1. A Comparison of the CHRNA2 and CHRNA4 Subunits In Smokers and Non-Smokers By Max Ciaglo and Jordan Smith

2. Do the alpha 2 and alpha 4 nicotine acetylcholine receptor subunits contribute to an individual’s susceptibility to nicotine addiction? The Question

3. If there are rare variations which can be uncovered by deep re-sequencing nicotine acetylcholine receptor genes in human DNA that contribute to nicotine addiction and if we re-sequence the alpha two and alpha four gene segments, using PCR, of smokers and non- smokers, and compare these specific regions of DNA then we will find that these regions contribute to the susceptibility of nicotine addiction. Hypothesis

4.  There are seventeen nicotine acetylcholine receptors that we know of in the human body.  These nicotinic receptors are made up of five subunits that surround a central ion channel.  This central ion channel is the main target and receptor of the nicotine compound. Background

6.  Nicotinic receptors are mainly located on presynaptic nerve terminals where they facilitate the release of dopamine and gamma- aminobutyric acid (GBA).  The release of dopamine and GBA can be activated when nicotine comes in contact with the central channel.

7. Background  GBA and dopamine are both neurotransmitters in the human body. Nicotine dependence starts when the body begins to crave and depend on these neurotransmitters and stops creating the correct amounts of them without nicotine.

8. Background  The genes for these nicotinic receptors are comprised of alpha and beta subunits.  Research shows that there may be a link between mutations in the alpha and beta subunits and likelihood for individuals to depend upon nicotine.

9. Background  These mutations are called SNPs (Single Nucleotide Polymorphisms)  A SNP is where one nucleotide in a DNA sequence differs from another of the same species.  This can be in the form of a  deletion where the base pair is removed  substitution where the base pair is changed  addition where one base pair is added to the sequence.

11.  We plan to examine the alpha 2 and alpha 4 nicotine acetylcholine receptor subunits (CHRNA2 and CHRNA4). We will prepare and optimize primers for PCR to amplify DNA for CHRNA2 and CHRNA4. Methods/ Procedures

12.  We will design primers using Primer 3 (an online program used to design primers) and we will then order these primers from Invitrogen. Isolated DNA from a previous non- identified human subject will be used to optimize the PCR products. Methods/ Procedures

13. Primers

14.  The PCR products will be run through Gel Electrophoresis to gauge the effectiveness of our PCR reaction.  The optimized PCR products will then be sent to a company for gene sequencing.  After we receive the sequencing data it will be used to identify and compare SNPs in smokers versus non-smokers.

15. Gel Electrophoresis

16. Methods/ Procedures  We will analyze these data for statistical significance using a t-test, and if consistent differences in SNPs are found between smokers and non-smokers it will suggest that these variations make an individual more or less susceptible to nicotine addiction.

17. Currently  All the PCR reactions have been optimized.  The reactions were carried out with ten pilot samples of DNA from ten random volunteers.  The reactions are being quantitated to be sent off to a company to be sequenced.

18. In the Future  These preliminary results will show whether or not our PCR reactions were effective.  Once we know that the reactions are optimally effective then we will carry out the same reactions with the hundred smoker’s and non smoker’s DNA.

19. References  1. Marissa A. Ehringer et al, Association of the Neuronal Nicotinic Receptor b2 Subunit Gene (CHRNB2) With Subjective Responses to Alcohol and Nicotine, American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:596-604 (2007)  2. Marissa A. Ehringer, Research Plan, (Not published in a journal as of yet)  3. Julia Gorey J.D., OHRP Letter  4. Joseph G. Rosse PH.D, OHRP Prisoner Certification Letter  5. Marissa A Ehringer, Protection of Human Subjects, Involvement of Human Subjects, (Not published in journal as of yet)  6. Hutchison KE, Allen D, Haughey H, Jepson C, Lerman C, Benowitz N, Stitzel J, Bryan A, Filbey F. in preparation. CHRNA4 and Tobacco Dependence: From gene regulation to treatment outcome.  7. Lueders KK, Hu S, McHugh L, Myakishev MV, Sirota LA, Hamer DH. 2002.Genetic and functional analysis of single nucleotide polymorphisms in the beta2-neuronal nicotinic acetylcholine receptor gene (CHRNB2). Nicotine Tob Res 4(1):115–125.

20. We would like to thank the MAE Lab at the Institute for Behavioral Genetics at Colorado University, especially Marissa Ehringer, Nicole Hoft, and Jill Miyamoto-Ditmon. Thanks!