Strategy for single-nucleotide polymorphism (SNP) selection plays a key role in association study outcome. In general, variants predicted to have functional consequences, eg, because they alter predicted amino acid sequence, have been favored for study; alternatively, researchers often try to capture most of the genetic variation at a locus via selection of haplotype tagging SNPs followed by haplotype reconstruction. It is important to recognize some of the limitations of these strategies at the outset. Although most common putatively functional SNPs are known, rarer SNPs may have large phenotypic effects, and there are many such variants yet to be discovered. Not all functional SNPs are easily recognized as such. SNPs vary by population, and populations differ in the extent to which common genetic variation has been identified. The same population variation is reflected in differences in haplotype structure. Finally, haplotype reconstruction is almost always accomplished via computer algorithms, and the results are estimated. With these limitations in mind, we discuss several examples of genetic associations with DD phenotypes, focusing on interesting physiological candidates and on replicated findings.
Association of variants that map at or near the D2 dopamine receptor (DRD2
locus) with drug or alcohol dependence was proposed many years ago and has been widely debated. We identified a “suggestive” linkage peak for ND at the region of chromosome 11 that includes the NCAM1-TTC12-ANKK1-DRD2
The inconsistent results with DRD2
may be attributable to an indirect effect - observed association could actually be mediated through variation at a nearby locus in linkage disequilibrium with DRD2.
To test this hypothesis, we genotyped 43 SNP markers in a region including DRD2
and the three adjacent genes, in an SD linkage sample of >1600 subjects. We found very strong evidence of association of multiple SNPs at TTC12
in two different populations, EAs and AAs (minimal P=0.0007 in AAs and minimal P=0.00009 in EAs), and highly significant association of a single haplotype (set of markers) spanning TTC12
to ND in the pooled sample (P=0.
0000001). Thus, a risk locus for ND maps to a region that spans TTC12
and ANKK1 .
The exact localization of the risk haplotype depends on the disease definition, and whether and which co-occurring diagnoses are present in the study sample.24
These results support the hypothesis that the DRD2
findings could be attributable to variants in nearby loci. Such variants could reflect either functional variation that affect those loci (and not DRD2),
or relatively distant regulatory regions important for DRD2
function. The ANKK1
finding in ND has been replicated.25
Another set of risk loci that are of interest in relation to the risk of drug dependence are those encoding proteins that regulate or mediate opioidergic function. All of the opioid receptor genes have been reported to be associated with substance dependence liability. A functional polymorphism in OPRM1
(Asn40 Asp), which encodes the mu-opioid receptor, has been the most extensively studied in this regard, though the association is controversial. Although multiple studies have shown a significant allelic association with DD, they are nearly evenly divided between those showing a significant excess of the Asp40 allele among cases26,27
and those showing a significant excess of the Asp40 allele among controls.28-30
Consequently, meta-analyses of that literature failed to show a reliable association of the SNP with either OD31
or any SD disorder.32
However, Zhang et al33
examined 13 SNPs spanning the coding region of OPRM1
in a sample of EAs with AD and/or DD and 338 EA healthy controls. The SNPs formed two haplotype blocks. There were significant differences between cases and controls in allele and/or genotype frequencies for SNPs in Block I and in Block II, after correction for multiple testing. Haplotypes constructed from five tag SNPs differed significantly in frequency between both AD and DD subjects and controls. Logistic regression analyses in which the sex and age of subjects and alleles, genotypes, haplotypes, or diplotypes of the five tag SNPs were considered confirmed the association between OPRM1
variants and SD.
Zhang et al34
also examined the genes encoding the other two opioidergic receptors: OPRD1
(which encodes the delta receptor) and OPRK1
(which encodes the kappa receptor). Eleven SNPs spanning OPRD1
were examined in EAs with AD, CD, and/or OD, and control subjects. Although nominally significant associations were observed for five SNPs with SD, only the association of the nonsynonymous variant G80T with OD remained significant after correction for multiple testing. Haplotype analyses with six tag SNPs indicated that a specific haplotype was significantly associated with AD and OD (P<0.001). In logistic regression analyses, controlling for sex and age, this haplotype had a risk effect on AD and, to a much greater extent, on OD. In addition, seven SNPs covering OPRK1
were examined in the majority of subjects and although there were no significant differences in allele, genotype, or haplotype frequency distributions between cases and controls, a specific OPRK1
haplotype was significantly associated with AD, but not DD. In summary, these findings demonstrated a robust positive association between OPRD1
variants and SD, particularly OD.
Finally, Zhang et al35
the gene that encodes pro-opioimelanocortin, from which functionally different peptides are derived via tissue-specific post-translational processing; of particular relevance here are two principal elements of the hypothalamic-pituitaryadrenal axis: adrenocorticotropin (ACTH) and p-endorphin. Five SNPs spanning POMC
were examined in independent family and case-control samples of EAs and AAs. The families were ascertained based on a pair of siblings affected with cocaine and/ or opioid dependence. Case-control studies included cases affected with AD, CD and/or OD and controls. Family-based analyses revealed an association of one SNP (rs6719226) with OD in AA families, and a different SNP (rs6713532) with CD in EA families. Case-control analyses demonstrated an association of rs6713532 with AD or CD. Moreover, the minor allele of a third SNP was a risk factor for CD or OD in AAs, and for AD, CD, or OD in EAs. Logistic regression analyses in which sex and age were considered and population stratification analyses confirmed these findings. Additionally, specific haplotypes increased risk for CD in AAs and OD in EAs.
In summary, as might be expected given that the brain's opioidergic system plays a central role in reinforcement, which has important implications for addiction,36
variation in a number of functional candidate genes encoding opioidergic proteins have been implicated in dependence on alcohol, cocaine, and opioids. Assuming independent replication of these findings, a key question to be addressed is the nature of gene-gene and gene by environment interactions to which risk of SD is attributable.
Other studies have demonstrated associations with the cannabinoid receptor gene (CNR1)
neurexin 1 (NRXN1)
and a set of alcohol-metabolizing enzymes.41
A clear pattern emerges from the examination of this sampling of candidate gene associations with SD: insofar as genes with known function are concerned, there are no big surprises with respect to physiology. (This can not be said about genes without clearly delineated functional roles, such as ANKK1,
which was identified, not incidentally, based on its position, rather than its function.) This highlights the limitations of the candidate gene approach, which is often inherently biased by prior knowledge about physiology. Unbiased studies have greater potential to reveal new mechanisms of addiction, and that is a key attraction of the genome -wide association study (GWAS) methodology discussed below.
GWASs are an alternative to linkage for locating genes anywhere in the genome without prior hypotheses.
GWAS designs are of interest due to their potential to identify risk loci of relatively small effect, much smaller than through linkage strategies. (In fact, one controversy engendered by the widespread adoption of GWAS designs is that often risk alleles are identified that have such a small effect - typically with odds ratios less than 1.2 - that it is hard to know what to do with them once they have been identified.) A second advantage of GWASs is that they may be based on case-control samples, which are easier to recruit than family sampling schemes, which must be deployed to prepare for linkage. Family samples are more difficult to recruit (markedly so for many kinds of SD because of the tendency of these disorders to fragment families) and can introduce certain kinds of bias. The first GWAS for a specific SD trait, excluding studies that used a pooling methodology exclusively (see ref 42), examined ND.43
This study employed a two-stage design; first pooled DNA was used to screen 2.4 million SNPs; second, >30 000 SNPs selected from the first stage were screened individually in ~1000 each cases and controls. Numerous genes were identified as possibly associated to ND, including both novel genes and genes that were previously considered candidates based on known physiology (eg, cholinergic receptor, nicotinic, beta 3, CHRNB3).
The latter finding has been confirmed in larger studies: subsequent GWASs have demonstrated highly significant associations between variation in the nicotinic receptor gene cluster CHRNA5-CHRNA3-CHRNB4
and ND and related traits44,45
and with lung cancer.46,47
In a hypothesis-generating study, we studied a set of 5633 SNP markers in 1699 subjects from 339 AA families and 334 EA families ascertained through a sib pair meeting DSM-IV criteria for either CD or OD. This is considered a sparse marker set for the purposes of GWAS. It is expected to interrogate <10% of the genome, thus, cannot be considered to be a study of truly genome -wide depth. Associations between these markers and five substance dependence traits (CD, OD, AD, ND, and cocaine-induced paranoia) were assessed by family -based association tests (FBAT). The top-ranked result was an association of a specific SNP in the MANEA
gene with cocaine-induced paranoia. This study provided an initial SD trait-specific blueprint of associated regions for future candidate gene studies. There are, at the time of this writing, no published GWAS studies for several of these traits. The MANEA
finding was replicated and extended in a larger sample.48