Associations of NLRP3 and CARD8 gene polymorphisms with alcohol dependence and commonly related psychiatric disorders: a preliminary study

The study included only male participants (to exclude the influence of sex differences, see ref. 22) aged from 18 to 65 years from the Slovenian (Caucasian) population. The first group (group 1; N=88) included hospitalised alcohol-dependent patients who met the criteria of alcohol dependence of the 4^th edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (23) and were no longer having major abstinence symptoms (after having spent at least two weeks in respective departments/units for addiction treatment). The second group (group 2; N=99) included abstinent alcohol-dependent individuals recruited from support group meetings who had maintained full abstinence for more than two years. Participants of either group were excluded if they had a history of abuse or dependence of psychoactive substances other than nicotine and other major mental or neurological disorders or significant medical illnesses in their medical records. The third group (group 3; N=94) included healthy controls recruited from blood donors, who answered a short structured clinical questionnaire through interview to exclude DSM-IV Axis I disorders or clinical problems with alcohol consumption.

All participants also gave information about their residence (rural or urban), partnership status (single or in partnership: either married or living in the extramarital union), and years of education and signed informed consent.

The following questionnaires were employed: Zung Depression (24) and Anxiety (25) scale and Brief Social Phobia Scale (BSPS) (26) to rate depression and anxiety symptoms, Alcohol Use Disorders Identification Test (AUDIT) (27) to define drinking habits and severity of alcohol problems and dependence, Yale-Brown Obsessive Compulsive Scale (YBOCS) (28) and Obsessive Compulsive Drinking Scale (OCDS) (29) to rate obsessive-compulsive traits, and Buss-Durkee Hostility Inventory (BDHI) (30) to rate symptoms of aggression and hostility. All the questionnaires were administered at the entry into the study by the same rater, who was blinded to the genotyping results.

The study was approved by the Slovenian National Medical Ethics Committee (approval No. 117/06/10 and 148/02/1011) and followed the latest version of the Declaration of Helsinki (31). Each participant received a code to protect all the personal and medical information. All biological and DNA samples were processed and analysed under these codes.

Table 1 shows participant demographic characteristics of interest. Hospitalised and abstinent alcohol-dependent patients were significantly older (P<0.001; df=2; F=46.080) and had fewer years of education than controls (P<0.001; df=2; F=46.080). Significantly more abstinent alcohol dependents and controls were in partnership than hospitalised alcohol dependents (P=0.004; df=2; F=5.601).

Genotype distribution of CARD8 did not deviate significantly from the Hardy-Weinberg equilibrium (HWE), but we found a deviation for NLPR3 in controls (Table 2). The three groups significantly differed in the distribution of the CARD8 rs2043211 genotypes (P=0.049; chi-squared=9.557; df=4) but not in the distribution of the NLPR3 rs35829419 genotypes.

No significant association was found between the NLPR3 genotypes and selected mental disorder symptoms (Table 3). On the other hand, CARD8 rs2043211 genotypes were significantly associated with Zung Anxiety Scale scoring among abstinent alcohol-dependent participants (P=0.048; df=2, F=3.140). In this group, CARD8 rs2043211 TT genotype carriers reached the highest mean scores on the Zung Anxiety Scale (Table 4). In controls the CARD8 rs2043211 genotype was associated with OCDS scoring (P=0.027; df=2; F=3.744). However, because control candidates with clinical problems with alcohol consumption were excluded from the study and because OCDS measures obsessive-compulsive symptoms related to alcohol drinking, this result may be considered a chance finding that has no clinical importance.

We found that CARD8 rs2043211 was associated with the risk of alcohol dependence, but found no such association for NLPR3 rs35829419. However, our findings are limited only to males. A recent study on rodents (22) reported NLPR3 inflammasome-dependent differences in alcohol consumption between male and female mice, but there are no reports of similar kind in humans.

Another significant association we did observe is the one between CARD8 rs2043211 and anxiety in abstinent alcohol-dependent participants. This finding supports the hypothesis that inflammasome could contribute to the development of psychiatric disorders associated with alcohol dependence (33, 34, 35). To the best of our knowledge, this is the first such finding, as no human study conducted so far reported a direct association between CARD8 and anxiety in alcohol dependents.

The limitation of our study is a relatively small but ethnically homogeneous sample. The advantage is that all the questionnaires were applied by the same rater.

In conclusion, our findings point to a link between the genes of the innate immune system and alcohol dependence. However, further studies with larger cohorts are required to confirm these preliminary findings.