Aryeh I. Herman

Interactive Effects of the Serotonin Transporter 5-HTTLPR Polymorphism and Stressful Life Events on College Student Drinking and Drug Use

BACKGROUND A common functional polymorphism, 5-HTTLPR, in the serotonin transporter gene has been associated with heavy drinking in college students. We examined this polymorphism as it interacted with negative life events to predict drinking and drug use in college students. METHODS Daily reports of drinking and drug use obtained using a daily web-based survey were combined with self-reports of past-year negative life events and 5-HTTLPR genotypes in a regression analysis of alcohol and nonprescribed drug use in a sample of 295 college students. RESULTS Genotype and negative life events significantly interacted in relation to drinking and drug use outcomes. Individuals homozygous for the short (s) allele who experienced multiple negative life events in the prior year reported more frequent drinking and heavy drinking, stronger intentions to drink, and greater nonprescribed drug use. In individuals homozygous for the long (l) allele, drinking and drug use were unaffected by past-year negative life events. Heterozygous subjects showed drinking outcomes that were intermediate to the two homozygous groups. CONCLUSIONS The 5-HTTLPR s-allele is associated with increased drinking and drug use among college students who have experienced multiple negative life events. The s-allele carriers may be at risk for a variety of adverse behavioral outcomes in response to stress.

Array-Based Profiling of DNA Methylation Changes Associated with Alcohol Dependence

BACKGROUND Epigenetic regulation through DNA methylation may influence vulnerability to numerous disorders, including alcohol dependence (AD). METHODS Peripheral blood DNA methylation levels of 384 CpGs in the promoter regions of 82 candidate genes were examined in 285 African Americans (AAs; 141 AD cases and 144 controls) and 249 European Americans (EAs; 144 AD cases and 105 controls) using Illumina GoldenGate Methylation Array assays. Association of AD and DNA methylation changes was analyzed using multivariate analyses of covariance with frequency of intoxication, sex, age, and ancestry proportion as covariates. CpGs showing significant methylation alterations in AD cases were further examined in a replication sample (49 EA cases and 32 EA controls) using Sequenoms MassARRAY EpiTYPER technology. RESULTS In AAs, 2 CpGs in 2 genes (GABRB3 and POMC) were hypermethylated in AD cases compared with controls (p ≤ 0.001). In EAs, 6 CpGs in 6 genes (HTR3A, NCAM1, DRD4, MBD3, HTR2B, and GRIN1) were hypermethylated in AD cases compared with controls (p ≤ 0.001); CpG cg08989585 in the HTR3A promoter region showed a significantly higher methylation level in EA cases than in EA controls after Bonferroni correction (p = 0.00007). Additionally, methylation levels of 6 CpGs (including cg08989585) in the HTR3A promoter region were analyzed in the replication sample. Although the 6 HTR3A promoter CpGs did not show significant methylation differences between EA cases and EA controls (p = 0.067 to 0.877), the methylation level of CpG cg08989585 was nonsignificantly higher in EA cases (26.9%) than in EA controls (18.6%; p = 0.139). CONCLUSIONS The findings from this study suggest that DNA methylation profile appears to be associated with AD in a population-specific way and the predisposition to AD may result from a complex interplay of genetic variation and epigenetic modifications.

Association study of the CNR1 gene exon 3 alternative promoter region polymorphisms and substance dependence

An alternative promoter producing a novel 5′‐untranslated region of cannabinoid receptor mRNA has recently been described in CNR1, the gene encoding the cannabinoid receptor protein. Single nucleotide polymorphisms (SNPs) adjacent to this site were reported to be associated with polysubstance abuse [Zhang et al., 2004 ]. We examined the association of 4 SNPs (rs6928499, rs806379, rs1535255, rs2023239) in the distal region of intron 2 of CNR1 both with individual substance dependence diagnoses (i.e., alcohol, cocaine, and opioids), as well as with polysubstance dependence. The study samples consisted of European‐American (EA) and African‐American (AA) subjects with drug and or alcohol dependence (n = 895), and controls (n = 472). Subjects were grouped as polysubstance dependent, opioid dependent, cocaine dependent, cannabis dependent, and alcohol dependent. There was a modest association of marker rs1535255 with alcohol dependence (P = 0.04), though with correction for multiple phenotype comparisons, this effect was not considered statistically significant. These findings fail to replicate the original report of an association between SNPs adjacent to an alternative CNR1 exon 3 transcription start site and polysubstance abuse.

Rapid nicotine clearance is associated with greater reward and heart rate increases from intravenous nicotine.

The ratio of nicotine metabolites (trans-3′-hydroxycotinine (3HC) to cotinine) correlates with nicotine clearance. In previous studies, high nicotine metabolite ratio (NMR) predicted poor outcomes for smoking cessation treatment with nicotine patch. The underlying mechanisms that associate NMR with treatment outcomes have not been fully elucidated. A total of 100 smokers were divided into quartiles based on their baseline plasma NMR. Following overnight abstinence, smokers received saline followed by escalating intravenous doses of nicotine (0.5 and 1.0 mg/70 kg) given 30 min apart. The effects of nicotine on subjective, plasma cortisol, heart rate, and systolic and diastolic blood pressure measures were obtained. Smokers in the first NMR quartile (slower metabolizers) had lower Fagerstrom Test for Nicotine Dependence (FTND) scores, suggesting lower levels of dependence. In contrast, smokers in the fourth NMR quartile (faster metabolizers) reported greater craving for cigarettes following overnight abstinence from smoking and reported greater ratings of nicotine-induced good drug effects, drug liking, and wanting more drug. Higher NMR was also associated with greater heart rate increases in response to nicotine. These results suggest that enhanced nicotine reward and cigarette craving may contribute to the poor treatment response in smokers with high NMR. These findings warrant further investigation, especially in treatment-seeking smokers undergoing cessation treatment.

Genome-wide Association Study of Cannabis Dependence Severity, Novel Risk Variants, and Shared Genetic Risks

IMPORTANCE Cannabis dependence (CAD) is a serious problem worldwide and is of growing importance in the United States because cannabis is increasingly available legally. Although genetic factors contribute substantially to CAD risk, at present no well-established specific genetic risk factors for CAD have been elucidated. OBJECTIVE To report findings for DSM-IV CAD criteria from association analyses performed in large cohorts of African American and European American participants from 3 studies of substance use disorder genetics. DESIGN, SETTING, AND PARTICIPANTS This genome-wide association study for DSM-IV CAD criterion count was performed in 3 independent substance dependence cohorts (the Yale-Penn Study, Study of Addiction: Genetics and Environment [SAGE], and International Consortium on the Genetics of Heroin Dependence [ICGHD]). A referral sample and volunteers recruited in the community and from substance abuse treatment centers included 6000 African American and 8754 European American participants, including some from small families. Participants from the Yale-Penn Study were recruited from 2000 to 2013. Data were collected for the SAGE trial from 1990 to 2007 and for the ICGHD from 2004 to 2009. Data were analyzed from January 2, 2013, to November 9, 2015. MAIN OUTCOMES AND MEASURES Criterion count for DSM-IV CAD. RESULTS Among the 14 754 participants, 7879 were male, 6875 were female, and the mean (SD) age was 39.2 (10.2) years. Three independent regions with genome-wide significant single-nucleotide polymorphism associations were identified, considering the largest possible sample. These included rs143244591 (β = 0.54, P = 4.32 × 10-10 for the meta-analysis) in novel antisense transcript RP11-206M11.7;rs146091982 (β = 0.54, P = 1.33 × 10-9 for the meta-analysis) in the solute carrier family 35 member G1 gene (SLC35G1); and rs77378271 (β = 0.29, P = 2.13 × 10-8 for the meta-analysis) in the CUB and Sushi multiple domains 1 gene (CSMD1). Also noted was evidence of genome-level pleiotropy between CAD and major depressive disorder and for an association with single-nucleotide polymorphisms in genes associated with schizophrenia risk. Several of the genes identified have functions related to neuronal calcium homeostasis or central nervous system development. CONCLUSIONS AND RELEVANCE These results are the first, to our knowledge, to identify specific CAD risk alleles and potential genetic factors contributing to the comorbidity of CAD with major depression and schizophrenia.

Subjective, Physiological, and Cognitive Responses to Intravenous Nicotine: Effects of Sex and Menstrual Cycle Phase

Nicotine dependence is a serious public health concern. Optimal treatment of nicotine dependence will require greater understanding of the mechanisms that contribute to the maintenance of smoking behaviors. A growing literature indicates sex and menstrual phase differences in responses to nicotine. The aim of this study was to assess sex and menstrual phase influences on a broad range of measures of nicotine response including subjective drug effects, cognition, physiological responses, and symptoms of withdrawal, craving, and affect. Using a well-established intravenous nicotine paradigm and biochemical confirmation of overnight abstinence and menstrual cycle phase, analyses were performed to compare sex (age 18–50 years; 115 male and 45 female) and menstrual cycle phase (29 follicular and 16 luteal) effects. Females had diminished subjective drug effects of, but greater physiological responses to, nicotine administration. Luteal-phase females showed diminished subjective drug effects and better cognition relative to follicular-phase women. These findings offer candidate mechanisms through which the luteal phase, wherein progesterone is dominant relative to estradiol, may be protective against vulnerability to smoking.

Genome-wide association study of nicotine dependence in American populations: Identification of novel risk loci in both African-Americans and European-Americans

BACKGROUND We report a genome-wide association study (GWAS) of nicotine dependence defined on the basis of scores on the Fagerström Test for Nicotine Dependence in European-American (EA) and African-American (AA) populations. METHODS Our sample, from the one used in our previous GWAS, included only subjects who had smoked >100 cigarettes lifetime (2114 EA and 2602 AA subjects) and an additional 927 AA and 2003 EA subjects from the Study of Addiction: Genetics and Environment project [via the database of Genotypes and Phenotypes (dbGAP)]. GWAS analysis considered Fagerström Test for Nicotine Dependence score as an ordinal trait, separately in each population and sample and by combining the results in meta-analysis. We also conducted analyses that were adjusted for other substance use disorder criteria in a single nucleotide polymorphism (SNP) subset. RESULTS In EAs, one chromosome 7 intergenic region was genome-wide significant (GWS): rs13225753, p = 3.48 × 10(-8) (adjusted). In AAs, GWS associations were observed at numerous SNPs mapped to a region on chromosome 14 of >305,000 base pairs (minimal p = 4.74 × 10(-10)). Two chromosome 8 regions were associated: p = 4.45 × 10(-8) at DLC1 SNP rs289519 (unadjusted) and p = 1.10 × 10(-9) at rs6996964 (adjusted for other substances), located between CSGALNACT1 and INTS10. No GWS associations were observed at the chromosome 15 nicotinic receptor gene cluster (CHRNA5-CHRNA3-CHRNB4) previously associated with nicotine dependence and smoking quantity traits. TSNAX-DISC1 SNP rs821722 (p = 1.46 × 10(-7)) was the most significant result with substantial contributions from both populations; we previously identified DISC1 associations with opioid dependence. Pathway analysis identified association with nitric oxide synthase and adenosine monophosphate-activated protein kinase pathways in EAs. CONCLUSIONS The key risk loci identified, which require replication, offer novel insights into nicotine dependence biology.

Hypermethylation of OPRM1 promoter region in European Americans with alcohol dependence

The μ-opioid receptor mediates rewarding effects of alcohol and illicit drugs. We hypothesized that altered DNA methylation in the μ-opioid receptor gene (OPRM1) might influence the vulnerability to alcohol dependence (AD). Genomic DNA was extracted from the peripheral blood of 125 European Americans with AD and 69 screened European American controls. Methylation levels of 16 CpGs in the OPRM1 promoter region were examined by bisulfite sequencing analysis. A multivariate analysis of covariance was conducted to analyze AD-associated methylation changes in the OPRM1 promoter region, using days of intoxication in the past 30 days, sex, age, ancestry proportion and childhood adversity (CA) as covariates. Three CpGs (80, 71, and 10 bp upstream of the OPRM1 translation start site) were more highly methylated in AD cases than in controls (CpG-80: P=0.033; CpG-71: P=0.004; CpG-10: P=0.008). Although these sites were not significant after correction for multiple comparisons, the overall methylation level of the 16 CpGs was significantly higher in AD cases (13.6%) than in controls (10.6%) (P=0.049). Sex and CA did not significantly influence OPRM1 promoter methylation levels. Our findings suggest that OPRM1 promoter hypermethylation may increase the risk for AD and other substance dependence disorders.

REVIEW: Cognitive effects of nicotine: genetic moderators

Cigarette smoking is the main preventable cause of death in developed countries, and the development of more effective treatments is necessary. Cumulating evidence suggests that cognitive enhancement may contribute to the addictive actions of nicotine. Several studies have demonstrated that nicotine enhances cognitive performance in both smokers and non‐smokers. Genetic studies support the role of both dopamine (DA) and nicotinic acetylcholine receptors (nAChRs) associated with nicotine‐induced cognitive enhancement. Based on knockout mice studies, β2 nAChRs are thought to be essential in mediating the cognitive effects of nicotine. α7nAChRs are associated with attentional and sensory filtering response, especially in schizophrenic individuals. Genetic variation in D2 type DA receptors and the catechol‐O‐methyltransferase enzyme appears to moderate cognitive deficits induced by smoking abstinence. Serotonin transporter (5‐HTT) gene variation also moderates nicotine‐induced improvement in spatial working memory. Less is known about the contribution of genetic variation in DA transporter and D4 type DA receptor genetic variation on the cognitive effects of nicotine. Future research will provide a clearer understanding of the mechanism underlying the cognitive‐enhancing actions of nicotine.

Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region.

The δ-opioid receptor mediates rewarding effects of many substances of abuse. We reported an increased frequency of the minor G-allele of single-nucleotide polymorphism (SNP) rs569356 (the only variant identified so far in the promoter region of the δ-opioid receptor gene (OPRD1)) in subjects with opioid dependence. In this study, we examined the functional significance of this variant. OPRD1 promoter region harboring SNP rs569356 was amplified by PCR and inserted into a firefly luciferase reporter vector. HEK293 cells were co-transfected with these constructs and a renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase (driven by the OPRD1 promoter) was measured by a dual luciferase reporter assay and normalized by renilla luciferase expression. Moreover, alleles altering expression were further assessed for binding of human brain nuclear proteins by electrophoretic mobility shift assay (EMSA). The minor G-allele was associated with significantly greater expression levels of firefly luciferase than the major A-allele of SNP rs569356 (P=0.003). EMSA also showed specific gel shift bands, suggesting that SNP rs569356 is situated in the binding site of potential transcription factors. These results suggest that the minor G-allele of SNP rs569356 may enhance transcription factor binding and increase OPRD1 expression.