Neuropsychopharmacology (2011) 36, 1142–1148 & 2011 American College of Neuropsychopharmacology. All rights reserved 0893-133X/11 $32.00 www.neuropsychopharmacology.org KCNJ6 is Associated with Adult Alcohol Dependence and Involved in Gene  Early Life Stress Interactions in Adolescent Alcohol Drinking ,1 2,3 4 4 2 Toni-Kim Clarke* , Manfred Laucht , Monika Ridinger , Norbert Wodarz , Marcella Rietschel , 5 6 1 7 1 Wolfgang Maier , Mark Lathrop , Anbarasu Lourdusamy , Ulrich S Zimmermann , Sylvane Desrivieres 1,5 and Gunter Schumann 1 2 Section of Addiction Biology, MRC-SGDP Centre, Institute of Psychiatry, King’s College London, London, UK; Department of Child and 3 Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health, Mannheim, Germany; Department of Psychology, Division of 4 Clinical Psychology and Psychotherapy, University of Potsdam, Potsdam, Germany; Department of Psychiatry and Psychotherapy, University of 5 6 Regensburg, Regensburg, Germany; Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany; Centre National de 7 Genotypage, Evry, France; Department of Psychiatry and Psychotherapy, University Hospital Carl Gustav Carus, Technische Universita¨t, Dresden, Germany Alcohol abuse and dependence have proven to be complex genetic traits that are influenced by environmental factors. Primate and human studies have shown that early life stress increases the propensity for alcohol abuse in later life. The reinforcing properties of alcohol are mediated by dopaminergic signaling; however, there is little evidence to indicate how stress alters alcohol reinforcement. KCNJ6 (the gene encoding G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2)) is a brain expressed potassium channel with inhibitory effects on dopaminergic tone. The properties of GIRK2 have been shown to be enhanced by the stress peptide corticotrophin-releasing hormone. Therefore, we sought to examine the role of KCNJ6 polymorphisms in adult alcohol dependence and stress-related alcohol abuse in adolescents. We selected 11 SNPs in the promoter region of KCNJ6, which were genotyped in 1152 adult alcohol dependents and 1203 controls. One SNP, rs2836016, was found to be associated with alcohol dependence (p ¼ 0.01, false discovery rate). We then assessed rs2836016 in an adolescent sample of 261 subjects, which were characterized for early life stress and adolescent hazardous drinking, defined using the Alcohol Use Disorders Identification Test (AUDIT), to examine gene–environment interactions. In the adolescent sample, the risk genotype of rs2836016 was significantly associated with increased AUDIT scores, but only in those individuals exposed to high levels of psychosocial stress in early life (p ¼ 0.01). Our findings show that KCNJ6 is associated with alcohol dependence and may moderate the effect of early psychosocial stress on risky alcohol drinking in adolescents. We have identified a candidate gene for future studies investigating a possible functional link between the response to stress and alcohol reinforcement. Neuropsychopharmacology (2011) 36, 1142–1148; doi:10.1038/npp.2010.247; published online 9 February 2011 Keywords: alcoholism; genetics; GIRK2; stress; gene  environment; KCNJ6 INTRODUCTION environmental factors are almost equally contributing to produce a complex phenotype. The dopaminergic meso- In 2002, the WHO’s World Health Report concluded that limbic system is one of the main neuronal networks alcohol was the fifth leading cause of death and disability responsible for inducing the reinforcing effects of drugs of worldwide, accounting for 9.2% of disability-adjusted life abuse (Robbins and Everitt, 1999, Tupala and Tiihonen, years in the developed world (Grimm, 2008). Alcoholism 2004). Ion channels are known to regulate dopaminergic is estimated to be between 50 and 60% heritable (Dick and neuronal signalling. One such family of ion channels Bierut, 2006), which suggests that both genetic and includes the G-protein-coupled inwardly rectifying potas- sium channels (GIRKs). GIRKs induce the postsynaptic inhibitory tone in response to the activation of the D2, D3 *Correspondence: Dr T-K Clarke, Translational Research Laboratory, Department of Psychiatry, School of Medicine, University of dopamine receptors, although their effects are not limited to Pennsylvania, Room 2109, 125 South 31st St Phila, Philadelphia, PA dopaminergic neurons (Kuzhikandathil et al, 1998). As the 19104, USA, Tel: + 1 215 746 3665, E-mail: [email protected] GIRK channel is opened, potassium ions permeate the cell, Received 14 September 2010; revised 16 November 2010; accepted 8 hyperpolarization ensues (Nicoll, 1988), and the neuronal December 2010 firing rate is reduced (Werner et al, 1996). KCNJ6 polymorphisms, stress and alcoholism T-K Clarke et al 1143 While dopaminergic signalling reinforces alcohol con- also assessed for nicotine dependence using diagnostic sumption, exposure to early life stress is known to promote criteria according to the DSM-IV and ICD-10. All partici- vulnerability to alcohol abuse in later life. This concept has pants were of German descent. Parents of the participants been extensively studied in Rhesus macaques, where it has were living in Bavaria and participants themselves were been shown that stress during early life increases alcohol born and raised in this area. Written informed consent consumption in adults (Fahlke et al, 2000). An enhanced was obtained for all participants before the investigation. sensitivity to stress in later life is also seen in these animals, Diagnosis was assessed after alcohol withdrawal by the who display increased salivary cortisol in response to social Composite International Diagnostic Interview (Wittchen isolation. Similar findings are observed in human beings et al, 1996) performed by trained staff who rated partici- with the number of adverse childhood experiences being pants independently. Patients with a lifetime history of correlated with an increased propensity for adolescent schizophrenia or an addiction to drugs other than tobacco alcohol abuse (Dube et al, 2006). Despite a well-established or alcohol were excluded from the study. link between early life stress and alcohol abuse in later life, In total, 1203 individuals were from the region of Bonn, there is currently no known mechanism showing how such Germany, and were recruited from 2001 to 2003 within the effects are mediated. Interestingly, the efficiency of the German National Research Project to serve as controls for GIRK channels has been shown to be increased by stress genetic studies in several neuropsychiatric phenotypes. peptides such as corticotrophin-releasing hormone (CRH) Population-based recruitment was performed in colla- (Beckstead et al, 2009). Inhibitory postsynaptic currents boration with the local consensus bureau. Participants were mediated by GIRK2 channels in D2-dopaminergic neurons screened for neurological and psychiatric disorders with of the mouse brain were found to be enhanced by CRH, and self-report questionnaires adapted from the German version this was dependent on the CRH-R1 receptor. Furthermore, of the inventory to diagnose depression (Kuhner, 1997); protein levels of GIRKs (GIRK1 and GIRK2) have been smoking and drinking were diagnosed with the Fagerstro¨m shown to be regulated by corticosterones in the brain of rats Tolerance Questionnaire (Fagerstrom, 1978) and the (Muma and Beck, 1999), providing evidence that GIRK2 Alcohol Use Disorders Identification Test (AUDIT) (Bohn interacts with neuronal stress systems. GIRK2 channels et al, 1995). More than 96% of the participants were of therefore may establish a link between brain stress systems, German or Western European origin as ascertained by place dopaminergic signalling, and thus reinforcement-related of birth of their grandparents. For all patients and controls, behavior. written informed consent was obtained before study Animal studies have analyzed the effects of ethanol on participation. GIRK2-null mice. They found that they could not establish a place preference or conditioned taste aversion for 2–3 g/kg Adolescent at-risk sample. Participants were members of of ethanol in mice lacking GIRK2 channels (Hill et al, 2003), the Mannheim Study of Children at Risk, an ongoing concluding that GIRK2 may be mediating the reinforcing epidemiological cohort study examining the outcome of and/or aversive motivational aspects of ethanol action. In early risk factors from infancy into adulthood (Laucht et al, human beings, only one study has examined GIRK2 and 2000). The initial sample comprised 384 children born addictive phenotypes. A large-scale candidate gene study between 1986 and 1988, of predominantly (499.0%) analyzed SNPs in 1050 nicotine-dependent individuals and European descent. Infants were recruited from two obstetric 879 controls (Saccone et al, 2007). An SNP in the promoter and six children’s hospitals of the Rhine-Neckar Region of of KCNJ6, the gene encoding GIRK2, provided one of the Germany and were included consecutively into the sample top signals for nicotine dependence in this study, suggesting according to a two-factorial design intended to enrich and that GIRK2 may be important in human nicotine addiction. control the status of the sample with regard to obstetric and Our approach was to explore the significance of genetic psychosocial risks (Laucht et al, 1997). Only firstborn variations in the promoter of KCNJ6 in alcohol dependence children with singleton births and