Clinical trial participant characteristics and saliva and DNA metrics.

BACKGROUND: Clinical trial and epidemiological studies need high quality biospecimens from a representative sample of participants to investigate genetic influences on treatment response and disease. Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of saliva biospecimen samples collected through the mail, trial participant demographic and behavioral characteristics, and their association with saliva and DNA quantity and quality. METHODS: Saliva biospecimens were collected using the Oragene(R) DNA Self-Collection Kits from participants in a National Cancer Institute funded smoking cessation trial. Saliva biospecimens from 565 individuals were visually inspected for clarity prior to and after DNA extraction. DNA samples were then quantified by UV absorbance, PicoGreen(R), and qPCR. Genotyping was performed on 11 SNPs using TaqMan(R) SNP assays and two VNTR assays. Univariate, correlation, and analysis of variance analyses were conducted to observe the relationship between saliva sample and participant characteristics. RESULTS: The biospecimen kit return rate was 58.5% among those invited to participate (n = 967) and 47.1% among all possible COMPASS participants (n = 1202). Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females. Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity. Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010). Genotyping completion rate was not influenced by saliva or DNA clarity. CONCLUSION: Findings from this study show that demographic and behavioral characteristics of smoking cessation trial participants have significant associations with saliva and DNA metrics, but not with the performance of TaqMan(R) SNP or VNTR genotyping assays. TRIAL REGISTRATION: COMPASS; registered as NCT00301145 at clinicaltrials.gov.

Gene-gene interactions between CYP2B6 and CYP2A6 in nicotine metabolism.

OBJECTIVES: CYP2A6 is the major enzyme involved in nicotine metabolism, yet large interindividual variations in the rate of nicotine metabolism exist within groups of individuals having the same CYP2A6 genotype. We investigated the influence of genetic variation in another potential nicotine-metabolizing enzyme, CYP2B6, and its interaction with CYP2A6, on the metabolism of nicotine. METHODS: Two hundred and twelve healthy Caucasian adult twin volunteers underwent an intravenous infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, for characterization of pharmacokinetic and metabolism phenotypes. Five CYP2B6 genetic polymorphisms causing amino acid substitutions (R22C, Q172 H, S259R, K262R, and R487C) were analyzed. RESULTS: We observed that the CYP2B6*6 haplotype (defined as having both Q172 H and K262R variants) was associated with faster nicotine and cotinine clearance, and that such associations were more prominent among individuals having decreased-activity CYP2A6 genotypes. Statistically significant interactions between CYP2B6 and CYP2A6 genotypes were observed (P<0.003 for nicotine clearance and P<0.002 for cotinine clearance). CONCLUSIONS: Our results indicate that CYP2B6 genetic variation is associated with the metabolism of nicotine and cotinine among individuals with decreased CYP2A6 activity. Further investigation of the roles of CYP2B6 and the interaction between CYP2B6 and CYP2A6 genotypes in mediating nicotine dependence and tobacco-related diseases is merited.