Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human factor IX variant in mice and rhesus macaques.

We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction efficiencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

Role of microbes in the pathogenesis of neuropsychiatric disorders.

Microbes inhabit different anatomical sites of the human body including oral cavity, gut, and skin. A growing literature highlights how microbiome variation is associated with human health and disease. There is strong evidence of bidirectional communication between gut and brain mediated by neurotransmitters and microbial metabolites. Here, we review the potential involvement of microbes residing in the gut and in other body sites in the pathogenesis of eight neuropsychiatric disorders, discussing findings from animal and human studies. The data reported provide a comprehensive overview of the current state of the microbiome research in neuropsychiatry, including hypotheses about the mechanisms underlying the associations reported and the translational potential of probiotics and prebiotics.

Association of purine asymmetry, strand-biased gene distribution and PolC within Firmicutes and beyond: a new appraisal.

BACKGROUND: The Firmicutes often possess three conspicuous genome features: marked Purine Asymmetry (PAS) across two strands of replication, Strand-biased Gene Distribution (SGD) and presence of two isoforms of DNA polymerase III alpha subunit, PolC and DnaE. Despite considerable research efforts, it is not clear whether the co-existence of PAS, PolC and/or SGD is an essential and exclusive characteristic of the Firmicutes. The nature of correlations, if any, between these three features within and beyond the lineages of Firmicutes has also remained elusive. The present study has been designed to address these issues. RESULTS: A large-scale analysis of diverse bacterial genomes indicates that PAS, PolC and SGD are neither essential nor exclusive features of the Firmicutes. PolC prevails in four bacterial phyla: Firmicutes, Fusobacteria, Tenericutes and Thermotogae, while PAS occurs only in subsets of Firmicutes, Fusobacteria and Tenericutes. There are five major compositional trends in Firmicutes: (I) an explicit PAS or G + A-dominance along the entire leading strand (II) only G-dominance in the leading strand, (III) alternate stretches of purine-rich and pyrimidine-rich sequences, (IV) G + T dominance along the leading strand, and (V) no identifiable patterns in base usage. Presence of strong SGD has been observed not only in genomes having PAS, but also in genomes with G-dominance along their leading strands - an observation that defies the notion of co-occurrence of PAS and SGD in Firmicutes. The PolC-containing non-Firmicutes organisms often have alternate stretches of R-dominant and Y-dominant sequences along their genomes and most of them show relatively weak, but significant SGD. Firmicutes having G + A-dominance or G-dominance along LeS usually show distinct base usage patterns in three codon sites of genes. Probable molecular mechanisms that might have incurred such usage patterns have been proposed. CONCLUSION: Co-occurrence of PAS, strong SGD and PolC should not be regarded as a genome signature of the Firmicutes. Presence of PAS in a species may warrant PolC and strong SGD, but PolC and/or SGD not necessarily implies PAS.

AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

ACE2 Netlas: In silico Functional Characterization and Drug-Gene Interactions of ACE2 Gene Network to Understand Its Potential Involvement in COVID-19 Susceptibility.

Angiotensin-converting enzyme-2 (ACE2) receptor has been identified as the key adhesion molecule for the transmission of the SARS-CoV-2. However, there is no evidence that human genetic variation in ACE2 is singularly responsible for COVID-19 susceptibility. Therefore, we performed an integrative multi-level characterization of genes that interact with ACE2 (ACE2-gene network) for their statistically enriched biological properties in the context of COVID-19. The phenome-wide association of 51 genes including ACE2 with 4,756 traits categorized into 26 phenotype categories, showed enrichment of immunological, respiratory, environmental, skeletal, dermatological, and metabolic domains (p < 4e-4). Transcriptomic regulation of ACE2-gene network was enriched for tissue-specificity in kidney, small intestine, and colon (p < 4.7e-4). Leveraging the drug-gene interaction database we identified 47 drugs, including dexamethasone and spironolactone, among others. Considering genetic variants within ± 10 kb of ACE2-network genes we identified miRNAs whose binding sites may be altered as a consequence of genetic variation. The identified miRNAs revealed statistical over-representation of inflammation, aging, diabetes, and heart conditions. The genetic variant associations in RORA, SLC12A6, and SLC6A19 genes were observed in genome-wide association study (GWAS) of COVID-19 susceptibility. We also report the GWAS-identified variant in 3p21.31 locus, serves as trans-QTL for RORA and RORC genes. Overall, functional characterization of ACE2-gene network highlights several potential mechanisms in COVID-19 susceptibility. The data can also be accessed at https://gpwhiz.github.io/ACE2Netlas/.

Drinking and smoking polygenic risk is associated with childhood and early-adulthood psychiatric and behavioral traits independently of substance use and psychiatric genetic risk.

Alcohol drinking and tobacco smoking are hazardous behaviors associated with a wide range of adverse health outcomes. In this study, we explored the association of polygenic risk scores (PRS) related to drinks per week, age of smoking initiation, smoking initiation, cigarettes per day, and smoking cessation with 433 psychiatric and behavioral traits in 4498 children and young adults (aged 8-21) of European ancestry from the Philadelphia neurodevelopmental cohort. After applying a false discovery rate multiple testing correction accounting for the number of PRS and traits tested, we identified 36 associations related to psychotic symptoms, emotion and age recognition social competencies, verbal reasoning, anxiety-related traits, parents' education, and substance use. These associations were independent of the genetic correlations among the alcohol-drinking and tobacco-smoking traits and those with cognitive performance, educational attainment, risk-taking behaviors, and psychopathology. The removal of participants endorsing substance use did not affect the associations of each PRS with psychiatric and behavioral traits identified as significant in the discovery analyses. Gene-ontology enrichment analyses identified several neurobiological processes underlying mechanisms of the PRS associations we report. In conclusion, we provide novel insights into the genetic overlap of smoking and drinking behaviors in children and young adults, highlighting their independence from psychopathology and substance use.

Epigenomic Profiles of African-American Transthyretin Val122Ile Carriers Reveals Putatively Dysregulated Amyloid Mechanisms.

BACKGROUND: The Val122Ile mutation in Transthyretin (TTR) gene causes a rare, difficult to diagnose hereditary form of cardiac amyloidosis. This mutation is most common in the United States and mainly present in people of African descent. The carriers have an increased risk of congestive heart failure, peripheral edema, and several other noncardiac phenotypes such as carpal tunnel syndrome, and arthroplasty which are top reasons for ambulatory/outpatient surgeries (OSs) in the country. METHODS: We conducted first-ever epigenome-wide association study using the Illumina's EPIC array, in Val122Ile carriers of African descent for heart disease and multiple OSs-an early disease indicator. Differential methylation across genome wide cytosine-phosphate guanine (CpG) sites was tested between carriers with and without heart disease and OS. Significant CpG sites were investigated for cis-mQTLs loci, followed by gene ontology and protein-protein interaction network. We also investigated the significant CpG sites in a secondary cohort of carriers for replication. RESULTS: Five differentially methylated sites (P≤2.1×10-8) in genes-FAM129B, SKI, WDR27, GLS, and an intergenic site near RP11-550A5.2, and one differentially methylated region containing KCNA6 and GALNT3 (P=1.1×10-12) were associated with heart disease. For OS, we observe 4 sites-2 sites in UBE2E3 and SEC14L5, and other 2 in intergenic regions (P≤1.8×10-7) and 3 regions overlapping SH3D21, EVA1B, LTB4R2, and CIDEB (P≤3.9×10-7). Functional protein-interaction module analysis identified ABCA1 (P=0.001) for heart disease. Six cis-mQTLs were associated with one of the significant CpG sites (FAM129B; P=4.1×10-24). We replicated 2 CpG sites (cg18546846 and cg06641417; P<0.05) in an external cohort of biopsy-confirmed cases of TTR (transthyretin) amyloidosis. The genes identified are involved in transport and clearance of amyloid deposits (GLS, ABCA1, FAM129B); cardiac fibrosis (SKI); and muscle tissue regulation (SKI, FAM129B). CONCLUSIONS: These findings highlight the link between a complex amyloid circuit and diverse symptoms of Val122Ile.

Sex-stratified gene-by-environment genome-wide interaction study of trauma, posttraumatic-stress, and suicidality.

Epidemiologic studies recognize that trauma and posttraumatic stress are associated with heightened suicidal behavior severity, yet examination of these associations from a genetic perspective is limited. We performed a multivariate gene-by-environment genome-wide interaction study (GEWIS) of suicidality in 123,633 individuals using a covariance matrix based on 26 environments related to traumatic experiences, posttraumatic stress, social support, and socioeconomic status. We discovered five suicidality risk loci, including the male-associated rs2367967 (CWC22), which replicated in an independent cohort. All GEWIS-significant loci exhibited interaction effects where at least 5% of the sample had environmental profiles conferring opposite SNP effects from the majority. We identified PTSD as a primary driving environment for GxE at suicidality risk loci. The male suicidality GEWIS was enriched for three middle-temporal-gyrus inhibitory neuron transcriptomic profiles: SCUBE- and PVALB-expressing cells (ß = 0.028, p = 3.74 × 10-4), OPRM1-expressing cells (ß = 0.030, p = 0.001), and SPAG17-expressing cells (ß = 0.029, p = 9.80 × 10-4). Combined with gene-based analyses (CNTN5 p association  = 2.38 × 10-9, p interaction  = 1.51 × 10-3; PSMD14 p association  = 2.04 × 10-7, p interaction  = 7.76 × 10-6; HEPACAM p association  = 2.43 × 10-6, p interaction  = 3.82 × 10-7) including information about brain chromatin interaction profiles (UBE2E3 in male neuron p = 1.07 × 10-5), our GEWIS points to extracellular matrix biology and synaptic plasticity as biological interactors with the effects of potentially modifiable lifetime traumatic experiences on genetic risk for suicidality. Characterization of molecular basis for the effects of traumatic experience and posttraumatic stress on risk of suicidal behaviors may help to identify novel targets for which more effective treatments can be developed for use in high-risk populations.

ACE2 Netlas: In-silico functional characterization and drug-gene interactions of ACE2 gene network to understand its potential involvement in COVID-19 susceptibility.

Angiotensin-converting enzyme-2 ( ACE2 ) receptor has been identified as the key adhesion molecule for the transmission of the SARS-CoV-2. However, there is no evidence that human genetic variation in ACE2 is singularly responsible for COVID-19 susceptibility. Therefore, we performed a multi-level characterization of genes that interact with ACE2 (ACE2-gene network) for their over-represented biological properties in the context of COVID-19. The phenome-wide association of 51 genes including ACE2 with 4,756 traits categorized into 26 phenotype categories, showed enrichment of immunological, respiratory, environmental, skeletal, dermatological, and metabolic domains (p<4e-4). Transcriptomic regulation of ACE2-gene network was enriched for tissue-specificity in kidney, small intestine, and colon (p<4.7e-4). Leveraging the drug-gene interaction database we identified 47 drugs, including dexamethasone and spironolactone, among others. Considering genetic variants within ± 10 kb of ACE2-network genes we characterized functional consequences (among others) using miRNA binding-site targets. MiRNAs affected by ACE2-network variants revealed statistical over-representation of inflammation, aging, diabetes, and heart conditions. With respect to variants mapped to the ACE2-network, we observed COVID-19 related associations in RORA, SLC12A6 and SLC6A19 genes. Overall, functional characterization of ACE2-gene network highlights several potential mechanisms in COVID-19 susceptibility. The data can also be accessed at https://gpwhiz.github.io/ACE2Netlas/.

Heterogeneity and Polygenicity in Psychiatric Disorders: A Genome-Wide Perspective.

Genome-wide association studies (GWAS) have been performed for many psychiatric disorders and revealed a complex polygenic architecture linking mental and physical health phenotypes. Psychiatric diagnoses are often heterogeneous, and several layers of trait heterogeneity may contribute to detection of genetic risks per disorder or across multiple disorders. In this review, we discuss these heterogeneities and their consequences on the discovery of risk loci using large-scale genetic data. We primarily highlight the ways in which sex and diagnostic complexity contribute to risk locus discovery in schizophrenia, bipolar disorder, attention deficit hyperactivity disorder, autism spectrum disorder, posttraumatic stress disorder, major depressive disorder, obsessive-compulsive disorder, Tourette's syndrome and chronic tic disorder, anxiety disorders, suicidality, feeding and eating disorders, and substance use disorders. Genetic data also have facilitated discovery of clinically relevant subphenotypes also described here. Collectively, GWAS of psychiatric disorders revealed that the understanding of heterogeneity, polygenicity, and pleiotropy is critical to translate genetic findings into treatment strategies.

Strand-biased gene distribution, purine assymetry and environmental factors influence protein evolution in Bacillus.

A strong purine asymmetry, along with strand-biased gene distribution and the presence of PolC, prevails in Bacillus and some other members of Firmicutes, Fusobacteria and Tenericutes. The analysis of protein features in 21 Bacillus species of diverse metabolic, virulence and ecological traits revealed that purine asymmetry in conjunction with lineage/niche specific constraints significantly influences protein evolution in Bacillus. All Bacillus species, except for Se-respiring Bacillus selenitireducens, display distinct strand-specific biases in amino acid usage, which may affect the isoelectric point or surface charge distribution of proteins with prevalence of acidic and basic residues in the leading and lagging strand proteins, respectively.