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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.

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.

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.

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, 6β-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.

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, 6β-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.

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.

Abstract Background The CHRNA5/A3/B4 gene locus is associated with nicotine dependence and other smoking related disorders. While the non-synonymous CHRNA5 variant rs16969968 appears to be the main risk factor, linkage disequilibrium (LD) bins in the gene cluster carry frequent variants that regulate expression. Pairwise LD and haplotype analyses had identified at least three haplotype tagging SNPs including rs16969968 as main genetic risk factors. Searching for variants with evidence of regulatory functions, we have reported interactions between CHRNA5 and CHRNA3 enhancer variants (tagged by rs880395 and rs1948, respectively) and rs16969968, forming 3-SNP haplotypes and diplotypes that may more accurately reflect the cluster’s combined effects on nicotine dependence (Barrie et al., Hum Mutat 38:112–9, 2017). Here we address further contributions by variants affecting CHRNB4, a possibly limiting component of nicotinic receptors. Results We identify an LD bin (tagged by rs4887074) associated with expression of CHRNB4. Additive logistic regression models indicate that rs4887074 is associated with nicotine dependence and modulates the effect of rs16969968 in GWAS datasets (COGEND, UW-TTURC, SAGE). 4-SNP haplotype and diplotype analyses (rs880395-rs16969968-rs1948 -rs4887074) yield nicotine dependence risk values that further differentiate those obtained with the 3-SNP model. Moreover, both the main G allele of rs16969968 and the minor G allele of rs4887074 (associated with reduced expression of CHRNB4), residing predominantly on common haplotypes that are protective, represent significant allele-specific variance QTLs, indicating that they interact with each other. Conclusions These results indicate rs4887074 is associated with CHRNB4 expression, and along with two regulatory variants of CHRNA3 and CHRNA5, modulates the effect of rs16969968 on nicotine dependence risk. Assignable to individuals because of strong LD structures, 4-SNP haplotypes and diplotypes serve to assess the combined genetic influence of this multi-gene cluster on complex traits, accounting for complex LD relationships and tissue-specific genetic effects (CHRNA5/3) relevant to the traits analyzed. The 4-SNP haplotypes account at least in part for previous tagging SNPs, including the highly GWAS-significant rs6495308, located in a distinct pair-wise LD bin but included in protective 4-SNP haplotypes. Our approach refines and integrates the cluster’s overall genetic influence, an important variable when integrating the genetics of multiple genomic loci.

Abstract Background Recently, 34 genes had been associated with differential expression relative to blood pressure (BP)/ hypertension (HTN). We hypothesize that some of the genes associated with BP/HTN are also associated with BP response to antihypertensive treatment with thiazide diuretics. Methods We assessed these 34 genes for association with differential expression to BP response to thiazide diuretics with RNA sequencing in whole blood samples from 150 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) and PEAR-2 studies. PEAR white and PEAR-2 white and black participants (n = 50 for each group) were selected based on the upper and lower quartile of BP response to hydrochlorothiazide (HCTZ) and to chlorthalidone. Results FOS, DUSP1 and PPP1R15A were differentially expressed across all cohorts (meta-analysis p-value

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 (6β-naltrexol), and mixed opioid agonist-antagonists (buprenorphine). Upon binding to MOR-μ*, naltrexone but not 6β-naltrexol suppresses MOR-μ*signaling. Naltrexone blocks opioid analgesia non-competitively at MOR-μ*with high potency, whereas 6β-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 6β-naltrexol potently prevents dependence, below doses affecting analgesia or causing withdrawal, possibly binding to MOR conformations relevant to opioid dependence. We propose that 6β-naltrexol is a biased opioid antagonist modulating opioid dependence at low doses, opening novel avenues for opioid pain therapy and use management.

Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq's reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

Tuberculosis (TB) is a global epidemic caused by the infection of human macrophages with the world’s most deadly single bacterial pathogen, Mycobacterium tuberculosis (M.tb). M.tb resides in a phagosomal niche within macrophages, where trace element concentrations impact the immune response, bacterial metal metabolism, and bacterial survival. The manipulation of micronutrients is a critical mechanism of host defense against infection. In particular, the human zinc transporter Zrt-/Irt-like protein 8 (ZIP8), one of 14 ZIP family members, is important in the flux of divalent cations, including zinc, into the cytoplasm of macrophages. It also has been observed to exist on the membrane of cellular organelles, where it can serve as an efflux pump that transports zinc into the cytosol. ZIP8 is highly inducible in response to M.tb infection of macrophages, and we have observed its localization to the M.tb phagosome. The expression, localization, and function of ZIP8 and other divalent cation transporters within macrophages have important implications for TB prevention and dissemination and warrant further study. In particular, given the importance of zinc as an essential nutrient required for humans and M.tb, it is not yet clear whether ZIP-guided zinc transport serves as a host protective factor or, rather, is targeted by M.tb to enable its phagosomal survival.

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.

Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.

Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.

Variation of the gene coding for D2 receptors (DRD2) has been associated with risk for schizophrenia and with working memory deficits. A functional intronic SNP (rs1076560) predicts relative expression of the two D2 receptors isoforms, D2S (mainly pre-synaptic) and D2L (mainly post-synaptic). However, the effect of functional genetic variation of DRD2 on striatal dopamine D2 signaling and on its correlation with prefrontal activity during working memory in humans is not known.Thirty-seven healthy subjects were genotyped for rs1076560 (G>T) and underwent SPECT with [123I]IBZM (which binds primarily to post-synaptic D2 receptors) and with [123I]FP-CIT (which binds to pre-synaptic dopamine transporters, whose activity and density is also regulated by pre-synaptic D2 receptors), as well as BOLD fMRI during N-Back working memory.Subjects carrying the T allele (previously associated with reduced D2S expression) had striatal reductions of [123I]IBZM and of [123I]FP-CIT binding. DRD2 genotype also differentially predicted the correlation between striatal dopamine D2 signaling (as identified with factor analysis of the two radiotracers) and activity of the prefrontal cortex during working memory as measured with BOLD fMRI, which was positive in GG subjects and negative in GT.Our results demonstrate that this functional SNP within DRD2 predicts striatal binding of the two radiotracers to dopamine transporters and D2 receptors as well as the correlation between striatal D2 signaling with prefrontal cortex activity during performance of a working memory task. These data are consistent with the possibility that the balance of excitatory/inhibitory modulation of striatal neurons may also affect striatal outputs in relationship with prefrontal activity during working memory performance within the cortico-striatal-thalamic-cortical pathway.

With recent advances in understanding of the neuroscience of risk taking, attention is now turning to genetic factors that may contribute to individual heterogeneity in risk attitudes. In this paper we test for genetic associations with risk attitude measures derived from both the psychology and economics literature. To develop a long-term prospective study, we first evaluate both types of risk attitudes and find that the economic and psychological measures are poorly correlated, suggesting that different genetic factors may underlie human response to risk faced in different behavioral domains. We then examine polymorphisms in a spectrum of candidate genes that affect neurotransmitter systems influencing dopamine regulation or are thought to be associated with risk attitudes or impulsive disorders. Analysis of the genotyping data identified two single nucleotide polymorphisms (SNPs) in the gene encoding the alpha 4 nicotine receptor (CHRNA4, rs4603829 and rs4522666) that are significantly associated with harm avoidance, a risk attitude measurement drawn from the psychology literature. Novelty seeking, another risk attitude measure from the psychology literature, is associated with several COMT (catechol-O-methyl transferase) SNPs while economic risk attitude measures are associated with several VMAT2 (vesicular monoamine transporter) SNPs, but the significance of these associations did not withstand statistical adjustment for multiple testing and requires larger cohorts. These exploratory results provide a starting point for understanding the genetic basis of risk attitudes by considering the range of methods available for measuring risk attitudes and by searching beyond the traditional direct focus on dopamine and serotonin receptor and transporter genes.