|(Fig. 1) Genetic disposition affects brain behavioural outcomes and behaviour |
Initially, addiction seems to arise directly as a consequence of an individual’s social setting. Though setting is an influence, there are factors that contribute to peoples’ personalities, behaviours and/or biological functionalities that can reel them an inch closer to addiction development. Meta-analysis of twin studies reveal that heritable genetic factors account for 48-66% of alcohol, 42-79% of cocaine and 51-59% of cannabis addiction. In this sense, genetics can suggest the possibility of addiction development when looking at polymorphic variants of genes for receptors, transporters and enzymes. The presence of different receptor subunits or transporter variants can modulate different amounts of neurotransmitter synthesis, and release affecting behaviours (Fig. 1). Consequently, external behaviours such as impulsiveness, aggression and depression can arise, increasing the risk of individuals resorting to substance-use and compulsory addiction. Some variants of subunits and enzymes can yield less efficient functionality resulting in aversive side effects of drugs (and less risk of addiction) or more efficient functioning driving an individual to consume higher doses of drugs for a satisfactory effect.
Regardless of whether a variant manifests to diminish or enhance risk of addiction development, it’s inevitably the interplay of gene-gene, gene-environment interactions and the polygenic nature that makes it so difficult to tease apart the causal associations of addiction development. Thus, genes thought to contribute to addiction development are upheld only as vulnerability factors and not causes since addiction is not as simple as one or a few genes giving rise to addiction.
|Genes and Addiction. Prod. by Seastage & Dr. T. Wilens
(Massachusetts General Hospital)
These candidate or implicated genes may increase susceptibility of an individual to addiction and provide an idea or path for future studies to explore but it does not determine whether he or she will develop addiction. Even if specific genes were directly correlated, there would still be uncertainties in predictive abilities because of the complex interactions giving different behaviours and gene expression. Many studies investigate and suggest possible genes associated with addiction but inconsistencies in results exist because of the elaborate influences of social, economic, and psychological environments on an individual’s genetic expression.
1. Receptors and Transporters
Drugs of abuse involve many different brain receptors and pathways but the common use of dopamine (DA) neurotransmitters and its prevalence in the reward pathway supports its importance in addiction development. As the medial-forebrain bundle, reaching the striatum modulates locomotor-arousal, the mesocorticolimbic pathway functions in pleasure-seeking behaviours, the evaluation of rewards and even the consolidation of memories associated with drug-use. Specific mechanisms and effects of drugs may differ but chronic drug administration generally increases DA levels at the synapses of limbic regions such as the NAc, enhancing the reinforcing and pleasurable effects and psychological craving of drugs. Because DA plays a crucial role, DA receptors have been examined to see which receptor genes and particular polymorphism variants yield higher vulnerability in developing addiction.
The dopamine receptor D1 (DRD1) is found in cortical pyramidal cells on dendritic spines and its association with addiction is evidenced by a DRD1 gene knockout (KO) study in mice performed by El-Ghundi et al. (1998).  KO mice experienced dramatic decrease in alcohol consumption and reinforcing effects of cocaine compared to the wild-type (WT). Its most studied polymorphisms are DdeI (48A/G), found within the 5’ UTR (untranslated region) and Bsp12861 or rs686 (T/C alleles), found within the 3’UTR.  
The DdeI G allele and rs686 T variants were found to associate with heavy smoking behaviour in a study conducted with 2037 African- and European- American ethnic individuals. Another study examined 11 different polymorphisms of DA receptors with 535 Korean alcoholics and found the DRD1 DdeI G allele significantly associated with alcohol-dependence, exploratory behaviour, persistence and harm avoidance. Furthermore, Batel P. et al. (2008) compared 134 alcohol-dependent Caucasian individuals to healthy controls and found the rs 686 T allele more frequent among the alcoholic individuals. The trend was more significant with increasing severity of alcohol addiction. The counter alleles for both polymorphisms were found to associate with disordered gambling addictions. However, for substance addiction, there have been consistent findings of the DdeI G allele and rs686 T allele more associated with addicted individuals than controls indicating that the DRD1 is a vulnerability factor in addiction development.
However, the most prominent DA receptor implicated in addiction is DRD2. This receptor is found distributed in all dopaminergic neurons, with its highest presence in the VTA and SN. DRD2 has two isoforms via alternative splicing: D2S and D2L. The D2S functions as a presynaptic autoreceptor (autoDRD2) that modulates DA firing, synthesis and release by exerting a negative feedback loop. In contrast, D2L is expressed post-synaptically and at slightly greater amounts than D2S.
|(Fig. 2) DRD2 Expression in cocaine and meth abusers
compared to healthy individuals 
Albeit, D2S is less scrutinized in addiction, Bello et al. (2011) used autoDRD2 KO mice and observed DA disinhibition, hyperactivity, increased motivation and sensitivity to cocaine. Lacking autoDRD2 can have significance in contributing to addiction by removing modulatory control of DA levels in the synapse.
TaqIA is the most studied single-nucleotide polymorphism (SNP) of the DRD2 gene on chromosome 11 with two variants: A1 and A2 alleles. It was previously thought to be located in the 3’ noncoding region of the DRD2 gene but recently, it’s been located to the coding region of the neighbouring ANKK1 gene. Le Foll et al. (2009) observed the association between DRD2 TaqI A1 allele and alcoholism using a meta-analysis of 40 case-control studies. This analysis revealed significantly higher frequency of the A1 allele in alcohol-dependent individuals than controls. The results were replicated with 69% correlation of A1 allele and alcoholics in contrast to 20% in non-alcoholics with the allele frequency increasing with the severity of alcoholism. The A1 allele results in less DRD2 activity but the controversy remains unresolved whether A1 produces less D2 receptors or decreases DRD2 binding affinitiy. The decreased availability of DRD2 receptors have also been implicated in cocaine and heroin-addicts (see Fig. 2). Further support is provided by Thanos et al. (2001) with overexpression of DRD2 reduced alcohol self-administration in mice. Interestingly, Limosin et al. (2003) studied the link between DRD2 TaqI A1 and impulsiveness by analyzing 92 alcohol addicts and the Barratt Impulsiveness Scale (BIS-10). The researchers found that participants with A2/A2 or A2/A1 genotypes had higher BIS-10 scores (more impulsive) than A1/A1. This result is contradictory given that comorbidity of impulsion and addiction is common. The role of DA receptors in addiction still remains unclear and speculation continues, as the addiction itself is multifactorial and difficult to tease apart the causal links. Nonetheless, reduced DRD2 (D2L isoform) activity associated with addiction is a consistent finding of many studies.
γ- Aminobutyric Acid receptors (GABAR) mediate the primary inhibitory role of GABA neurotransmitters in the CNS and/or PNS by different mechanisms depending on which type of GABA receptor.
GABAA receptors (GABAAR) are ionotropic as they directly gate Cl- channels. When bound by two GABA molecules, there’s an influx of Cl- resulting in hyperpolarization of the cell. It consists of 5 subunits including both alpha and beta subunits. Because of its pivotal role in inhibition, Nowak et al. (1998) microinjected agonists and antagonists into the VTA (i.e. bicuculline and picrotoxin) and found that agonists of GABAA by inhibiting DA neuron firing, increased ethanol intake to achieve a pleasurable effect. Antagonists excited DA neurons by disinhibition and decreased ethanol intake and increased locomotor activity. This suggests that the VTA’s DA neurons and GABA receptors are involved in alcohol-consumption behaviour dependent on GABA’s inhibition. Also, it was found that mild inhibition of GABAAR had an anxiolytic effect and decreased anxiety in mice while more intense inhibition of GABAAR induced general anesthesia. Increased anxiety could predispose an individual more to alcohol or substance abuse to relieve overwhelming emotions, assuming there’s face validity in the animal models. It is thought that rs279826 and rs279858 regions in GABAAR alpha 2 gene is associated with alcoholism and impulsivity. However, Drgon et al. (2006) located different regions when they used linkage, association- genome scans and fine mapping. By genome-wide association scanning (GWAS) two samples of polysubstance abusers, they found a significant link between region rSA3 on chromosome 4p12 and addiction vulnerability. This region encodes the subunits alpha 2, alpha 4, beta 1 and gamma 1 of GABAAR and is expressed in the reward-pathway. Then by convergent data and fine mapping the alpha 2 gene region, they found the most frequent SNP among European American polysubstance abusers to be rs270858.  Nonetheless, there were vast inconsistencies in results based on ethnicity and age group. Delineating the precise loci of GABAAR associated with increased susceptibility to alcoholism or substance abuse and still remains a challenge.
|(Fig. 3) Synaptic cascade of GABAB Receptors |
GABAB receptors (GABABR) are coupled to G-proteins and act within a slower metabotropic pathway; they indirectly affect Ca2+ and K+ channels via second messenger systems. Structurally, the receptor exists as a heterodimer with B1 and B2 subunits and is found in both the central and peripheral nervous systems. This receptor differs from GABAAR because it’s localized on both pre- and post-synaptic membranes and hyperpolarizes the cell even more by increasing post-synaptic K+ conductance (efflux) and decreasing Ca2+ influx (see Fig. 3). Previously, it was found that chronic alcohol consumption impairs presynaptic GABABR function in the hippocampus implicating its involvement in alcohol addiction. Also, injecting GABABR agonist, baclofen, decreased cocaine self-administration in rats and pre-injecting the agonist before any drug-intake suppressed cocaine administration. In contrast, antagonist, CGP 56433A reversed baclofen’s effects and increased cocaine administration supporting that GABABR is involved in the reinforcing effects of cocaine. To study potential genetic markers, single-strand confirmation analysis was used to reveal a polymorphic 1974 T/C allele (rs29230) in the GABABR B1 subunit gene located on chromosome 6p21.3.18 There were higher frequencies of the T allele in alcoholics than healthy individuals. However, Kohnke et al (2006), identified alcoholic and control subjects’ genotypes using polymerase chain reactions and found no significant association of the T allele to alcoholism. GABAB contributes to drug consumption and consequent neuronal plasticity but there are no consistently supported candidate genes to serve as a genetic marker.
Serotonin (5-HT) is an important monoamine or neuromodulator that’s been implicated to affect sleep, appetite and mood.
The serotonin transporter (5-HTT) functions to re-uptake released 5-HT from the synapse back into the pre-synaptic terminal to be repackaged into vesicles. 5-HT is implicated in addiction, as it seems to be important in risk-inducing behaviours like depression, impulsivity and aggression especially under the influence of stressful situations. The 5-HTT gene is polymorphic in the promoter region with either the Short (s) or Long (L) allele.
|(Fig. 4) The S allele has a truncated promotor region giving less
5-HTT at the synapse. The L allele has more 5-HTT expressed. 
The S allele is 447-bp long with 14 tandem repeats of G-C rich sequences and the L allele is 528-bp long with 16 repeats. Thus, the S allele has a truncated promoter region yielding less expressed 5-HTT, less 5-HTT functioning and less 5-HT reuptake at the synapse (see Fig. 4). An experiment with 110 French male alcoholics that were also depressed, suicidal or anxious revealed S allele frequencies to be highest among those whom attempted suicide at least once (78.2%) compared to the control group (60.6%). This S allele frequency was directly proportionate to the number of suicide attempts made by alcohol dependent individuals. The relationship suggests that the S allele (S/L or S/S) interacts with suicidal, addicted patients more so than merely addicted individuals. In accordance, Caspi et al. (2003) using a longitudinal prospective study showed that people with one or two copies of the S allele experienced greater activity in the amygdala to fearful stimuli compared to those with homozygous L alleles. They concluded that patients with the S allele (S/S or S/L) were more likely to develop depression and more vulnerable of addiction than L/L individuals. The logic follows that since the S allele expresses less 5-HTT at synapse, less 5-HT is taken up, more is degraded and eventually levels of 5-HT decrease over time compared to homozygous L/L individuals. Another experiment also showed that the S allele was significantly associated with alcohol dependence, dysphoria, anti-social behaviour and suicide attempts, with the correlation dependent on severity (see Fig. 5). When the same experimenters tried to observe the link of S allele and anxiety using a meta-analysis of twenty-three case-control studies, they found no significant association.
|Behaviours associated with addiction|
|(Fig. 5) The S allele is seen with higher frequently
with depressed, suicidal individuals 
However, there have been many variable results in the association of S allele of the 5-HTT gene and behaviours more susceptible to addiction development. Schuckit et al. (1999) performed a prospective study with 41 men and measured their level of response (LR) to alcohol and examined the correlation between their genotypes and potential addiction development for 15 years. They proposed that individuals with lower LR were more likely to develop alcohol addiction within the 15 year duration because they’ll drink more to compensate for a more satisfactory effect of alcohol. Results showed that people around age 20 with genotype L/L had lower LR scores and were more likely to develop alcohol addiction while S/S individuals had the highest LR scores and were less likely to become alcoholics. The difficulty in achieving consistent results across studies can be accountable to the complex interaction between alleles and genes as well as between the consequent behaviours and the environment yielding the possibility of addiction development.
5-HT 1B Receptor
Another regulator of 5-HT is the 5-HT 1B receptor (HTR1B). This receptor is widely distributed within the basal ganglia, hippocampus and other cortical regions. The receptor functions as a post-synaptic and presynaptic autoreceptor that modulates 5-HT synthesis and release. The gene for HTR1B is located on chromosome 9 within several alcohol dependence-implicated quantitative-trait loci in mice. When this gene was K/O, the mice displayed increased aggression, alcohol preference, exploratory behaviour, decreased anxiety and enhanced locomotor activity to cocaine self-administration. When HTR1B agonists were injected, there was decreased cocaine self-administration. It suggests that HTR1B contributes to reward-seeking and relapse-prone behaviours. The HTR1B gene has one single exon with 4 SNPs. Cao et al. (2012) investigated the 4 SNPs most commonly reported with alcohol, cocaine and heroin addictions: -261 T/G (rs11568817), -161A/T (rs130058), 1180 G/A (rs6297) and 861 G/C (rs6296). The experimenters conducted meta-analyses using GWAS and forest blots with 35 case-control studies of substance abusers of European (non-Hispanic), Hispanic, Asian and African ethnicities. The only significantly associated SNPs were -161 A/T and -261T/G in which there was on average, higher incidence of the -161T allele in all groups and -261G allele in the European and Hispanic populations. The latter SNP varied greatly for the Asian and African ethnicities. However, the analyses were based on a relatively small sample with varying amounts of individuals per abused substance that the results are difficult to be generalized to a grander scheme of the population. It does, nonetheless, provide increments of information useful for future studies to further explore linked possibilities.
Contradictory and weakly associated results of studies on the 5-HTT and HTR1B genes have made it difficult to locate a functional locus that’s associated with addiction. It rather suggests predispositions to certain behaviours (i.e. impulsiveness, aggression, depression) that may lead to addiction development.
Proper transcription and translation of genes encoding enzymes determine whether enzymes function optimally, improper or at all. Nearby the enzyme genes are important regulatory regions influencing gene expression and amount of enzyme protein present. Thus, knowing gene variants are useful in analyzing addiction development because some enzymes are substance-specific and contribute to the side effects of drugs. If these side effects are too aversive, it'll be a deterrent to the substance whereas if there are no effects of the drugs then it may evoke more substance consumption.
ALDH2 & ADH1B
ALDH2 and ADH1B are two genes encoding enzymes responsible in consecutive steps of alcohol metabolism. ADH1B metabolizes ethanol to acetaldehyde then ALDH2 breaks down acetaldehyde to acetate. These enzymes have been consistently shown to partake in the “flushing syndrome” often manifested in Asian populations after alcohol consumption. The flushing syndrome includes aversive effects that reflect those of disulfiram used to prevent relapse in addicted individuals. These effects include facial redness, headaches, nausea, and vomiting.
|Genetic profiles of alcoholics and controls|
|(Fig. 6) 6 Alcoholics, 6 Controls have different gene
expression levels. Red: high expression, blue: low expression. 
ALDH2 gene has a polymorphism Glu 487Lys in which the Lys allele variant displays dominance and inactivates the ALDH2 enzyme (arbitrarily labeled; Glu: A allele, Lys: B allele). This relationship is supported by in vitro and in vivo studies that showed ALDH2 B/B genotype having no enzyme activity in the liver while ALDH2 A/B had 12-20% the enzyme activity of ALDH2 A/A.25 The ADH1B gene has a polymorphism His47Arg in which both alleles act co-dominantly. The Arg variant induces hyperactivity in the ADH1B enzyme (His: A allele, Arg: B allele).
Because these enzymes are substance-specific to alcohol, their particular alleles and gene expression have been consistently implicated in alcohol addiction (see Fig. 6). The flushing syndrome is invariably an unpleasant effect, which often causes a negative association with alcohol intake and decreased risk of alcohol-dependence. It has been proposed that this “flushing” is exerted through either low ALDH2 activity (via B allele) or high ADH1B activity (via B allele) presumably by the accumulation of acetaldehyde. Therefore, individuals with genotypes ALDH2 A/A and ADH1B A/A seem to be at highest risk and ALDH2 B/B and ADH1B B/B to be most protected from alcoholism. However, a meta-analysis with five Chinese, four Japanese and six Caucasian groups conducted by Whitfield (2002) showed that the protective effects of the B alleles manifested to different extents by ethnicity. Chinese and Japanese individuals having ADH1B A/B genotype had only 1/5 risk of alcoholism compared to ADH1B A/A whereas Caucasian individuals having the same genotype (A/B alleles) had ½ risk of alcohol dependence. Nonetheless, carrying even one allele of ALDH2 B lowered the rates of alcohol dependence in the general population. Moreover, population genotype samples revealed that the ALDH2 B allele was highly prevalent in Asian, moderately prevalent in Russian and rare in Western and Central European Caucasian ethnicities. Despite the high frequency of alcohol protective alleles among Asians, Japanese and Korean populations have reported high rates of alcohol consumption and addiction presumably due to prevalent social pressures and cultural norms of (particularly males) socializing and drinking as a bonding tradition.
Catechol-O-methyltransferase (COMT) gene encodes an enzyme responsible for metabolizing norepinephrine, dopamine (DA) and other catecholamines and regulates the amount of neurotransmitter present at the synapse. It’s mostly localized in regions like the pre-frontal cortex (PFC) and mesolimbic reward systems where DA transporters are less abundant. COMT enzyme’s function is apparent with gene K/O studies which increases DA levels dramatically within the PFC. Since drug use and subsequent drug addiction involves elevated dopamine release in the reward system, COMT polymorphisms have been investigated for any associations.
The gene has a polymorphism Val158Met in which both alleles are expressed co-dominantly and affect the enzyme’s stability and activity. The Val allele is noted to express 3-4 fold more COMT activity yielding less DA levels at the synapse, less efficiency of PFC function, higher stress resiliency but reduced cognitive performance evidenced by neurocognitive tests and Wisconsin sorting tasks. The reduced DA function in the PFC is suggested to impair the control of compulsive behaviour in substance use. In contrast, the Met allele expresses less COMT activity, better cognitive function but increased anxiety and decreased stress resiliency. For example, when two different populations of women were studied, individuals with homozygous Met/Met genotypes had increased anxiety, decreased stress resiliency and pain-thresholds. Given the functional outcomes, the warrior vs. worrier model was branded to represent these alleles: the warrior allele corresponds to the Val variant and the worrier allele corresponds to the Met variant. This model tries to explain the balancing selection and conservation of both alleles across the general population.
|Environment & Genetic Interactions|
|(Fig. 7) Genes affect disposition and environment affects genes. Complex interactions exist to define
overall behaviour 
There have been contradictory findings in regards to addiction vulnerability as to which allele is more prevalent. Li et al. (2004) performed a case-control study with 416 methamphetamine abusers and 435 healthy Han Chinese individuals from Taiwan. They found a strong correlation of the high COMT activity, Val variant in methamphetamine addicts than in controls. This variant was also found in excess among heroin-dependent individuals. They found that the Met allele induced aversive effects on the PFC functioning, providing a protective effect against methamphetamine dependence. However, in another study conducted by Tiihonen et al. (1999), the low activity Met allele was associated with late-onset alcoholics and frequent social drinkers. This suggests that both alleles have potential to predispose an individual to substance use by externalizing (Val allele: extroversion, disinhibition, impulsion) or internalizing (Met allele: anxiety, depression, easily stressed) effects (see Fig. 7). 
The genes contributing to addiction development is never straightforward due to the constant level of interaction with the environment. A very interesting and informative study conducted by Schellekens et al. (2012) tried to observe the genetic and environmental interactions between COMT variants, DRD2/ANKK1 TaqI A1 allele and stressful childhood experiences (i.e. abuse, neglect) in influencing alcohol addiction. These variables were of interest because genetic composition often determines stress resiliency and behaviours that may lead to more frequent alcohol consumption. In turn, a stressful environment can influence or exacerbate the minute effects of numerous genes. Also, since both COMT variants and DRD2 TaqIA1 have effects on DA presence in the reward pathway, the experimenters assumed gene-gene interactions to exist.
|Probability of Alcoholism given COMT variants|
|(Fig. 8) COMT Met/Met correlated with increased severity of childhood
adversity in predicting alcohol- dependence 
Schellekens et al. (2012) used a case control study with 110 alcoholics and 99 controls of European ethnicity. They used the Addiction Severity Index to consistently measure the onset age, duration and severity of alcohol addiction. Genotypes were categorized as Val/Val, Val/Met, Met/Met for COMT gene and as A1/- and A2/A2 for DRD2/ANKK1 gene because of the paucity of A1/A1 individuals. Childhood Trauma Questionnaire, Stressful and Traumatic Events Questionnare, and Parental Acceptance and Rejection Questionnaire were used to measure the severity, subjective experience and frequency of adverse childhood experiences. Results indicated that childhood adversity interacted with COMT Met/Met genotype to significantly predict alcohol addiction. With increasing exposure of stressful childhood events, there was increased probability of alcohol dependence and decreased age onset of alcohol addiction. However, the DRD2/ANKK1 Taq1A variant did not interact, at least not as much, with childhood experiences and did not predict alcohol addiction. Because Met/Met yield low COMT activity and increase PFC DA levels and stressful stimuli, affecting gene expression via epigenetics, also increase PFC DA levels, the experimenters postulated that having both conditions causes exaggerated DA responses and increased sensitivity to stressful events. Thus, Met/Met genotype and severely stressful childhood-experienced individuals are more vulnerable to alcohol addiction development (see Fig. 8). Limitations to this study include potential false memories while retrospective reporting of stressful childhood experiences, difficulty in quantitatively measuring emotional experiences and the use of only male subjects. However, the study achieved a feat in showing a correlation in childhood environmental interactions with genes to predict addiction development. Future studies should try to replicate the experimental design but investigate the 5-HTT S allele and COMT variants to see if varying COMT activity levels and reduced 5-HT transporters interact to predict more severe cases of depression in individuals. These findings could then be elaborated to question if an association exists with severe depression and substance-dependence in individuals.
3. External links
- Mouse Party for Specific Drug Pathways
- Genetic Science Learning Center, Univ. of Utah; Addiction
- Genetic Science Learning Center; DNA, Genes, Proteins, Technology
- NIDA Summary of Genetics and Addiction