Association of the OPRM1 Variant rs1799971 (A118G) with Non-Specific Liability to Substance Dependence in a Collaborative de novo Meta-Analysis of European-Ancestry Cohorts.

The mu1 opioid receptor gene, OPRM1, has long been a high-priority candidate for human genetic studies of addiction. Because of its potential functional significance, the non-synonymous variant rs1799971 (A118G, Asn40Asp) in OPRM1 has been extensively studied, yet its role in addiction has remained unclear, with conflicting association findings. To resolve the question of what effect, if any, rs1799971 has on substance dependence risk, we conducted collaborative meta-analyses of 25 datasets with over 28,000 European-ancestry subjects. We investigated non-specific risk for "general" substance dependence, comparing cases dependent on any substance to controls who were non-dependent on all assessed substances. We also examined five specific substance dependence diagnoses: DSM-IV alcohol, opioid, cannabis, and cocaine dependence, and nicotine dependence defined by the proxy of heavy/light smoking (cigarettes-per-day >20 vs. ≤ 10). The G allele showed a modest protective effect on general substance dependence (OR = 0.90, 95% C.I. [0.83-0.97], p value = 0.0095, N = 16,908). We observed similar effects for each individual substance, although these were not statistically significant, likely because of reduced sample sizes. We conclude that rs1799971 contributes to mechanisms of addiction liability that are shared across different addictive substances. This project highlights the benefits of examining addictive behaviors collectively and the power of collaborative data sharing and meta-analyses.

Activation domains for controlling plant gene expression using designed transcription factors.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

Protein isoprenylation: the fat of the matter.

Protein isoprenylation refers to the covalent attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl moiety to a cysteine residue at or near the carboxyl terminus. This post-translational lipid modification, which mediates protein-membrane and protein-protein interactions, is necessary for normal control of abscisic acid and auxin signaling, meristem development, and other fundamental processes. Recent studies have also revealed roles for protein isoprenylation in cytokinin biosynthesis and innate immunity. Most isoprenylated proteins are further modified by carboxyl terminal proteolysis and methylation and, collectively, these modifications are necessary for the targeting and function of isoprenylated proteins.

The CaaX specificities of Arabidopsis protein prenyltransferases explain era1 and ggb phenotypes.

BACKGROUND: Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. This modification, which mediates protein-membrane and protein-protein interactions, is characterized by the covalent attachment of a fifteen-carbon farnesyl or twenty-carbon geranylgeranyl group to the cysteine residue of a carboxyl terminal CaaX motif. In Arabidopsis, era1 mutants lacking protein farnesyltransferase exhibit enlarged meristems, supernumerary floral organs, an enhanced response to abscisic acid (ABA), and drought tolerance. In contrast, ggb mutants lacking protein geranylgeranyltransferase type 1 exhibit subtle changes in ABA and auxin responsiveness, but develop normally. RESULTS: We have expressed recombinant Arabidopsis protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type 1 (PGGT1) in E. coli and characterized purified enzymes with respect to kinetic constants and substrate specificities. Our results indicate that, whereas PFT exhibits little specificity for the terminal amino acid of the CaaX motif, PGGT1 exclusively prenylates CaaX proteins with a leucine in the terminal position. Moreover, we found that different substrates exhibit similar K(m) but different k(cat) values in the presence of PFT and PGGT1, indicating that substrate specificities are determined primarily by reactivity rather than binding affinity. CONCLUSIONS: The data presented here potentially explain the relatively strong phenotype of era1 mutants and weak phenotype of ggb mutants. Specifically, the substrate specificities of PFT and PGGT1 suggest that PFT can compensate for loss of PGGT1 in ggb mutants more effectively than PGGT1 can compensate for loss of PFT in era1 mutants. Moreover, our results indicate that PFT and PGGT1 substrate specificities are primarily due to differences in catalysis, rather than differences in substrate binding.

Childhood maltreatment, subsequent antisocial behavior, and the role of monoamine oxidase A genotype.

BACKGROUND: A functional promoter polymorphism in monoamine oxidase A (MAOA) has been implicated as a moderating factor in the relationship between childhood maltreatment and later adolescent and adult antisocial behavior. Despite wide interest in this hypothesis, results remain mixed from the few attempts at replication. METHODS: Regression-based analyses were conducted to test for a genotype-environment interaction using self-reported physical abuse and MAOA genotype to predict later antisocial behavior and arrests for violence by participants in the National Youth Survey Family Study. We also examined the interaction using a measure of violent victimization. The analysis sample included 277 Caucasian male respondents, aged 11-15 in 1976, who provided buccal swab DNA samples and who were successfully genotyped for the variable number tandem repeat (VNTR) in the MAOA promoter using polymerase chain reaction. RESULTS: Maltreatment by a parent during adolescence was a risk factor for adolescent and adult antisocial and violence related behavioral problems. Tests for the main effect of MAOA and a MAOA-maltreatment interaction were nonsignificant. Similar results were obtained using the measure of adolescent violent victimization. CONCLUSIONS: Findings from this general population sample could not confirm the hypothesis that MAOA moderates the relationship between adolescent maltreatment and adolescent or adult antisocial behavior.

Farnesylcysteine lyase is involved in negative regulation of abscisic acid signaling in Arabidopsis.

The Arabidopsis FCLY gene encodes a specific farnesylcysteine (FC) lyase, which is responsible for the oxidative metabolism of FC to farnesal and cysteine. In addition, fcly mutants with quantitative decreases in FC lyase activity exhibit an enhanced response to ABA. However, the enzymological properties of the FCLY-encoded enzyme and its precise role in ABA signaling remain unclear. Here, we show that recombinant Arabidopsis FC lyase expressed in insect cells exhibits high selectivity for FC as a substrate and requires FAD and molecular oxygen for activity. Arabidopsis FC lyase is also shown to undergo post-translational N-glycosylation. FC, which is a competitive inhibitor of isoprenylcysteine methyltransferase (ICMT), accumulates in fcly mutants. Moreover, the enhanced response of fcly mutants to ABA is reversed by ICMT overexpression. These observations support the hypothesis that the ABA hypersensitive phenotype of fcly plants is the result of FC accumulation and inhibition of ICMT.

Using trajectory analyses to refine phenotype for genetic association: conduct problems and the serotonin transporter (5HTTLPR).

BACKGROUND: Conduct disorder is a serious, relatively common disorder of childhood and adolescence. Findings from genetic association studies searching for genetic determinants of the liability toward such behaviors have been inconsistent. One possible explanation for differential results is that most studies define phenotype from a single assessment; for many adolescents conduct problems decrease in severity over time, whereas for others such behaviors persist. Therefore, longitudinal datasets offer the opportunity to refine phenotype. METHODS: We used Caucasians that were first assessed during adolescence from the National Youth Survey Family Study. Nine waves of data were used to create latent growth trajectories and test for associations between trajectory class and 5HTTLPR genotype. RESULTS: For the full sample, 5HTTLPR was not associated with conduct problem phenotypes. However, the short (s) allele was associated with chronic conduct problems in females; a nominally significant sex by 5HTTLPR genotype interaction was noted. CONCLUSION: Longitudinal studies provide unique opportunities for phenotypic refinement and such techniques, with large samples, may be useful for phenotypic definition with other study designs, such as whole genome association studies.

A discrete-time survival analysis of the relationship between truancy and the onset of marijuana use.

OBJECTIVE: Despite truancy being a common behavior among teenagers, little research has assessed its deleterious effects. In this study, the effect of truancy on the initiation of marijuana use was examined. METHOD: Using data from the Rochester Youth Development Study (a longitudinal sample of predominantly minority youth), discrete time survival analyses were estimated to assess the effect of truancy on the subsequent initiation of marijuana use. The current analyses used 5 years of panel data collected from youth and their primary caregiver every 6 months throughout adolescence. RESULTS: Truancy was a significant predictor of the initiation of marijuana use during each subsequent 6-month period. The effect was more robust in earlier compared with later adolescence. These effects persisted after controlling for potential risk factors that are shared by both truancy and drug use, including commitment to school, grade-point average, delinquent values, prior involvement in delinquency, peer reactions to delinquency, parental monitoring, affective ties to the child, and positive parenting. CONCLUSIONS: We argue that the effect is, in part, the result of reduced social control (i.e., disengagement from pro-social entities such as school) and, in part, the result of the unsupervised, unmonitored time afforded by truancy. Prevention initiatives aimed at reducing truancy also may have a beneficial impact on preventing the initiation of drug use among adolescents.

Genetic association of the CHRNA6 and CHRNB3 genes with tobacco dependence in a nationally representative sample.

Neuronal nicotinic acetylcholine receptors are activated by both endogenous acetylcholine and exogenous nicotine, making sequence variations in these receptors likely candidates for association with tobacco phenotypes. Previous studies have found evidence for significant association between single nucleotide polymorphisms (SNPs) in the genomic region containing the CHRNA6 and CHRNB3 genes and tobacco behaviors. In this study, we provide support for an association between these genes and tobacco dependence in the National Youth Survey Family Study wave 10, a nationally representative sample of households. Eight SNPs in the CHRNA6 and CHRNB3 genomic region were genotyped in 1051 subjects, approximately half of whom are members of sibling pairs. Genetic association with DSM-IV dependence was assessed using a family-based approach as implemented in the statistical package PBAT. Individual SNPs were tested for association with quit attempts and overall dependence. Variation in CHRNA6 was found to be associated with tobacco dependence (p=0.007 in Caucasians). SNPs in CHRNB3 were found to be associated with the number of quit attempts (p=0.0024). Together these results further implicate the region downstream of CHRNA6 and the region upstream of CHRNB3 in risk of nicotine dependence.

Isoprenylcysteine methylation and demethylation regulate abscisic acid signaling in Arabidopsis.

Isoprenylated proteins bear an isoprenylcysteine methyl ester at the C terminus. Although isoprenylated proteins have been implicated in meristem development and negative regulation of abscisic acid (ABA) signaling, the functional role of the terminal methyl group has not been described. Here, we show that transgenic Arabidopsis thaliana plants overproducing isoprenylcysteine methyltransferase (ICMT) exhibit ABA insensitivity in stomatal closure and seed germination assays, establishing ICMT as a negative regulator of ABA signaling. By contrast, transgenic plants overproducing isoprenylcysteine methylesterase (ICME) exhibit ABA hypersensitivity in stomatal closure and seed germination assays. Thus, ICME is a positive regulator of ABA signaling. To test the hypothesis that ABA signaling is under feedback regulation at the level of isoprenylcysteine methylation, we examined the effect of ABA on ICMT and ICME gene expression. Interestingly, ABA induces ICME gene expression, establishing a positive feedback loop whereby ABA promotes ABA responsiveness of plant cells via induction of ICME expression, which presumably results in the demethylation and inactivation of isoprenylated negative regulators of ABA signaling. These results suggest strategies for metabolic engineering of crop species for drought tolerance by targeted alterations in isoprenylcysteine methylation.

School-related risk and protective factors associated with truancy among urban youth placed at risk.

Truancy is a serious concern in the United States. Its negative effects are so pervasive that in 2003 the Office of Juvenile Justice and Delinquency Prevention named truancy prevention a national priority. Effective prevention of truancy requires a thorough understanding of the characteristics that describe truant youth as well as factors that may put them at risk for truancy. Unfortunately, surprisingly little is known about the correlates and/or causes of truancy. In this paper we explore associations between truancy and several salient school-related risk and protective factors among a sample of youth who grew up in socially disorganized neighborhoods of Denver, CO. We demonstrate that several school-related risk and protective factors are associated with truancy. Perhaps most importantly, we identify that the two most robust predictors are school performance and involvement with delinquent peers, and that these two variables form a synergistic relationship in which the relationship between delinquent peer association and truancy is mitigated among students who perform well in school. EDITORS' STRATEGIC IMPLICATIONS: The authors use data from a large probability sample drawn from neighborhoods with high crime rates to identify key correlates of truancy. They also draw attention to the dearth of efficacious truancy prevention efforts in spite of the magnitude of the problem.

Truancy's effect on the onset of drug use among urban adolescents placed at risk.

PURPOSE: To examine the relationship between truancy and the onset of drug use. METHODS: Discrete time survival analysis was used to assess the effect of truancy on initiation of drug use after adjusting for several potential confounders from age 11 to 15 years, using data from the Denver Youth Survey, a longitudinal sample of youth who grew up in socially disorganized neighborhoods of Denver, CO. RESULTS: In this population, truancy was a significant predictor of initiation of alcohol, tobacco, and marijuana use. The robust effect of truancy persisted after controlling for potential confounders, including school performance, school isolation, association with delinquent peers, personal delinquent values, parental monitoring, and family attachment. CONCLUSIONS: Although this study cannot point to a causal relationship, we argue that the effect may be at least in part due to the unsupervised, unmonitored time with peers that truancy affords a young person. Truancy prevention is a field of research that needs much more attention. Keeping youth in school every day is likely to have many beneficial effects, and effective truancy prevention efforts may also help to prevent or delay the onset of drug use among adolescents.

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine.

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.

Between- and within-family association test of the dopamine receptor D2 TaqIA polymorphism and alcohol abuse and dependence in a general population sample of adults.

OBJECTIVE: Dopaminergic dysfunction has been hypothesized to play an important role in the etiology of alcohol-use disorders. A restriction fragment length polymorphism (RFLP) in the 3' untranslated region (3'UTR) of the DRD2 gene affects gene expression and has been implicated as a risk factor for alcohol dependence. This polymorphism (TaqIA) has been reported as positively associated with alcohol-use disorders in case-control samples, but these results have not been replicated in family-based association studies. The mixed results of association between the DRD2 TaqIA polymorphism and alcohol-use disorders may be the result of differences in sample size, phenotype definition, heterogeneity of the samples, and genetic admixture. METHOD: We conducted tests of association in a sample of 838 adults participating in the National Youth Survey Family Study (NYSFS). We examined whether the DRD2 TaqIA polymorphism was associated with a symptom-count measure of alcohol abuse and dependence derived from the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, and the Craving Withdrawal Model. RESULTS: Tests of association were nonsignificant across each classification system examined. Power calculations suggested that these results were despite the ability to detect an effect size of 1%. CONCLUSIONS: This study supports other family-based association tests that have reported no association between the DRD2 TaqIA polymorphism and alcohol abuse and dependence.