Thomas J. Payne

Multiple Independent Loci at Chromosome 15q25.1 Affect Smoking Quantity: a Meta-Analysis and Comparison with Lung Cancer and COPD

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10−35 and <10−8 respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10−6). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10−20) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Assessing nicotine dependence: a comparison of the Fagerström Tolerance Questionnaire (FTQ) with the Fagerström Test for Nicotine Dependence (FTND) in a clinical sample.

A recent effort to improve the psychometric properties of the Fagerström Tolerance Questionnaire (FTQ) resulted in the revised Fagerström Test for Nicotine Dependence (FTND). We endeavored to replicate and extend findings suggesting the superiority of the FTND by examining the psychometric properties of both instruments, as well as their relationship to self-report and biochemical variables associated with nicotine dependence in 110 smoking clinic participants. Results verified that the FTND represents a modest improvement over the FTQ, however, the need for continued development of self-report measures of nicotine dependence is noted.

Exposure to smoking-relevant cues: effects on desire to smoke and topographical components of smoking behavior.

The effects of exposure to two classes of smoking-relevant cues (environmental; negative affect) on desire to smoke and smoking topography were evaluated. Sixty chronic smokers were randomly assigned to one of six groups in a two-way ANOVA design in which the salience of environmental cues and presence of negative affective cues were manipulated. This was followed by a 20-minute interval during which ad libitum smoking was videotaped in an unobtrusive manner. Results indicated that the experimental manipulations differentially influenced ratings of desire and topographical components of smoking behavior. Implications are raised regarding the role of cue reactivity in the maintenance of smoking behavior.

Significant association of catechol-O-methyltransferase (COMT) haplotypes with nicotine dependence in male and female smokers of two ethnic populations.

The catechol-O-methyltransferase (COMT) gene plays a prominent role in dopaminergic circuits central to drug reward. Allelic variants within the COMT gene are therefore potential candidates for examining interindividual differences in vulnerability to nicotine dependence (ND). We analyzed five single nucleotide polymorphisms (SNPs), including the Val/Met variant (rs4680), which results in a three- to four-fold difference in enzyme activity within COMT, for association with the three ND measures, SQ, HSI, and FTND, in 602 nuclear families of African-American (AA) or European-American (EA) origin. The Val/Met variant showed a significant association with the three ND measures in the pooled and EA samples and with FTND in the AA sample. Haplotype analysis revealed a major protective A-G-T haplotype (frequency 23.6%) for rs740603-rs4680-rs174699 in the AA sample (minimum Z=−3.35; P=0.0005 for FTND), a major protective T-G-T haplotype (frequency 15.2%; minimum Z=−2.92; P=0.003 for FTND) in the EA sample, and a high-risk C-A-T haplotype (frequency 16.9%; minimum Z=3.16; P=0.002 for SQ) in the AA sample for rs933271-rs4680-rs174699. Furthermore, we found that the significant haplotypes within COMT were gender-specific and the significantly associated A-G-T is protective in AA females only, whereas T-G-T is protective in EA males only. Moreover, we found a major high-risk T-A-T haplotype (frequency 56.7%) that showed significant association with the three ND measures in EA males. Further examination of two protective haplotypes, A-G-T in AAs and T-G-T in EAs, indicated that the low COMT enzyme activity Met allele is protective to become nicotine dependent. In summary, our results provide evidence for a role of COMT in the susceptibility to ND and further confirm that its effect is ethnic and gender specific.

Significant association of ANKK1 and detection of a functional polymorphism with nicotine dependence in an African-American sample.

The dopaminergic system in the brain plays a critical role in nicotine addiction. Genetic variants in the dopaminergic system, including those in dopamine receptor genes, represent plausible candidates for the genetic study of nicotine dependence (ND). We investigated various polymorphisms in the dopamine D2 receptor gene (DRD2) and its neighboring ankyrin repeats and kinase domain containing 1 gene (ANKK1) to determine whether they were associated with ND. We examined 16 single nucleotide polymorphisms (SNPs) at DRD2 and 7 SNPs at ANKK1 in our Mid-South Tobacco Family cohort, which consisted of 2037 participants representing two distinct American populations. Several SNPs (rs7131056, rs4274224, rs4648318, and rs6278) in DRD2, along with the Taq IA polymorphism (rs1800497) in ANKK1, revealed initial significant associations with ND in European Americans, but not after correction for multiple testing, indicating a weak association of DRD2 with ND. In contrast, associations for ANKK1 with ND in the African-American and pooled samples, specifically for SNP rs2734849, remained significant after correction. With a non-synonymous G to A transition, rs2734849 produces an amino-acid change (arginine to histidine) in C-terminal ankyrin repeat domain of ANKK1. Using the luciferase reporter assay, we further demonstrated that the variant alters expression level of NF-κB-regulated genes. Since DRD2 expression is regulated by transcription factor NF-κB, we suspect that rs2734849 may indirectly affect dopamine D2 receptor density. We conclude that ANKK1 is associated with ND and polymorphism rs2734849 in ANKK1 represents a functional causative variant for ND in African-American smokers.

A genome-wide scan to identify loci for smoking rate in the Framingham Heart Study population

BackgroundAlthough many years of genetic epidemiological studies have demonstrated that genetics plays a significant role in determining smoking behavior, little information is available on genomic loci or genes affecting nicotine dependence. Several susceptibility chromosomal regions for nicotine dependence have been reported, but few have received independent confirmation. To identify susceptibility loci for nicotine dependence, 313 extended pedigrees selected from the Framingham Heart Study population were analyzed by both the GENEHUNTER and S.A.G.E. programs.ResultsAfter performing linkage analyses on the 313 extended Framingham Heart Study families, the EM Haseman-Elston method implemented in GENEHUNTER provided evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 9, 14, and 17. Multipoint sib-pair regression analysis using the SIBPAL program of S.A.G.E. on 1389 sib pairs that were split from the 313 extended families identified suggestive linkage of smoking rate to chromosomes 4, 7, and 17. Of these identified positive regions for nicotine dependence, loci on chromosomes 7, 11, and 17 were identified by both GENEHUNTER and S.A.G.E. programs.ConclusionOur genome-wide scan results on the Framingham Heart Study data provide evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 4, 7, 9, 14, and 17. These findings suggest that some of these regions may harbor susceptibility loci for nicotine dependence, and warrant further investigation in this and other populations.

Significant association of DRD1 with nicotine dependence

Epidemiologic studies have strongly implicated genetics in smoking behavior. Genes in the dopaminergic system, which mediates the reinforcing and dependence-producing properties of nicotine, are plausible candidates for roles in nicotine dependence (ND). In this study, we examined five single-nucleotide polymorphisms (SNPs) within or near the dopamine D1 receptor gene (DRD1) for their association with ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerström Test for ND (FTND). The samples were obtained from 2,037 participants representing 200 European American (EA) and 402 African American (AA) families. Although we found significant associations of SNPs rs265973, rs686, and rs4532 in the AA sample; of rs4532 in the EA sample; and of rs265975, rs686, and rs4532 in the pooled sample with various ND measures, only the association of rs686 in the AA sample and of rs686 and rs4532 in the pooled sample remained significant after correction for multiple testing. Haplotype-based association analysis revealed that haplotype C-T-A, formed by rs265973, rs265975, and rs686, was significantly associated with all three ND measures in both the AA and the pooled sample. Another haplotype, T-A-T, formed by rs265975, rs686, and rs4532, showed a significant association with FTND in the pooled sample. Furthermore, in a luciferase reporter assay, rs686, located in the 3′ untranslated region, caused differential luciferase activities, indicating that rs686 is a functional polymorphism affecting expression of DRD1.

Significant association of BDNF haplotypes in European-American male smokers but not in European-American female or African-American smokers

Brain‐derived neurotrophic factor (BDNF) influences dopamine and serotonin neurotransmission in the brain, both of which are involved in the reward system of addiction. The BDNF gene is located in a genomic region on chromosome 11p where we and others have found ‘significant’ linkage to nicotine dependence (ND). We tested the potential role of variants within BDNF in vulnerability to ND, which was assessed by Smoking Quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerström Test for ND (FTND). Six single nucleotide polymorphisms (SNPs) in BDNF were analyzed in an extensively phenotyped cohort of 602 nuclear families with smokers and non‐smokers of African‐American (AA) or European‐American (EA) ancestry. Individual SNP analysis revealed that two SNPs in the pooled male and three SNPs in the EA male samples were significantly associated with at least one adjusted ND measure. However, none of these associations remained significant after correction for multiple testing. Haplotype analysis of rs6484320‐rs988748‐rs2030324‐rs7934165 revealed that a major T–C–T–G haplotype was significantly associated, even after Bonferroni correction, with the three ND measures in the pooled and EA male samples (maximum Z = 3.00, P = 0.002 and maximum Z = 3.13, P = 0.0009 for SQ, respectively). No significant association of a major haplotype with ND was found in the AA or EA female smokers. The significant association of BDNF variants with ND implies that this gene plays a role in the etiology of ND in EAs and that its involvement is gender specific. BDNF may warrant further investigation in ND.

Significant association of the neurexin-1 gene (NRXN1) with nicotine dependence in European- and African-American smokers

The neurexin-1 gene (NRXN1) has been shown to play a fundamental role in synaptogenesis and synaptic maintenance, as well as Ca(2+) channel and NMDA receptor recruitment. A recent study reported that NRXN1 is associated with nicotine dependence (ND); this, together with the intriguing physiological functions of the gene, motivated us to investigate the involvement of NRXN1 with ND in independent samples. In this study, we analyzed 21 single nucleotide polymorphisms (SNPs) within NRXN1 for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerström test for ND (FTND). Individual SNP and haplotype association tests were carried out in a sample consisting of 2037 individuals from 602 nuclear families of African-American (AA) or European-American (EA) origin. Individual SNP analysis revealed significant associations of rs2193225 with SQ, HSI and FTND (P = 0.00014-0.0010) in the EA sample and with SQ (P = 0.0019) in the pooled sample under the dominant model and rs6721498 with SQ, HSI and FTND in the AA (P = 0.000090-0.0000086) and pooled (P = 0.0010-0.00099) samples under the additive model, following correction for multiple testing. Haplotype analysis revealed six major haplotypes in the AA sample (minimum P-value = 0.000079), one major haplotype in the EA sample (P = 0.0062) and five major haplotypes in the pooled sample (minimum P-value = 0.00083), which showed significant association with all three ND measures; all of these contained one specific allele from one of the two aforementioned SNPs. Based on our findings that NRXN1 has significant association with ND in two independent samples, recent findings that NRXN1 plays an important role in synaptic development, and the previous report of association, we conclude that this gene represents a strong candidate for involvement in the etiology of ND.

Reactivity to smoking cues: Mediating roles of nicotine dependence and duration of deprivation

This study examined the roles of nicotine dependence and duration of smoking deprivation as mediators of smoking cue reactivity in 117 individuals enrolled in treatment. Participants were randomly assigned to 15, 90, or 180 min of deprivation; all were exposed to smoking cues (observing a research assistant smoke a cigarette) while a variety of self-report, behavioral, and psychophysiological measurements were obtained. Results indicated that in general, psychophysiological reactivity to smoking-relevant cues was greater with increasing deprivation; heart rate analysis revealed an interaction between nicotine dependence and duration of deprivation. Smoking desire, negative affect, physical withdrawal symptoms, and other ratings were interrelated during cue exposure, and generally demonstrated predicted relationships with the independent variables. Multiple regression analysis revealed that desire to smoke during cue exposure was predicted by a combination of psychophysiological, self-report, and demographic variables. These findings have implications for methodological considerations and theoretical underpinnings of smoking cue reactivity research.