Using an informative panel of single nucleotide polymorphisms (SNPs) that were typed in a subset of individuals from the Collaborative Study on the Genetics of Alcoholism (COGA), we conducted multipoint linkage analyses for DSM-IV criteria of alcohol and drug dependence. Linkage peaks on chromosome 2 and 10 were noted for alcohol dependence and for an average score of alcohol and drug dependence.
Prior linkage studies, especially those utilizing COGA data and a categorical phenotype (i.e. affected sibling pairs), have noted linkage peaks on chromosome 2 for a variety of substance-related phenotypes but these peaks have converged to 120-135 cM (
Bierut et al., 2004;
Dick et al., 2004;
Foroud et al., 2000;
Hesselbrock et al., 2004;
Reich et al., 1998). Our findings, in contrast, occur at 230-260 cM, where Schuckit et al., (
Schuckit et al., 2001) report linkage for SRE (Subjective Response to Ethanol) scores during the first five times alcohol was consumed (FIRST 5) and by
Nurnberger et al. (2001) for comorbid alcoholism and depression. In addition,
Straub et al. (1999) report linkage in this 2q region for nicotine dependence in their Christchurch sample.
Gelernter et al., (2005,
2006) have also reported linkage for late-onset cocaine dependence and for DSM-IV opioid dependence at 221 cM on chromosome 2, but only in their African-American pedigrees.
Zubenko et al., (2003a,
b) find linkage in this region for Depression Spectrum Disorder, and recently,
Kuo et al., (2006) report linkage for an alcohol withdrawal criteria factor score. While there is considerable phenotypic, and potentially genetic overlap, across the phenotypes assessed in our manuscript and those used in the above referenced studies, it is also possible that imprecise localization of linkage signals, or random chance, may have contributed to convergence of linkage signals.
Our highest LOD score for alcohol dependence was on chromosome 10. The elevations at 60 and 116 cM are in regions where several studies have reported linkage for correlated psychiatric traits.
Li and colleagues (2006) report a LOD of 4.17 for a peak ranging from 60-100 cM for number of cigarettes smoked per day in a sample of African-American families ascertained for nicotine dependence. In contrast,
Uhl et al., 2001, in their genomewide association study found evidence for association between a polymorphism (WIAF-3336) at 86 cM on chromosome 10 and substance dependence vulnerability in their European-American cases and controls alone. While the African-American families contributed to our LOD score on chromosome 10, re-doing the linkage analyses without these families did not greatly reduce the LOD score (LOD was reduced to 3.5), suggesting that our finding on chromosome 10 is not specific to African-American families.
Vink et al. (2004) showed LOD scores of similar magnitude of both smoking initiation and quantity at 50-60 cM on chromosome 10. Strikingly, Zubenko and colleagues have reported linkage for mood disorders at 76 and 113 cM in their family study of recurrent unipolar major depressive disorder (
Zubenko et al., 2003b). The latter peak at 116 cM also maps to within 10 cM of the region where
Schuckit et al, (2005) report linkage for the Subjective High Assessment Scale (SHAS), a measure of alcohol response.
We did not, however, find significant evidence for linkage peaks on chromosome 3, 9 or 17, where
Stallings et al., (2003) report linkage for average scores of alcohol and drug dependence, as well as for cannabis dependence, in their sample of adolescent probands from treatment samples, and matched controls. In fact, our highest LOD score for cannabis dependence criteria (LOD 1.92) occurred on chromosome 14. There may be several reasons for this including different ascertainment strategies between the two studies (i.e. COGA used alcohol dependent probands and dense families, while Stallings et al., used probands with substance use or conduct problems) and the higher information content afforded by using SNP panels instead of microsatellites.
It is also worth noting that some linkage peaks previously identified in COGA on chromosome 4 and 7 (
Saccone et al., 2000;
Saccone et al., 2005;
Foroud et al., 2000;
Reich et al., 1998;
Nurnberger, Jr. et al., 2001) were not identified in the current study. Those peaks harbor genes such as
GABRA2, ADH (on chromosome 4) and
CHRM2 and
hTAS2R16 (on chromosome 7), which have subsequently found to be associated with risk for alcoholism (
Edenberg et al.,2004;
Wang et al., 2004;
Edenberg et al., 2006a; Hinrichs et al., 2006). We did not detect these peaks in our analyses. In other samples, however, quantitative indices of alcohol dependence similar to those used here have yielded linkage on chromosome 4 (see
Prescott et al. 2006 for summary). For instance, in the Irish high-density sample of alcoholics,
Prescott and colleagues (2006) report linkage (LOD 4.5) on chromosome 4. Whether the lack of these linkage findings in the present study is related to the phenotype used here or is a false negative is unknown. While the non-replication poses some interpretive challenges, it is not unusual in the extant linkage literature for substance-related phenotypes.
Before considering plausible candidates in the genomic regions encompassing our highest LOD scores, some study limitations need to be considered. First, there is significant overlap between the high risk families that contributed to the linkage signal for alcohol dependence criteria and the families for illicit drug dependence criteria. In our sample, nearly half of those with alcoholism also meet criteria for DSM-IV drug dependence and less than 25% of those with drug dependence do not report a lifetime history of alcohol dependence. Therefore, the overlap of linkage findings is likely due to the high comorbidity in this sample. Second, due to the relatively small proportion of African-American families, we did not have sufficient power to conduct linkage analyses independently in these families. Differences in allele frequencies, however, were maintained by creating pedigrees in each racial group and using pedmerge to combine them. Third, the community-based sample was ascertained from various sources: driver’s license registries, dental clinics and health maintenance organizations. While these families were not enriched for substance dependence or psychiatric disorders, they were selected to be large, therefore they may provide limited representation of the general population. Notwithstanding this limitation, using community-based estimates is highly recommended for regression-based linkage analyses. Furthermore, re-doing the analyses without using the community sample (as discussed below) did not significantly alter our findings. Lastly, diagnostic criteria for nicotine dependence were not included in the initial COGA assessments, and hence dependence vulnerability to tobacco was not included in these analyses.
Several subsidiary linkage analyses were conducted to validate the robustness of our LOD scores. First, analyses were performed using means and variances from the high-risk sample (i.e. without scoring) and without regressing out the influence of covariates. Second, analyses were replicated using varying estimates of heritability (0.45-0.55). Third, analyses were also conducted using DSM-IIIR dependence criteria for alcohol and illicit drugs. Fourth, linkage analyses were repeated in the full panel of 4,596 SNPs and were also conducted using the original microsatellite panel. Across all subsidiary analyses, LOD scores remained consistent, although modest magnitude changes were noted in some instances. In addition, LOD scores remained unchanged when particular large families were trimmed from the analysis.
Our strongest evidence for linkage was observed on 2q and 10p-q. On the q-arm of chromosome 2, putative candidate genes include
HTR2B (5-Hydroxytryptamine receptor 2B) receptors. Recently,
Lin et al. (2004) reported association between 3 coding SNPs (2 non-synonymous, resulting in double-mutants of the protein, and one synonymous) in
HTR2B and substance abuse vulnerability. Other possible candidate genes include
GPR55 (G protein-coupled receptor 55), which is expressed in human brain tissue and involved in extracellular-intracellular signal transduction, and
HDAC4 (histone deacetylase 4) which participates in epigenetic modification of core histones and. Work by Zubenko and colleagues has also suggested a role of
CREB1 on mood disorders; this lies 20 cM centromeric to our linkage peak (
Zubenko et al., 2003a). These authors have also identified a sex-specific association between the 124bp repeat allele of D2S944 and recurrent, early-onset major depression and with anxious depression in an independent U.S. and Dutch sample (
Beem et al., 2006;
Philibert et al., 2003;
Zubenko et al., 2002). Due to the high level of comorbidity between alcohol and drug dependence and major depression, and as indexed by a high LOD score of 3.49 for comorbid alcoholism and major depression in prior linkage analyses using COGA data (
Nurnberger, Jr. et al., 2001), this region on chromosome 2 may be tapping into a region of susceptibility for both disorders.
The linkage peak on chromosome 10 spans from 42 to 65 cM.
GAD2 (glutamic acid decarboxylase 2), a possible candidate in this region, has been putatively implicated, although not unequivocally, for its role in acute ethanol consumption and withdrawal, in animal models (
Fehr et al., 2003). Two recent association studies in humans, however report negative findings for the role of
GAD2 in the risk for alcoholism while another found limited evidence for the role of this gene in association with anxiety disorders, depression and neuroticism (
Hettema et al., 2006;
Lappalainen et al., 2007;
Loh et al., 2006). A smaller linkage peak at 90-110 cM encompasses candidate genes such as
HTR7 (5-Hydroxytryptamine receptor 2B) and VMAT2 (vesicular monoamine oxidase 2), as well as
KCNMA1 (calcium activated channel). SNPs in
VMAT2 and
KCNMA1 have been shown to be associated with alcoholism and the endophenotype of subjective responses to ethanol (
Lin et al., 2005;
Schuckit et al., 2005). Also located at 83 cM is
CTNNA3 (catenin alpha 3) which was recently identified in genomewide association studies of substance dependence vulnerability and nicotine dependence (
Bierut et al., 2007;
Liu et al., 2006).
Finally, our finding on chromosome 14 for cannabis dependence, while modest, is in a region of biological interest. Our linkage peak on chromosome 14 harbors candidate genes, such as GPR68 (G-protein coupled receptor 68) involved in cAMP regulation, CKB (creatine kinase, brain), encoding a cytoplasmic enzyme involved in energy homeostasis, as well as SERPINA1 and SERPINA2 (serine-peptidase inhibitor, clade A, members 1 and 2), which were previously identified in the genomewide association study for substance abuse vulnerability by Liu and colleagues (and also by Bierut et al.,61 for SERPINA1 and nicotine dependence).
In the past, linkage analyses in COGA have been extremely successful in identifying putative candidate genes which have led to positive association results (
Dick et al., 2006;
Edenberg et al., 2006b). We have now identified additional genomic regions that may harbor genetic loci that contribute to the etiology of alcohol and illicit drug dependence. Future efforts will target candidate genes in these regions for association analyses. This continued effort at understanding the biological basis of alcohol and drug use disorders is critical as, even today, the challenge posed by the considerable morbidity and mortality due to alcohol and drug problems is a substantial concern (
Centers for Disease Control (CDC), 2004;
Williams et al., 1988).
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= r + 1/1001, where r was the number of times a simulated LOD score exceeded an observed LOD score value (