CYP2A6 associates with respiratory disease risk and younger age of diagnosis: a phenome-wide association Mendelian Randomization study.

CYP2A6, a genetically variable enzyme, inactivates nicotine, activates carcinogens, and metabolizes many pharmaceuticals. Variation in CYP2A6 influences smoking behaviors and tobacco-related disease risk. This phenome-wide association study examined associations between a reconstructed version of our weighted genetic risk score (wGRS) for CYP2A6 activity with diseases in the UK Biobank (N = 395 887). Causal effects of phenotypic CYP2A6 activity (measured as the nicotine metabolite ratio: 3'-hydroxycotinine/cotinine) on the phenome-wide significant (PWS) signals were then estimated in two-sample Mendelian Randomization using the wGRS as the instrument. Time-to-diagnosis age was compared between faster versus slower CYP2A6 metabolizers for the PWS signals in survival analyses. In the total sample, six PWS signals were identified: two lung cancers and four obstructive respiratory diseases PheCodes, where faster CYP2A6 activity was associated with greater disease risk (Ps < 1 × 10-6). A significant CYP2A6-by-smoking status interaction was found (Psinteraction < 0.05); in current smokers, the same six PWS signals were found as identified in the total group, whereas no PWS signals were found in former or never smokers. In the total sample and current smokers, CYP2A6 activity causal estimates on the six PWS signals were significant in Mendelian Randomization (Ps < 5 × 10-5). Additionally, faster CYP2A6 metabolizer status was associated with younger age of disease diagnosis for the six PWS signals (Ps < 5 × 10-4, in current smokers). These findings support a role for faster CYP2A6 activity as a causal risk factor for lung cancers and obstructive respiratory diseases among current smokers, and a younger onset of these diseases. This research utilized the UK Biobank Resource.

Genotyping, characterization, and imputation of known and novel CYP2A6 structural variants using SNP array data.

CYP2A6 metabolically inactivates nicotine. Faster CYP2A6 activity is associated with heavier smoking and higher lung cancer risk. The CYP2A6 gene is polymorphic, including functional structural variants (SV) such as gene deletions (CYP2A6*4), duplications (CYP2A6*1 × 2), and hybrids with the CYP2A7 pseudogene (CYP2A6*12, CYP2A6*34). SVs are challenging to genotype due to their complex genetic architecture. Our aims were to develop a reliable protocol for SV genotyping, functionally phenotype known and novel SVs, and investigate the feasibility of CYP2A6 SV imputation from SNP array data in two ancestry populations. European- (EUR; n = 935) and African- (AFR; n = 964) ancestry individuals from smoking cessation trials were genotyped for SNPs using an Illumina array and for CYP2A6 SVs using Taqman copy number (CN) assays. SV-specific PCR amplification and Sanger sequencing was used to characterize a novel SV. Individuals with SVs were phenotyped using the nicotine metabolite ratio, a biomarker of CYP2A6 activity. SV diplotype and SNP array data were integrated and phased to generate ancestry-specific SV reference panels. Leave-one-out cross-validation was used to investigate the feasibility of CYP2A6 SV imputation. A minimal protocol requiring three Taqman CN assays for CYP2A6 SV genotyping was developed and known SV associations with activity were replicated. The first domain swap CYP2A6-CYP2A7 hybrid SV, CYP2A6*53, was identified, sequenced, and associated with lower CYP2A6 activity. In both EURs and AFRs, most SV alleles were identified using imputation (>70% and >60%, respectively); importantly, false positive rates were <1%. These results confirm that CYP2A6 SV imputation can identify most SV alleles, including a novel SV.

Influence of CYP2A6 Genetic Variation, Nicotine Dependence Severity, and Treatment on Smoking Cessation Success.

INTRODUCTION: Genetic variation in Cytochrome P450 2A6 (CYP2A6), the major nicotine metabolizing enzyme, is associated with nicotine dependence and smoking cessation. Nicotine dependence severity also predicts smoking cessation. Our goals were to determine how CYP2A6 variation and nicotine dependence alter smoking cessation, and whether dependence could refine CYP2A6-based treatment recommendations. AIMS AND METHODS: Adult smokers treated for 12 weeks with placebo, nicotine patch, or varenicline (NCT01314001) were grouped as CYP2A6 normal (n = 567) or slow (n = 432) nicotine metabolizers based on a CYP2A6 weighted genetic risk score. Fagerström test for nicotine dependence scores were measured at baseline and biochemically verified smoking cessation was assessed at end of treatment. RESULTS: Dependence neither mediated nor moderated an association between CYP2A6 variation and smoking cessation overall, within any treatment arm, or after stratifying by ancestry (n = 591 European, n = 408 African ancestry) or sex (n = 444 women, n = 555 men). In within-treatment analyses, the mediation effect odds ratio (OR) ranged from 0.95 to 1.00 and the bias-corrected 95% confidence interval contained 1. Moderation (i.e. interaction) effect ORs ranged from 0.88 to 1.61 (p = .397-.828). For CYP2A6 normal metabolizers, quit rates on varenicline were similar for those with high (41.1%) and low (43.4%) dependence, while quit rates were lower for those with high versus low dependence on both patch (16.5 vs. 29.7%) and placebo (8.9 vs. 18.5%). CYP2A6 slow metabolizers with high versus low dependence had lower quit rates in all three treatment arms. CONCLUSIONS: Although nicotine dependence severity neither mediated nor moderated CYP2A6 associations with smoking cessation, incorporating information on dependence may optimize the choice of smoking cessation treatment aid in CYP2A6 normal and slow metabolizers. IMPLICATIONS: Variation in CYP2A6 and nicotine dependence severity alter smoking cessation success. Our findings suggest that while nicotine dependence severity is unlikely to mediate or moderate CYP2A6 associations with cessation, incorporating patient information on both CYP2A6 and nicotine dependence severity may lead to improved smoking cessation strategies.

Accuracy and applications of sequencing and genotyping approaches for CYP2A6 and homologous genes.

OBJECTIVES: We evaluated multiple genotyping/sequencing approaches in a homologous region of chromosome 19, and investigated associations of two common 3'-UTR CYP2A6 variants with activity in vivo. METHODS: Individuals (n = 1704) of European and African ancestry were phenotyped for the nicotine metabolite ratio (NMR), an index of CYP2A6 activity, and genotyped/sequenced using deep amplicon exon sequencing, SNP array, genotype imputation and targeted capture sequencing. Amplicon exon sequencing was the gold standard to which other methods were compared within-individual for CYP2A6, CYP2A7, CYP2A13, and CYP2B6 exons to identify highly discordant positions. Linear regression models evaluated the association of CYP2A6*1B and rs8192733 genotypes (coded additively) with logNMR. RESULTS: All approaches were ≤2.6% discordant with the gold standard; discordant calls were concentrated at few positions. Fifteen positions were discordant in >10% of individuals, with 12 appearing in regions of high identity between homologous genes (e.g. CYP2A6 and CYP2A7). For six, allele frequencies in our study and online databases were discrepant, suggesting errors in online sources. In the European-ancestry group (n = 935), CYP2A6*1B and rs8192733 were associated with logNMR (P < 0.001). A combined model found main effects of both variants on increasing logNMR. Similar trends were found in those of African ancestry (n = 506). CONCLUSION: Multiple genotyping/sequencing approaches used in this chromosome 19 region contain genotyping/sequencing errors, as do online databases. Gene-specific primers and SNP array probes must consider gene homology; short-read sequencing of related genes in a single reaction should be avoided. Using improved sequencing approaches, we characterized two gain-of-function 3'-UTR variants, including the relatively understudied rs8192733.

Genome-wide association meta-analysis of nicotine metabolism and cigarette consumption measures in smokers of European descent.

Smoking behaviors, including amount smoked, smoking cessation, and tobacco-related diseases, are altered by the rate of nicotine clearance. Nicotine clearance can be estimated using the nicotine metabolite ratio (NMR) (ratio of 3'hydroxycotinine/cotinine), but only in current smokers. Advancing the genomics of this highly heritable biomarker of CYP2A6, the main metabolic enzyme for nicotine, will also enable investigation of never and former smokers. We performed the largest genome-wide association study (GWAS) to date of the NMR in European ancestry current smokers (n = 5185), found 1255 genome-wide significant variants, and replicated the chromosome 19 locus. Fine-mapping of chromosome 19 revealed 13 putatively causal variants, with nine of these being highly putatively causal and mapping to CYP2A6, MAP3K10, ADCK4, and CYP2B6. We also identified a putatively causal variant on chromosome 4 mapping to TMPRSS11E and demonstrated an association between TMPRSS11E variation and a UGT2B17 activity phenotype. Together the 14 putatively causal SNPs explained ~38% of NMR variation, a substantial increase from the ~20 to 30% previously explained. Our additional GWASs of nicotine intake biomarkers showed that cotinine and smoking intensity (cotinine/cigarettes per day (CPD)) shared chromosome 19 and chromosome 4 loci with the NMR, and that cotinine and a more accurate biomarker, cotinine + 3'hydroxycotinine, shared a chromosome 15 locus near CHRNA5 with CPD and Pack-Years (i.e., cumulative exposure). Understanding the genetic factors influencing smoking-related traits facilitates epidemiological studies of smoking and disease, as well as assists in optimizing smoking cessation support, which in turn will reduce the enormous personal and societal costs associated with smoking.

Stability of Varenicline Concentration in Saliva Over 21 Days at Three Storage Temperatures.

INTRODUCTION: Varenicline is the most efficacious drug for smoking cessation; saliva varenicline concentrations can be useful for the evaluation of adherence in smoking cessation trials. Saliva is a useful noninvasive matrix for mail-in specimen collection, if stable. We investigated the stability of varenicline in saliva at different storage temperatures simulating the time it takes to mail in a sample. METHODS: We evaluated the concentrations of varenicline, nicotine, cotinine, 3'-hydroxycotinine, and 3'-hydroxycotinine/cotinine (3HC/COT) ratio in quality control saliva samples (and after repeated freezing and thawing), and in smokers' saliva samples, stored for up to 21 days at room temperature (~25°C), 4°C, and -80°C. RESULTS: In saliva quality control samples, concentrations of varenicline, nicotine, cotinine, 3'-hydroxycotinine, and 3HC/COT remained unchanged and showed little within-sample variation (CV ≤ 5.5%) for up to 21 days at the three storage temperatures; they were also not altered after three thaw-freeze cycles. In smokers' saliva, a significant main effect of storage duration, but not temperature, was observed for varenicline, cotinine, and 3'-hydroxycotinine, but not for nicotine or the 3HC/COT ratio. However, these changes were within analytical (i.e., equipment) variation resulting in little within-sample variation (CV ≤ 5.8%) for all analytes in smokers' saliva. CONCLUSIONS: Varenicline, the other analytes, and the 3HC/COT ratio remained stable in saliva during storage for 21 days at all temperatures tested and after repeated freezing and thawing with only minor changes in concentration over time. These findings support the potential use of mail-in approach for saliva samples in varenicline smoking cessation clinical trials. IMPLICATIONS: Assessing saliva varenicline concentrations can be useful for the evaluation of adherence in smoking cessation trials. Saliva is a noninvasive matrix suitable for mail-in specimen collection. This is the first investigation of stability of varenicline in saliva. Varenicline, nicotine, cotinine, 3'-hydroxycotinine, and 3HC/COT were stable in saliva for up to 21 days at room temperature (~25°C), 4°C, and -80°C, supporting the use of a mail-in approach for saliva specimen in smoking cessation trials.

A Genome-Wide Association Study of Nausea Incidence in Varenicline-Treated Cigarette Smokers.

INTRODUCTION: Varenicline is the most efficacious smoking cessation treatment; however, long-term cessation rates tend to be <25%. Nausea, the most common side effect of varenicline, observed in ~28% of individuals treated, peaks early following treatment initiation and reduces cessation success. Genetic variation influences treatment response, however genetic contributors to individual differences in side effects are less understood. METHODS: We conducted a genome-wide association study of nausea incidence at 1 week following the initiation of varenicline treatment (corresponding to the target quit date) in 189 cigarette smokers of European ancestry (NCT01314001). Additive genetic models examining the likelihood of experiencing any versus no nausea controlled for population substructure, age, and sex. Variants with minor allele frequencies (MAF)≥10% were considered. RESULTS: Fifty-seven (30.2%) out of 189 participants reported nausea. The top variant associated with nausea was rs1568209 (odds ratio [OR] = 2.61 for A vs. G allele; 95% confidence interval [CI] = 1.65,4.15; p = 2.1e-7; MAF = 48.7%), mapping to the SLCO3A1 drug transporter gene on chromosome 15. In the same trial, rs1568209 was not associated with nausea in either the nicotine patch (p = .56; n = 181) or placebo (p = .59; n = 174) arms. In varenicline-treated smokers, the incidence of nausea was higher in females (44.6%; n = 74) versus males (20.9%; n = 115) (p = .001), however there was no evidence of a difference in the influence of rs1568209 on nausea between the sexes (p for sex*genotype interaction = .36). Future studies in larger samples are required to test the robustness of this finding. CONCLUSIONS: Variation in SLCO3A1 may influence the risk for developing nausea in varenicline-treated smokers, which may alter adherence and cessation. IMPLICATIONS: Varenicline-associated nausea reduces adherence and limits cessation success. Previous candidate gene association studies showed genetic factors influence nausea on varenicline. This pilot genome-wide investigation of nausea, the most common side effect associated with varenicline treatment and an important cause of treatment discontinuation, suggests the potential involvement of common variation in the SLCO3A1 drug transporter gene.

Impact of CYP2A6 Activity on Nicotine Reinforcement and Cue-Reactivity in Daily Smokers.

INTRODUCTION: Variation in CYP2A6, the primary enzyme responsible for nicotine metabolism, is associated with nicotine dependence, cigarette consumption, and abstinence outcomes in smokers. The impact of CYP2A6 activity on nicotine reinforcement and tobacco cue-reactivity, mechanisms that may contribute to these previous associations, has not been fully evaluated. AIMS AND METHODS: CYP2A6 activity was indexed using 3 genetic approaches in 104 daily smokers completing forced-choice and cue-induced craving tasks assessing nicotine reinforcement and tobacco cue-reactivity, respectively. First, smokers were stratified by the presence or absence of reduced/loss-of-function CYP2A6 gene variants (normal vs. reduced metabolizers). As nicotine metabolite ratio (NMR) is a reliable biomarker of CYP2A6 activity, our second and third approaches used additional genetic variants identified in genome-wide association studies of NMR to create a weighted genetic risk score (wGRS) to stratify smokers (fast vs. slow metabolizers) and calculate a wGRS-derived NMR. RESULTS: Controlling for race and sex, normal metabolizers (vs. reduced) selected a greater proportion of puffs from nicotine-containing cigarettes (vs. denicotinized) on the forced-choice task (p = .031). In confirmatory analyses, wGRS-based stratification (fast vs. slow metabolizers) produced similar findings. Additionally, wGRS-derived NMR, which correlated with actual NMR assessed in a subset of participants (n = 55), was positively associated with the proportion of puffs from nicotine-containing cigarettes controlling for race and sex (p = .015). None of the CYP2A6 indices were associated with tobacco cue-reactivity in minimally deprived smokers. CONCLUSIONS: Findings suggest increased nicotine reinforcement is exhibited by smokers with high CYP2A6 activity, which may contribute to heavier smoking and poorer cessation outcomes previously reported in faster metabolizers. IMPLICATIONS: CYP2A6 activity is a key determinant of smoking behavior and outcomes. Therefore, these findings support the targeting of CYP2A6 activity, either therapeutically or as a clinically relevant biomarker in a precision medicine approach, for tobacco use disorder treatment.

Evaluation of a weighted genetic risk score for the prediction of biomarkers of CYP2A6 activity.

The nicotine metabolite ratio (NMR; 3-hydroxycotinine/cotinine) is an index of CYP2A6 activity. CYP2A6 is responsible for nicotine's metabolic inactivation and variation in the NMR/CYP2A6 is associated with several smoking behaviors. Our aim was to integrate established alleles and novel genome-wide association studies (GWAS) signals to create a weighted genetic risk score (wGRS) for the CYP2A6 gene for European-ancestry populations. The wGRS was compared with a previous CYP2A6 gene scoring approach designed for an alternative phenotype (C2/N2; cotinine-d2/(nicotine-d2 + cotinine-d2)). CYP2A6 genotypes and the NMR were assessed in European-ancestry participants. The wGRS training set included N = 933 smokers recruited to the Pharmacogenetics of Nicotine Addiction and Treatment clinical trial [NCT01314001]. The replication cohort included N = 196 smokers recruited to the Quit 2 Live clinical trial [NCT01836276]. Comparisons between the two CYP2A6 phenotypes and with fractional clearance were made in a laboratory-based pharmacokinetic study (N = 92 participants). In both the training and replication sets, the wGRS, which included seven CYP2A6 variants, explained 33.8% (P < 0.001) of the variance in NMR, providing improved predictive power to the NMR phenotype when compared with other CYP2A6 gene scoring approaches. NMR and C2/N2 were strongly correlated to nicotine clearance (ρ = 0.70 and ρ = 0.79, respectively; P < 0.001), and to one another (ρ = 0.82; P < 0.001); however reduced function genotypes occurred in slow NMR but throughout C2/N2. The wGRS was able to predict smoking quantity and nicotine intake, to discriminate between NMR slow and normal metabolizers (AUC = 0.79; P < 0.001), and to replicate previous NMR-stratified cessation outcomes showing unique treatment outcomes between metabolizer groups.

The Nicotine Metabolite Ratio is Associated With Early Smoking Abstinence Even After Controlling for Factors That Influence the Nicotine Metabolite Ratio.

INTRODUCTION: The decrease in smoking rates in North America has plateaued, underscoring the need for new approaches to treat nicotine dependence. Inter-individual differences in smoking behavior result, in part, from variation in the rate of CYP2A6-mediated nicotine metabolism. A phenotypic measure of CYP2A6 activity is the nicotine metabolite ratio (NMR), the ratio of 3'hydroxycotinine/cotinine. The NMR is associated with smoking cessation. However, the NMR is also associated with genetic (eg, CYP2A6 genotype) and other (eg, sex and ethnicity) factors. Here we aimed to determine if previously identified non-CYP2A6 sources of variation in the NMR mitigated the association between the NMR and short-term abstinence. METHODS: The NMR was determined from blood samples collected at intake from daily smokers aged 18-65. Biochemically-verified point prevalence abstinence (exhaled carbon monoxide level ≤ 8 ppm) was measured at 1 week following the target quit date in participants from a smoking cessation clinical trial (NCT01314001). Analyses were restricted to N = 462 blacks and N = 693 whites in the intent-to-treat sample. RESULTS: Lower NMR (<0.31) was associated with a higher likelihood of 1-week abstinence (OR = 1.43; 95% CI = 1.12, 1.84). NMR was associated with abstinence even after controlling for treatment arm (nicotine patch or varenicline) and factors previously associated with NMR variation including sex, ethnicity, estrogen-containing hormonal therapy, body mass index, alcohol, and cigarette consumption. CONCLUSIONS: NMR was associated with 1-week smoking abstinence; NMR may be a useful addition to medication screening approaches evaluating treatments for nicotine dependence.

Test-Retest Reliability and Stability of the Nicotine Metabolite Ratio Among Treatment-Seeking Smokers.

INTRODUCTION: The nicotine metabolite ratio (NMR), the ratio of 3-hydroxycotinine to cotinine, is a biomarker used in smoking cessation research, with several retrospective studies suggesting that NMR predicts treatment outcome. To be maximally useful in tailoring treatment, estimates of NMR should be stable over time. The present study is the first to examine the short-term test-retest reliability of NMR among treatment-seeking smokers. METHODS: Blood NMR was assessed at two time points, approximately 2-3 weeks apart and prior to intervention, among 72 healthy adult smokers (49% female; 35% non-White) enrolled in a cessation trial (http://ClinicalTrials.gov ID: NCT01314001). RESULTS: Mean NMR was stable from Time-1 to Time-2, with no significant change between assessments; test-retest reliability for NMR values was excellent (ICC[2,1] = 0.87). Test-retest reliability remained acceptable to high when NMR was categorized, as in recent clinical trials. Classification of participants as slow (quartile 1, NMR ≤ 0.24) or normal/fast NMR (quartiles 2-4, NMR ≥ 0.25) was consistent from Time-1 to Time-2 for 96% of participants (κ = 0.89). Though classification of participants into NMR quartiles was less consistent from Time-1 to Time-2 (67% agreement; weighted κ = 0.73), all reclassifications occurred between adjacent quartiles. CONCLUSIONS: Overall, these data support the use of a single NMR assessment for association studies with smoking phenotypes and in smokers seeking to quit, and they encourage large-scale efforts to determine optimal NMR cutpoints for tailoring treatment selection.

Variation in P450 oxidoreductase (POR) A503V and flavin-containing monooxygenase (FMO)-3 E158K is associated with minor alterations in nicotine metabolism, but does not alter cigarette consumption.

The rates of nicotine metabolism differ widely, even after controlling for genetic variation in the major nicotine-metabolizing enzyme, CYP2A6. Genetic variants in an additional nicotine-metabolizing enzyme, flavin-containing monooxygenase (FMO)-3, and an obligate microsomal CYP-supportive enzyme, cytochrome P450 oxidoreductase (POR), were investigated. We examined the impact of FMO3 E158K and POR A503V before and after stratifying by CYP2A6 metabolism group. In 130 nonsmokers of African descent who received 4 mg oral nicotine, FMO3 158K trended toward slower nicotine metabolism in reduced CYP2A6 metabolizers (P=0.07) only, whereas POR 503V was associated with faster CYP2A6 activity (nicotine metabolite ratio) in normal (P=0.03), but not reduced, CYP2A6 metabolizers. Neither FMO3 158K nor POR 503V significantly altered the nicotine metabolic ratio (N=659), cigarette consumption (N=667), or urine total nicotine equivalents (N=418) in smokers of African descent. Thus, FMO3 E158K and POR A503V are minor sources of nicotine metabolism variation, insufficient to appreciably alter smoking.

IL-15 can signal via IL-15Rα, JNK, and NF-κB to drive RANTES production by myeloid cells.

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8(+) memory T cells expressing IL-15/IL-2Rß and common γ chain (γ(c)). Previously, we showed that the prophylactic delivery of IL-15 to Rag2(-/-)γ(c)(-/-) mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2(-/-)γ(c)(-/-) mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rß and γ(c) expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells, but the presence of γ(c) did not increase bone marrow cell sensitivity to IL-15. This IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells occurred independently of the IL-15/IL-2Rß and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15-mediated IFN-γ production by NK cells but reduces IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.

Associating CYP2A6 structural variants with ovarian and lung cancer risk in the UK Biobank: replication and extension.

CYP2A6 is a polymorphic enzyme that inactivates nicotine; structural variants (SVs) include gene deletions and hybrids with the neighboring pseudogene CYP2A7. Two studies found that CYP2A7 deletions were associated with ovarian cancer risk. Using their methodology, we aimed to characterize CYP2A6 SVs (which may be misidentified by prediction software as CYP2A7 SVs), then assess CYP2A6 SV-associated risk for ovarian cancer, and extend analyses to lung cancer. An updated reference panel was created to impute CYP2A6 SVs from UK Biobank array data. Logistic regression models analyzed the association between CYP2A6 SVs and cancer risk, adjusting for covariates. Software-predicted CYP2A7 deletions were concordant with known CYP2A6 SVs. Deleterious CYP2A6 SVs were not associated with ovarian cancer (OR = 1.06; 95% CI: 0.80-1.37; p = 0.7) but did reduce the risk of lung cancer (OR = 0.44; 95% CI: 0.29-0.64; p < 0.0001), and a lung cancer subtype. Replication of known lung cancer associations indicates the validity of array-based SV analyses.

Pharmacogenetics of Biochemically Verified Abstinence in an Opioid Agonist Therapy Randomized Clinical Trial of Methadone and Buprenorphine/Naloxone.

Methadone and buprenorphine/naloxone are opioid agonist therapies for opioid use disorder treatment. Genetic factors contribute to individual differences in opioid response; however, little is known regarding genetic associations with clinical outcomes in people receiving opioid agonist therapies. Participants diagnosed with opioid use disorder, principally consisting of prescription opioids (licit or illicit), were randomized to methadone or buprenorphine/naloxone for 24 weeks of daily treatment (NCT03033732). Urine was collected at 12 biweekly study visits and analyzed for non-treatment opioids. Variants in genes involved in methadone metabolism (CYP2B6, CYP2C19, and CYP3A4), buprenorphine metabolism (CYP3A4 and UGT2B7), and µ-opioid receptor function (OPRM1) were genotyped and analyzed for their association with the number of non-treatment opioid-free urine screens. Primary analyses focused on the last 12 weeks (6 study visits, post-titration) of treatment among those reporting White ethnicity. Additional sensitivity and exploratory analyses were performed. Among methadone-treated participants (n = 52), the OPRM1 rs1799971 AA genotype (vs. G-genotypes, i.e., having one or two G alleles) was associated with greater opioid-free urine screens (incidence rate ratio = 5.24, 95% confidence interval (CI) = 2.43-11.26, P = 0.000023); longitudinal analyses showed a significant genotype-by-time interaction over the full 24 weeks (12 study visits, ß = -0.28, 95% CI = -0.45 to -0.11, P = 0.0015). Exploratory analyses suggest an OPRM1 rs1799971 genotype effect on retention. No evidence of association was found between other genetic variants, including in metabolic variants, and non-treatment opioid-free urine screens in the methadone or buprenorphine/naloxone arms. Those with the OPRM1 rs1799971 G-genotypes may have a poorer response to methadone maintenance treatment, an effect that persisted through 24 weeks of treatment.

Genetic Prediction of Smoking Cessation Medication Side Effects: A Genome-Wide Investigation of Abnormal Dreams on Varenicline.

Varenicline, the most efficacious smoking cessation monotherapy, produces abnormal dreams. Although genetic contributions to varenicline-associated nausea and cessation have been identified, the role of genetics in abnormal dreams is unknown. We conducted a genomewide association study (GWAS) of abnormal dreams in 188 European ancestry smokers treated with varenicline (NCT01314001). Additive genetic models examined the likelihood of experiencing abnormal dreams 2 weeks following varenicline initiation. For the top locus, we tested for selectivity to varenicline, effects on cessation, replication, and generalizability to African ancestry (AA) individuals. The top GWAS variant associated with abnormal dreams was rs901886, mapping to intron 2 of ICAM5 on chromosome 19. The prevalence of abnormal dreams in those with rs901886 CC, CT, and TT genotypes was 15%, 36%, and 62%, respectively (odds ratio = 2.94 for T vs. C, 95% confidence interval = 1.92-4.55, P = 2.03e-7; T allele frequency = 52%). This rs901886 association was selective to varenicline (P values > 0.05 on nicotine patch and placebo). There were also positive associations for rs901886 T (vs. C allele, P = 0.03) and for abnormal dreams (P = 0.06) with varenicline-aided cessation. Neither rs901886 (P = 0.40) nor abnormal dreams (P = 0.24) were associated with adherence. A similar direction of effect of rs901886 on abnormal dreams was observed in a second varenicline trial (NCT01836276). In AA individuals (n = 137), rs901886 was not associated with abnormal dreams (P = 0.41), but there was an association for a variant located ~ 74.4 kb 5' of ICAM5 (P = 2.56e-3). Variation in ICAM5 may influence abnormal dreams and cessation on varenicline. These findings provide additional support for genetically optimized smoking cessation approaches.

CYP2A6 slow nicotine metabolism is associated with increased quitting by adolescent smokers.

Variation in the CYP2A6 gene, which decreases the rate of nicotine metabolic inactivation, is associated with higher adult smoking cessation rates during clinical trials. We hypothesized that slow metabolism is associated with increased quitting during adolescence. White adolescent smokers (N=308, aged 12-17, 36.3% male) from a cohort study were genotyped for CYP2A6, resulting in 7.8% slow, 14.0% intermediate and 78.2% normal metabolizers. Overall, 144 smokers quit smoking, as indicated by being abstinent for at least 12 months. In logistic regression analyses, the odds ratio for quitting was 2.25 (95% confidence interval 1.05, 4.80; P=0.037) for slow metabolizers relative to normal metabolizers. A linear trend toward increased quitting with decreasing CYP2A6 activity was also observed (odds ratio=1.44, 95% confidence interval 1.02, 2.01; P=0.034). Thus, CYP2A6 slow metabolism is associated with increased adolescent smoking cessation, indicating that even early in the smoking history, genetic variation is influencing smoking cessation.

Critical role of natural killer cells in lung immunopathology during influenza infection in mice.

Influenza viral infection results in excessive pulmonary inflammation that has been linked to the damage caused by immune responses and viral replication. The multifunctional cytokine interleukin (IL-15), influences the proliferation and maintenance of immune cells such as CD8(+) T cells and natural killer (NK) cells. Here we show that IL-15(-/-) mice are protected from lethal influenza infection. Irrespective of the mouse strains, the protection observed was linked to the lack of NK cells. Increased survival in the IL-15(-/-) or NK1.1(+) cell-depleted wild-type mice was associated with significantly lower lung lesions as well as decreased mononuclear cells and neutrophils in the airway lumen. Levels of interleukin 10 were significantly higher and levels of proinflammatory cytokines, including interleukin 6 and interleukin 12, were significantly lower in the bronchoalveolar lavage fluid from IL-15(-/-) and NK1.1(+) cell-depleted wild-type mice than in that from control mice. Our data suggest that NK cells significantly augment pulmonary inflammation, contributing to the pathogenesis of influenza infection.

Genetic variation in fatty acid amide hydrolase (FAAH): Associations with early drinking and smoking behaviors.

BACKGROUND: The endocannabinoid system is implicated in psychiatric disorders and drug dependence. Within this system, fatty acid amide hydrolase (FAAH) metabolizes endocannabinoids. Individuals with A-group genotypes (C/A or A/A) of a common FAAH variant (rs324420; C > A; Pro129Thr) have slower enzymatic activity compared to C-group individuals (C/C genotype). Slow FAAH activity is differentially associated with alcohol and nicotine use. METHODS: Among European-ancestry participants in the NDIT study (n = 249-607), genotype associations with past-year binge drinking in young adults were estimated in logistic regression models. In adolescents, hazard ratios (HR) were estimated from Cox proportional hazards models to assess the FAAH genotype group association with time to drinking initiation and attaining drinking frequency outcomes. HR were also used to assess genotype effect on time to smoking initiation and attaining early smoking milestones (e.g., first inhalation, ICD-10 dependence). RESULTS: Compared to those in the C-group, those in the A-group had higher odds of binge drinking at ages 20 (Odds ratio (OR) = 2.16, 95 % CI 1.36-3.42) and 30 (OR = 1.61, 95 % CI 1.10-2.36). Time to initiation of drinking and daily drinking was faster in adolescents in the A-group (HR = 1.39, 95 % CI 1.09-1.77 and HR = 2.24, 95 % CI 1.05-4.76, respectively). Time to smoking initiation was faster in the A-group (HR = 1.20, 95 % CI 1.04-1.39); however, time to smoking milestones among adolescent smokers was not consistently different for the A- versus C-groups (HR = 0.43 to 1.13). CONCLUSIONS: Slow FAAH activity (A-group) was associated with greater risks for binge drinking, drinking initiation and escalation, and cigarette smoking initiation, but had little impact on the escalation in cigarette smoking behaviors.

Examining the role of mitochondrial genetic variation in nicotine dependence.

Nicotine dependence (ND) has a heritability rate of ∼50%, suggesting genetic factors contribute to underlying mechanisms. Here, we aimed to examine variants within both mtDNA and the nuclear genome to determine if mitochondrial genes are associated with ND. A total of 129 mtDNA SNPs and 1136 nuclear-encoded mitochondrial genes in a sample of N = 374 Caucasians were selected for analysis. Age of onset of first, occasional, and daily smoking and Fagerström Test for Nicotine Dependence were used as outcomes for the analysis. Linear regression was used to test common variants. Gene analyses were performed using MAGMA. One nuclear mitochondrial SNP, rs78417112 found in the HSD17B4 gene, was significantly associated with the age of onset of occasional smoking. Additionally, one nuclear mitochondrial gene, PRKACA, was significantly associated with age of onset of both first and occasional smoking. Replication testing of the mtDNA m.1700T>C SNP, nominally associated with age of onset of daily smoking, was available in the PNAT2 clinical trial (N = 930 Caucasians). A meta-analysis showed this SNP was associated with age of onset of daily smoking (p-value = 0.004). Overall, the findings suggest mitochondrial genetic variation may contribute to variability in smoking phenotypes, although replication in larger samples is required.