Alcohol dependence and genetic variability in serotonin pathway among currently and formerly alcohol-dependent males
Anja Plemenitas, MDa; Matej Kastelic, PhDb; Stefano Porcelli, MD, PhDc; Alessandro Serretti MD, PhDc; Vita Dolžan MD, PhDb; Blanka Kores Plesnicar, MD, PhDd*
a Department of Psychiatry, University Clinical Center Maribor, Maribor, Slovenia
b Pharmacogenetics Laboratory, Institute of Bichemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
c Department of Biomedical and NeuroMotor Sciences, University of Bologna, Bologna, Italy
d University Psychiatric Clinic Ljubljana, Ljubljana, Slovenia
Background: Genes involved in the serotonin pathway may determine susceptibility for alcohol dependence and its severity. The present study explored whether specific polymorphisms in the serotonin pathway could be associated with alcohol dependence or alcohol-related psychopathological symptoms.
Methods: The cohort comprised 101 currently and 100 formerly alcohol-dependent males, as well as 97 male healthy blood donors. The following questionnaires were employed: AUDIT, Zung Depression and Anxiety Scale, Brief Social Phobia Scale, Yale-Brown Obsessive Compulsive Scale and Obsessive Compulsive Drinking Scale and Buss-Durkee Hostility Inventory. Subjects were genotyped for bi-and tri-allelic SLC6A4 5-HTTLPR, HTR1A rs6295 and HTR1B rs13212041.
Results: Statistic differences in bi-and tri-allelic SLC6A4 5-HTTLPR genotypes distribution were observed among the three groups investigated (p = 0.008 and p = 0.023 respectively), however no gene-dose effect was observed. The severity of alcohol problems was higher in currently alcohol-dependent subjects with 5-HTTLPR LL (p = 0.039) and L’L’ genotype (p = 0.027). Formerly dependent subjects with 5-HTTLPR S’S’ genotype showed more traits of social anxiety, depressive and anxiety traits (p = 0.009, p = 0.006 and p = 0.036 respectively). Healthy controls with 5-HTTLPR SS genotype showed more traits of social anxiety (p = 0.020).
Conclusions: Our findings suggest that bi-and tri-allelic SLC6A4 5-HTTLPR has some effects on the severity of alcohol dependence. The tri-allelic 5-HTTLPR was associated with social anxiety, anxiety and depressive traits in alcohol dependent subjects.
Alcohol dependence is characterized by frequent, compulsive and uncontrolled consumption of alcohol associated with behaviour of maladaptation and destruction. It is an etiologically and clinically heterogeneous syndrome, moderately to highly heritable, and caused by interaction of genes and environment. Alcohol dependence is related to other psychiatric diseases by common neurobiological pathways, including those that modulate reward, behavioural control as well as anxiety and stress response. A central serotonin 5-hydroxytryptamine or 5-HT deficit is thought to be involved in the pathogenesis of alcohol dependence by modulating motivational behaviour, neuroadaptive processes, and resulting emotional disturbance. 5-HT-related impulsive, aggressive, and suicidal behaviour has been linked to a primordial personality that is susceptible to alcohol dependence . Altered serotonergic neurotransmission is hypothesized to be central also to the pathogenesis of alcohol dependence besides a wide range of other central nervous system disorders .
5-HT is a monoamine neurotransmiter and is synthesized from the amino acid L-tryptophan via a short metabolic pathway that involves tryptophan hydroxylase (TPH). A 5-HT transporter protein called 5-HTT (SLC6A4 or SERT) acts as a regulator of 5-HT levels and plays a critical role in determining magnitude and duration of serotoninergic neurotransmission through 5-HT reuptake into presynaptic neurons . Although variations in many of the genes that encode receptors, enzymes, and transporters of the 5-HT system have been tested as risk factors for alcohol dependence, genetic analyses of 5-HT signaling in alcohol dependence have mainly been focused on the 5-HTT (SLC6A4) gene . The 5-HTT insertion/deletion (indel) promoter variant (5-HTTLPR or rs4795541) consists of a series of 22 base pairs (bp) repeats (Figure 1). A short allele (S) has 14 repeats and a long allele (L) has 16 repeats, resulting in a 44-bp insertion or deletion polymorphism . The long allele (L) is associated with twice the SERT expression at basal state compared to S allele. A functional polymorphism, rs25531 (A>G) within the L allele gives rise to a triallelic polymorphism (LA, LG, S). As LG and S allele were shown to be similar in terms of transcriptional activity , it is important to account for this polymorphism in association studies.
5-HTT (SLC6A4) gene has been extensively investigated in neuropsychiatric diseases because it is believed to be the principal site of action of the selective serotonin reuptake inhibitors (SSRIs). Genetic variability of SLC6A4 have been associated with response to antidepressants and antipsychotics [5, 6]
Figure 1. SLC6A4 location and allelic variation of the indel promoter polymorphism.
MAOA: Monoamine oxidase A. Copied with permission from Future Medicine Ltd 
Although 5-HTT (SLC6A4) gene has also been extensively investigated in alcohol dependence and alcohol related psychosymptomatology, the triallelic polymorphism has not been considered in most of these studies. We have recently performed an association study to explore among others whether bi- or tri-allelic (considering rs25531) 5-HTTLPR polymorphisms could be associated with alcohol dependence and/or with comorbid alcohol-related psychopathological symptoms . This study included 101 currently and 100 formerly alcohol-dependent subjects and also 97 healthy controls. Socio-demographic characteristics and assessment of hazardous and harmful alcohol consumption for the subjects included in the study are presented in Table 1.
Table 1. Socio-demographic characteristics of the subjects and assessment of hazardous and harmful alcohol consumption
Means ± standard deviations are given; or number of participants for categorical variables, AUDIT – Alcohol Use Disorders Identification Test
In our study we have observed statistically significant differences in SLC6A4 5-HTTLPR genotypes distribution among the currently and formerly dependent subjects and healthy controls (p = 0.009, Chi-square = 13.569, df = 4) . When we have merged both groups shared the diagnosis of alcohol dependence – either current or former, this merged group differed significantly from healthy controls regarding the frequency distribution of both bi- and tri-allelic 5-HTTLPR genotypes (p = 0.005, Chi-square = 10.739, df = 2 and p = 0.027, Chi-square = 12.640, df = 5, respectively). Furthermore, subjects with present or past alcohol dependence with 5-HTTLPR LL and L’L’ genotype expressed the most severe alcohol problems .
We have also applied AUDIT (Alcohol Use Disorders Identification Test) questionnaire that defines drinking habits and severity of alcohol problems and dependence in a group of currently dependent subjects. As shown in Figures 2 and 3 both bi- and tri-allelic 5-HTTLPR genotypes were significantly associated with the score on the AUDIT questionnaire. Among currently alcohol dependent subjects 5-HTTLPR L’ and L allele carrier status was associated with more severe alcohol problems. Furthermore, we have observed that the triallelic 5-HTTLPR genotype was also associated with social anxiety, anxiety, and depressive traits in alcohol-dependent subjects .
Figure 2: The associations between AUDIT questionnaire and bi-allelic 5-HTTLPR genotypes in currently dependent subjects
Figure 3: The associations between AUDIT questionnaire and predicted tri-alellic 5-HTTLPR genotypes (considering rs25531) in currently dependent patients
The observations of our recent study  are in agreement with several former studies investigating the effects of the serotonin pathway genetic polymorphisms on alcohol dependence and reporting that 5-HTTLPR L allele was significantly related to alcoholism and frequent comorbid psychopathological symptoms in alcohol dependence, although some studies reported conflicting results. Recently published meta-analysis showed that individuals with the homozygous S allele of 5-HTTLPR polymorphism are at increased risks of alcohol dependence , although another meta-analysis showed no such association . Only few studies were conducted considering tri-allelic 5-HTTLPR polymorphism. Recently published study on Italian population found no statistically significant differences for 5-HTTLPR and rs25531 polymorphisms in terms of either genotypes or alleles frequencies, but showed earlier onset of alcohol dependence in male carriers of low-functional S or LG allele , which supports the influence of possible gene × environmental interactions and gender differences. Another recent study suggested that the effects of 5-HTTLPR genotypes are sensitive to environmental interactions with low expression genotypes being more sensitive to negative stimuli, in particular when accompanied with poor social support .
It should be advisable to increase the number of alcohol dependent subjects and include both genders in future studies, with the aim to further evaluate the genetic basis and molecular mechanisms of alcohol dependence. Further studies are required to investigate the association between 5-HTTLPR polymorphism and alcohol dependence as well as gene × environmental interactions. Better understanding of the role of genetic variability in serotonergic pathway as well as the related gene-gene and gene-environment interactions may also have important implications for pharmacological treatment of alcohol dependence and alcohol-related psychosymptomatology with serotonergic drugs .
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The study was financially supported by the Ministry of Education, Science, and Sport of the Republic of Slovenia (Grant Nos P1-0170 and P3-0366).