Ligand-Free Signaling of G-Protein-Coupled Receptors: Physiology, Pharmacology, and Genetics.

G-protein-coupled receptors (GPCRs) are ubiquitous sensors and regulators of cellular functions. Each GPCR exists in complex aggregates with multiple resting and active conformations. Designed to detect weak stimuli, GPCRs can also activate spontaneously, resulting in basal ligand-free signaling. Agonists trigger a cascade of events leading to an activated agonist-receptor G-protein complex with high agonist affinity. However, the ensuing signaling process can further remodel the receptor complex to reduce agonist affinity, causing rapid ligand dissociation. The acutely activated ligand-free receptor can continue signaling, as proposed for rhodopsin and µ opioid receptors, resulting in robust receptor activation at low agonist occupancy with enhanced agonist potency. Continued receptor stimulation can further modify the receptor complex, regulating sustained ligand-free signaling-proposed to play a role in opioid dependence. Basal, acutely agonist-triggered, and sustained elevated ligand-free signaling could each have distinct functions, reflecting multi-state conformations of GPCRs. This review addresses basal and stimulus-activated ligand-free signaling, its regulation, genetic factors, and pharmacological implications, focusing on opioid and serotonin receptors, and the growth hormone secretagogue receptor (GHSR). The hypothesis is proposed that ligand-free signaling of 5-HT2A receptors mediate therapeutic effects of psychedelic drugs. Research avenues are suggested to close the gaps in our knowledge of ligand-free GPCR signaling.

Human alveolar macrophage response to Mycobacterium tuberculosis: immune characteristics underlying large inter-individual variability.

Background: Mycobacterium tuberculosis (M.tb), the causative bacterium of tuberculosis (TB), establishes residence and grows in human alveolar macrophages (AMs). Inter-individual variation in M.tb-human AM interactions can indicate TB risk and the efficacy of therapies and vaccines; however, we currently lack an understanding of the gene and protein expression programs that dictate this variation in the lungs. Results: Herein, we systematically analyze interactions of a virulent M.tb strain H37Rv with freshly isolated human AMs from 28 healthy adult donors, measuring host RNA expression and secreted candidate proteins associated with TB pathogenesis over 72h. A large set of genes possessing highly variable inter-individual expression levels are differentially expressed in response to M.tb infection. Eigengene modules link M.tb growth rate with host transcriptional and protein profiles at 24 and 72h. Systems analysis of differential RNA and protein expression identifies a robust network with IL1B, STAT1, and IDO1 as hub genes associated with M.tb growth. RNA time profiles document stimulation towards an M1-type macrophage gene expression followed by emergence of an M2-type profile. Finally, we replicate these results in a cohort from a TB-endemic region, finding a substantial portion of significant differentially expressed genes overlapping between studies. Conclusions: We observe large inter-individual differences in bacterial uptake and growth, with tenfold variation in M.tb load by 72h.The fine-scale resolution of this work enables the identification of genes and gene networks associated with early M.tb growth dynamics in defined donor clusters, an important step in developing potential biological indicators of individual susceptibility to M.tb infection and response to therapies.

Pharmacogenomics: Driving Personalized Medicine.

Personalized medicine tailors therapies, disease prevention, and health maintenance to the individual, with pharmacogenomics serving as a key tool to improve outcomes and prevent adverse effects. Advances in genomics have transformed pharmacogenetics, traditionally focused on single gene-drug pairs, into pharmacogenomics, encompassing all "-omics" fields (e.g., proteomics, transcriptomics, metabolomics, and metagenomics). This review summarizes basic genomics principles relevant to translation into therapies, assessing pharmacogenomics' central role in converging diverse elements of personalized medicine. We discuss genetic variations in pharmacogenes (drug-metabolizing enzymes, drug transporters, and receptors), their clinical relevance as biomarkers, and the legacy of decades of research in pharmacogenetics. All types of therapies, including proteins, nucleic acids, viruses, cells, genes, and irradiation, can benefit from genomics, expanding the role of pharmacogenomics across medicine. Food and Drug Administration approvals of personalized therapeutics involving biomarkers increase rapidly, demonstrating the growing impact of pharmacogenomics. A beacon for all therapeutic approaches, molecularly targeted cancer therapies highlight trends in drug discovery and clinical applications. To account for human complexity, multicomponent biomarker panels encompassing genetic, personal, and environmental factors can guide diagnosis and therapies, increasingly involving artificial intelligence to cope with extreme data complexities. However, clinical application encounters substantial hurdles, such as unknown validity across ethnic groups, underlying bias in health care, and real-world validation. This review address the underlying science and technologies germane to pharmacogenomics and personalized medicine, integrated with economic, ethical, and regulatory issues, providing insights into the current status and future direction of health care. SIGNIFICANCE STATEMENT: Personalized medicine aims to optimize health care for the individual patients with use of predictive biomarkers to improve outcomes and prevent adverse effects. Pharmacogenomics drives biomarker discovery and guides the development of targeted therapeutics. This review addresses basic principles and current trends in pharmacogenomics, with large-scale data repositories accelerating medical advances. The impact of pharmacogenomics is discussed, along with hurdles impeding broad clinical implementation, in the context of clinical care, ethics, economics, and regulatory affairs.

Transcription Factors and ncRNAs Associated with CYP3A Expression in Human Liver and Small Intestine Assessed with Weighted Gene Co-Expression Network Analysis.

CYP3A4, CYP3A5, and CYP3A7, which are located in a multigene locus (CYP3A), play crucial roles in drug metabolism. To understand the highly variable hepatic expression of CYP3As, regulatory network analyses have focused on transcription factors (TFs). Since long non-coding RNAs (lncRNAs) likely contribute to such networks, we assessed the regulatory effects of both TFs and lncRNAs on CYP3A expression in the human liver and small intestine, main organs of CYP3A expression. Using weighted gene co-expression network analysis (WGCNA) of GTEx v8 RNA expression data and multiple stepwise regression analysis, we constructed TF-lncRNA-CYP3A co-expression networks. Multiple lncRNAs and TFs displayed robust associations with CYP3A expression that differed between liver and small intestines (LINC02499, HNF4A-AS1, AC027682.6, LOC102724153, and RP11-503C24.6), indicating that lncRNAs contribute to variance in CYP3A expression in both organs. Of these, HNF4A-AS1 had been experimentally demonstrated to affect CYP3A expression. Incorporating ncRNAs into CYP3A expression regulatory network revealed additional candidate TFs associated with CYP3A expression. These results serve as a guide for experimental studies on lncRNA-TF regulation of CYP3A expression in the liver and small intestines.

Ligand-Free Signaling of G-Protein-Coupled Receptors: Relevance to µ Opioid Receptors in Analgesia and Addiction.

Numerous G-protein-coupled receptors (GPCRs) display ligand-free basal signaling with potential physiological functions, a target in drug development. As an example, the µ opioid receptor (MOR) signals in ligand-free form (MOR-µ*), influencing opioid responses. In addition, agonists bind to MOR but can dissociate upon MOR activation, with ligand-free MOR-µ* carrying out signaling. Opioid pain therapy is effective but incurs adverse effects (ADRs) and risk of opioid use disorder (OUD). Sustained opioid agonist exposure increases persistent basal MOR-µ* activity, which could be a driving force for OUD and ADRs. Antagonists competitively prevent resting MOR (MOR-µ) activation to MOR-µ*, while common antagonists, such as naloxone and naltrexone, also bind to and block ligand-free MOR-µ*, acting as potent inverse agonists. A neutral antagonist, 6ß-naltrexol (6BN), binds to but does not block MOR-µ*, preventing MOR-µ activation only competitively with reduced potency. We hypothesize that 6BN gradually accelerates MOR-µ* reversal to resting-state MOR-µ. Thus, 6BN potently prevents opioid dependence in rodents, at doses well below those blocking antinociception or causing withdrawal. Acting as a 'retrograde addiction modulator', 6BN could represent a novel class of therapeutics for OUD. Further studies need to address regulation of MOR-µ* and, more broadly, the physiological and pharmacological significance of ligand-free signaling in GPCRs.

Association of IRGM promoter region polymorphisms and haplotype with pulmonary tuberculosis in Pakistani (Punjab) population.

Single nucleotide polymorphisms (SNPs) in IRGM are reported to affect Mycobacterium tuberculosis (M.tb) degradation pathway. Here, we aim to screen promoter-region regulatory SNPs of IRGM in Pakistani population. DNA extracted from blood of cohort containing 70 TB patients (TB) and 30 controls subjects (Ctrl), was amplified for IRGM promoter region, followed by DNA sequencing. Group-specific variations were found in allelic frequencies at four loci. Allele T (p-value = 0.03) at -1161T/C, allele G (p-value = 0.027) at -1133G/A; allele C (p-value = 0.029) at -1049C/T; and allele G (p-value = 0.02) at -708G/A, showed higher associations with TB in our cohort. These SNPs display strong linkage disequilibrium (LD) in Pakistani population. Haplotype analysis showed a significant association of haplotype -1161T/-1133G/-1049C/-708G (p-value = 0.007) to TB. This 4-SNP haplotype also represents an expression quantitative trait locus (eQTL), associated with Crohn's disease and chronic inflammatory diseases. Our findings show that variants -1161T/C, -1133G/A, -1049C/T, and -708G/A are associated with IRGM expression and susceptibility to TB in a Pakistani population.

Interpreting coronary artery disease GWAS results: A functional genomics approach assessing biological significance.

Genome-wide association studies (GWAS) have implicated 58 loci in coronary artery disease (CAD). However, the biological basis for these associations, the relevant genes, and causative variants often remain uncertain. Since the vast majority of GWAS loci reside outside coding regions, most exert regulatory functions. Here we explore the complexity of each of these loci, using tissue specific RNA sequencing data from GTEx to identify genes that exhibit altered expression patterns in the context of GWAS-significant loci, expanding the list of candidate genes from the 75 currently annotated by GWAS to 245, with almost half of these transcripts being non-coding. Tissue specific allelic expression imbalance data, also from GTEx, allows us to uncover GWAS variants that mark functional variation in a locus, e.g., rs7528419 residing in the SORT1 locus, in liver specifically, and rs72689147 in the GUYC1A1 locus, across a variety of tissues. We consider the GWAS variant rs1412444 in the LIPA locus in more detail as an example, probing tissue and transcript specific effects of genetic variation in the region. By evaluating linkage disequilibrium (LD) between tissue specific eQTLs, we reveal evidence for multiple functional variants within loci. We identify 3 variants (rs1412444, rs1051338, rs2250781) that when considered together, each improve the ability to account for LIPA gene expression, suggesting multiple interacting factors. These results refine the assignment of 58 GWAS loci to likely causative variants in a handful of cases and for the remainder help to re-prioritize associated genes and RNA isoforms, suggesting that ncRNAs maybe a relevant transcript in almost half of CAD GWAS results. Our findings support a multi-factorial system where a single variant can influence multiple genes and each genes is regulated by multiple variants.

Functional CYP3A variants affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients.

The aim of this study was to identify novel genetic variants affecting tacrolimus trough blood concentrations. We analyzed the association between 58 single nucleotide polymorphisms (SNPs) across the CYP3A gene cluster and the log-transformed tacrolimus concentration/dose ratio (log (C0/D)) in 819 renal transplant recipients (Discovery cohort). Multivariate linear regression was used to test for associations between tacrolimus log (C0/D) and clinical factors. Luciferase reporter gene assays were used to evaluate the functions of select SNPs. Associations of putative functional SNPs with log (C0/D) were further tested in 631 renal transplant recipients (Replication cohort). Nine SNPs were significantly associated with tacrolimus log (C0/D) after adjustment for CYP3A5*3 and clinical factors. Dual luciferase reporter assays indicated that the rs4646450 G allele and rs3823812 T allele were significantly associated with increased normalized luciferase activity ratios (p < 0.01). Moreover, CYP3A7*2 was associated with higher TAC log(C0/D) in the group of CYP3A5 expressers. Age, serum creatinine and hematocrit were significantly associated with tacrolimus log (C0/D). CYP3A7*2, rs4646450, and rs3823812 are proposed as functional SNPs affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients. Clinical factors also significantly affect tacrolimus metabolism.

Sulfatase 2 Is Associated with Steroid Resistance in Childhood Nephrotic Syndrome.

Glucocorticoid (GC) resistance complicates the treatment of ~10-20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF's reported role as a downstream mediator of SULF2's effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Association of ANRIL Polymorphism With Overall Survival in Adult Patients With Hematologic Malignancies After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND/AIM: Genetic variations of the non-coding RNA gene, ANRIL, have been associated with human diseases including cancer, type-2 diabetes, and atherosclerosis. In the present study, we investigated the potential associations of select ANRIL single nucleotide polymorphisms (SNPs) with overall survival and other clinical outcomes in adult patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation (allo-HSCT). PATIENTS AND METHODS: Genomic DNA was extracted from whole blood samples from 103 adult patients with hematologic malignancies who had received allo-HSCT followed by oral tacrolimus therapy. The genotypes of four select ANRIL SNPs, rs564398, rs1063192, rs2151280, and rs2157719 were determined using qRT-PCR-based genotyping assays. RESULTS: rs2151280 (C->T) in ANRIL was associated with worse overall survival in these patients (CT/CC vs. TT). Contrarily, rs2151280 and the other select ANRIL SNPs were not associated with death at Day-100 after transplantation, the incidence of graft-versus-host disease (GVHD), acute kidney injury (AKI), and neurotoxicity in the study cohort. CONCLUSION: rs2151280 represents a potential prognostic biomarker for overall survival in adult patients with hematologic malignancies after allo-HSCT.

Biased Opioid Antagonists as Modulators of Opioid Dependence: Opportunities to Improve Pain Therapy and Opioid Use Management.

Opioid analgesics are effective pain therapeutics but they cause various adverse effects and addiction. For safer pain therapy, biased opioid agonists selectively target distinct µ opioid receptor (MOR) conformations, while the potential of biased opioid antagonists has been neglected. Agonists convert a dormant receptor form (MOR-µ) to a ligand-free active form (MOR-µ*), which mediates MOR signaling. Moreover, MOR-µ converts spontaneously to MOR-µ* (basal signaling). Persistent upregulation of MOR-µ* has been invoked as a hallmark of opioid dependence. Contrasting interactions with both MOR-µ and MOR-µ* can account for distinct pharmacological characteristics of inverse agonists (naltrexone), neutral antagonists (6ß-naltrexol), and mixed opioid agonist-antagonists (buprenorphine). Upon binding to MOR-µ*, naltrexone but not 6ß-naltrexol suppresses MOR-µ*signaling. Naltrexone blocks opioid analgesia non-competitively at MOR-µ*with high potency, whereas 6ß-naltrexol must compete with agonists at MOR-µ, accounting for ~100-fold lower in vivo potency. Buprenorphine's bell-shaped dose-response curve may also result from opposing effects on MOR-µ and MOR-µ*. In contrast, we find that 6ß-naltrexol potently prevents dependence, below doses affecting analgesia or causing withdrawal, possibly binding to MOR conformations relevant to opioid dependence. We propose that 6ß-naltrexol is a biased opioid antagonist modulating opioid dependence at low doses, opening novel avenues for opioid pain therapy and use management.

Challenges of Immune Response Diversity in the Human Population Concerning New Tuberculosis Diagnostics, Therapies, and Vaccines.

Universal approaches to the prevention and treatment of human diseases fail to take into account profound immune diversity resulting from genetic variations across populations. Personalized or precision medicine takes into account individual lifestyle, environment, and biology (genetics and immune status) and is being adopted in several disease intervention strategies such as cancer and heart disease. However, its application in infectious diseases, particularly global diseases such as tuberculosis (TB), is far more complex and in a state of infancy. Here, we discuss the impact of human genetic variations on immune responses and how they relate to failures seen in current TB diagnostic, therapy, and vaccine approaches across populations. We offer our perspective on the challenges and potential for more refined approaches going forward.

Pharmacological Prevention of Neonatal Opioid Withdrawal in a Pregnant Guinea Pig Model.

Newborns exposed to prenatal opioids often experience intense postnatal withdrawal after cessation of the opioid, called neonatal opioid withdrawal syndrome (NOWS), with limited pre- and postnatal therapeutic options available. In a prior study in pregnant mice we demonstrated that the peripherally selective opioid antagonist, 6ß-naltrexol (6BN), is a promising drug candidate for preventive prenatal treatment of NOWS, and a therapeutic mechanism was proposed based on preferential delivery of 6BN to fetal brain with relative exclusion from maternal brain. Here, we have developed methadone (MTD) treated pregnant guinea pigs as a physiologically more suitable model, enabling detection of robust spontaneous neonatal withdrawal. Prenatal MTD significantly aggravates two classic maternal separation stress behaviors in newborn guinea pigs: calling (vocalizing) and searching (locomotion) - natural attachment behaviors thought to be controlled by the endogenous opioid system. In addition, prenatal MTD significantly increases the levels of plasma cortisol in newborns, showing that cessation of MTD at birth engages the hypothalamic-pituitary-adrenal (HPA) axis. We find that co-administration of 6BN with MTD prevents these withdrawal symptoms in newborn pups with extreme potency (ID50 ∼0.02 mg/kg), at doses unlikely to induce maternal or fetal withdrawal or to interfere with opioid antinociception based on many prior studies in rodents and non-human primates. Furthermore, we demonstrate a similarly high potency of 6BN in preventing opioid withdrawal in adult guinea pigs (ID50 = 0.01 mg/kg). This high potency appears to run counter to our pharmacokinetic studies showing slow 6BN transit of both the placenta and maternal blood brain barrier in guinea pigs, and calls into question the preferential delivery mechanism. Rather, it suggests a novel receptor mechanism to account for the selectively high potency of 6BN to suppress opioid dependence at all developmental stages, even in adults, as compared to its well-established low potency as a classical opioid antagonist. In conclusion, 6BN is an attractive compound for development of a preventive therapy for NOWS.

Association of Regulatory Genetic Variants for Protein Kinase Cα with Mortality and Drug Efficacy in Patients with Heart Failure.

PURPOSE: Protein kinase C alpha (gene: PRKCA) is a key regulator of cardiac contractility. Two genetic variants have recently been discovered to regulate PRKCA expression in failing human heart tissue (rs9909004 [T → C] and rs9303504 [C → G]). The association of those variants with clinical outcomes in patients with heart failure (HF), and their interaction with HF drug efficacy, is unknown. METHODS: Patients with HF in a prospective registry starting in 2007 were genotyped by whole genome array (n = 951). The primary outcome was all-cause mortality. Cox proportional hazards models adjusted for established clinical risk factors and genomic ancestry tested the independent association of rs9909004 or rs9303504 and the variant interactions with cornerstone HF pharmacotherapies (beta-blockers or angiotensin-converting enzyme inhibitors/angiotensin receptor blockers) in additive genetic models. RESULTS: The minor allele of rs9909004, but not of rs9303504, was independently associated with a decreased risk for all-cause mortality: adjusted HR = 0.81 (95% CI = 0.67-0.98), p = 0.032. The variants did not significantly interact with mortality benefit associated with cornerstone HF pharmacotherapies (p > 0.1 for all). CONCLUSIONS: A recently discovered cardiac-specific regulatory variant for PRKCA (rs9909004) was independently associated with a decreased risk for all-cause mortality in patients with HF. The variant did not interact with mortality benefit associated with cornerstone HF pharmacotherapies.

Ligand-Free Estrogen Receptor α (ESR1) as Master Regulator for the Expression of CYP3A4 and Other Cytochrome P450 Enzymes in the Human Liver.

Cytochrome P450 3A4 isoform (CYP3A4) transcription is controlled by hepatic transcription factors (TFs), but how TFs dynamically interact remains uncertain. We hypothesize that several TFs form a regulatory network with nonlinear, dynamic, and hierarchical interactions. To resolve complex interactions, we have applied a computational approach for estimating Sobol's sensitivity indices (SSI) under generalized linear models to existing liver RNA expression microarray data (GSE9588) and RNA-seq data from genotype-tissue expression (GTEx), generating robust importance ranking of TF effects and interactions. The SSI-based analysis identified TFs and interacting TF pairs, triplets, and quadruplets involved in CYP3A4 expression. In addition to known CYP3A4 TFs, estrogen receptor α (ESR1) emerges as key TF with the strongest main effect and as the most frequently included TF interacting partner. Model predictions were validated using small interfering RNA (siRNA)/short hairpin RNA (shRNA) gene knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation of ESR1 in biliary epithelial Huh7 cells and human hepatocytes in the absence of estrogen. Moreover, ESR1 and known CYP3A4 TFs mutually regulate each other. Detectable in both male and female hepatocytes without added estrogen, the results demonstrate a role for unliganded ESR1 in CYP3A4 expression consistent with unliganded ESR1 signaling reported in other cell types. Added estrogen further enhances ESR1 effects. We propose a hierarchical regulatory network for CYP3A4 expression directed by ESR1 through self-regulation, cross regulation, and TF-TF interactions. We also demonstrate that ESR1 regulates the expression of other P450 enzymes, suggesting broad influence of ESR1 on xenobiotics metabolism in human liver. Further studies are required to understand the mechanisms underlying role of ESR1 in P450 regulation. SIGNIFICANCE STATEMENT: This study focuses on identifying key transcription factors and regulatory networks for CYP3A4, the main drug metabolizing enzymes in liver. We applied a new computational approach (Sobol's sensitivity analysis) to existing hepatic gene expression data to determine the role of transcription factors in regulating CYP3A4 expression, and used molecular genetics methods (siRNA/shRNA gene knockdown and CRISPR-mediated transcriptional activation) to test these interactions in life cells. This approach reveals a robust network of TFs, including their putative interactions and the relative impact of each interaction. We find that ESR1 serves as a key transcription factor function in regulating CYP3A4, and it appears to be acting at least in part in a ligand-free fashion.

Significant association of DRD2 enhancer variant rs12364283 with heroin addiction in a Pakistani population.

The dopamine D2 receptor encoded by DRD2 has been implicated in multiple psychiatric disorders, mediated at least in part by two intronic variants affecting mRNA splicing, rs1076560 and rs2283265, and a less frequent enhancer variant, rs12364283, which increases DRD2 mRNA expression. This study tests whether these functionally validated variants confer susceptibility toward heroin addiction in a Pakistani population. A total of 540 heroin addicts and 467 healthy controls were genotyped, basic allele and genotype tests were performed. Neither rs1076560 nor rs2283265 significantly associated with heroin addiction. The enhancer rs12364283 occurs more frequently in heroin-dependent cases than controls (MAF 13% vs. 7%, respectively), revealing significant association with heroin addiction (p = 3.0E-06, OR 2.1). This study identifies rs12364283 of DRD2 as a potential risk factor for heroin addiction in the Pakistani study population. This enhancer variant had been shown to increase DRD2 mRNA expression, a possible factor in increased vulnerability to heroin addiction. Further studies are needed to validate this association of rs12364283.

Association of Genetic Polymorphisms in the Beta-1 Adrenergic Receptor with Recovery of Left Ventricular Ejection Fraction in Patients with Heart Failure.

Two common genetic polymorphisms in the beta-1 adrenergic receptor (ADRB1 Ser49Gly [rs1801252] and Arg389Gly [rs1801253]) significantly affect receptor function in vitro. The objective of this study was to determine whether ADRB1 Ser49Gly and Arg389Gly are associated with recovery of left ventricular ejection fraction (LVEF) in patients with heart failure. Patients with heart failure and baseline LVEF ≤ 40% were genotyped (n = 98), and retrospective chart review assessed the primary outcome of LVEF recovery to ≥ 40%. Un/adjusted logistic regression models revealed that Ser49Gly, but not Arg389Gly, was significantly associated with LVEF recovery in a dominant genetic model. The adjusted odds ratio for Ser49 was 8.2 (95% CI = 2.1-32.9; p = 0.003), and it was the strongest predictor of LVEF recovery among multiple clinical variables. In conclusion, patients with heart failure and reduced ejection fraction that are homozygous for ADRB1 Ser49 were significantly more likely to experience LVEF recovery than Gly49 carriers.

CYP2D6 haplotypes with enhancer single-nucleotide polymorphism rs5758550 and rs16947 (*2 allele): implications for CYP2D6 genotyping panels.

INTRODUCTION: CYP2D6 metabolizes ∼25% of all clinically used drugs, with numerous genetic polymorphisms affecting enzyme activity and drug response. Clinical utility of current CYP2D6 genotyping is partially compromised the unresolved complex haplotype structure of the CYP2D6 locus. We have identified a distal enhancer single-nucleotide polymorphism rs5758550 that robustly increases CYP2D6 expression, whereas rs16947 (CYP2D6*2), previously considered inert, reduces correct mRNA splicing and expression, thereby affecting presumed activity of other alleles on the *2 haplotype. OBJECTIVE: This study aims to determine the structure and frequency of haplotypes containing either rs5758550 or rs16947, or both, together with other relevant CYP2D6 alleles, assigning predictive enzyme activity scores to each, and addressing ambiguities in estimating diplotypes in different populations. METHODS: The structure and frequency of haplotypes containing rs5758550 and/or rs16947 in different populations were determined by using phased genotype data from 'The 1000 Genomes Project'. The assigned haplotype-phenotype relationship was tested by associating assigned CYP2D6 activity score with CYP2D6 enzyme activity in a cohort of 122 human liver microsomes. RESULTS: Addition of enhancer single-nucleotide polymorphism rs5758550 and *2 to a CYP2D6 panel improves prediction of CYP2D6 activity. Moreover, the haplotype containing rs5758550 and rs16947 predict extensive CYP2D6 activity more accurately than CYP2D6*2A, a surrogate marker for extensive activity. CONCLUSION: With further studies, the results support possible incorporation of rs5758550 and rs16947 into CYP2D6 biomarker panels for more accurate prediction of CYP2D6 metabolizer status.

IL-13-regulated Macrophage Polarization during Granuloma Formation in an In Vitro Human Sarcoidosis Model.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).