I.-G. Choi, Department of Neuropsychiatry, Hallym University, Hangang Sacred Heart Hospital, 94-200 Youngdungpo-Dong, Youngdungpo-Gu, Seoul 150-719, Republic of Korea. E-mail: firstname.lastname@example.org
The cholinergic muscarinic 2 receptor (CHRM2) gene has been considered a candidate gene for the alcohol dependence in that it might underpin certain risk factors for this condition. This study examined variations in the CHRM2 between the patients with alcohol dependence and population controls in Korean and explored the associations between CHRM2 polymorphisms and severity of symptoms in the patients with alcohol dependence. One hundred and fifty-five patients with alcohol dependence, defined by the Alcohol Use Disorders Identification Test (AUDIT) and the Alcohol Dependence Scale (ADS) to measure the severity of symptoms, and one hundred and ninety-five population controls were drawn in the study. Three single nucleotide polymorphisms (SNPs) of CHRM2 were genotyped using the TaqMan assay and analyzed with the severity of symptoms of alcohol dependence. We found that although SNP rs324650 showed marginal association with the risk of alcohol dependence (P = 0.03), the significance of the result was not sustained after multiple corrections. SNP rs1824024 was significantly associated with the AUDIT and ADS scores in patients (P = 0.005 and 0.003, respectively). These findings suggested that the muscarinic acetylcholine function might be related not with alcohol dependence itself but with the severity of alcohol dependence in Korean population.
Alcohol dependence has both genetic and environmental risk factors, and numerous candidate genes for the neurotransmitter systems, including opioidergic, serotonergic, glutamatergic, dopaminergic, GABAergic and cholinergic system, have been suggested for alcohol dependence (Heinz et al. 2004). Some studies of genome-wide linkage analyses have reported a locus for genetic susceptibility to alcohol dependence on the long arm of chromosome 7 (Foroud et al. 2000; Reich et al. 1998), where the cholinergic muscarinic 2 receptor (CHRM2) gene is located.
The muscarinic acetylcholine receptors belong to a class of metabotropic receptors, which use G proteins as their signaling mechanism. They are distributed throughout the central nervous system and influence a multitude of signaling pathways important for modulating neuronal excitability, synaptic plasticity and feedback of acetylcholine release (Volpicelli & Levey 2004). These cholinergic neurons project to cortical and limbic structures and they are considered to be involved in many cognitive brain functions, including attention, learning and memory, by optimizing cortical information processing (Baxter & Chiba 1999).
Three single nucleotide polymorphisms (SNPs) in introns 4 and 5 downstream of the CHRM2 have been reported to increase the risk of alcohol dependence as part of the Collaborative Study of the Genetics of Alcoholism (COGA) (Wang et al. 2004). Using a regression framework, Luo et al. (2005) provided support for an association between variations in CHRM2 gene and alcohol dependence in subjects with European-American and African-American, although they did not show a significant association with SNPs of the CHRM2. They reported Hardy–Weinberg disequilibrium for CHRM2 rs1824024 in several subgroups, which were divided by disease (alcohol dependence, drug dependence and affective disorder) and race. The CHRM2 rs1824024 is located in intron 4 and associated with alcohol dependence in the previous result from COGA sample as well. These findings suggest strong associations between SNPs of the CHRM2 and alcohol dependence. In addition, Luo et al. (2007) reported with a subsample of their previous study including that CHRM2 variations associated with lower conscientious scores and higher agreeableness scores assessed by Neuroticism-Extroversion-Openness (NEO) Five-Factor inventory (Costa & McCrae 1997).
Based on the evidence noted above, CHRM2 might underlie the risk of alcohol dependence, and it could be considered as a candidate gene for examining phenotypes of this condition. In the present study, we examined the variations in the CHRM2 between the patients with alcohol dependence and controls drawn from Korean population. In addition, we investigated the relationships between CHRM2 polymorphisms and the severity of symptoms in patients with alcohol dependence.
Materials and methods
Study subjects and genotyping of polymorphisms
We recruited 155 patients (male/female: 150/5; mean age: 47.56 ± 9.98) who met the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) criteria for alcohol dependence (American Psychiatric Association 1994). Participants were recruited from Hangang Sacred Heart Hospital, Yong-In Mental Hospital, Hanmaum Hospital, Gumin Hospital and Hando Hospital. The patients who were a subgroup of our previous study had neither major medical nor comorbid psychiatric illnesses other than alcohol-related disorders (Joe et al. 2008; Kim et al. 2008). The population controls were unrelated healthy employees of the Center for Health Promotion of Seoul National University Hospital (Seoul, Republic of Korea) with an age range of 28–80 years (n = 195, male/female: 109/86; mean age: 53.58 ± 10.05). The institutional review board of each hospital approved the study, and all subjects provided informed consent.
The Alcohol Use Disorders Identification Test (AUDIT) (Bohn et al. 1995) and the Alcohol Dependence Scale (ADS) were used to measure the severity of alcohol-related symptoms (Kivlahan et al. 1989). The AUDIT is a 10-item instrument used to identify harmful or hazardous patterns of alcohol consumption. This instrument assesses quantity and frequency of drinking, alcohol intake, symptoms of dependence and tolerance and alcohol-related problems during the past 12 months. Each item is scored from 0 to 4. The ADS is one of the most widely used instruments for the severity of alcohol dependence and consists of a 25-item multiple-choice questionnaire, yielding a score from 0 to 40.
Three SNP loci in the CHRM2 gene, including rs1824024 G>T, rs324650 T>A and rs1424548 G>A (CHRM2 in previous studies), were genotyped using the TaqMan method (Livak 1999), which has been described in our previous work (Kim et al. 2008).
The χ2 tests were used to determine if the individual variant was in Hardy–Weinberg equilibrium (HWE). A widely used measure of the linkage disequilibrium between all pairs of biallelic loci, Lewontin's D′ (|D′|) (Hedrick 1987; Hedrick & Kumar 2001) was used, and r2 was calculated. Haplotypes of each individual were inferred using the phase software version 2.1 (Stephens et al. 2001). Using this software, the phase probabilities of all polymorphic sites for haplotypes were calculated for each individual. Individuals with phase probabilities less than 97% were excluded from the analysis. The genetic effects of the inferred haplotypes were analyzed in the same way as were the polymorphisms. Logistic regression analyses were used to calculate the P-values for case–control analysis while adjusting age and sex as covariates, so the regression analysis was used to examine the associations between the polymorphisms and alcoholic phenotypes while adjusting for age. All statistical analyses were performed using sas 9.1 (SAS Institute, Cary, NC, USA). P-value was corrected for six times using Bonferroni's correction and P < 0.008 (0.05/6) was considered as statistically significant.
The three polymorphisms in CHRM2, which accounted for more than 10% of the minor allele frequencies in Asian population of International HapMap DB among the six SNPs (Luo et al. 2005), were genotyped and analyzed for the replication in Korean population in the present study. The allele frequency, heterozygosity and HWE coefficient were assessed (Table 1). Linkage disequilibria among SNPs were measured by calculating Lewontin's D′ and r2 values. CHRM2 rs324650 T>A and rs1424548 G>A showed tight linkage with each other, whereas rs1824024 G>T showed no linkage with the other two SNPs. Haplotypes of CHRM2 were constructed with only rs324650 T>A and rs1424548 G>A. Two major haplotypes accounted for over 98.1% of the distribution (Fig. 1). CHRM2 haplotypes were not analyzed because CHRM2-ht1 and -ht2 were almost equivalent to CHRM2 rs324650 T>A, and CHRM2-ht3 and -ht4 showed very low frequency [minor allele frequency (MAF) <0.02].
Table 1. Allele frequencies and Hardy–Weinberg equilibrium (HWE) of CHRM2 polymorphisms in Korean subjects
In the initial analysis, the allele frequencies for each polymorphism were analyzed for the alcoholic patients and population controls using logistic regression models. We found that the CHRM2 rs324650 T>A showed marginal association with the risk of alcohol dependence, but the significance of this association was not sustained after multiple correction (Table 2). In additional analyses of alcohol-related phenotypes using multiple regression models, CHRM2 rs1824024 G>T was significantly associated with the AUDIT and ADS scores of alcoholic patients (Table 3). Patients with the ‘GG’ genotype of CHRM2 rs1824024 G>T showed higher AUDIT and ADS scores than did those with other variations (P = 0.005 and 0.003, respectively).
Table 2. SNP genotype and allele distributions of CHRM2 gene polymorphisms in patients with alcohol dependence and population controls in Korean (controlling age and sex; n = 350)
C/C, C/R and R/R indicate common homozygotes, heterozygotes and minor homozygotes, respectively. Data are given as the number of patients with alcohol dependence (means ± SD of AUDIT or ADS scores). Significant values (P < 0.008) are highlighted in bold.
*P-values of codominant models.
57 (19.74 ± 7.90)
71 (24.65 ± 9.16)
13 (24.23 ± 7.73)
100 (22.92 ± 8.97)
41 (21.90 ± 8.50)
134 (22.69 ± 8.76)
7 (21.43 ± 10.58)
57 (13.74 ± 10.30)
68 (19.88 ± 11.30)
12 (20.58 ± 9.86)
98 (17.74 ± 11.27)
39 (16.49 ± 10.89)
130 (17.32 ± 11.11)
7 (18.71 ± 12.46)
We found that the common homozygote of CHRM2 rs1824024 was significantly associated with the AUDIT and ADS scores of patients with alcohol dependence in Korean population. Although CHRM2 rs324650 T>A was also significantly associated with the risk of alcohol dependence in Korean population, the significance of this result was not sustained after controlling confounding variables. The SNP rs1824024, which was found to be significantly associated with the severity of alcohol dependence, had been reported to be associated with alcohol dependence in COGA sample, of which 82% is Caucasian and 15% is African-American (Wang et al. 2004). They had examined 11 SNPs spanning the CHRM2 gene and reported that 3 SNPs including rs1824024 had been significantly associated with alcohol dependence, that is rare alleles of 3 SNPs of the CHRM2 gene are relatively more frequent in the patients with alcohol dependence. This is in the same direction of our result, because the rare allele of rs1824024 is relatively more frequent in the severe form of alcohol dependence in our sample, although the rare alleles of rs1824024 were different between the previous COGA population and this Korean population. It is the same phenomenon as the association of the functional serotonin transporter promoter polymorphism with alcohol dependence. The rare variants (short allele in Caucasians, long allele in Korean population) of the serotonin transporter linked polymorphic region (5-HTTLPR) were relatively more frequent in the patients with alcohol dependence (Feinn et al. 2005; Kweon et al. 2005). A case–control study among European-American and African-American patients with alcohol dependence also had found that CHRM2 might be important in the development of this disorder (Luo et al. 2005).
Luo et al. (2007) suggested that CHRM2 might contribute to the genetic component of variation in personality traits. They proposed that CHRM2 polymorphisms might control the properties of the CHRM2 receptor, and affect directly cholinergic activity and indirectly cholinergic–dopaminergic or cholinergic–adrenergic balance, which might contribute to the development of personality and the behavioral effects of drugs of abuse including alcohol dependence.
In the previous study, the effects of the alcohol and aldehyde dehydrogenase genes (ADH1B and ALDH2) had huge impacts on disease phenotype of alcohol dependence in Korean population (Chai et al. 2005; Kim et al. 2008). The impact of these genes on alcohol dependence overrides the effects of any other neurotransmitter genes on alcohol dependence, especially in Asian population whose genes for alcohol metabolism have unique alleles that strongly prohibit alcohol drinking and prevent alcoholism, different from other ethnic groups such as Caucasian and African (Kimura et al. 2009). Thus, our findings that common homozygote of CHRM2 rs1824024 is significantly associated with the severity of alcohol dependence support other previous results, and we speculate that SNPs of CHRM2 gene are associated with alcohol consumption behavior or severity of alcohol dependence rather than alcohol dependence itself in East Asian ethnic group.
Our study had some methodological limitations, including the disproportionate distribution of sex and age between the patient and control groups and the population-based association design. More specifically, nearly all of the patients with alcohol dependence were men, and there was a lack of characterization of population controls including consumption of alcohol, so we calculated the association between CHRM2 gene and severity of alcoholism only in the patients with alcohol dependence. We used logistic regression analysis in order to correct any possible error of judgment. On the other hand, a report that the population-based association design was more powerful than the family-based design for the detection of gene–gene interactions should also be noted in this regard (Wang & Zhao 2003). This study also has a rather serious limitation, because the sample size is quite a small for genetic analysis. However, it still has a value of cross-population replication.
In conclusion, although CHRM2 rs324650 was marginally associated with the risk of alcohol dependence in Korean population, no significant associations emerged in this regard. The common homozygote of CHRM2 rs1824024 was significantly associated with the AUDIT and ADS scores. It is suggested that the muscarinic acetylcholine function might be related not with alcohol dependence itself but with the severity of alcohol dependence. Further investigation in other ethnic groups is needed to clarify the relationship between the muscarinic cholinergic function and the alcohol dependence and/or its severity.
This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A084589). All authors declare they have no conflict of interest.