Geographic variation of mutagenic exposures in kidney cancer genomes.

International differences in the incidence of many cancer types indicate the existence of carcinogen exposures that have not yet been identified by conventional epidemiology make a substantial contribution to cancer burden1. In clear cell renal cell carcinoma, obesity, hypertension and tobacco smoking are risk factors, but they do not explain the geographical variation in its incidence2. Underlying causes can be inferred by sequencing the genomes of cancers from populations with different incidence rates and detecting differences in patterns of somatic mutations. Here we sequenced 962 clear cell renal cell carcinomas from 11 countries with varying incidence. The somatic mutation profiles differed between countries. In Romania, Serbia and Thailand, mutational signatures characteristic of aristolochic acid compounds were present in most cases, but these were rare elsewhere. In Japan, a mutational signature of unknown cause was found in more than 70% of cases but in less than 2% elsewhere. A further mutational signature of unknown cause was ubiquitous but exhibited higher mutation loads in countries with higher incidence rates of kidney cancer. Known signatures of tobacco smoking correlated with tobacco consumption, but no signature was associated with obesity or hypertension, suggesting that non-mutagenic mechanisms of action underlie these risk factors. The results of this study indicate the existence of multiple, geographically variable, mutagenic exposures that potentially affect tens of millions of people and illustrate the opportunities for new insights into cancer causation through large-scale global cancer genomics.

Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Evaluating interactions of polygenic risk scores and NAT2 genotypes with tobacco smoking in bladder cancer risk.

Tobacco smoking is the most important risk factor for bladder cancer. Previous studies have identified the N-acetyltransferase (NAT2) gene in association with bladder cancer risk. The NAT2 gene encodes an enzyme that metabolizes aromatic amines, carcinogens commonly found in tobacco smoke. In our study, we evaluated potential interactions of tobacco smoking with NAT2 genotypes and polygenic risk score (PRS) for bladder cancer, using data from the UK Biobank, a large prospective cohort study. We used Cox proportional hazards models to measure the strength of the association. The PRS was derived using genetic risk variants identified by genome-wide association studies for bladder cancer. With an average of 10.1 years of follow-up of 390 678 eligible participants of European descent, 769 incident bladder cancer cases were identified. Current smokers with a PRS in the highest tertile had a higher risk of developing bladder cancer (HR: 6.45, 95% CI: 4.51-9.24) than current smokers with a PRS in the lowest tertile (HR: 2.41, 95% CI: 1.52-3.84; P for additive interaction = <.001). A similar interaction was found for genetically predicted metabolizing NAT2 phenotype and tobacco smoking where current smokers with the slow NAT2 phenotype had an increased risk of developing bladder cancer (HR: 5.70, 95% CI: 2.64-12.30) than current smokers with the fast NAT2 phenotype (HR: 3.61, 95% CI: 1.14-11.37; P for additive interaction = .100). Our study provides support for considering both genetic and lifestyle risk factors in developing prevention measures for bladder cancer.

A Novel Four­Gene Biomarker for Tobacco Smoking-Induced Colorectal Cancer Progression.

INTRODUCTION: Cigarette smoking greatly promotes the progression and poor prognosis of colorectal cancer (CRC) patients, with the molecular mechanism still not fully clear. METHODS: In this study, CRC cells were exposed to tobacco-specific nitrosamine 4­(methylnitrosamino)­1­(3­pyridyl)-1­butanone (NNK), and the differentially expressed smoking-related genes were identified based on both NNK-induced CRC cells and a total of 763 CRC tissues from The Cancer Genome Atlas cohort. Cox regression analysis, receiver operating characteristic curve and Kaplan-Meier plot were used to establish the risk score model for CRC prognosis. Moreover, quantitative real-time-PCR, western blotting, colony formation, migration, and invasion assays were performed to verify the core differentially expressed smoking-related gene and its molecular function in NNK-induced CRC progression. RESULTS: Results indicated NNK significantly enhanced CRC cell proliferation, migration and invasion. Moreover, a four-gene signature containing AKR1B10, CALB2, PLAC1, and GNA15 was established as a CRC prognosis marker. Among these four genes, AKR1B10 was further validated as the core gene, and its expression was significantly inhibited after NNK exposure in CRC cells. Results of gene enrichment analysis and western blotting suggested AKR1B10 might reduce the malignant progression of NNK-induced CRC cells by inhibiting the Wnt signaling pathway by promoting E-Cadherin expression and inhibiting the expression of N-Cadherin, ß-Catenin, Vimentin, and Snail. CONCLUSIONS: In conclusion, new four smoking-related genes can be jointly used as prognostic markers for CRC. AKR1B10 served as a tumor suppressor, and can be used as a potential target to inhibit NNK-induced CRC malignant progression by regulating the Wnt signaling pathway. IMPLICATIONS: This study demonstrates that tobacco-derived NNK dependence would promote the malignant progression of colorectal cancer by regulating the expressions of the AKR1B10/Wnt signaling pathway. A novel four-gene signature is established for the prognosis prediction of smoking CRC patients. These findings have important translational implications given the continued use of tobacco and the difficulty in smoking cessation worldwide, which can be applied to alleviate the adverse effects induced by tobacco dependence on colorectal cancer patients.

Genetic mutations in smoking-associated prostate cancer.

OBJECTIVES: Tobacco smoking is known to cause cancers potentially predisposed by genetic risks. We compared the frequency of gene mutations using a next generation sequencing database of smokers and nonsmokers with prostate cancer (PCa) to identify subsets of patients with potential genetic risks. MATERIALS AND METHODS: Data from the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE) registry was analyzed. The GENIE registry contains clinically annotated sequenced tumor samples. We included 1832 men with PCa in our cohort, categorized as smokers and nonsmokers, and compared the frequency of mutations (point mutations, copy number variations, and structural variants) of 47 genes with more than 5% mutation rate between the two categories and correlated with overall survival using logistic regression analysis. RESULTS: Overall, 1007 (55%) patients were nonsmokers, and 825 (45%) were smokers. The mutation frequency was significantly higher in smokers compared to nonsmokers, 47.6% and 41.3%, respectively (p = 0.02). The median tumor mutational burden was also significantly higher in the samples from smokers (3.59 mut/MB) compared to nonsmokers (1.87 mut/MB) (p < 0.001). Patients with a smoking history had a significantly higher frequency of PREX2, PTEN, AGO2, KMT2C, and a lower frequency of adenomatous polyposis coli (APC) and KMT2A mutations than compared to nonsmokers. The overall mortality rate (28.5% vs. 22.8%) was significantly higher among smokers (p = 0.006). On a multivariate logistic regression analysis, the presence of metastatic disease at the time of diagnosis (OR: 2.26, 95% CI: 1.78-2.89, p < 0.001), smoking history (OR: 1.32, 95% CI: 1.05-1.65, p = 0.02), and higher frequency of PTEN somatic gene mutation (OR: 1.89, 95% CI: 1.46-2.45, p < 0.001) were independent predictors of increased overall mortality among patients with PCa. Patients with PTEN mutation had poorer overall survival compared to men without PTEN mutations: 96.00 (95% CI: 65.36-113.98) and 120.00 (95% CI: 115.05-160.00) months, respectively (p < 0.001) irrespective of smoking history although the G129R PTEN mutation was characteristically detected in smokers. CONCLUSIONS: PCa patients with a tobacco smoking history demonstrated a significantly higher frequency of somatic genetic mutations. Whereas mutations of PREX2, KMT2C, AGO2, and PTEN genes were higher in smokers, the APC and KMT2A mutations were higher in nonsmokers. The PTEN somatic gene mutation was associated with increased overall mortality among patients with PCa irrespective of smoking history. We found that G129R PTEN mutation known to reduce the PTEN phosphatase activity and K267Rfs*9 a frameshift deletion mutation in the C2 domain of PTEN associated with membrane binding exclusively detected in smokers and nonsmokers, respectively. These findings may be used to further our understanding of PCa associated with smoking.

A polygenic approach to the association between smoking and schizophrenia.

Smoking prevalence in schizophrenia is considerably larger than in general population, playing an important role in early mortality. We compared the polygenic contribution to smoking in schizophrenic patients and controls to assess if genetic factors may explain the different prevalence. Polygenic risk scores (PRSs) for smoking initiation and four genetically correlated traits were calculated in 1108 schizophrenic patients (64.4% smokers) and 1584 controls (31.1% smokers). PRSs for smoking initiation, educational attainment, body mass index and age at first birth were associated with smoking in patients and controls, explaining a similar percentage of variance in both groups. Attention-deficit hyperactivity disorder (ADHD) PRS was associated with smoking only in schizophrenia. This association remained significant after adjustment by psychiatric cross-disorder PRS. A PRS combining all the traits was more explanative than smoking initiation PRS alone, indicating that genetic susceptibility to the other traits plays an additional role in smoking behaviour. Smoking initiation PRS was also associated with schizophrenia in the whole sample, but the significance was lost after adjustment for smoking status. This same pattern was observed in the analysis of specific SNPs at the CHRNA5-CHRNA3-CHRNB4 cluster associated with both traits. Overall, the results indicate that the same genetic factors are involved in smoking susceptibility in schizophrenia and in general population and are compatible with smoking acting, directly or indirectly, as a risk factor for schizophrenia that contributes to the high prevalence of smoking in these patients. The contrasting results for ADHD PRS may be related to higher ADHD symptomatology in schizophrenic patients.

Smoking induces sex-specific changes in the small airway proteome.

INTRODUCTION: Cigarette smoke triggers many cellular and signaling responses in the lung and the resulting inflammation plays a central role in smoke-related lung diseases, such as COPD. We explored the effects of smoking on the small airway proteome in samples obtained by collection of exhaled particles with the aim to identify specific proteins dysregulated by smoking. METHODS: Exhaled particles were obtained from 38 current smokers, 47 former smokers and 22 healthy controls with the PExA method. 120 ng of sample was collected from individual subjects and analyzed with the SOMAscan proteomics platform. General linear model-based statistics were performed. RESULTS: Two hundred and three proteins were detected in at least half of 107 total samples. Active smoking exerted a significant impact on the protein composition of respiratory tract lining fluid (RTLF), with 81 proteins altered in current smokers compared to never smokers (p < 0.05, q < 0.124). Among the proteins most clearly discriminating between current and never smokers were sRAGE, FSTL3, SPOCK2 and protein S, all of them being less abundant in current smokers. Analysis stratified for sex unveiled sex differences with more pronounced proteomic alterations due to active smoking in females than males. Proteins whose abundance was altered by active smoking in women were to a larger extent related to the complement system. The small airway protein profile of former smokers appeared to be more similar to that observed in never smokers. CONCLUSIONS: The study shows that smoking has a strong impact on protein expression in the small airways, and that smoking affects men and women differently, suggesting PExA sampling combined with high sensitivity protein analysis offers a promising platform for early detection of COPD and identification of novel COPD drug targets.

Deep molecular phenotypes link complex disorders and physiological insult to CpG methylation.

Epigenetic regulation of cellular function provides a mechanism for rapid organismal adaptation to changes in health, lifestyle and environment. Associations of cytosine-guanine di-nucleotide (CpG) methylation with clinical endpoints that overlap with metabolic phenotypes suggest a regulatory role for these CpG sites in the body's response to disease or environmental stress. We previously identified 20 CpG sites in an epigenome-wide association study (EWAS) with metabolomics that were also associated in recent EWASs with diabetes-, obesity-, and smoking-related endpoints. To elucidate the molecular pathways that connect these potentially regulatory CpG sites to the associated disease or lifestyle factors, we conducted a multi-omics association study including 2474 mass-spectrometry-based metabolites in plasma, urine and saliva, 225 NMR-based lipid and metabolite measures in blood, 1124 blood-circulating proteins using aptamer technology, 113 plasma protein N-glycans and 60 IgG-glyans, using 359 samples from the multi-ethnic Qatar Metabolomics Study on Diabetes (QMDiab). We report 138 multi-omics associations at these CpG sites, including diabetes biomarkers at the diabetes-associated TXNIP locus, and smoking-specific metabolites and proteins at multiple smoking-associated loci, including AHRR. Mendelian randomization suggests a causal effect of metabolite levels on methylation of obesity-associated CpG sites, i.e. of glycerophospholipid PC(O-36: 5), glycine and a very low-density lipoprotein (VLDL-A) on the methylation of the obesity-associated CpG loci DHCR24, MYO5C and CPT1A, respectively. Taken together, our study suggests that multi-omics-associated CpG methylation can provide functional read-outs for the underlying regulatory response mechanisms to disease or environmental insults.

The effect of SLC6A3 variable number of tandem repeats and methylation levels on individual susceptibility to start tobacco smoking and on the ability of smokers to quit smoking.

OBJECTIVE: Nicotine acts through the dopamine pathway in the brain affecting reward processing through cigarette consumption. Thus, both genetic and epigenetic factors related to dopamine metabolism may influence individual's smoking behavior. MATERIALS AND METHODS: We studied variations of two variable numbers of tandem repeats (VNTRs), 40 and 30 bp in length, in SLC6A3 gene together with six DNA methylation sites located in a first intron of the gene in relation to several smoking-related phenotypes in a study population consisting of 1230 Whites of Russian origin. RESULTS: Both the 5R allele of 30 bp VNTR and the 9R allele of 40 bp VNTR in SLC6A3 were associated with a reduced risk to tobacco smoking [odds ratio (OR) 0.53, 95% confidence interval (CI) 0.37-0.75; OR 0.62, 95% CI 0.43-0.88]. Although the carriers of 9R allele also had high Fagerström test for nicotine dependence scores (OR 1.65, 95% CI 1.04-2.60), they were still more likely to succeed in smoking cessation (OR 0.59, 95% CI 0.40-0.88). Also, current smokers had more than 2.5-fold likelihood to have increased SLC6A3 methylation levels than former smokers (OR 2.72, 95% CI 1.63-4.53). CONCLUSION: The SLC6A3 5R of 30 bp and 9R of 40 bp VNTR variants may lead to a reduced risk to start smoking through decreased dopamine availability, and can also affect the success in subsequent smoking cessation attempts. Moreover, the elevated mean methylation values in the first intron of SLC6A3 may be related to nicotine dependence via a more active dopamine transporter.

Is Brain-Derived Neurotrophic Factor Associated With Smoking Initiation? Replication Using a Large Finnish Population Sample.

INTRODUCTION: Brain-derived neurotrophic factor (BDNF) is a growth factor in the central nervous system. There is evidence for the involvement of BDNF in addictions and mental disorders. We aimed to replicate the earlier reported association of a functional genetic variant of BDNF with smoking initiation (SI) using a large population-based sample and to test whether the association is independent of depression. METHODS: Our sample was drawn from the Finnish population-based FINRISK surveys conducted in 1992, 1997, 2002, and 2007. We had nonmissing data on the genotype BDNF  Val66Met (G/A) variant (rs6265) and self-reported never (n = 10 619) versus ever (n = 16 028) smoking among 26 647 adults aged 25-74 years. The association between BDNF  Val66Met and SI was modeled using logistic regression adjusted for age and sex, and in secondary analyses also for depression. Depression was defined as self-reported depression diagnosed or treated by physician during the past year. RESULTS: The sex- and age-adjusted analysis confirmed that the major (Val) allele increased the risk of being a lifetime ever smoker (per allele odds ratio [OR] = 1.07; 95% CI = 1.01 to 1.12; p = .01). When depression, which itself was significantly associated with SI (OR = 1.58; 95% CI = 1.37 to 1.82; p < .001), was added to the model, the association of the gene with SI remained significant (per allele OR = 1.06; 95% CI = 1.01 to 1.12; p = .01). Exclusion of depressed individuals did not change the results (OR = 1.06; 95% CI = 1.01 to 1.12; p = .02). CONCLUSIONS: In a Finnish population sample, we replicated the earlier reported association of BDNF Val66Met with SI. Our data further suggest that this association is independent of depression. IMPLICATIONS: Earlier finding about the association between the BDNF gene and smoking initiation is replicated and shown to be independent of depression within Finnish adult population.

2018 Langley Award for Basic Research on Nicotine and Tobacco: Bringing Precision Medicine to Smoking Cessation.

INTRODUCTION: Large segments of the world population use combustible cigarettes, and our society pays a high price for smoking, through increased healthcare expenditures, morbidity and mortality. The development of combustible cigarette smoking requires the initiation of smoking and a subsequent chain of behavioral transitions from experimental use, to established regular use, to the conversion to addiction. Each transition is influenced by both environmental and genetic factors, and our increasing knowledge about genetic contributions to smoking behaviors opens new potential interventions. METHODS: This review describes the journey from genetic discovery to the potential implementation of genetic knowledge for the treatment of tobacco use disorder. RESULTS AND CONCLUSIONS: The field of genetics applied to smoking behaviors has rapidly progressed with the identification of highly validated genetic variants that are associated with different smoking behaviors. The large scale implementation of this genetic knowledge to accelerate smoking cessation represents an important clinical challenge in precision medicine.

Smoking-Related DNA Methylation is Differentially Associated with Cadmium Concentration in Blood.

Tobacco smoking, a risk factor for several human diseases, can lead to alterations in DNA methylation. Smoking is a key source of cadmium exposure; however, there are limited studies examining DNA methylation alterations following smoking-related cadmium exposure. To identify such cadmium exposure-related DNA methylation, we performed genome-wide DNA methylation profiling using DNA samples from 50 smokers and 50 non-smokers. We found that a total of 136 CpG sites (including 70 unique genes) were significantly differentially methylated in smokers as compared to that in non-smokers. The CpG site cg05575921 in the AHRR gene was hypomethylated (Δ ß > - 0.2) in smokers, which was in accordance with previous studies. The rs951295 (within RNA gene LOC105370802) and cg00587941 sites were under-methylated by > 15% in smokers, whereas cg11314779 (within CELF6) and cg02126896 were over-methylated by ≥ 15%. We analyzed the association between blood cadmium concentration and DNA methylation level for 50 smokers and 50 non-smokers. DNA methylation rates of 307 CpG sites (including 207 unique genes) were significantly correlated to blood cadmium concentration (linear regression P value < 0.001). The four significant loci (cg05575921 and cg23576855 in AHRR, cg03636183 in F2RL3, and cg21566642) were under-methylated by > 10% in smokers compared to that in non-smokers. In conclusion, our study demonstrated that DNA methylation levels of rs951295, cg00587941, cg11314779, and cg02126896 sites may be new putative indicators of smoking status. Furthermore, we showed that these four loci may be differentially methylated by cadmium exposure due to smoking.

Association of Non-Hodgkin lymphoma risk with CYP1A1, GSTM1 and GSTT1 gene variants, in tobacco addicted patients of Pashtun ethnicity of Khyber Pakhtunkhwa, Pakistan.

The current study determines the possible antitumor and immunomodulatory effects of thymosin against the in vivo and in vitro growth of tumor-derived cell line in mice. Peritoneal phagocytes count, Ehrlich ascites tumor (EAT) cells, T- lymphocytes, and B- lymphocytes activities were determined. In addition, serum level of interleukin 2 (IL-2) and liver functions were measured. In animal testing, thymosin at doses of 0.50 and 1mg activated the phagocytic function of macrophages, as well as T- and B- cell function. Thymosin caused a marked shortage in the proliferation of EAT cells in the peritoneal fluid with dose 0.50g as compared with that of the corresponding control group. Furthermore, treatment with thymosin caused effectively elevate in serum level of IL-2, on the contrary reduce in serum levels of ALT, AST and total proteins. The size of solid Ehrlich tumor was significantly decreased, as measured morphologically with the doses 0.50 and 1 mg (P<0.01). These results confirmed that many biological activities attributed to thymosin and is as an adjuvant for immune enhancement.

Interactions between monoamine oxidase A rs1137070 and smoking on brain structure and function in male smokers.

The monoamine oxidase A (MAOA) enzyme metabolizes monoamine neurotransmitters such as dopamine, serotonin and norepinephrine, and its genetic polymorphism (rs1137070) influences its activity level and is associated with smoking behaviors. However, the underlying neural mechanisms of the gene × environment interactions remain largely unknown. In this study, we aimed to explore the interactive effects of the rs1137070 and cigarette smoking on gray matter volume (GMV) and functional connectivity strength (FCS). A total of 81 smokers and 42 nonsmokers were enrolled in the present study. Voxel-based morphometry analysis showed a significant rs1137070 genotype × smoking effect on the GMV of the left orbitofrontal cortex (OFC), such that individuals with risk allele had greater GMV among nonsmokers but not smokers. Meanwhile, rs1137070 variant and nicotine dependence interactively altered the FCS of the right hippocampus, the left inferior parietal lobule (IPL), the left dorsolateral prefrontal cortex and bilateral OFC. In addition, the FCS in the left IPL was correlated with smoking initiation and smoking years in smokers with the risk allele. These findings suggest that MAOA rs1137070 contributes to the susceptibility to nicotine dependence through its influence on brain circuits involved in reward and attention, and interacts with smoking in the progression.

Neurogenetic determinants and mechanisms of addiction to nicotine and smoked tobacco.

The single most preventable cause of disease, disability, and death in the United States is tobacco use. Decades of study show that the risk of becoming addicted to smoked cigarettes varies greatly amongst individuals and is heritable, yet environmental factors are also important contributors. In this review, we consider a wide range of methodologies and key published reports that have defined the inheritance of different stages of nicotine-dependent smoking behavior, including preference, initiation, regular use, withdrawal and dependence as well as cessation and relapse. Major findings from both animal and human studies are discussed. Current findings converge primarily on the role of nicotinic cholinergic receptor subunits, although other neurotransmitter systems as well as nicotine metabolism enzymes are implicated. Various stages of nicotine addiction may share common genetic mechanisms, yet several lines of evidence indicate that each stage also has its own unique genetic determinants. Studies on the heritability of smoking initiation demonstrate substantial evidence for gene-environment interaction, although the precise molecular genetic mechanism(s) remains unknown. Considering the relatively few genes identified so far and the small to modest fraction of the variance in risk for a particular smoking phenotype (e.g., smoking initiation in late adolescence) attributable to these genes, a large gap remains to be filled in order to account for the heritability of key phenotypes involved in each stage of addiction to smoked tobacco. Looking forward, new research strategies involving both human and animal studies will produce the fundamental genetic insights that are the foundation for the precision medical treatment of individuals addicted to smoked tobacco.

Specific induction of the unique GPR15 expression in heterogeneous blood lymphocytes by tobacco smoking.

Purpose: In the peripheral blood, it has been shown that smoking is, to date, the only specific condition leading to an increase in GPR15+ T cells. We, therefore, aimed to characterize GPR15-expressing blood T cells in more detail. Materials and Methods: The whole transcriptome by RNAseq as a proxy for protein expression was analyzed in GPR15+ and GPR15- T cells. A deep immuno-phenotyping was conducted for the identification of T cell subtypes. Results: The expression of GPR15 seemed to be unique, not concomitantly accompanied with the expression of another protein. According to different T cell subtypes, there is no single cell type prominently represented in GPR15+ T cells. The individually different proportions of GPR15+ cells among each GPR15-expressing T cell subtypes in blood were strongly associated with chronic smoking. Indeed, the frequency of GPR15+ T cell subtypes can be effectively used as a highly convincing biomarker for tobacco smoking. Conclusions: While the chronic smoking-induced enrichment of GPR15+ T cells in blood might indicate a systemic inflammation, by the widespread presence in different T cell subtypes, GPR15 could feature a general impact on maintaining the systemic homeostasis to putatively prevent harm from smoking.

Relationships of Long-Term Smoking and Moist Snuff Consumption With a DNA Methylation Age Relevant Smoking Index: An Analysis in Buccal Cells.

INTRODUCTION: Currently, there is no widely accepted, non-self-report measure that simultaneously reflects smoking behaviors and is molecularly informative of general disease processes. Recently, researchers developed a smoking index (SI) using nucleated blood cells and a multi-tissue DNA methylation-based predictor of chronological age and disease (DNA methylation age [DNAm-age]). To better understand the utility of this novel SI in readily accessible cell types, we used buccal cell DNA methylation to examine SI relationships with long-term tobacco smoking and moist snuff consumption. METHODS: We used a publicly available dataset composed of buccal cell DNA methylation values from 120 middle-aged men (40 long-term smokers, 40 moist snuff consumers, and 40 nonsmokers). DNAm-age (353-CpGs) and SI (66-CpGs) were calculated using CpG sites measured using the Illumina HumanMethylation450 BeadChip. We estimated associations of tobacco consumption habits with both SI and DNAm-age using linear regression models adjusted for chronological age, race, and methylation technical covariates. RESULTS: In fully adjusted models with nonsmokers as the reference, smoking (ß = 1.08, 95% CI = 0.82 to 1.33, p < .0001) but not snuff consumption (ß = .06, 95% CI = -0.19 to 0.32, p = .63) was significantly associated with SI. SI was an excellent predictor of smoking versus nonsmoking (area under the curve = 0.92, 95% CI = 0.85 to 0.98). Four DNAm-age CpGs were differentially methylated between smokers and nonsmokers including cg14992253 [EIF3I], which has been previously shown to be differentially methylated with exposure to long-term fine-particle air pollution (PM2.5). CONCLUSIONS: The 66-CpG SI appears to be a useful tool for measuring smoking-specific behaviors in buccal cells. Still, further research is needed to broadly confirm our findings and SI relationships with DNAm-age. IMPLICATIONS: Our findings demonstrate that this 66-CpG blood-derived SI can reflect long-term tobacco smoking, but not long-term snuff consumption, in buccal cells. This evidence will be useful as the field works to identify an accurate non-self-report smoking biomarker that can be measured in an easily accessible tissue. Future research efforts should focus on (1) optimizing the relationship of the SI with DNAm-age so that the metric can maximize its utility as a tool for understanding general disease processes, and (2) determining normal values for the SI CpGs so that the measure is not as study sample specific.

Lack of Association between Opioid-Receptor Genotypes and Smoking Cessation Outcomes in a Randomized, Controlled Naltrexone Trial.

AIMS: The present study examined how variation in mu- (OPRM1), kappa- (OPRK), and delta- (OPRD) opioid receptor genes may influence the efficacy of naltrexone in the context of a smoking cessation trial. METHODS: The study's primary objective was to examine the association of the Asn40Asp OPRM1 single nucleotide polymorphism (SNP) with naltrexone's effects on smoking quit rate, weight gain, and heavy drinking behavior during a double-blind, randomized clinical trial in 280 adult DSM-IV nicotine-dependent participants. The secondary goal of the study was to examine the relationship of 20 additional SNPs of OPRM1, OPRK, and OPRD with the aforementioned outcomes. RESULTS: Results indicated a null association between any opioid-receptor gene SNP and naltrexone's effects on smoking quit rate, weight gain, and heavy drinking behavior in this sample of nicotine dependent participants. CONCLUSIONS: In sum, these results do not suggest that genetic variation in opioid-receptors is related to treatment responses to naltrexone in a smoking cessation trial.

Genome-wide association study in Finnish twins highlights the connection between nicotine addiction and neurotrophin signaling pathway.

The heritability of nicotine dependence based on family studies is substantial. Nevertheless, knowledge of the underlying genetic architecture remains meager. Our aim was to identify novel genetic variants responsible for interindividual differences in smoking behavior. We performed a genome-wide association study on 1715 ever smokers ascertained from the population-based Finnish Twin Cohort enriched for heavy smoking. Data imputation used the 1000 Genomes Phase I reference panel together with a whole genome sequence-based Finnish reference panel. We analyzed three measures of nicotine addiction-smoking quantity, nicotine dependence and nicotine withdrawal. We annotated all genome-wide significant SNPs for their functional potential. First, we detected genome-wide significant association on 16p12 with smoking quantity (P = 8.5 × 10-9 ), near CLEC19A. The lead-SNP stands 22 kb from a binding site for NF-κB transcription factors, which play a role in the neurotrophin signaling pathway. However, the signal was not replicated in an independent Finnish population-based sample, FINRISK (n = 6763). Second, nicotine withdrawal showed association on 2q21 in an intron of TMEM163 (P = 2.1 × 10-9 ), and on 11p15 (P = 6.6 × 10-8 ) in an intron of AP2A2, and P = 4.2 × 10-7 for a missense variant in MUC6, both involved in the neurotrophin signaling pathway). Third, association was detected on 3p22.3 for maximum number of cigarettes smoked per day (P = 3.1 × 10-8 ) near STAC. Associating CLEC19A and TMEM163 SNPs were annotated to influence gene expression or methylation. The neurotrophin signaling pathway has previously been associated with smoking behavior. Our findings further support the role in nicotine addiction.

Not all smokers appear to seek nicotine for the same reasons: implications for preclinical research in nicotine dependence.

Tobacco use leads to 6 million deaths every year due to severe long-lasting diseases. The main component of tobacco, nicotine, is recognized as one of the most addictive drugs, making smoking cessation difficult, even when 70 percent of smokers wish to do so. Clinical and preclinical studies have demonstrated consistently that nicotine seeking is a complex behavior involving various psychopharmacological mechanisms. Evidence supports that the population of smokers is heterogeneous, particularly as regards the breadth of motives that determine the urge to smoke. Here, we review converging psychological, genetic and neurobiological data from clinical and preclinical studies supporting that the mechanisms controlling nicotine seeking may vary from individual to individual. It appears timely that basic neuroscience integrates this heterogeneity to refine our understanding of the neurobiology of nicotine seeking, as tremendous progress has been made in modeling the various psychopharmacological mechanisms driving nicotine seeking in rodents. For a better understanding of the mechanisms that drive nicotine seeking, we emphasize the need for individual-based research strategies in which nicotine seeking, and eventually treatment efficacy, are determined while taking into account individual variations in the mechanisms of nicotine seeking.