# Presentation: 095294

## A Hybird Approach:  Genome Wide Association and Candidate Gene Study for Nicotine Addiction

- Laura Jean Bierut, MD
- and the NICSNP Consortium

## Nicotine Dependence - A many step process

- Initiation

**Notes:**
The process of becoming nicotine dependence is a many step process-
The first step is the initiation of smoking, in other words, who will take a first puff of a cigarette.  Many antismoking campaigns focus on this first step of initiation, encouraging adolescents not to smoke.

## Nicotine Dependence - A many step process

- Initiation

- Smoking

**Notes:**
The process of becoming nicotine dependence is a many step process-
It involves the initiation of smoking, in other words, who will take a first puff of a cigarette.  Many antismoking campaigns focus on this first step of initiation, encouraging adolescents not to smoke.
The next step on the pathway to nicotine dependence is the development of regular smoking.  Regular smoking is defined in many studies as smoking 100 cigarettes or more.  Most individuals who pass this threshold of smoking 100 or more cigarettes go on to smoke daily.
Nicotine dependence represents the step where individuals who are regular smokers develop tolerance and withdrawal.  Tolerance develops so that the individual needs to smoke more in order to get the same effect.   Withdrawal is where characteristics unpleasant symptoms develop when nicotine no longer used.

## Nicotine Dependence - A many step process

- Initiation

- Smoking

- Nicotine Dependence

**Notes:**
The process of becoming nicotine dependence is a many step process-
It involves the initiation of smoking, in other words, who will take a first puff of a cigarette.  Many antismoking campaigns focus on this first step of initiation, encouraging adolescents not to smoke.
The next step on the pathway to nicotine dependence is the development of regular smoking.  Regular smoking is defined in many studies as smoking 100 cigarettes or more.  Most individuals who pass this threshold of smoking 100 or more cigarettes go on to smoke daily.
Nicotine dependence represents the step where individuals who are regular smokers develop tolerance and withdrawal.  Tolerance develops so that the individual needs to smoke more in order to get the same effect.   Withdrawal is where characteristics unpleasant symptoms develop when nicotine no longer used.

## NICSNP Project - A Hybrid Project

- Genome Wide Association Study
- Candidate Gene Study

**Notes:**
The identification of millions of genetic variants and the decreased cost of genotyping has ushered in the option of large scale genetic association studies. 
This approach takes on an agnostic view of the genome and screens hundreds of thousands genetic variants (SNPs) in a case control design.
The candidate gene approach is hypothesis driven, and a prior understanding of biology or previous findings lead to the targeting of a gene or gene family.

## Nicotine Studies

- Collaborative Study of the Genetics of Nicotine Dependence
- 	Principal Investigator:	Laura Jean Bierut  (P01 CA 089392)
- The Genetics of Vulnerability to Nicotine Addiction
- 	Principal Investigator:	Pamela Madden (R01 DA 012854)
- Genes for Smoking in Related and Unrelated Individuals
- 	Principal Investigator:	Ovide Pomerleau (R01 DA 017640)
- Pharmacokinetics of Nicotine in Twins
- 	Principal Investigator:	Gary Swan (R01 DA 011170)
- NIDA Phenotypic Repository:  	John Rice

## Phenotype Definition

- Case:  Nicotine dependent defined by a Fagerström Test for Nicotine Dependence (FTND) _>_ 4
- Control:  Individual who has smoked 100 or more cigarettes and never had any symptoms of nicotine dependence (Lifetime FTND = 0).

## Subjects

- Sample: 1050 cases and 879 controls
- Case FTND:  Mean 6.3 (range 4-10)
- Control FTND:  0

## Subjects

- Cases:  52% women and 48% men
- Controls:  66% women and 34% men
- Age:  Mean 37 years
- All subjects are of European descent

## Overview -Genome Wide Association Study

- Genotyping of 2.4 million SNPs in pooled case (N=500) and control (N=500) samples.
-  An evaluation of differences in allele frequencies between pools.
- Individual genotyping of the selected 40,000 SNPs in the case (N=1,000) and control (N=900) samples to further examine genetic association.

## Overview Candidate Gene Study

- Selection and ranking of candidate genes
- Examination of SNP coverage
- 4,000 SNPs allocated to candidate genes

## Candidate Genes

## Candidate Genes

- Rank A:  These genes will be followed with individual genotyping regardless of the pooled results.
- Nicotinic Receptors
- Rank B: All the rest

## Data Analysis

- Pooled genotyping (N=500 cases and 500 controls) was used to identify SNPs most likely associated with nicotine dependence in the genome wide association study.
- Individual genotyping is then examined in the entire sample (N=1000 cases and 900 controls).

- Skol et al, 2006

**Notes:**
The strategy for analysis.
We used the strategy described by Skol and colleagues.  Approximately half the sample was used to identify SNPs for individual genotyping.  The entire sample will then be used in analysis of results.

## Different Prior Probability

- The Genome Wide Association Study and the Candidate Gene Study have different prior probabilities.
- Correction for multiple testing varies between these two components of the study.

**Notes:**
The Genome Wide Association Study has no prior hypothesis about regions potentially involved in the development of nicotine dependence.  
The Candidate Gene Study has a prior hypothesis and knowledge about genes involved in the development of nicotine dependence.  This prior knowledge allows a different threshold for significance.

## Data Analysis

- There will be many significant differences between cases and controls.
- What findings will be “true” differences between cases and controls?
- More complicated analyses incorporating covariates.

## Analytic Model

- Logistic Regression Model
- Gender + Site + Genotype +Gender*Genotype
- Gender + Site

## Slide 18

## Slide 19

## Evidence of True Findings

- Enrichment of small p-values
- Sign agreement between pooled samples and additional samples (30 out of 35 SNPs) p-value < 10-4
- Convergence of results

## Summary

- Genome Wide Association Study - Nominates novel genes involved in nicotine dependence and a known candidate gene
- Candidate Gene Study - Numerous variants across several genes are associated with nicotine dependence

## Data Sharing

- Share with all in the NIDA Genetics Consortium.
- Website has been developed as a source of information.
- Share with the scientific community after 1 year.

## Future Directions

- Other replication samples will be key, including the study of parent offspring trios.
- Examine findings in different ethnic groups, such as the African American population.
- Move forward with laboratory studies.

## Thanks

- Dennis Ballinger, Andrew Bergen, Naomi Breslau, Gary Chase, Joseph Cubells, Danielle Dick, Dani Fallen, Mark Gold, Dorothy Hatsukami, Anthony Hinrichs, Eric Johnson, Ken Krauter, Mary Jeanne Kreek, Pamela Madden, Sharon Murphy, Cynthia Pomerleau, Ovide Pomerleau, John Rice, Huijun Ring, Nancy Saccone, Scott Saccone, Pak Sham, Gary Swan, Ming Tsuang, Michael Vanyukov, Kirk Wilhelmsen, Lei Yu, Hongyu Zhao and Joni Rutter