Department of Psychiatry, Ludwig-Maximilians Universität München, Nußbaumstr. 7, 80336 München, Germany
Received 16 May 2001; in revised form 20 July 2001; accepted 22 August 2001
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ABSTRACT |
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INTRODUCTION |
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Group I metabotropic glutamatergic receptors comprise of mGluR1 and mGluR5, which are linked via G-proteins to activation of phospholipase C. Group II (mGluR2 and mGluR3) and Group III (mGluR4 to mGluR8) are both negatively-linked to adenylyl cyclase activation (Chapman, 2000). Pharmacological activation of these group II and III receptors by selective agonists appears to have mixed convulsant (Ghauri et al., 1996
) and anticonvulsant action (Tizzano et al., 1995
; Tang et al., 1997
). The Group III receptors mGluR7 and mGluR8 are reportedly located within the presynaptic grid of the glutamatergic synapse (Shigemoto et al., 1996
) and thus may provide significant control of glutamatergic synaptic transmission (Friedl et al., 1999
).
The Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release (Gasparini et al., 1999). Experimental absence seizures were reported to be associated with perturbations in the presynaptic release of gamma-aminobutyric acid (GABA) and glutamate within thalamocortical circuitry. The release of both glutamate and GABA is regulated by Group III mGluR (Snead et al., 2000
).
Experimental studies in vitro and in vivo have given evidence that severity of withdrawal and seizures might be genetically influenced (Sander et al., 1997; Dvirskii, 1999
). For instance, a previous study reported an association between dopamine transporter polymorphisms and withdrawal seizures or delirium tremens in 293 alcohol-dependent individuals (Sander et al., 1997
). Further evidence that mGluR might be involved in the pathogenesis of alcohol withdrawal-induced seizures and delirium tremens come from animal studies. A mGluR7-knockout mouse has been bred and found to develop spontaneous seizures, supporting an anticonvulsant role for this receptor (Bushell et al., 1996
).
For the mGluR7, a Tyr433Phe polymorphism with possible functional relevance has been previously reported (Makoff et al., 1996) and was confirmed as a polymorphism by SbspEI restriction enzyme assay (Bolonna et al., 2001
). For the mGluR8 receptor a C2756T polymorphism from an association study investigating the association between mGluR7 and mGluR8 polymorphisms with idiopathic epilepsy was reported (Goodwin et al., 2000
).
Of the Group III mGluR, only mGluR7 has a wide distribution throughout the CNS including the hippocampus (Ohishi et al., 1998), whereas mGluR8 is also expressed in the hippocampus, particularly the lateral perforant path (Shigemoto et al., 1996
). This makes it a prime candidate in hippocampal kindling which may be an important factor generating alcohol withdrawal-induced seizures and delirium tremens (Dietrich et al., 1997
).
The aim of this study was to investigate the association between mGluR7 and mGluR8 polymorphisms and a history of epileptic seizures or delirium tremens during alcohol withdrawal in an inpatient alcohol-dependent sample.
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MATERIALS AND METHODS |
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Age of onset of alcohol dependence was assessed by computing the mean of retrospectively obtained first alcohol dependence age of onset criteria as mentioned in DSM-IV by the SSAGA: higher consumption of alcohol than intended, attempts to stop or control alcohol consumption, significant time spent to consume alcohol or recover from alcohol intake, regular withdrawal symptoms during important daily obligations like school or work, reduction of important occupational or private activities because of alcohol intake, continued alcohol consumption despite the occurrence of psychological or physical harm and occurrence of 50% higher tolerance to alcohol effects. Daily alcohol intake was obtained using the typical daily average alcohol consumption of one week during the last 30 days before admission. Pure alcohol intake was computed in g/day. Duration of alcohol dependence was computed as the difference between age and age of onset.
Control persons of the general population were recruited from different locations (e.g. libraries, road-works, big stores), representing different social groups from unskilled worker to university graduates. In all control persons, a comprehensive medical and psychiatric assessment was carried out together with routine laboratory screening to exclude severe physical or psychiatric axis I or axis II disorders such as schizophrenia, depression, personality disorders and substance dependence including alcohol dependence.
In order to assess psychological problems, personality traits and possible present psychopathological symptoms were evaluated using the MMPI (Minnesota Multiphasic Personality Inventory; Endler et al., 1989), one of the most commonly used personality questionnaires in clinical practice. All persons with first-degree relatives suffering from any axis I disorders or alcohol dependence were excluded. All patients and controls were Caucasian and of German descent to avoid ethnic stratification effects.
Genotyping
Genomic DNA was isolated from whole blood according to standard procedures. The mGluR7 and mGluR8 polymorphisms were detected by PCR amplification, restriction enzyme digestion, electrophoresis on 3% agarose gels and visualization by ethidium bromide staining with the following conditions: mGluR7 (Tyr433Phe): forward primer: 5'-GAC AGG AGA GAA TTG GAA AAG ATT C-3'; reverse primer: 5'-ATC TCT GGG CAG ACA CTC CGG-3'. PCR was carried out in a final volume of 25 µl containing 50 ng genomic DNA, 200 µM of each dNTP, 100 µM 7-deaza-GTP, 5% DMSO, 0.5 µM of the forward and reverse primer and 0.625 units Hot Start Taq DNA polymerase. After an initial denaturation step of 95°C for 10 min, there were 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min. A final step was performed at 72°C for 7 min. The 144 bp product was digested with 10 units BspEI according to the manufacturer's instructions leading to products of 144 bp (A allele, wildtype) or 122 bp and 22 bp (T allele). For mGluR8 (C2756T): forward primer: 5prime;-TTC CTC TAC CAA GAC AAC AT-3prime;; reverse primer: 5'-GGG TTT CTT CAC TCC CCG TT-3'. PCR and cycling conditions were the same as for the mGluR7 polymorphism except for an annealing temperature of 53°C. The 233 bp PCR product was digested with 10 units BsmAI leading to products of 132 bp, 68 bp and 33 bp (C allele, wildtype) or 132 bp and 101 bp (T allele).
Statistics
Statistics were performed using SPSS software (Statistical Package for Social Sciences, Version 9.0, SPSS Inc, Chicago, 1997). All continuous data were tested for normal distribution.
The relationships between age, alcohol dependence criteria such as age of onset, duration of alcohol dependence and daily alcohol intake across mGluR7 and mGluR8 genotypes were computed using one-way analysis of variance. Post hoc tests (Scheffé's test) were applied when necessary.
The relationships between the frequencies of seizures and delirium tremens with genotypes were tested using 2 statistics. A two-tailed
-significance level of P < 0.05 was defined to be statistically significant.
Ethical standards
Informed consent was obtained from patients and controls after complete and extensive description of the study. The ethical committee of the Ludwig-Maximilians-University of Munich approved the study. All patients signed a written informed consent.
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RESULTS |
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Genotype results
Genotyping the DNA in our sample, we observed a frequency for the mGluR7 A allele of 58 and of 42% for the T allele. Homozygosity for the AA genotype was found in 40% (n = 72) of alcohol-dependent individuals, 45% had the AT genotype (n = 82) and 15% (n = 28) were homozygous for the TT genotype (Table 2). Our data followed the HardyWeinberg equilibrium (
2 = 0.321, df 2, P = 0.852). The frequency of the mGluR8 C allele was 45%, whereas that for the T allele was 55%. Homozygosity for the CC genotype was found in 19% (n = 34) of alcohol-dependent individuals, 53% had the CT genotype (n = 96) and 28% (n = 52) were homozygous for the TT genotype (Table 2
). Again, our data followed the Hardy Weinberg equilibrium (
2 = 0.807, df 2, P = 0.668). These allele frequencies were slightly different from the results obtained by Goodwin et al. (2000), who reported genotype frequencies of the mGluR7 (AA: 31%; AT: 56%; TT: 13%) and the mGluR8 polymorphism (CC: 19%; CT: 39%; TT: 42%) from a sample of epileptic patients in England. Thus, there might be a greater variability between several ethnic samples within Europe.
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Clinical parameters like age of onset, mean duration of alcohol dependence or patients mean daily alcohol intake showed no significant association with allelic distributions (see Table 2).
Alcohol withdrawal-induced seizures and mGluR7 and mGluR8 polymorphisms
Alcohol-dependent individuals with a history of alcohol withdrawal-induced seizures showed a weak relationship with the mGluR7 T allele compared to healthy controls [2 = 4.519, df 2, P (two-sided) = 0.037, Table 3
]. No association was found between mGluR7 and mGluR8-alleles and alcohol-dependent individuals with a history of alcohol withdrawal-induced seizures and those without (Table 3
).
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DISCUSSION |
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First, a weak association between mGluR7 Tyr433Phe T-allele and alcohol withdrawal-induced seizures was found. Regarding the multiple testing between mGluR polymorphism, delirium tremens and alcohol withdrawal-induced seizures performed in our sample, a more stringent criterion should be used for statistical significance than the conventional P < 0.05 level. A recent article (Perenger, 1998) reviewed a number of reasons why Bonferroni adjustments of the level of significance are unnecessary and deleterious to sound statistical inference, such as the increase of type II errors and the number of tests which might have been performed with data. These reasons are also valid for this study and thus no Bonferroni correction was performed. However, in a recent article on problems with the candidate gene approach (Buckland, 2001
), significantly lower alpha-significance levels of P = 0.0000125 were reported to be required to get a 95% likelihood of a true positive result in association tests. Hence, no significant association between phenotype and mGluR7 polymorphism can be recognized at this significance level. The influence of this polymorphism on alcohol withdrawal-induced seizures or delirium tremens remains doubtful. However, due to its possible functional relevance from subsequent amino-acid exchange (Tyr
Phe) it might provide further evidence in larger samples of alcohol-dependents with a history of alcohol withdrawal complications.
This result indirectly supports previous research (Goodwin et al., 2000) which reported no association between the two polymorphisms and idiopathic generalized epilepsy (IGE). Subjects with a known idiopathic generalized epilepsy and alcohol withdrawal are reported to be at a higher risk for alcohol withdrawal-induced seizures during alcohol withdrawal and have even been suggested to have a possible common genetic background (e.g. Marchal, 1999
; Samochowiec et al., 1999
).
As reported for IGE (e.g. Sander, 1996), a complex inheritance and genetic heterogeneity of the human susceptibility to both IGE and alcohol withdrawal-induced seizures may be present. The influence of a single gene or polymorphism might be very small and difficult to reveal. Another important candidate gene from the glutamate system, such as a glutamate transporter (EAAT2) polymorphism has been reported to have no association with alcohol withdrawal-induced seizures (Sander et al., 2000
).
Recent reports on the same sample from the Berlin research group (Samochowiec et al., 1999; Sander et al., 2000
) did not reveal an association of 5-HT2C receptor and glutamate transporter (EAAT2) polymorphisms with alcohol withdrawal-induced seizures. Thus, in the light of the lack of definitive results, the genetic background of alcohol withdrawal-induced seizures and the possible role of glutamate system candidate gene polymorphisms remains to be explored.
No association between alcohol withdrawal-induced delirium tremens and the mGluR7 and mGluR8 polymorphisms was found in the present work. This is the first study conducted on metabotropic glutamate receptor polymorphisms and alcohol withdrawal-induced seizures. Previous studies have reported an association between dopamine transporter polymorphisms and withdrawal seizures or delirium tremens in 293 alcohol-dependent subjects (Sander et al., 1997). In mice, an association between quantitative trait loci in proximity to genes that directly or indirectly affect gamma-aminobutyric acid (GABAA) receptor-mediated transmission and withdrawal intensity have been reported (Buck and Finn, 2001
). Decreased GABAA-mediated inhibition during alcohol withdrawal has been related to increased glutamate-mediated excitability and both may be critical mechanisms of alcohol withdrawal-induced seizures (Faingold et al., 2000
).
Limitations of this study include a retrospective design. Our data were obtained retrospectively from inpatient alcoholics after alcohol withdrawal using a semi-structured interview and later crosschecked with inpatients' clinical files.
Furthermore, the number of alcohol-dependent patients with delirium tremens and seizures was a subgroup of the patients investigated. Consequently, the number of patients with this phenotype compared to the control group was small (seizures: n = 46, 25%; delirium tremens: n = 37, 20% of the patients). In our sample, at an -level of 0.05 with an
of 0.147 (Cohen, 1988
), a sample size of n = 443 would be needed to achieve a power of 0.80. Thus, a larger sample size is needed to detect significant differences in this association at a low to moderate effect size.
Due to the exploratory character of this study, it should be mentioned that, in a phenotype with a complex inheritance, a number of genes and polymorphisms might be involved. Each of these genes may contribute in a minor fashion to the phenotype and might be only detected in very large samples with sufficient power if a very low significance level is applied. Thus, general conclusions about the specificity of our findings should be drawn with care. A second independent sample with increased power is needed to confirm the possible role of the mGluR7 and mGluR8 receptors in alcohol withdrawal-induced seizures and delirium tremens.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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