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Apolipoprotein E gene polymorphism and previous alcohol withdrawal seizures
JOURNAL OF PSYCHIATRIC RESEARCH
Journal of Psychiatric Research 41 (2007) 871–875
www.elsevier.com/locate/jpsychires
Apolipoprotein E gene polymorphism and previous alcohol withdrawal seizures Julia Wilhelm a
a,*
, Nicolas von Ahsen b, Thomas Hillemacher a, Kristina Bayerlein a, Helge Frieling a, Johannes Kornhuber a, Stefan Bleich a
Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, D-91054 Erlangen, Germany b Department of Clinical Chemistry, University of Go¨ttingen, Germany Received 14 May 2006; received in revised form 9 July 2006; accepted 19 July 2006
Abstract Aim of this study was to investigate the possible association of apolipoprotein E (ApoE) gene polymorphism with a history of alcohol withdrawal seizures. We included 194 patients with alcohol dependence who were divided into patients with (SZ+) and without (SZ ) previous alcohol withdrawal seizures. ApoE genotypes were determined using PCR. For statistical analysis we examined the number of ApoE alleles (ApoE2: n = 36; ApoE3: n = 311; ApoE4: n = 41). A significant positive association with a positive history of withdrawal seizures (SZ+) was found in the ApoE3 allele group (Fisher’s exact test: p = 0.006) while a significant negative association was observed in the ApoE2 allele group (Fisher’s exact test: p = 0.029). For the ApoE4 allele group no significant differences were found regarding a history of withdrawal seizures. Our findings suggest an association between the apolipoprotein E3 gene variant and an elevated risk of alcohol withdrawal seizures. These preliminary results must be validated in further studies. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Apolipoprotein E; Alcohol withdrawal; Seizures; Alcoholism
1. Introduction Alcohol withdrawal seizures are a common and severe complication of alcohol detoxification. Therefore, predicting the course of alcohol withdrawal and alcohol withdrawal seizures seems to be useful and tempting. Several studies have been conducted in order to identify pathophysiological markers for alcohol dependent patients being at elevated risk of developing withdrawal seizures such as homocysteine and prolactin (Bayerlein et al., 2005; Bleich et al., 2000; Hillemacher et al., 2006a). Moreover, further * Corresponding author. Tel.: +49 9131 8533001; fax: +49 9131 8534105. E-mail address: [email protected] (J. Wilhelm).
0022-3956/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2006.07.011
risk factors and/or predictors such as repeated alcohol withdrawals (‘kindling’ model) (Brown et al., 1988) or alcohol level on admission (Petrakis et al., 2005) have been discussed. Recent research has been attracting increasing attention concerning the pathophysiological role of the apolipoprotein E gene (ApoE gene) and its alleles (ApoE2, ApoE3, ApoE4) in different psychiatric and neurological disorders. The results of various studies show that the apolipoprotein E4 allele has to be regarded as a vulnerability factor for a higher incidence of neurodegenerative diseases as well as for a reduced capacity for neuronal regeneration. In contrast to ApoE4-negative carriers ApoE4-positive carriers are more likely to develop the sporadic (Noguchi et al., 1993; Poirier et al., 1993; Sitharthan et al., 1992) and the familial ‘‘late onset’’ form (Corder et al., 1993; Payami
872
J. Wilhelm et al. / Journal of Psychiatric Research 41 (2007) 871–875
et al., 1993) of Alzheimer‘s disease and the so-called tauopathy group of diseases (Kalman et al., 2000; Schneider et al., 1995). Moreover, the ApoE4 gene variant is associated with an increased risk of ischemic cerebral infarction (McCarron et al., 1999) and predicts a poor outcome after both intracerebral haemorrhage (Alberts et al., 1995) and traumatic brain injury (Friedman et al., 1999). Recently, a significant relationship between the ApoE4 allele and hippocampal atrophy in females with alcoholism has been observed (Bleich et al., 2003). Several studies investigated the pathophysiological role of apolipoprotein E alleles in epilepsy with controversial findings. For example, it has been observed that ApoE polymorphism is not associated with the development of major seizure disorders including temporal lobe epilepsy (Gambardella et al., 1999; Kilpatrick et al., 1996; Yeni et al., 2005). Nevertheless, an earlier onset of chronic temporal lobe epilepsy (Briellmann et al., 2000) as well as an increased risk of late posttraumatic seizures (Diaz-Arrastia et al., 2003) in patients carrying the ApoE4 genotype has also been reported. However, no association between the ApoE4 allele and age of epilepsy onset, seizure type, febrile seizures, family history of epilepsy, surgical outcome and neuropathological findings in patients with temporal lobe epilepsy has been detected (Blu¨mcke et al., 1997). The present study was performed in order to investigate whether there is an association between apolipoprotein E polymorphism and a history of withdrawal seizures in patients suffering from alcohol dependence. Taking into account that the ApoE4 allele seems to be a general risk factor for the brains integrity and plasticity the hypothesis was that alcohol dependent patients carrying the ApoE4 genotype are more likely to report a positive history of alcohol withdrawal seizures than ApoE4-negative patients. 2. Subjects and methods The present investigation was approved by the local ethical committee on human experimentation in accordance with the ethical standards Declaration of Helsinki of 1975, as revised in 1983. Written informed consent was obtained from all 194 patients (156 men, 38 women). The study was part of the FARS (Franconian Alcoholism Research Studies), a larger prospective investigation studying different neurobiological aspects in patients undergoing alcohol withdrawal (Bleich et al., 2005; Hillemacher et al., 2004; Wilhelm et al., 2006). The ApoE frequencies of 172 and 192 of these patients have been reported earlier (Hillemacher et al., in press; Wilhelm et al., 2005). All patients suffered from alcohol dependence according to criteria of the DSM-IV. Patients with other illnesses and patients with a concomitant psychiatric disorder apart from alcohol and nicotine dependence were not included into the study. Furthermore, all patients underwent a detailed neurological examination. Patients with known neurological disorders (including epilepsy or seizures of other origin than alcohol
withdrawal seizures, cerebral diseases or lesions) and patients with relevant neurological deficits were excluded. Preceding alcohol withdrawals (PW), withdrawal seizures and further socio-demographic and personal data, such as known somatic illnesses, the daily ethanol intake in grams (DI), the period of drinking (YD; in years) and lifetime drinking (LD; calculated by estimating an average daily ethanol intake in kg, multiplied by 365 and by the years of drinking) in kilograms of ethanol were obtained in a standardized self-structured interview according to Wetterling et al. (1999). The interview was uniformly conducted during the whole study for all subjects by the same trained observer (K.B.). The blood alcohol concentration (BAC) at admission was measured using an enzymatic essay (Roche/HitachiÒ Ethyl Alcohol, Mannheim/Germany, 904/911: ACN 270). In the majority of cases, the information given by the patients could be objectified by using the medical records as most patients had been treated in the same detoxification unit before. In addition, patients were classified according to Lesch’s typology (subtypes 1–4). Allocation was done using a computer program according to the decision tree (Lesch et al., 1990; Lesch and Walter, 1996). Classification was not possible in five patients because of missing necessary data. For ApoE genotyping peripheral blood was collected from the patients, and leukocyte DNA was extracted according to a routine method and measurement (Genomix DNA extraction kit). Genotyping was done by real-time PCR using hybridisation probes on a LightCycler (Roche Biochemica) using an established protocol (Mollenhauer et al., 2006) as described recently (Wilhelm et al., 2005). Participants were divided into two subgroups: patients with (SZ+) and without (SZ ) a history of alcohol withdrawal seizures. For statistical analysis we compared the ApoE alleles of the included patients. In total, subgroups consisted of 36 ApoE2 alleles, 311 ApoE3 alleles and 41 ApoE4 alleles. Analysis of Hardy–Weinberg equilibrium and comparisons between populations were done using the GENEPOP software V3.4 (Raymond and Rousset, 1995b). The rare ApoE2 and ApoE4 alleles resulted in cell frequencies less than 5 and therefore exact tests were performed as appropriate (Goudet et al., 1996; Louis and Dempster, 1987; Raymond and Rousset, 1995a). The odds ratio was calculated according to Miettinen and Nurminen (1985). For all statistical tests we applied a significance level of a = 0.05. Data were analyzed using SPSSä for Windows 12.0 (SPSS Inc., Chicago, IL). 3. Results The demographic characteristics of the study population and the distribution of ApoE genotypes are presented in Table 1. The observed ApoE allele frequencies were 0.093 (ApoE2), 0.802 (ApoE3) and 0.106 (ApoE4) in the total group (n = 194). Both, cases and German controls (Heyser et al., 1997; Riemenschneider et al., 2002) were
J. Wilhelm et al. / Journal of Psychiatric Research 41 (2007) 871–875 Table 1 Demographic characteristics and distribution of ApoE genotypes SZ
(n = 142)
SZ+ (n = 52)
t-test
Mean (SD)
Mean (SD)
df = 192
Age YD LD DI PW BAC
43.7 (9.3) 18.4 (11.0) 1622.2 (2052.5) 230.6 (186.4) 9.6 (14.3) 128.7 (110.0)
43.5 (8.5) 17.8 (8.7) 1652.3 (1149.2) 262.7 (150.8) 11.3 (11.0) 146.7 (144.1)
n.s. n.s. n.s. n.s. n.s. n.s.
Apo E genotype E2/E2 E2/E3 E2/E4 E3/E3 E3/E4 E4/E4 Lesch’s type 1/2/3/4a
1 25 5 84 25 2 20/73/31/15
1 2 0 42 7 0 14/22/6/8
Total 2 27 5 126 32 2 189
SD: standard deviation; YD: years of drinking; LD: lifetime drinking (kg); DI: daily ethanol intake (g/d); PW: number of preceding withdrawals; BAC: blood alcohol concentration (mg/dl). Difference between groups SZ and SZ+ tested with the t-test for independent samples (p < .05, n.s.: not significant). Statistical details are summarized in Section 3. a No significant difference tested with Pearson’s v2-test (p = 0.068); five patients were not classified because of missing data.
in Hardy–Weinberg equilibrium (p = 0.775 and p = 0.885), respectively. They did not differ with regard to allele frequencies (p = 0.510) or genotypes (p = 0.511). However, the distribution of ApoE alleles and genotypes differed significantly between cases with a positive (SZ+, n = 142) or negative (SZ , n = 52) history of alcohol withdrawal seizures (Fisher’s method, genotypes: p = 0.016; alleles: p = 0.011). Following these global associations we further analyzed these groups by using Fisher’s exact test to infer for significant differences. A significant positive association with a positive history of withdrawal seizures (SZ+) was found in the ApoE3 allele group (p = 0.006; n = 311). In contrast, analysis revealed a significant negative association with SZ+ in the ApoE2 allele group (p = 0.029; n = 36). However, for the ApoE4 allele group no significant differences were found regarding a history of withdrawal seizures (p = 0.191; n = 41). Details of the analysis using Fisher’s exact test are shown in Table 2. Given the fact that the ApoE3 allele has to be regarded as the predominant allele, we translated our results into odds ratios to test for a possible protective effect of the
Table 2 Fisher’s exact test based on the number of alleles
Apo E4 Not Apo E4 Apo E3 Not Apo E3 Apo E2 Not Apo E2
SZ
SZ+
Fisher’s exact test
34 250 218 66 32 252
7 97 93 11 4 100
p = 0.191 p = 0.006 p = 0.029
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ApoE2 and ApoE4 alleles. Comparing E2 with E3 we found an odds ratio of 0.29 (95% CI 0.10–0.85; p = 0.018), while the ApoE4 allele only showed a significant trend to be protective (OR: 0.48, 95%CI 0.21–1.13; p = 0.099). 4. Discussion Aim of the present study was to investigate the possible association of apolipoprotein E gene polymorphism with a history of alcohol withdrawal seizures in patients suffering from alcohol dependence. In the present study, none of the patients suffered from an alcohol withdrawal seizure during hospitalization since all patients were prophylactically treated with anti-seizure medication (clomethiazole and carbamazepine). Recent research has focused on how to predict alcohol withdrawal seizures as a severe complication of alcohol withdrawal. Up to now, various risk factors and/or predictors such as repeated alcohol withdrawals (‘kindling’ model) (Brown et al., 1988), alcohol level on admission (Petrakis et al., 2005), hazardous alcohol drinking (Brathen et al., 1999), genetic factors (Schaumann et al., 1994) and reduced white matter volume in the temporal lobes (Sullivan et al., 1996) have been suggested. Moreover, recent investigations have shown that prolactin serum levels may serve as a pre-ictal marker (Hillemacher et al., 2006a) and that elevated homocysteine serum levels predict first-onset withdrawal seizures in patients with alcoholism (Bleich et al., 2000; Kurth et al., 2001). The authors concluded that determining homocysteine levels in patients undergoing alcohol detoxification might be a useful screening method to identify patients with a higher risk of developing withdrawal seizures (Bleich et al., 2006). The present results point towards a pathophysiological role of apolipoprotein E polymorphism in alcohol withdrawal seizures. However, it is not the ApoE4 gene variant which is associated with a history of seizures under the conditions of alcohol withdrawal. Instead we found a significant association with the ApoE3 allele. According to our results patients carrying the ApoE2 allele were unlikely to report a history of withdrawal seizures during the course of their disease. Regarding the fact that the ApoE3 allele is the predominating allele in a typical Caucasian population as well as in our study group, carrying the ApoE3 allele could be assumed to be the ‘‘normal’’ situation. Under these circumstances the ApoE2 allele may indicate a significantly lower risk of developing alcohol withdrawal seizures, while the ApoE4 allele shows a trend. These preliminary findings have to be interpreted carefully but they hint towards the possibility that in patients with diagnosis of alcohol dependence the ApoE2 genotype might have a protective effect concerning the risk of developing alcohol withdrawal seizures. A protective effect of the E2 allele has also been reported in other neurological disorders such as ischemic cerebrovascular diseases and vascular dementia (Lin et al., 2004), age at onset of
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J. Wilhelm et al. / Journal of Psychiatric Research 41 (2007) 871–875
amyotrophic lateral sclerosis (Li et al., 2004) and episodic memory decline in older persons (Wilson et al., 2002). However, the underlying pathophysiological mechanisms remain unclear. From recent studies it is known that patients with type 4 of Lesch’s typology suffer from more repeated withdrawals while the kindling phenomenon is more important in patients of type 1 (Hillemacher et al., 2006b). Also, type 1 and type 4 patients show elevated levels of glutamic acid if compared to type 2 and type 3 patients (Walter et al., 2006). In our study sample we found no significant difference regarding the different subgroups of Lesch’s typology, comparing patients with or without history of withdrawal seizures. However, we found a trend towards an influence of different subgroups, which shows that using typologies is of high importance in addiction research. Further studies are needed in order to verify our findings and to clarify the pathophysiological role of apolipoprotein E polymorphism in alcohol withdrawal seizures. Acknowledgements We gratefully acknowledge the support by a Grant (S.B.) from Axis Shield, Norway (CT-H2003). The sponsor did not participate in study design, data collection, data analysis, interpretation, or writing the report. Also we thank Dr. Brigitte Mugele, Klinik fu¨r Sucht und Psychotherapeutische Medizin, Klinikum am Europakanal, Erlangen, Germany, for her support in recruiting patients. We also thank Dr. G. Beck for laboratory support. References Alberts MJ, Graffagnino C, McClenny C, DeLong D, Strittmatter W, Saunders AM, et al. ApoE genotype and survival from intracerebral haemorrhage. Lancet 1995;346:575. Bayerlein K, Hillemacher T, Reulbach U, Mugele B, Sperling W, Kornhuber J, et al. Alcohol-associated hyperhomocysteinemia and previous withdrawal seizures. Biological Psychiatry 2005;57:1590–3. Bleich S, Degner D, Bandelow B, von Ahsen N, Ru¨ther E, Kornhuber J. Plasma homocysteine is a predictor of alcohol withdrawal seizures. Neuroreport 2000;11:2749–52. Bleich S, Wilhelm J, Graesel E, Degner D, Sperling W, Rossner V, et al. Apolipoprotein E epsilon 4 is associated with hippocampal volume reduction in females with alcoholism. Journal of Neural Transmission 2003;110:401–11. Bleich S, Carl M, Bayerlein K, Reulbach U, Biermann T, Hillemacher T, et al. Evidence of increased homocysteine levels in alcoholism: The Franconian Alcoholism Research Studies (FARS). Alcoholism – Clinical and Experimental Research 2005;29:334–6. Bleich S, Bayerlein K, Hillemacher T, Degner D, Kornhuber J, Frieling H. An assessment of the potential value of elevated homocysteine in predicting alcohol withdrawal seizures. Epilepsia 2006;47:934–8. Blu¨mcke I, Brockhaus A, Scheiwe C, Rollbrocker B, Wolf HK, Elger CE, et al. The apolipoprotein E epsilon 4 allele is not associated with early onset temporal lobe epilepsy. Neuroreport 1997;8:1235–7. Brathen G, Brodtkorb E, Helde G, Sand T, Bovim G. The diversity of seizures related to alcohol use. A study of consecutive patients. European Journal of Neurology 1999;6:697–703. Briellmann RS, Torn-Broers Y, Busuttil BE, Major BJ, Kalnins RM, Olsen M, et al. APOE epsilon4 genotype is associated with an earlier onset of chronic temporal lobe epilepsy. Neurology 2000;55:435–7.
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Journal of Psychiatric Research 41 (2007) 871–875
www.elsevier.com/locate/jpsychires
Apolipoprotein E gene polymorphism and previous alcohol withdrawal seizures Julia Wilhelm a
a,*
, Nicolas von Ahsen b, Thomas Hillemacher a, Kristina Bayerlein a, Helge Frieling a, Johannes Kornhuber a, Stefan Bleich a
Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Schwabachanlage 6, D-91054 Erlangen, Germany b Department of Clinical Chemistry, University of Go¨ttingen, Germany Received 14 May 2006; received in revised form 9 July 2006; accepted 19 July 2006
Abstract Aim of this study was to investigate the possible association of apolipoprotein E (ApoE) gene polymorphism with a history of alcohol withdrawal seizures. We included 194 patients with alcohol dependence who were divided into patients with (SZ+) and without (SZ ) previous alcohol withdrawal seizures. ApoE genotypes were determined using PCR. For statistical analysis we examined the number of ApoE alleles (ApoE2: n = 36; ApoE3: n = 311; ApoE4: n = 41). A significant positive association with a positive history of withdrawal seizures (SZ+) was found in the ApoE3 allele group (Fisher’s exact test: p = 0.006) while a significant negative association was observed in the ApoE2 allele group (Fisher’s exact test: p = 0.029). For the ApoE4 allele group no significant differences were found regarding a history of withdrawal seizures. Our findings suggest an association between the apolipoprotein E3 gene variant and an elevated risk of alcohol withdrawal seizures. These preliminary results must be validated in further studies. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Apolipoprotein E; Alcohol withdrawal; Seizures; Alcoholism
1. Introduction Alcohol withdrawal seizures are a common and severe complication of alcohol detoxification. Therefore, predicting the course of alcohol withdrawal and alcohol withdrawal seizures seems to be useful and tempting. Several studies have been conducted in order to identify pathophysiological markers for alcohol dependent patients being at elevated risk of developing withdrawal seizures such as homocysteine and prolactin (Bayerlein et al., 2005; Bleich et al., 2000; Hillemacher et al., 2006a). Moreover, further * Corresponding author. Tel.: +49 9131 8533001; fax: +49 9131 8534105. E-mail address: [email protected] (J. Wilhelm).
0022-3956/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2006.07.011
risk factors and/or predictors such as repeated alcohol withdrawals (‘kindling’ model) (Brown et al., 1988) or alcohol level on admission (Petrakis et al., 2005) have been discussed. Recent research has been attracting increasing attention concerning the pathophysiological role of the apolipoprotein E gene (ApoE gene) and its alleles (ApoE2, ApoE3, ApoE4) in different psychiatric and neurological disorders. The results of various studies show that the apolipoprotein E4 allele has to be regarded as a vulnerability factor for a higher incidence of neurodegenerative diseases as well as for a reduced capacity for neuronal regeneration. In contrast to ApoE4-negative carriers ApoE4-positive carriers are more likely to develop the sporadic (Noguchi et al., 1993; Poirier et al., 1993; Sitharthan et al., 1992) and the familial ‘‘late onset’’ form (Corder et al., 1993; Payami
872
J. Wilhelm et al. / Journal of Psychiatric Research 41 (2007) 871–875
et al., 1993) of Alzheimer‘s disease and the so-called tauopathy group of diseases (Kalman et al., 2000; Schneider et al., 1995). Moreover, the ApoE4 gene variant is associated with an increased risk of ischemic cerebral infarction (McCarron et al., 1999) and predicts a poor outcome after both intracerebral haemorrhage (Alberts et al., 1995) and traumatic brain injury (Friedman et al., 1999). Recently, a significant relationship between the ApoE4 allele and hippocampal atrophy in females with alcoholism has been observed (Bleich et al., 2003). Several studies investigated the pathophysiological role of apolipoprotein E alleles in epilepsy with controversial findings. For example, it has been observed that ApoE polymorphism is not associated with the development of major seizure disorders including temporal lobe epilepsy (Gambardella et al., 1999; Kilpatrick et al., 1996; Yeni et al., 2005). Nevertheless, an earlier onset of chronic temporal lobe epilepsy (Briellmann et al., 2000) as well as an increased risk of late posttraumatic seizures (Diaz-Arrastia et al., 2003) in patients carrying the ApoE4 genotype has also been reported. However, no association between the ApoE4 allele and age of epilepsy onset, seizure type, febrile seizures, family history of epilepsy, surgical outcome and neuropathological findings in patients with temporal lobe epilepsy has been detected (Blu¨mcke et al., 1997). The present study was performed in order to investigate whether there is an association between apolipoprotein E polymorphism and a history of withdrawal seizures in patients suffering from alcohol dependence. Taking into account that the ApoE4 allele seems to be a general risk factor for the brains integrity and plasticity the hypothesis was that alcohol dependent patients carrying the ApoE4 genotype are more likely to report a positive history of alcohol withdrawal seizures than ApoE4-negative patients. 2. Subjects and methods The present investigation was approved by the local ethical committee on human experimentation in accordance with the ethical standards Declaration of Helsinki of 1975, as revised in 1983. Written informed consent was obtained from all 194 patients (156 men, 38 women). The study was part of the FARS (Franconian Alcoholism Research Studies), a larger prospective investigation studying different neurobiological aspects in patients undergoing alcohol withdrawal (Bleich et al., 2005; Hillemacher et al., 2004; Wilhelm et al., 2006). The ApoE frequencies of 172 and 192 of these patients have been reported earlier (Hillemacher et al., in press; Wilhelm et al., 2005). All patients suffered from alcohol dependence according to criteria of the DSM-IV. Patients with other illnesses and patients with a concomitant psychiatric disorder apart from alcohol and nicotine dependence were not included into the study. Furthermore, all patients underwent a detailed neurological examination. Patients with known neurological disorders (including epilepsy or seizures of other origin than alcohol
withdrawal seizures, cerebral diseases or lesions) and patients with relevant neurological deficits were excluded. Preceding alcohol withdrawals (PW), withdrawal seizures and further socio-demographic and personal data, such as known somatic illnesses, the daily ethanol intake in grams (DI), the period of drinking (YD; in years) and lifetime drinking (LD; calculated by estimating an average daily ethanol intake in kg, multiplied by 365 and by the years of drinking) in kilograms of ethanol were obtained in a standardized self-structured interview according to Wetterling et al. (1999). The interview was uniformly conducted during the whole study for all subjects by the same trained observer (K.B.). The blood alcohol concentration (BAC) at admission was measured using an enzymatic essay (Roche/HitachiÒ Ethyl Alcohol, Mannheim/Germany, 904/911: ACN 270). In the majority of cases, the information given by the patients could be objectified by using the medical records as most patients had been treated in the same detoxification unit before. In addition, patients were classified according to Lesch’s typology (subtypes 1–4). Allocation was done using a computer program according to the decision tree (Lesch et al., 1990; Lesch and Walter, 1996). Classification was not possible in five patients because of missing necessary data. For ApoE genotyping peripheral blood was collected from the patients, and leukocyte DNA was extracted according to a routine method and measurement (Genomix DNA extraction kit). Genotyping was done by real-time PCR using hybridisation probes on a LightCycler (Roche Biochemica) using an established protocol (Mollenhauer et al., 2006) as described recently (Wilhelm et al., 2005). Participants were divided into two subgroups: patients with (SZ+) and without (SZ ) a history of alcohol withdrawal seizures. For statistical analysis we compared the ApoE alleles of the included patients. In total, subgroups consisted of 36 ApoE2 alleles, 311 ApoE3 alleles and 41 ApoE4 alleles. Analysis of Hardy–Weinberg equilibrium and comparisons between populations were done using the GENEPOP software V3.4 (Raymond and Rousset, 1995b). The rare ApoE2 and ApoE4 alleles resulted in cell frequencies less than 5 and therefore exact tests were performed as appropriate (Goudet et al., 1996; Louis and Dempster, 1987; Raymond and Rousset, 1995a). The odds ratio was calculated according to Miettinen and Nurminen (1985). For all statistical tests we applied a significance level of a = 0.05. Data were analyzed using SPSSä for Windows 12.0 (SPSS Inc., Chicago, IL). 3. Results The demographic characteristics of the study population and the distribution of ApoE genotypes are presented in Table 1. The observed ApoE allele frequencies were 0.093 (ApoE2), 0.802 (ApoE3) and 0.106 (ApoE4) in the total group (n = 194). Both, cases and German controls (Heyser et al., 1997; Riemenschneider et al., 2002) were
J. Wilhelm et al. / Journal of Psychiatric Research 41 (2007) 871–875 Table 1 Demographic characteristics and distribution of ApoE genotypes SZ
(n = 142)
SZ+ (n = 52)
t-test
Mean (SD)
Mean (SD)
df = 192
Age YD LD DI PW BAC
43.7 (9.3) 18.4 (11.0) 1622.2 (2052.5) 230.6 (186.4) 9.6 (14.3) 128.7 (110.0)
43.5 (8.5) 17.8 (8.7) 1652.3 (1149.2) 262.7 (150.8) 11.3 (11.0) 146.7 (144.1)
n.s. n.s. n.s. n.s. n.s. n.s.
Apo E genotype E2/E2 E2/E3 E2/E4 E3/E3 E3/E4 E4/E4 Lesch’s type 1/2/3/4a
1 25 5 84 25 2 20/73/31/15
1 2 0 42 7 0 14/22/6/8
Total 2 27 5 126 32 2 189
SD: standard deviation; YD: years of drinking; LD: lifetime drinking (kg); DI: daily ethanol intake (g/d); PW: number of preceding withdrawals; BAC: blood alcohol concentration (mg/dl). Difference between groups SZ and SZ+ tested with the t-test for independent samples (p < .05, n.s.: not significant). Statistical details are summarized in Section 3. a No significant difference tested with Pearson’s v2-test (p = 0.068); five patients were not classified because of missing data.
in Hardy–Weinberg equilibrium (p = 0.775 and p = 0.885), respectively. They did not differ with regard to allele frequencies (p = 0.510) or genotypes (p = 0.511). However, the distribution of ApoE alleles and genotypes differed significantly between cases with a positive (SZ+, n = 142) or negative (SZ , n = 52) history of alcohol withdrawal seizures (Fisher’s method, genotypes: p = 0.016; alleles: p = 0.011). Following these global associations we further analyzed these groups by using Fisher’s exact test to infer for significant differences. A significant positive association with a positive history of withdrawal seizures (SZ+) was found in the ApoE3 allele group (p = 0.006; n = 311). In contrast, analysis revealed a significant negative association with SZ+ in the ApoE2 allele group (p = 0.029; n = 36). However, for the ApoE4 allele group no significant differences were found regarding a history of withdrawal seizures (p = 0.191; n = 41). Details of the analysis using Fisher’s exact test are shown in Table 2. Given the fact that the ApoE3 allele has to be regarded as the predominant allele, we translated our results into odds ratios to test for a possible protective effect of the
Table 2 Fisher’s exact test based on the number of alleles
Apo E4 Not Apo E4 Apo E3 Not Apo E3 Apo E2 Not Apo E2
SZ
SZ+
Fisher’s exact test
34 250 218 66 32 252
7 97 93 11 4 100
p = 0.191 p = 0.006 p = 0.029
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ApoE2 and ApoE4 alleles. Comparing E2 with E3 we found an odds ratio of 0.29 (95% CI 0.10–0.85; p = 0.018), while the ApoE4 allele only showed a significant trend to be protective (OR: 0.48, 95%CI 0.21–1.13; p = 0.099). 4. Discussion Aim of the present study was to investigate the possible association of apolipoprotein E gene polymorphism with a history of alcohol withdrawal seizures in patients suffering from alcohol dependence. In the present study, none of the patients suffered from an alcohol withdrawal seizure during hospitalization since all patients were prophylactically treated with anti-seizure medication (clomethiazole and carbamazepine). Recent research has focused on how to predict alcohol withdrawal seizures as a severe complication of alcohol withdrawal. Up to now, various risk factors and/or predictors such as repeated alcohol withdrawals (‘kindling’ model) (Brown et al., 1988), alcohol level on admission (Petrakis et al., 2005), hazardous alcohol drinking (Brathen et al., 1999), genetic factors (Schaumann et al., 1994) and reduced white matter volume in the temporal lobes (Sullivan et al., 1996) have been suggested. Moreover, recent investigations have shown that prolactin serum levels may serve as a pre-ictal marker (Hillemacher et al., 2006a) and that elevated homocysteine serum levels predict first-onset withdrawal seizures in patients with alcoholism (Bleich et al., 2000; Kurth et al., 2001). The authors concluded that determining homocysteine levels in patients undergoing alcohol detoxification might be a useful screening method to identify patients with a higher risk of developing withdrawal seizures (Bleich et al., 2006). The present results point towards a pathophysiological role of apolipoprotein E polymorphism in alcohol withdrawal seizures. However, it is not the ApoE4 gene variant which is associated with a history of seizures under the conditions of alcohol withdrawal. Instead we found a significant association with the ApoE3 allele. According to our results patients carrying the ApoE2 allele were unlikely to report a history of withdrawal seizures during the course of their disease. Regarding the fact that the ApoE3 allele is the predominating allele in a typical Caucasian population as well as in our study group, carrying the ApoE3 allele could be assumed to be the ‘‘normal’’ situation. Under these circumstances the ApoE2 allele may indicate a significantly lower risk of developing alcohol withdrawal seizures, while the ApoE4 allele shows a trend. These preliminary findings have to be interpreted carefully but they hint towards the possibility that in patients with diagnosis of alcohol dependence the ApoE2 genotype might have a protective effect concerning the risk of developing alcohol withdrawal seizures. A protective effect of the E2 allele has also been reported in other neurological disorders such as ischemic cerebrovascular diseases and vascular dementia (Lin et al., 2004), age at onset of
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amyotrophic lateral sclerosis (Li et al., 2004) and episodic memory decline in older persons (Wilson et al., 2002). However, the underlying pathophysiological mechanisms remain unclear. From recent studies it is known that patients with type 4 of Lesch’s typology suffer from more repeated withdrawals while the kindling phenomenon is more important in patients of type 1 (Hillemacher et al., 2006b). Also, type 1 and type 4 patients show elevated levels of glutamic acid if compared to type 2 and type 3 patients (Walter et al., 2006). In our study sample we found no significant difference regarding the different subgroups of Lesch’s typology, comparing patients with or without history of withdrawal seizures. However, we found a trend towards an influence of different subgroups, which shows that using typologies is of high importance in addiction research. Further studies are needed in order to verify our findings and to clarify the pathophysiological role of apolipoprotein E polymorphism in alcohol withdrawal seizures. Acknowledgements We gratefully acknowledge the support by a Grant (S.B.) from Axis Shield, Norway (CT-H2003). The sponsor did not participate in study design, data collection, data analysis, interpretation, or writing the report. Also we thank Dr. Brigitte Mugele, Klinik fu¨r Sucht und Psychotherapeutische Medizin, Klinikum am Europakanal, Erlangen, Germany, for her support in recruiting patients. We also thank Dr. G. Beck for laboratory support. References Alberts MJ, Graffagnino C, McClenny C, DeLong D, Strittmatter W, Saunders AM, et al. ApoE genotype and survival from intracerebral haemorrhage. Lancet 1995;346:575. Bayerlein K, Hillemacher T, Reulbach U, Mugele B, Sperling W, Kornhuber J, et al. Alcohol-associated hyperhomocysteinemia and previous withdrawal seizures. Biological Psychiatry 2005;57:1590–3. Bleich S, Degner D, Bandelow B, von Ahsen N, Ru¨ther E, Kornhuber J. Plasma homocysteine is a predictor of alcohol withdrawal seizures. Neuroreport 2000;11:2749–52. Bleich S, Wilhelm J, Graesel E, Degner D, Sperling W, Rossner V, et al. Apolipoprotein E epsilon 4 is associated with hippocampal volume reduction in females with alcoholism. Journal of Neural Transmission 2003;110:401–11. Bleich S, Carl M, Bayerlein K, Reulbach U, Biermann T, Hillemacher T, et al. Evidence of increased homocysteine levels in alcoholism: The Franconian Alcoholism Research Studies (FARS). Alcoholism – Clinical and Experimental Research 2005;29:334–6. Bleich S, Bayerlein K, Hillemacher T, Degner D, Kornhuber J, Frieling H. An assessment of the potential value of elevated homocysteine in predicting alcohol withdrawal seizures. Epilepsia 2006;47:934–8. Blu¨mcke I, Brockhaus A, Scheiwe C, Rollbrocker B, Wolf HK, Elger CE, et al. The apolipoprotein E epsilon 4 allele is not associated with early onset temporal lobe epilepsy. Neuroreport 1997;8:1235–7. Brathen G, Brodtkorb E, Helde G, Sand T, Bovim G. The diversity of seizures related to alcohol use. A study of consecutive patients. European Journal of Neurology 1999;6:697–703. Briellmann RS, Torn-Broers Y, Busuttil BE, Major BJ, Kalnins RM, Olsen M, et al. APOE epsilon4 genotype is associated with an earlier onset of chronic temporal lobe epilepsy. Neurology 2000;55:435–7.
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