutical treatment for anxiety disorders.