1 Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, 2 Department of Epidemiology, National Institute for Longevity Sciences, Ohbu, 3 Division of Gastroenterology, National Kyushu Cancer Center, Fukuoka, 4 Institute of Clinical Research, National Alcoholism Center, 5 National Alcoholism Center, Kurihama Hospital and 6 Department of Biochemistry and Cell Biology, Institute of Gerontology, Nippon Medical School, Kanagawa, Japan
* Author to whom correspondence should be addressed at: Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho Itabashiku, Tokyo 173-0015, Japan. Tel.: +81 3964 3241 (ext. 3088); Fax: +81 3579 4776; E-mail: miyasaka{at}tmig.or.jp
(Received 20 May 2003; first review notified 31 July 2003; in revised form 20 August 2003; accepted 26 August 2003)
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ABSTRACT |
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INTRODUCTION |
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Recent reports (Blum et al., 1990; Muramatsu et al., 1996
) in human subjects showed an association of polymorphisms of the dopamine D2 and/or D4 receptor gene with alcohol dependence, although results have been equivocal. In contrast, Okubo et al. (2000)
reported that the CCK gene polymorphism does not play a major role in alcohol withdrawal symptoms. Based on our recent finding of two sequence changes in the promoter region (a G to T change in nucleotide 128 and an A to G change in nucleotide 81; GenBank database accession number D85606; Funakoshi et al., 2000
), in the present study, we examined the association between CCK-AR gene polymorphisms and alcohol dependence.
Liver mitochondrial aldehyde dehydrogenase-2 (ALDH2) is responsible for metabolizing the acetaldehyde produced from ethanol into acetate. More than 40% of Asians have the inactive form of ALDH2, encoded either as heterozygous ALDH2*1/2*2 or homozygous ALDH2*2 (Higuchi et al., 1995), while the majority of Caucasians possess the active form of ALDH2 (2*1/2*1). A previous report (Murayama et al., 1998
) showed that the clinical characteristics of alcoholic patients having inactive ALDH2 differed from those of alcoholic patients with active ALDH2. In this study, we excluded subjects with inactive ALDH2 to avoid the influence of its overwhelming effect as a negative risk factor for alcoholism.
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SUBJECTS AND METHODS |
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The subjects consisted of 435 (aged 3274 years) Japanese male alcoholics who had been consecutively hospitalized at Kurihama Hospital. They were diagnosed as having DSM-III-R (American Psychiatric Association, 1987) alcohol dependence, based on the Structured Clinical Interview for DSM-III-R (SCID) assessment (Spitzer et al., 1990
).
The age-matched control subjects consisted of 1134 male participants in the NILS Longitudinal Study of Aging (LSA) (Shimokata et al., 2000) and 356 males who were Institute employees. They were free of alcohol dependence, based on the results of the Kurihama Alcoholism Screening Test, the most widely used alcoholism screening test in Japan, which was administered to potential controls before entering into this study.
First, the genotype of the ALDH2 gene was determined by mismatched polymerase chain reaction restriction fragment length polymorphism (PCRRFLP) method reported previously (Kamino et al., 2000). Then the CCK-AR gene polymorphism was determined in the subjects with ALDH2*1/2*1 (384 alcoholics and 792 controls).
Genotyping procedures
The polymorphism in the promoter region of CCK-AR gene was examined using a mismatched PCRRFLP method (Funakoshi et al., 2000). In brief, a pair of primers (sense primer = 5'-CATATGTACACATGTGTGTAAAAAGCAGCC-AGAC-3' and anti-sense primer = 5'-GCCCTTTCCTGGG-CCAGACT-3'), were designed to amplify the 103-bp product, which was subsequently digested with restriction enzyme HinfI, and analysed by 12% polyacrylamide gel electrophoresis. Six genotypes were identified: a wild type (81A/A, 128G/G); heterozygous mutant types (81A/G, 128G/G), (81A/G, 128G/T), (81G/G, 128G/G), (81G/G, 128G/T); and a homozygous mutant type (81G/G, 128T/T).
Clinical data
We used a structured clinical interview for DSM-III-R to diagnose alcohol dependence and antisocial personality disorder (Spitzer et al., 1990). We also used a structured interview to obtain responses to questions on social background as well as history of drinking and alcohol withdrawal. Family histories of alcohol dependence among all biological first-degree relatives were evaluated by using the Family History Research Diagnostic Criteria (Andreasen et al., 1977
). Age at onset of alcoholism was defined as the age at which the individual first met the DSM-III-R diagnostic criteria for alcohol dependence.
Statistical analyses
Statistical differences between alcohol-dependent and control subjects were assessed using the chi-squared test. A continuity correction was performed when the frequency of at least one cell was less than 5. An odds ratio (OR) with a 95% confidence interval (CI) was calculated to evaluate the genotype frequencies between groups. Probability differences of P < 0.05 were considered statistically significant. To assess the linkage disequilibrium between the two polymorphisms of the CCK-AR gene, we calculated the D value and its significance, using the ASSOCIAT program (downloaded from the website of J. Ott: ftp://linkage.rockefeller.edu/software/utilities/). All statistical computations were carried out using the Statistical Analysis System package, version 6.12 (SAS Institute, 1998).
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RESULTS |
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DISCUSSION |
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The comorbidity rate of antisocial personality disorder was only 2.3% and an average age at onset of alcohol dependence was around 40 years in our samples. These figures are substantially different from those of US alcoholic samples recruited from inpatient treatment settings (Hesselbrock et al., 1986; Raimo et al., 1999
). Although reasons are not clear, we have observed a relatively low comorbidity rate of antisocial personality disorder in Japanese alcoholic samples (Yoshino and Kato, 1996
; Murayama et al., 1998
). In addition, age of onset of our alcoholic samples is comparable to that of other Japanese alcoholic inpatients. (Murayama et al., 1998
). These comparisons suggest that our samples did not deviate from general Japanese alcoholic samples.
There have been several previous reports of CCK-AR gene polymorphisms (Inoue et al., 1997; Tachikawa et al., 2000
; Okubo et al., 2002
). Okubo et al. (2002)
determined five mutations, 388 (GT)8/(GT)9, 333G/T, 286A/G, 241G/A, and 85C/G in the promoter region of the CCK-AR gene, and reported a significant association between 85C to G change and alcoholic patients with hallucinations. However, once we had determined the transcriptional start site of the CCK-AR gene (Funakoshi et al., 2000
), we discovered that the 85 is not in the promoter region, but is in the 5' untranslated region. Okubo et al. (2002)
numbered not from the transcriptional start site but from the initial site of the coding region of exon 1. We examined CCK-AR gene polymorphisms in 50 patients with gallstone and 300 patients with diabetes mellitus before the establishment of the RFLP method (Funakoshi et al., 2000
). We found one case with G to A in intron 1, and another case with C to G in exon 3, without any change in amino acid (Thr). The polymorphisms of the promoter region (between 351 and +176) were also examined, and no polymorphisms other than 81A to G and 128G to T were detected. Those designated as 333G/T and 286A/G by Okubo et al. (2002)
were identical to 128G/T and 81A/G in the present study, respectively. No association of these polymorphisms (128G/T and 81A/G) with alcohol dependence was observed (Okubo et al., 2002
). One possible explanation for the differences between the study by Okubo et al. (2002)
and our study is that Okubo et al. (2002)
did not exclude subjects with inactive ALDH2. Inactive ALDH2 (2*1/2*2 and 2*2/2*2) is a strong negative risk factor for alcohol dependence (Higuchi et al. 1995
). Tachikawa et al. (2000)
reported an association of the 201A allele (201A/G is identical to 81A/G in the present study) of the CCK-AR gene with schizophrenia. Given the potential differences between alcohol dependence and other psychiatric disorders, our results do not completely contradict their findings.
We recently reported that functional comparison of the A and G variants of the 81 A/G polymorphism by luciferase assay demonstrated a slight decrease in the G variant, but no significant difference (Takata et al., 2002). However, we used STC-1 (Rindi et al., 1990
), established from a transgenic mouse expressing a viral oncogene under the control of the insulin promoter, because no human-derived cell line expressing CCK-AR was available. Further studies employing various experimental conditions are needed before conclusions can be drawn regarding the effect of this polymorphism on expression of the CCK-AR gene.
A recent report mapped the CCK-AR gene to chromosome 4 (4p15.215.1), in the vicinity of the dopamine D5 receptor gene (4p16.115.1) (Beischlag et al., 1995). The dopamine D5 receptor binds dopamine with a 10-fold greater affinity than that of dopamine receptor 1. The dopamine D5 receptor protein is also localized in the prefrontal cortex. Thus, alterations in the CCK-AR gene may lead to some modification of dopamine release, and alteration of dopaminergic neurotransmission may be involved in alcohol misuse (Crawley, 1991
; Marshall et al., 1991
; Woodruff et al., 1991
; Kalivas, 1993
; Ladurelle et al., 1994
; Hamilton and Freeman, 1995
; Self and Nestler, 1995
; Wank, 1995
).
In summary, the CCK-AR gene 81A/G polymorphism was found to be associated with alcohol dependence, and the 81G allele of the CCK-AR gene to be possibly associated with intractable alcohol dependence.
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ACKNOWLEDGEMENTS |
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