1 Center for Genomics and Bioinformatics and 2 Department of Clinical Neuroscience, Division of Psychiatry, Karolinska Institutet, Stockholm, Sweden
* Author to whom correspondence should be addressed at: Karolinska Institutet, Neurotec Department, Division of Psychiatry, Huddinge University Hospital, M57, SE-141 86 Stockholm, Sweden. Tel.: +46 8 58 58 6666; Fax: +46 8 58 58 5785; E-mail: markus.heilig{at}neurotec.ki.se
(Received 24 May 2004; first review notified 28 May 2004; in revised form 24 June 2004; accepted 29 June 2004)
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ABSTRACT |
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INTRODUCTION |
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Alcohol dependence is a heterogeneous diagnostic category. Late-onset alcoholism or Type 1 found in both men and women, is characterized by a late age of onset of heavy drinking, and anxious personality traits, and has a less severe course. Early-onset alcoholism (defined as onset of heavy alcohol consumption before 25 years of age) or Type 2 is more common in males, and affects 25% of all male alcoholics. Early-onset alcoholics are frequently characterized by a history of hyperactivity, antisocial personality traits, alcoholic fathers, lower mean platelet monoamine oxidase activity, and a strong urge to consume alcohol in response to an intravenous infusion of the serotonin 2C receptor agonist meta-chlorophenyl-piperazine (George et al., 1997; Hommer et al., 1997
).
Serotonergic disturbances and loss of control are common features in suicide attempters (Asberg, 1997), violent offenders (Linnoila et al., 1994
; Virkkunen and Linnoila, 1997
) and alcoholics (Ballenger et al., 1979
). Depressed alcoholics, compared with never-depressed alcoholics, have been shown to have a higher daily alcohol intake, more lifetime diagnoses of other anxiety and affective disorders and drug abuse, more suicide attempts, and more reported alcoholism in both parents. There seems to be evidence for a shared serotonergic pathway between obesity and depression (Roberts et al., 2000
). Feeding disorders, described as a loss of control, have been reported to involve serotonin receptor genes and associations observed for polymorphisms within these genes (Ricca et al., 2002
; Rosmond et al., 2002
; von Meyenburg et al., 2003
; Murphy et al., 2004
; Pooley et al., 2004
).
The prevalence of alcohol dependence is markedly gender dependent, with an approximately 3:1 male to female ratio (Kessler et al., 1994). This indicates the possible involvement of sex-related factors in the pathogenesis of this syndrome. Additional gender differences may also exist. Women show higher peak blood-alcohol levels than men on the same alcohol dose (Thomasson, 1995
). A number of imaging studies have investigated gender-specific vulnerability of the brain to alcohol (Jacobson, 1986
; Mann et al., 1992
; Hommer et al., 1997
; Agartz et al., 1999
; Hommer et al., 2001
). These studies of alcohol dependent subjects have suggested that alcoholic women have an equal or greater degree of brain damage compared to alcoholic men despite fewer years of heavy drinking. However, studies indicating lack of evidence for a gender difference in the vulnerability of the brain to the effects of alcohol have also been published (Pfefferbaum et al., 2001
).
Considering the gender differences in the prevalence of alcohol dependence and in responses to alcohol, and a pivotal role of the serotonergic system in behavioural traits related to alcoholism, substance abuse and loss of control, we investigated the role of the gene of an X chromosome localized serotonergic neuroreceptor in an alcohol dependent population of Nordic origin. We designed a classical casecontrol association study for four promoter single nucleotide polymorphisms and one promoter microsatellite of the serotonin 2C receptor gene (HTR2C). Of the four SNPs, one (rs3813928) (Yuan et al., 2000) has earlier been implicated in low BMI in Japanese population. The polymorphisms were genotyped in a population of 317 females (cases = 127; controls = 190) and 392 males (cases = 309; controls = 83), for possible haplotype and allele associations.
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SUBJECTS AND METHODS |
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Diagnostic categories
The main categories for analysis were alcohol dependent (i.e. confirmed DSM-III-R diagnosis of alcohol dependence as described above), or unaffected (i.e. lack of any substance related diagnosis). A subgroup analysis was carried out by independently examining a possible association in Type 1 and Type 2 alcoholics, respectively. For the 127 female cases, 91 could be classified as Type I and 26 as Type II. For the 309 male cases, 145 could be classified as Type I and 90 as Type II. The Structured Clinical Interview for DSM-III-R (SCID) was administered to both alcoholics and controls to establish or exclude a diagnosis of alcohol dependence, respectively. In the dependent subjects, sufficient clinical information was available to additionally allow sub-typing into Type 1 (late onset) and Type 2 (early onset) alcoholism according to Cloninger et al. (1981) using the revised criteria published in Hallman et al. (1996)
. All subjects provided informed consent before entering the study. All the subjects were 17 years or older at the time of the psychiatric interview. These protocols, including patient information and consent forms, were approved by the Stockholm South (patients) and Stockholm North (controls) Human Subjects Ethics Committee.
Genotyping
The genomic sequence and allele information for SNPs in 5-HT2Rc were obtained from the SNP database at the US National Center for Biotechnology Information (NCBI). The SNPs were referred to by their Reference Cluster ID (rs#) as given at the NCBI website (www.ncbi.nlm.nih.gov/SNP/). Genotyping employed the dynamic allele-specific hybrid- ization (DASH) method (Howell et al., 1999). Genotypes were scored from fluorescence curves as previously described. Table 2 shows the sequences of the primers and probes used for the DASH assay. The primer sequences used for PCR amplification, were modified to amplify a DNA product with the least stable secondary structures, using Michael Zuker's program mFold (www.bioinfo.math.rpi.edu/~mfold/dna/form1.cgi). Each of the two alleles were probed independently using specific oligo-primers (allelic variations are underlined in Table 2). All PCR reactions were performed on a MJ Research PTC-225 (Wellesley, MA). The DNA polymerase (AmpliTAQGoldTM, Applied Biosystems, Foster City, CA) was activated by heating the PCR mixture at 98°C for 10 min. The amplification was carried out by 40 cycles of 98°C (10 s) and 55°C (30 s). The HTR2C GT microsatellite was genotyped using FAM labeled primers flanking the repeat designed based on Genbank entry GI:4753292, using CPrimer and ordered from Life TechnologiesTM (Table 2). PCRs were carried out implementing the same instrumentation as above, however, denaturing at 95°C and with extension at 72°C. Genotyping was performed at the National Swedish Center for Large-Scale Genotyping (www.genomecenter.uu.se/) using an ABI PRISM® 3700 Capillary DNA Genotyper. Allelic lengths were determined using GeneScan® Genotyper® software (Applied Biosystems).
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RESULTS |
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DISCUSSION |
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Individual SNP analysis in this study showed no significant association with alcohol dependence, neither when the whole population was considered, nor when sub-groups of males, females, Type 1 and Type 2 were individually tested.
The frequencies of the most common haplotypes for the gender-based subgroups of the cases and controls were calculated but failed to show significant differences between the groups. On correction for multiple testing, no significant association with alcohol-dependence could be observed for any of the haplotypes in any of the groups within this population. The power of this study to detect weak genetic effects of HTR2C in alcohol dependence in both males and females was hampered by the initial sample size. Under these conditions, considering a dominant model in males the power to detect genetic risks of 1.6 (OR) was 37%. However, if controls were increased to match the number of cases there would be sufficient power (81%) to detect such genetic influences. In conclusion, although this study reports a lack of association between the markers tested and alcohol dependency, there seems to be a clear tendency indicative of a haplotype selection in males Type I which could be significantly apparent in a follow up study with more power.
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ACKNOWLEDGEMENTS |
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