DNA was extracted from saliva or blood samples using Oragene kits (DNA Genotek Inc., Kanata, ON, Canada) and QIAamp blood kits (Qiagen Inc., Valencia, CA, USA), respectively. Genotyping for CYP2A6 variant alleles was performed using a previously validated two-step gene and allele specific polymerase chain reaction [7], or an allele-specific TaqMan single nucleotide polymorphism genotyping assay (Applied Biosystems) and real-time polymerase chain reaction. Individuals were genotyped for CYP2A6*2, CYP2A6*4, CYP2A6*9, and CYP2A6*12 alleles, which were selected based on their known role in decreasing nicotine metabolism [3]. Subjects were categorized as normal, intermediate, or slow CYP2A6 genotype metabolizers based on the metabolic impact of CYP2A6 genotype [3]. Normal metabolizers (NM) had no variant alleles (100% CYP2A6 activity), while intermediate metabolizers (IM) had 1 copy of a decreased-function variant allele (CYP2A6*9 or CYP2A6*12; ~80% CYP2A6 activity), and slow metabolizers (SM) had 2 copies of a decreased-function allele or 1 or 2 copies of a loss-of-function variant allele (CYP2A6*2 or CYP2A6*4; ≤50% CYP2A6 enzymatic activity).
