Abstract
Onset of alcohol use at an early age increases the risk for later alcohol dependence. We investigated the role of the glucocorticoid receptor (GR) gene (NR3C1) in onset of alcohol use and abuse in 14-year-old adolescents (n = 4534). Several NR3C1 polymorphisms were associated with onset of alcohol drinking or drunkenness at this age. Strongest associations were observed in females, with one marker (rs244465) remaining significant after correction for multiple testing (Padj = 0.0067; odds ratio = 1.7, for drunkenness). Our data provide the first evidence that GR modulates initiation of alcohol abuse and reveal a polymorphism that might contribute to susceptibility to addiction.
Keywords: Addiction, adolescent, alcohol, glucocorticoid receptor, NR3C1, polymorphism
Age of onset of alcohol drinking is an important determinant in the addiction process. Early drinking has been found to be a strong predictor of later alcohol abuse and dependence (Grant & Dawson 1997; DeWit et al. 2000). The rates of lifetime alcohol dependence drops from more than 40% among adolescents who started drinking at ages 14 or younger to approximately 10% among individuals who started drinking after age 20 (Grant & Dawson 1997); the odds of dependence decreasing by 14% with each increasing year of age at onset of use. Thus, the age range (11–14 years) has been identified as critical for mediating elevated risk of developing alcohol-related disorders (DeWit et al. 2000). Given the link between early onset of drinking and later alcohol problems, it is essential to develop a better understanding of the factors that influence consumption patterns in adolescence.
Environmental influences are a more important determinant of alcohol consumption during adolescence than in adulthood (Kendler et al. 2008). In addition to a well-documented role in mediating responses to environmental factors like stress, a recent study demonstrated role for glucocorticoid receptor (GR) in the reinforcing properties of cocaine (Ambroggi et al. 2009). However, a role for GR in initiation of drug use has not yet been elucidated.
We performed a genetic screen in humans to investigate if genetic variations of NR3C1, the gene encoding GR, are associated with early onset of harmful drinking. For this, we analysed 14-year-old adolescents, a subset from the prospective general population-based Northern Finland Birth Cohort 1966 (NFBC 1966; Rantakallio 1988). Genotyping was described previously (Sabatti et al. 2009). For the current analyses, 26 single nucleotide polymorphisms (SNPs) covering the NR3C1 locus were selected from the whole genome data. Phenotypic characteristics of the sample and list of SNPs investigated are displayed in Table 1 and Supporting Information Table 1, respectively.
Table 1.
Phenotypic characterization of the Northern Finland Birth Cohort 1966 sample at age 14.
| Variable | N(%) |
|---|---|
| Gender | |
| Males | 2151(47.44) |
| Females | 2383(52.56) |
| Total | 4534(100) |
| Alcohol drinking | |
| Never drunk any | 1857(40.96) |
| Males | 902(41.93) |
| Females | 955(40.08) |
| Tasted once | 1562 (34.45) |
| Males | 753 (35.01) |
| Females | 809 (33.95) |
| Drunk a few times | 1004 (22.14) |
| Males | 461 (21.43) |
| Females | 543 (22.79) |
| Drink alcohol monthly | 99 (2.18) |
| Males | 31 (1.44) |
| Females | 68 (2.85) |
| Drink alcohol weekly | 29(0.64) |
| Males | 10 (0.46) |
| Females | 19(0.80) |
| Drunkenness | |
| Never been drunk | 3399(74.97) |
| Males | 1631 (75.83) |
| Females | 1768(74.19) |
| Once, slightly | 425(9.37) |
| Males | 218(10.13) |
| Females | 207 (8.69) |
| More often, slightly | 370 (8.16) |
| Males | 151 (7.02) |
| Females | 219(9.19) |
| Once very much | 143 (3.15) |
| Males | 71 (3.30) |
| Females | 72 (3.02) |
| 2–4 times, very much | 137(3.02) |
| Males | 57 (2.65) |
| Females | 80 (3.36) |
| More often, very much | 60 (1.32) |
| Males | 23 (1.07) |
| Females | 37(1.55) |
Association between SNPs and alcohol-related phenotypes was performed within gender using logistic regression analysis (additive model) using PLINK v1.06 (http://pngu.mgh.harvard.edu/~purcell/plink/) (Purcell et al. 2007). Threshold for filters were 0.01 for allele frequency, 0.1 for missingness per individual, 0.1 for missingness per marker and 0.01 for the Hardy–Weinberg equilibrium. Benjamini & Hochberg’s (1995) step-up false discovery rate procedure was used to estimate uncertainty due to multiple testing within the NR3C1 locus. These analyses revealed several markers showing nominal, gender-specific association with alcohol drinking behaviour (Table 2, Supporting Information Fig. S1). Effects of polymorphisms on early onset of alcohol use were assessed by comparing adolescents who had never drunk alcohol or tasted only once (i.e. non-drinkers) versus those who had repeatedly drunk alcohol (drunk a few times or more often). This revealed strongest associations for three markers (rs244465, rs4607376 and rs7721458) in females. While the minor alleles at rs244465 (A) and rs7721458 (A) were more common among repeated alcohol drinkers than female non-drinkers [P value, odds ratio (OR) [95% confidence interval (95% CI)]: P = 0.0021, 1.56 (1.18–2.07) and P = 0.0180, 1.20 (1.03–1.40), respectively,], minor allele at rs4607376 (A) appeared to be protective [P value, OR (95% CI): P = 0.0083, 0.84 (0.74–0.96)]. Likewise, comparison of adolescents who had never been drunk versus those having been drunk at least once uncovered markers that associated with drunkenness in females, not in males. As for initiation of drinking, the strongest association was at rs244465, an association that remained significant after gene-wide correction for multiple testing (Padj = 0.0067; OR (95% CI), 1.69 (1.28–2.25); Table 2).
Table 2.
NR3C1 polymorphisms associated with alcohol drinking patterns in 14-year-old adolescents.
| OR | SE | 195 | U95 | STAT | P | OR | SE | 195 | U95 | STAT | P | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|
||||||||||||
| SNP | Al | Alcohol drinking females (629 drinkers, 1761 non-drinkers) | Alcohol drinking males (502 drinkers, 1654 non-drinkers) | ||||||||||
| rs860465 | G | 1.06 | 0.08 | 0.91 | 1.24 | 0.75 | 0.4546 | 1.19 | 0.09 | 1 | 1.41 | 1.97 | 0.0491 |
| rs853184 | A | 1.07 | 0.08 | 0.91 | 1.25 | 0.83 | 0.4057 | 1.19 | 0.09 | 1.01 | 1.41 | 2.06 | 0.0391 |
| rs244465 | A | 1.56 | 0.14 | 1.18 | 2.07 | 3.08 | 0.0021 | 0.84 | 0.18 | 0.59 | 1.2 | −0.95 | 0.3405 |
| rs2963154 | G | 1.09 | 0.08 | 0.93 | 1.27 | 1.02 | 0.3102 | 1.2 | 0.09 | 1.02 | 1.43 | 2.14 | 0.032 |
| rs9324918 | G | 1.08 | 0.08 | 0.92 | 1.26 | 0.95 | 0.3428 | 1.23 | 0.09 | 1.04 | 1.46 | 2.41 | 0.0159 |
| rsl0052957 | A | 1.03 | 0.07 | 0.89 | 1.19 | 0.35 | 0.7228 | 1.18 | 0.08 | 1.01 | 1.38 | 2.12 | 0.0344 |
| rs4607376 | A | 0.84 | 0.07 | 0.74 | 0.96 | −2.64 | 0.0083 | 0.93 | 0.07 | 0.81 | 1.07 | −1.05 | 0.2926 |
| rs7721458 | A | 1.2 | 0.08 | 1.03 | 1.4 | 2.37 | 0.018 | 0.91 | 0.08 | 0.77 | 1.07 | −1.16 | 0.2469 |
|
| |||||||||||||
| Drunkenness, females (614 at least once, 1765 never) | Drunkenness, males (520 at least once, 1631 never) | ||||||||||||
|
| |||||||||||||
| rs864354 | A | 1.15 | 0.07 | 1.01 | 1.31 | 2.11 | 0.0348 | 0.88 | 0.07 | 0.77 | 1.02 | −1.73 | 0.0833 |
| rs244465 | A | 1.69 | 0.14 | 1.28 | 2.25 | 3.65 | 0.0003* | 0.87 | 0.18 | 0.61 | 1.23 | −0.8 | 0.423 |
| rs33383 | A | 1.16 | 0.07 | 1.01 | 1.32 | 2.15 | 0.0314 | 0.91 | 0.07 | 0.79 | 1.04 | −1.38 | 0.1663 |
| rs6877893 | A | 1.15 | 0.07 | 1.01 | 1.31 | 2.08 | 0.0376 | 0.9 | 0.07 | 0.78 | 1.04 | −1.48 | 0.1386 |
| rs2963156 | A | 0.84 | 0.08 | 0.72 | 0.98 | −2.19 | 0.0284 | 0.98 | 0.09 | 0.83 | 1.16 | −0.22 | 0.8294 |
| rs7721458 | A | 1.19 | 0.08 | 1.03 | 1.39 | 2.28 | 0.0227 | 1.02 | 0.08 | 0.87 | 1.19 | 0.19 | 0.8493 |
Associations attaining nominal significance (P ≤ 0.05) are highlighted in bold. Those attaining the threshold level for gene-wide false discovery rate (FDR ≤ 0.05) are marked by an asterisk.
Al = minor allele name (based on whole sample); L95 = lower bound of 95% confidence interval for odds ratio; OR = estimated odds ratio for Al; P = asymptotic P value for t-statistic; STAT = t-statistic; U95 = upper bound of 95% confidence interval for odds ratio.
These results indicate that genetic variation in NR3C1 may contribute to establishment of alcohol abuse, and that the minor (A) allele at rs244465 might increase this risk in females. Such genetic variation could potentially alter not only stress-related behaviours, but directly influence the motivation to drink alcohol. Existence of such independent and dissociable function of GR is supported by a recent study (Ambroggi et al. 2009) showing that, while inactivation of Nr3c1 in the entire mouse brain decreases both stress-related behaviours and motivation to self-administer cocaine, inactivation of Nr3c1 in dopaminoceptive neurons specifically inhibits cocaine self-administration.
A recent survey (WHO 2009) indicated that although drinking among adolescents increased markedly in recent years in Europe with large variations across countries, patterns of drinking observed in the NFBC 1966 cohort persisted over the years. While frequency of drinking in early adolescence is low in Finland, with 3% of 13-year-olds having reported drinking alcohol at least weekly, early onset of alcohol abuse is more common in Finland compared with most other countries, with 23% boys and 22% girls having been drunk by the age of 13 (WHO 2009). These numbers, similar to results displayed in Table 1, indicate that the NFBC 1966 still reflects some current adolescent drinking patterns. Further studies using appropriately selected samples are required to replicate our findings. In addition, taking into consideration gene–environment interactions by including covariates like adverse life events, parental substance use and peer influence, which might play a significant role in the development of alcohol use disorders (Sher, Grekin & Williams 2005), will help to definitely gauge role of GR in alcoholism.
Supplementary Material
Alleles and frequencies of single nucleotide polymorphisms (SNPs) investigated.
Associations between alcohol drinking and drunkenness during adolescence and single nucleotide polymorphisms in the NR3C1 locus. The SNPs most significantly associated with alcohol-related phenotypes in females (A) and males (B) are indicated. SNPs showing high r2 values with rs9324918 are colour coded (orange: r2 = 0.46 with rs10052957; red: r2 = 0.94 with rs2963154). (C), Genomic organisation of two of the NR3C1 isoforms on chromosome 5 and linkage disequilibrium map of the region was obtained from UCSI genome browser using NCBI build 35 coordinates.
Acknowledgements
This work was supported by the UK Department of Health NIHR Biomedical Centre for Mental Health and the EU-funded FP6 Integrated Project IMAGEN (LSHM-CT-2007-037286). The Northern Finland Birth Cohort 1966 study program (NFBC 1966) received financial support from the Academy of Finland (project grants 104781, 120315, 110143, 132071; Sigrid Juselius Foundation, NARSAD, Stanley Medical Research Institute and Centre of Excellence in Complex Disease Genetics), University Hospital Oulu, Biocenter, University of Oulu, Finland, NHLBI NIH grant 1-R01HL087679-01,through the STAMPEED program, NIMH NIH grant (1RL1MH083268-01), ENGAGE project and grant agreement HEALTH-F4-2007201413, the Medical Research Council (studentship grant G0500539, centre grant G0600705), the Wellcome Trust (project grant GR069224), UK, and the Research Council UK fellowship. The DNA extractions, sample quality controls, biobank up-keep and aliquotting were performed in the National Public Health Institute, Biomedicum Helsinki, Finland and supported financially by the Academy of Finland and Biocentrum Helsinki. Genotyping was supported by grant 5RL1MH083268 from the National Institute of Mental Health.
Footnotes
SUPPORTING INFORMATION
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Supplementary Materials
Alleles and frequencies of single nucleotide polymorphisms (SNPs) investigated.
Associations between alcohol drinking and drunkenness during adolescence and single nucleotide polymorphisms in the NR3C1 locus. The SNPs most significantly associated with alcohol-related phenotypes in females (A) and males (B) are indicated. SNPs showing high r2 values with rs9324918 are colour coded (orange: r2 = 0.46 with rs10052957; red: r2 = 0.94 with rs2963154). (C), Genomic organisation of two of the NR3C1 isoforms on chromosome 5 and linkage disequilibrium map of the region was obtained from UCSI genome browser using NCBI build 35 coordinates.