FTND, covariates and SNPs in the treatment seeking smoker cohort
The treatment seeking smoker cohort consists of 828 self-identified white treatment seeking smokers, with a mean (SD) baseline FTND score of 5.3 (2.1) (). Participants are about equally divided between college graduates and non-graduates, women and men, and recruitment site. Older age, a higher depression symptom score, lower educational achievement, male sex and two of three sites are significantly associated with higher FTND scores in this cohort (). The multivariate model indicates that these covariates account for 10% of the variance of FTND in the treatment seeking smoker cohort (F827=18.01, P<0.0001, r2=0.0987).
Treatment Seeking Smoker Cohort Covariate Associations with the FTND
73 SNPs at 22 candidate genes were identified in the treatment seeking smoker cohort with a PLRT value < 0.05 (see Table S1 for all SNP GLM results). Five SNPs at three genes (DRD2, SLC6A3 and NR4A2) were identified with a PACT < 0.10 (). These SNPs were evaluated in detail using genomic annotation, literature review and genotyping in a second cohort of multiplex ever smoker pedigrees.
Top Ranked SNPs Associated with the FTND in the Treatment Seeking Smoker Cohort
The top-ranked DRD2 SNP rs10891552 is associated with a ~0.9 unit increase of FTND score and with ~1.3% of FTND variance in an additive or dominant model, as, in this sample, the minor allele homozygote is not observed (). rs10891552 is located in a linkage disequilibrium (LD) block within the large first intervening sequence (IVS1), but exhibits only weak LD (r2 < 0.23) with SNPs in the local LD block in this sample (Figure S1). There are three additional DRD2 SNPs (rs2440390, rs4586205 and rs1076562) with PLRT values <0.05 (Table S1), located an average of 37 kilo base pairs (kbp) 3’ of rs10891552 within IVS1 of DRD2, and within a LD block that spans IVS1 through the coding region of DRD2, but these SNPs have PACT values ~ 0.6 (Table S1), and rs10891552 has an r2 < 0.01 with these SNPs (Figure S1).
Unadjusted FTND Distributions by Genotype in the Treatment Seeking Smoker Cohort
The top-ranked three SLC6A3 SNPs (rs2975226, rs2652510 and rs2652511) identified as associated with FTND () are highly correlated with each other (mean r2 ~ 0.97) in a LD block that extends in this sample from rs12652860, 61 kbp 5’ of SLC6A3 to rs6350 in exon 2 of SLC6A3, and are only modestly correlated (r2~0.2) with SNPs in two 3’ LD blocks extending from IVS1 through IVS7 (). SNP FTND association model characteristics are similar for the three SLC6A3 SNPs, i.e., for each of these SNPs, the presence of the minor allele is associated with a ~0.5 unit increase of FTND score and with ~1% of FTND variance in the additive model (). Inspection of all SLC6A3 SNP FTND association results (Table S1) identifies rs12652860, also in the 5’ linkage block, with a PLRT < 0.01 and a PACT = 0.20, and with similar effects on FTND score and variance (data not shown), while the remaining SLC6A3 SNPs exhibit PLRT values > 0.15 and PACT values > 0.95 (Table S1). Estimation of haplotypes in the 5’ linkage block results in five haplotypes with frequency > 0.01, with the minor allele at each of the three top-ranked SLC6A3 SNPs associated with increased FTND found in three haplotypes with combined frequency 39% ().
SLC6A3 SNPs, LD and haplotypes, treatment seeking smoker cohort.
The top-ranked NR4A2 SNP rs834829 was identified in the initial association analysis as most significantly associated with a dominant model (), and while both additive and dominant models are significantly associated with increased FTND score, inspection of FTND distributions by genotype in post-hoc analysis identifies the heterozgote genotype as significantly associated with increased FTND scores, accounting for a ~0.4 unit increase of mean FTND score and ~0.8% of FTND variance (). The NR4A2 SNP associated with FTND is found ~12 kbp 5’ of the 5’ UTR of the NR4A2 gene, within a LD block that extends from 5’ of the gene, across the entire gene to the 3’ flanking region in this sample (Figure S2). Inspection of all NR4A2 SNP FTND association results (Table S1) identifies rs1150144 and rs1150143, ~2.5 kbp 3’ of the 3’UTR, with PLRT values < 0.05, strong r2 with rs834829 (0.79 and 0.94, respectively) and modest PACT values (0.12 and 0.21, respectively).
In post-hoc power analyses, the sample size of 821 treatment seeking smokers has power of 82% to detect beta coefficients of 78%, 42% and 43% for minor allele frequencies of 4% (DRD2), 29% (NR4A2) and 39% (SLC6A3), respectively, for genetic effects in a dominant model with an r2 of 0.01 and the observed FTND mean and standard deviation () at an alpha value of 0.05 with a two-sided test.
SLC6A3 NR4A2 interaction analysis
We evaluated gene-gene interaction between rs2652511 at SLC6A3 and rs834829 at NR4A2, using a continuous model of FTND score, adjusted for age, depressive symptoms, education, sex and site in a sample of N=794 individuals with complete FTND, covariate and genotype data. We chose these two SNPs to perform the interaction analysis because the gene product of NR4A2, NURR1, is known to be required for SLC6A3 gene transcription (see Discussion), and because these two SNPs are highly ranked in our GLM results (). We evaluated training (prediction) accuracy (TA) of three GMDR analyses to predict the distribution of covariate adjusted normalized FTND score within genotype classes for rs2652511, for rs834829, and for both rs2652511 and rs834829 (Figure S2). While each SNP independently predicted FTND score (for rs2652511, TA=0.56, TA Odds Ratio (OR)=1.71, 95CI 1.37–2.14, P<0.0001, cross validation sign test P=0.02; for rs834829, TA=0.56, TA OR=1.64, 95 CI 1.31–2.04, P<0.0001, cross validation sign test P=0.83), only the analysis with both rs2652511 and rs834829 resulted in a highly significant cross validation sign test, in addition to increased training accuracy and odds ratio (TA=0.59, TA OR=2.15, 95CI 1.72–2.70, P<0.0001, cross validation sign test P=0.001).
Multiplex smoking pedigree cohort analysis
FTND score, age, and the proportion of individuals with 16 or more years of education were significantly lower in the multiplex smoking pedigree cohort than in the treatment seeking smoker cohort (all P < 0.0001). Age, depressive symptoms, education and sex exhibited significant association with FTND in the multiplex smoking pedigree cohort in the same direction as in the treatment seeking smoker cohort (). The multivariate model of covariate association with FTND indicates that these covariates account for 15% of the variance of FTND in the multiplex smoking pedigree cohort (F405=18.21, P<0.0001, r2=0.1537). With one exception (rs834829), each of the five top-ranked SNPs in the treatment seeking smoker cohort () exhibited nominally significant (P < 0.05) association with FTND in the multiplex smoking pedigree cohort in FBAT analyses uncorrected for multiple testing (). For rs10891552 (DRD2), the minor allele (in additive, dominant or genotype models) was associated with increased FTND. For the SLC6A3 SNPs, the minor allele (in the genotype model) was associated with increased FTND. For rs834829, the FBAT with the smallest P value was the heterozygote genotype model (P = 0.0511). We evaluated statistical interaction between rs2652511 at SLC6A3 and rs834829 at NR4A2 in 411 individuals with FTND scores from N=175 pedigrees using PGMDR (Figure S3). The PGMDR analysis with both rs2652511 and rs834829 had the greatest training (prediction) accuracy, but did not exhibit a significant cross validation sign test P value (TA=0.56, TA OR=1.65, 95 CI 1.24, 2.21, P=0.0006, cross validation sign test P=0.82). The increase in TA over the PGMDR analysis of rs2652511 (TA=0.54, TA OR= 1.66, 95 CI 1.15, 2.40, P=0.0063, cross validation sign test P=0.38) was modest. The PGMDR analysis of rs834829 had a non-significant training accuracy estimate and OR (data not shown).
Multiplex Ever Smoker Pedigree Cohort Covariate Associations with the FTND
FBAT of the FTND in the Multiplex Ever Smoker Pedigree Cohort