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. 2007 Jan 1;16(1):24-35.
doi: 10.1093/hmg/ddl441. Epub 2006 Dec 7.

Novel Genes Identified in a High-Density Genome Wide Association Study for Nicotine Dependence

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Novel Genes Identified in a High-Density Genome Wide Association Study for Nicotine Dependence

Laura Jean Bierut et al. Hum Mol Genet. .
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Abstract

Tobacco use is a leading contributor to disability and death worldwide, and genetic factors contribute in part to the development of nicotine dependence. To identify novel genes for which natural variation contributes to the development of nicotine dependence, we performed a comprehensive genome wide association study using nicotine dependent smokers as cases and non-dependent smokers as controls. To allow the efficient, rapid, and cost effective screen of the genome, the study was carried out using a two-stage design. In the first stage, genotyping of over 2.4 million single nucleotide polymorphisms (SNPs) was completed in case and control pools. In the second stage, we selected SNPs for individual genotyping based on the most significant allele frequency differences between cases and controls from the pooled results. Individual genotyping was performed in 1050 cases and 879 controls using 31 960 selected SNPs. The primary analysis, a logistic regression model with covariates of age, gender, genotype and gender by genotype interaction, identified 35 SNPs with P-values less than 10(-4) (minimum P-value 1.53 x 10(-6)). Although none of the individual findings is statistically significant after correcting for multiple tests, additional statistical analyses support the existence of true findings in this group. Our study nominates several novel genes, such as Neurexin 1 (NRXN1), in the development of nicotine dependence while also identifying a known candidate gene, the beta3 nicotinic cholinergic receptor. This work anticipates the future directions of large-scale genome wide association studies with state-of-the-art methodological approaches and sharing of data with the scientific community.

Figures

Figure 1
Figure 1
P values of genome-wide association scan for genes that affect the risk of developing nicotine dependence. -log10 (p) is plotted for each SNP in chromosomal order. The spacing between SNPs on the plot is based on physical map length. The horizontal lines show P values for logistic analysis. The vertical lines show chromosomal boundaries. Black diamonds represent SNPs that result in non-synonymous amino acid changes.
Figure 2
Figure 2
a) Distribution of p-values from the Stage I sample of the 31,960 individually genotyped SNPs that were selected from pooled genotyping stage. The distribution shows that the pooled genotyping produced an enrichment of SNPs with small p-values. A uniform distribution from 0-1 would be expected if there were no correlation between pooled genotyping and individual genotyping. b) Distribution of p-values from the additional samples added in Stage II. The distribution is fairly uniform with only a slight enrichment of small p-values.
Figure 2
Figure 2
a) Distribution of p-values from the Stage I sample of the 31,960 individually genotyped SNPs that were selected from pooled genotyping stage. The distribution shows that the pooled genotyping produced an enrichment of SNPs with small p-values. A uniform distribution from 0-1 would be expected if there were no correlation between pooled genotyping and individual genotyping. b) Distribution of p-values from the additional samples added in Stage II. The distribution is fairly uniform with only a slight enrichment of small p-values.
Figure 3
Figure 3
Scatter plot of the allele frequencies from pooling and individual genotyping from the Stage I sample.
Figure 4
Figure 4
Plot of distributions of standard errors of SNPs selected using different criteria. The plot illustrates that delta p^ cutoff selects preferentially SNPs with high standard errors of delta p^, regular t-test preferentially selects SNPs with low standard errors and the corrected t-test is centered on the standard error distribution from all SNPs.
Figure 5
Figure 5
Q-Q plot of logistic regression ANOVA deviance produced from samples added to Stage I samples at Stage II. Because these samples are independent of Stage I samples used for the SNP selection from pooled genotyping the test statistic is expected to largely follow the null distribution (Chi-square distribution with 2 degrees of freedom). Due to the lower power of this sample set compared to the combined set of samples and the small effect sizes found in this study, any possible associations are not expected to cluster together at low p-values, thereby changing the linear shape of this Q-Q plot. The dotted line represents 95% point-wise confidence envelope of expected null distribution.

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