We have found a novel, genomewide significant association of a composite substance dependence phenotype with a SNP in the PKNOX2 gene in White women. PKNOX2, PBX/knotted 1 homeobox 2, belongs to the three-amino-acid loop extension (TALE) homeobox family. Homeodomain proteins are highly conserved transcription regulators. Imoto et al. [31] identified PKNOX2 as a novel TALE homeodomain-encoding gene, located at 11q24 in humans and it functions as a nuclear transcription factor indicated by its structure and sub-cellular localization. Later, PKNOX2 was identified as one of the cis-regulated genes for alcohol addiction in mice [22]. However, PKNOX2 has not been reported to be associated with any substance dependence phenotype in humans to date.

The composite dichotomous substance dependence variable reflects cases with two or more addictions where the top three categories are alcohol, nicotine and cocaine (Figure 1). Among the cases, 47% have been diagnosed with alcohol dependence in combination with other substance dependence outcomes. Our results, which show a strong association of this composite substance dependence variable with PKNOX2 gene in a human sample, support the experimental findings in mice by Mulligan et al [22]. Thus our findings make an important contribution in reporting PKNOX2 as a novel candidate gene for substance dependence in humans, particularly for White women in the SAGE sample.

Interestingly, among our most significant SNPs, we do not observe those genes previously reported for alcoholism or nicotine. Rather we find a new set of genes among the top SNPs. When each substance dependence outcome was individually analyzed for association with the 8 most significant SNPs, we found no association that reached the genomewide significance. This suggests that substance dependence or addiction as a whole has different risk genes compared to any single addiction outcome. It may also mean that there is more power in detecting common genes acting upon co-morbid addiction outcomes as a whole.

For many complex diseases, different ethnic groups have vastly different underlying genetics, and these differences may confound association results when they are pooled together as one in the analysis. Previously, racial differences in the prevalence of substance abuse have been reported [32], [33], [34]. More recently, Luo et al. [24] have reported that genetic differences between Black smokers and White smokers influence the nature of their nicotine dependence. Their analysis suggested that Black smokers become dependent at a lower threshold (number cigarettes per day) than Whites. On the other side, in the presence of subjects in different ethnic populations in the data, it is crucial to investigate and control for potential confounding by stratification and admixture due to disequilibrium between pairs of unlinked loci [26]. Thus we investigated these two major ethnic groups separately in our analysis. In addition, we stratified our analysis by gender; based on the premise that gender may be a confounding factor for the substance dependence outcome – men may be socially more prone to environmental influences promoting substance use, and thus more vulnerable to addiction, compared to women [35]. Our results from the two ethnic groups do not corroborate each other, which underscores the underlying genetic differences in White and Black samples. In fact, strong association signals are observed only in the White woman sample. With a heterogeneous population like SAGE, one must be cautious in analyzing and interpreting the results.

The identification of PKNOX2 as a candidate gene for substance use disorders underscores two important issues: (a) this has not been possible in the past due to limited sample size; and (b) we have considered a composite trait of six substance dependence outcomes as a whole. The association becomes less significant if individual substance addictions are considered. Thus, this result highlights the importance of studying highly comorbid disorders or those which might otherwise have a common pathway. However, our study is limited to the information in the available data, and we acknowledge the difficulty in operationalizing substance dependence; whether our operationalization of addiction to two or more substances, truly reflects the strength of the addiction phenotype is open to question. Indeed, it may simply reflect the extent of access to drugs. We also recognize that dependence on one substance shows different characteristics from dependence on another, and it is valuable and necessary to study them as individual entities. However, our call for more attention to comorbidity and the combinatorial study of these disorders should be viewed as a valuable complementary effort.