1 Unidad de Medicina Molecular, Departamento de Medicina, 2 Unidad de Alcoholismo. Servicio de Medicina Interna, Hospital Universitario de Salamanca and 3 Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain
* Author to whom correspondence should be addressed at: Rogelio Gonzalez-Sarmiento, Unidad de Medicina Molecular, Departamento de Medicina, Avda. de Alfonso X el Sabio s/n, 37007 Salamanca, Spain. Tel.: +34 923294553; Fax: +34 923394593; E-mail: Gonzalez{at}usal.es
(Received 25 November 2004; first review notified 14 December 2004; in revised form 23 February 2005; accepted 8 March 2005; Advance Access publication 29 March 2005)
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ABSTRACT |
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INTRODUCTION |
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Recent work conducted by our group has shown that the presence of the IL1RN*1 allele is not associated with ALD but with greater risk of alcoholism in Spanish men (Pastor et al., 2000). Thus, we seek to investigate whether the association of IL1RN gene polymorphism and alcoholism in Spanish men can be extended to other members of the IL1 gene family located on chromosome 2q (Steinkasserer et al., 1992
; Nicklin et al., 2002
), such as IL1A, which encodes IL-1
, ILB, which encodes IL-1ß, and ILRI, which encodes the IL1 type I receptor.
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SUBJECTS AND METHODS |
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Histopathological examination of the livers of 47 cirrhotic patients in which biopsy was performed revealed micronodular cirrhosis. Liver biopsy was not performed in the remaining 10 patients owing to blood coagulation abnormalities. In these cases, the diagnosis of cirrhosis was established on the basis of the presence of the physical stigmata of chronic liver disease, a history of ascitis, variceal bleeding or hepatic encephalopathy, as well as on gastroscopic and/or ultrasonographic findings (Erlinger and Benhamou, 1999). None of the alcoholic patients without ALD had the physical stigmata of chronic liver disease (i.e. cutaneous signs, hepatosplenomegaly, gynaecomastia, testicular atrophy). Haemoglobin concentration, prothrombin time and serum albumin level were normal in all subjects; ALT and AST serum levels were required to be less than twice the upper normal limits (40 IU/l). Liver biopsy was not performed for ethical reasons, liver disease being excluded on the basis of ultrasonographic studies. Hepatitis B surface antigen (HbsAg) and antibodies to hepatitis C virus were negative in all alcoholic patients.
Eighty-one age- and sex-matched healthy volunteers were included in the study. All of them consumed <10 g of ethanol/day and none had a history of alcohol abuse or alcohol dependence and neither did their first-degree relatives.
Patients and control subjects gave informed consent. The study was approved by the Ethical Committee of the University Hospital of Salamanca.
Genetic analysis
Genomic DNA was extracted from nucleated peripheral blood cells using standard proteinase K digestion, phenol chloroform extraction and ethanol precipitation, and was stored at 20°C.
The C/T transition at position 889 in the promoter region of the IL1A gene was analysed after PCR amplification using the primers 5'-AAGCTTGTTCTACCACCTGAACT AGGC-3' and 5'-TTACATATGAGCCTTCCAATG-3'. PCR conditions consisted of 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 70°C for 1 min followed by a final extension step at 70°C for 5 min. The PCR products were digested overnight at 37°C with the NcoI restriction enzyme (Fermentas UAB, Vilnius, Lithuania) and the fragments were separated on 4% agarose gels (McDowell et al., 1995).
The region containing the C/T transition at position 511 in the IL1B gene promoter was amplified by PCR using the primers 5'-TGGCATTGATCTGGTTCATC-3' and 5'-GTTTA GGAATCTTCCCATTT-3'. PCR conditions were similar to those described for the amplification of the VNTR of the ILIRN gene (Pastor et al., 2000). The PCR products were digested overnight at 37°C with the AvaI restriction enzyme (Fermentas UAB, Vilnius, Lithuania) and the fragments were visualized on 2.5% agarose gels (Di Giovine et al., 1992
).
The region containing the silent T/C transition at position +3953 within exon 5 of the IL1B gene was amplified using the primers 5'-GTTGTCATCAGACTTTGACC-3' and 5'-TTCAGTTCATATGGACCAGA-3'. DNA samples were amplified under the same conditions as above. The PCR products were digested overnight at 65°C with the TaqI restriction enzyme (Fermentas UAB, Vilnius, Lithuania) and the fragments were visualized on 3% agarose gels (Pociot et al., 1992).
The region containing the G/A transition in the 5'UTR of the IL1R1 gene was amplified using the primers 5'-CAGGGATGACAGTCTCCACCTT-3' and 5'-GTGTCCAAATGGCAGTTCTGAA-3'. Denaturing was performed at 95° for 30 s, annealing was performed at 60° for 45 s and extension at 72° for 90 s. The PCR products were digested overnight at 37°C with the HinfI restriction enzyme (Fermentas UAB, Vilnius, Lithuania) and the fragments were visualized on 2.5% agarose gels (Bergholdt et al., 2000).
Molecular analysis of the intron 2 VNTR of the IL1RN gene was performed as reported previously (Pastor et al., 2000).
Statistical analysis
The results are presented as genotypic, allelic and haplotypic frequencies. The different groups were compared using the chi-square and Fisher's exact probability tests, with Yates correction for small numbers. Continuous variables were compared using the non-parametric MannWhitney U- and KruskalWallis tests, thereby avoiding any distributional assumptions. P < 0.05 was regarded as significant. The odds ratio and 95% confidence intervals (OR 95% CI) were also calculated. All analyses were performed using SPSS v.11.0.
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RESULTS |
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DISCUSSION |
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As shown in Tables 1 and 4, our results indicate that alcoholism is not associated with changes in the allelic frequency of polymorphisms located in the promoter region of the IL1A gene (position 889) or in the promoter region of the IL1R1 gene. However, as shown in Table 2, allele 511 IL1B*1 was more frequent in Spanish alcoholics than in healthy subjects, whereas alleles of the +3953 IL1B polymorphism had a similar distribution in both groups (Table 3). Moreover, the IL1RN*1 allele was over-represented in the alcoholics, in keeping with previous data from our group (Pastor et al., 2000) (Table 5). In addition, our results show that both the IL1RN*1/511 ILB*1 and the IL1RN*1/511 IL1B*1/+3953 IL1B*1 haplotypes were more frequent in the alcoholics than in the control subjects (Table 6).
The lack of association of alcoholism with polymorphisms in both the IL1A and IL1R1 genes indicates that the relationship between IL1RN and IL1B gene variants and alcoholic disease cannot be attributed to linkage disequilibrium with other gene or genes located on chromosome 2 q. The functional significance of allele 1 of IL1RN is unknown. Allele IL1RN*2 has been linked to an increased synthesis of IL1Ra and a decreased production of IL1 in stimulated monocytes (Danis et al., 1995
). Moreover, healthy carriers of the IL1RN*2 allele have significantly higher plasma levels of IL1-Ra than non-carriers (Hurme and Santtila, 1998
). The fact that we found a decrease in the frequency of allele 2 in Spanish alcoholics with and without liver disease suggests that the IL1Ra protein would not be involved in the development of ALD in Spanish alcoholics.
Interestingly, when we analysed the groups of alcoholics separately, we found that allele +3953 IL1B2 (Table 3) and both haplotypes IL1RN*1/+3953 IL1B*2 and ILRN*1/511 IL1B*1/+3953 IL1B*2 (Table 6) were more frequent in the alcohol abusers than in the alcohol-dependent subjects, suggesting that the carriers of allele +3953 IL1B*2 may be less susceptible to developing alcohol dependence. Furthermore, the presence of allele +3953 IL1B*2 has been associated with an increase in IL1ß secretion in vitro after lipopolysaccharide-induced activation of peripheral blood cells obtained from healthy subjects (Pociot et al., 1992), although in another report the authors failed to find such a relationship (Stokkers et al., 1998
). No differences were detected between the groups included in our study with respect to the frequency of haplotype ILRN*2/+3953 IL1B*1, which is associated with a higher production of IL1ß in immune cells (Hurme and Santtila, 1998
; Santtila et al., 1998
). In contrast to the findings reported for Japanese alcoholic patients (Takamatsu et al., 2000
), we did not find any association between haplotype 511IL1B*2/+3953 IL1B*1 and ALC.
Our results show a genetic association between alcoholism and genes that encode proteins involved in the immune response. The relationship between cytokines and the central nervous system (CNS) is well known (Reichlin, 1993; Hopkins and Rothwell, 1995
; Rothwell and Hopkins, 1995
; Allan and Rotwell, 2001
), and there is also evidence that several members of the cytokine family are present in the brain. Thus, IL-1 activity in the brain has been related to the differentiation of dopaminergic neurons (Ling et al., 1998
) and the modulation of central monoaminergic activity (MohanKumar et al., 1998
). Similarly, IL-1, IL-2 and IL-6 can modify the release of neurotransmitters from the nucleus accumbens and other brain regions of the CNS that play a central role in alcohol abuse and alcohol dependence (Koob et al., 1994
; Song et al., 1999
). It has also been reported that allele 511 IL1B*2 may be involved in the development of the structural changes seen in the CNS of subjects with schizophrenia (Katila et al., 1999
), and that some alleles of the IL1RN gene have been associated with attention deficit hyperactivity disorder (Segman et al., 2002
). Our results confirm that the IL1RN gene may be involved in susceptibility to alcoholism in Spanish subjects, and show for the first time that IL1B gene may also play a role in determining individual susceptibility to alcoholism. The mechanisms that define this relationship are yet to be determined.
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ACKNOWLEDGEMENTS |
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