Disease associated with cigarette smoking remains the largest cause of preventable death. A large margin for improvement in smoking cessation treatment exists, with great potential to benefit public health: more than half of American smokers attempt to quit every year, but only ~6% succeed annually. Differences in smoking phenotypes, including cessation, have significant genetic components, but the large majority of this influence is unexplained. Genetic studies of smoking behavior that reveal the mechanisms underlying variation in these traits will identify further targets for pharmacotherapy and aid in improving personalized cessation treatment. Utilizing measurements from an in vivo nicotine metabolism experiment, I developed a predictive genetic model that explains >70% of the variation in oral nicotine C-oxidation (the primary nicotine metabolism pathway), based on CYP2A6 genotype. Model predictions were significantly associated with different measures of cigarette consumption and smoking cessation success in further subjects. The model also allowed me to demonstrate two key novel findings: the independent influences upon smoking behaviors of polymorphisms in 1) EGLN2, a.k.a. Hypoxia Inducible Factor Prolyl Hydroxylase, which initiates a transcriptional cascade in response to cellular hypoxia, and 2) in FMO3 and CYP2B6, further nicotine metabolism genes that may have important extra-hepatic activity. The specific aims of this grant are: 1) Develop a comprehensive predictive genetic model of nicotine metabolism incorporating all three nicotine metabolism pathways and their associated genes, focusing on CYP2A6, the UGTs and FMOs. The improved model will then be applied in further samples to determine the influence of heritable differences in nicotine metabolism upon smoking phenotypes; 2) Identify variants that alter nicotine metabolism gene function and demonstrate the mechanisms of their effects. I will focus especially on protein and mRNA expression, and splicing, in human brain and liver samples; 3) Identify variants in EGLN2 and other hypoxia-response candidate genes, and determine the mechanisms of their effects on smoking phenotypes. My preliminary data indicate an EGLN2 variant associated with nicotine dependence and cigarette consumption alters the relative expression of different EGLN2 5'UTR mRNA splice-variants. I will identify differences in gene and protein expression influenced by EGLN2 genotype in cells cultured under normal and hypoxic conditions. The goal of this K01 application is to obtain further training in statistical human genetics and cell culture under special conditions and to apply this expertise to problems of substance abuse.