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A gene for alcohol toxicity, not drinking behavior
Medical Hypotheses I Medical &pothms (1992) 38.203-205 O~GmupUKLsd1992
A Gene for Alcohol Toxicity, not Drinking Behavior R. M. GOODMAN 1402 Astor Ave, Bronx, NY 10469, USA
Abstract-Apparently contradictory results concerning association of an allele of a human dopamine receptor gene with alcoholism are explained by the gene’s being involved with ethanol toxicity, not drinking behavior.
Introduction ‘Alcoholism’ has two meanings. The internist and neurologist use the ‘-ism’ suffix, similarly to ‘-osis’, to denote pathologic condition. Therefore as with salicylism, cinchonism, etc, alcoholism refers to toxic effects of alcohol overdose. Acute alcoholism is drunkenness. Chronic alcoholism is one or more of hepatitis, cirrhosis of the liver, pancreatitis, gastritis, cardiomyopathy, peripheral neuropathy, and encephalopathy. I will call this ‘alcoholism 1’. The psychiatrist uses ‘-ism’ as in bruxism, communism, Judaism, and athleticism, to indicate belief, tendency, practice, etc. Therefore alcoholism refers to a behavior pattern of excessive alcohol consumption. I will call this ‘alcoholism 2’. Although the two are strongly associated, alcoholisms 1 and 2 are not the same, and trouble arises when their meanings are conflated. For instance, there is the inference that the behavior pattern in alcoholism 2 is a pathology as in alcoholism 1 (1). It is also my hypothesis that a gene contributing to alcoholism 1 has been discovered and wrongly implicated in alcoholism 2, leading to apparently contradictory findings. Bunzow et al used a hamster be&-adrenergic receptor gene coding region to probe a rat genome liDate received 4 November 1991 Date accepted 23 December 1991
brary (2). A low stringency Southern blot detected a clone called RGB-2. This hybridized in Southems after a high stringency wash (0.2X SSC, 0.1% SDS, 65°C) to a 2.5 kb cDNA from rat brain, and in Northems to mRNA from various rat brain tissues wherein the D2 dopamine receptor is known to be expressed. The coding sequence is consistent with the molecular weight of the D2 dopamine receptor. An extra 87 bp of coding sequence are seen in some cDNAs of mature transcripts, suggesting alternative splicing (3). When RGB-2 is put in a eucaryotic expression system, binding of ligands to the cell membrane is similar to that seen in rat tissue containing this receptor (2). Using this rat cDNA as a probe, Grandy et al did a similar analysis to identify a human pituitary cDNA and test ligand binding properties of its expression product (3). On the basis of sequence similarities and product ligand binding properties, this was identified as the human D2 dopamine receptor gene (although great similarities are seen between the sequence of dopaminergic and adrenergic receptor proteins in rodents), named DRD2, and localized by hybridization to a place on chromosome 11 (4). The 87 bp variably present in the rat message appear in a distinct exon of the human gene (3).
203
204 Restriction of DRD2 by TaqI showed fragment length bimorphism, identifying alleles Al and A2 (4). The fragment containing the TaqI site contains coding and non-coding regions corresponding to the 3’ end of the mRNA. The alleles produce different average amounts of receptor sites in human caudate nuclei, but with equal affinity for ligand (5)-results suggesting a difference in a non-coding region, affecting translation efficiency, although an amino acid difference affecting efficiency of post-translational processing or stability is not ruled out. Whether the amounts of mRNA are comparable has not been established. Human cadaver studies by Blum and Noble associated allele Al with retrospective diagnoses of alcoholism (5, 6). This finding was hailed as portending prediction of alcoholism as a phenotype by genotyping (7). Differences were also shown in binding affinity and number of sites in caudate nuclei for a dopaminergic ligand between alcoholic and nonalcoholic cadaver populations, although with great variation between individuals (5). The difference in number of binding sites was consistent with that between cadavers homozygous for Al, heterozygous, and homozygous for A2. However, genotyping of living known alcoholics versus control populations led other authors to conclude that there was no link between DRD2 allele Al and alcoholism (8,9). The discrepancy has led to controversy and guesses at how to reconcile the data (10). Discussion The methods and results in the supposedly contradictory studies suggest that Blum and Noble found a marker for susceptibility to alcoholism 1, and that Bolos and Parsian showed this marker not to be associated with alcoholism 2. In other words, allele A2 confers relative resistance, and Al, relative susceptibility, to death or illness from chronic alcohol overdose, and the gene does not affect alcoholic behavior. Although the retrospective diagnoses were made by psychiatrists, for ‘a large majority of alcoholics’ in Blum’s initial study (a), ‘the cause of death was primarily attributed to the chronic damaging effects of alcohol on their bodily systems’. ‘The causes of death included accidents, gunshot wounds, myocardial infarction, heart failure, cancer, hepatorenal failure, gastrointestinal bleeding, suicide, and pneumonia.’ The selected population was similar or identical in the second paper (5). It would be interesting to separate the accident, gunshot, and suicide cases to reanalyze the remainder.
MEDICAL HYPOTHESES
Bolos’ study used psychiatric selection for alcoholism, but excluded subjects with ‘an acutely active medical disorder’ (8). Behavioral history was therefore determining. Parsian’s study used similar behavioral criteria, but did not exclude patients with medical problems (9). Neither study found association between alcoholism in general and presence of allele Al. But Parsian also separately studied alcoholics ‘classified as severe because of serious medical problems’. These included 8 cases comprising cirrhosis of the liver, pancreatitis, gastritis, organic brain syndrome, and peripheral netuopathy, and 2 cases without those somatic conditions, but with shakes or seizures on withdrawal of alcohol. There were 6 of these cases out of the total of 16 subjects in which the Al allele was present, and 4 of these cases out of the total of 41 without Al. The difference is significant at P = 0.05 in a two tailed fourfold table test. Parsian et al saw in this result ‘the possibility that the Al allele is not associated with increased risk of alcoholism in general,’ meaning alcoholism 2, ‘but only modifies the probability that an alcoholic will develop severe physical complications,’ meaning alcoholism 1 (9). They then reported results of a family study, wherein 8 of 17 of those with Al, but only 4 of 18 without Al, had severe medical problems associated with alcoholism (9). The difference, although suggestive, is not statistically significant in the fourfold table test. However, the sample in this test was smaller than in the former, and the failure to find a significant difference does not discount the previous positive result. Since Parsian did not make clear whether the populations in the 2 studies overlapped, I don’t know whether to combine them for statistical reanalysis. The effort to find a cause for alcoholism 2 has been under way for some time, and the line of inquiry cited here extends the work of others with rodents in attempts to model human behavior. My opinion is that the factors causing persons to practice alcoholism 2 are like those causing the practice of such other ‘isms’ as Judaism. Among these factors are the perception by the actor of personal risks in such behavior (11). The determination of such risks is promised by discoveries such as that hypothesized here of a genetic screen for alcoholism 1. Persons can have similar histories of alcohol consumption, yet one will get sick, and the other not. If we could predict susceptibility better, we could help some heavy drinkers cut back, and allay the fears of others.
205
A GENE FOR ALCOHOL TOXlClTY, NOT DRINKING BEHAVIOR
Conclusion
References
For this point mutation in DRD2 to contribute to alcohol toxicity in organs other than brain, one of two possibilities must exist. A receptor in brain tissue could mediate, via autonomic or hormonal mechanism, damage to distant organs, or differences in ethanol metabolism-unlikely. Or the gene could be expressed outside the brain. Dopaminergic transmission is identified with the central nervous system. However, a systematic search for mRNAs or cDNAs from outside the brain which hybridize to the probes has not been undertaken, but should be. Perhaps the scatter in ligand binding data from human cadavers could be eliminated by studies in the clone expression system used by Grandy et al (3). Such a system could also be used to assay any difference in ethanol or acetaldehyde cytotoxicity conferred by Al versus A2 in some ‘neutral’ host. However, failure to find a difference would not rule out a difference in their native habitat, wherein interference with normal operation of a dopamine receptor may be telling. Epidemiology should henceforth concentrate on retrospective studies of individuals with similar drinking histories. Is there an A2 vs Al edge among those who have drunk heavily for long periods, and escaped morbidity? A large enough sample population would provide separate data on the various target organs.
1. Goodman R M. Addiction? ~~278-81 in The Great Issues of Drug Policy (A S Trebach, K B Zeese. eds) Drug Policy Foundation, Washington, 1990. 2. Bunzow J R, Van To1 H H M, Grandy D K et al. Cloning and expression of a rat 4 dcpamine receptor cDNA. Nature 336: 783-7, 1988. 3. Grandy D K, Marchionni M A, Makam H et al. Cloning of the cDNA and gene for a human & dopamine receptor. Proc Nat Acad Sci USA 86: 97626, 1989. 4. Grandy D K, Litt M. Allen L et al.‘Ihe human D2 dcpamine receptor gene is located on chromosome 11 at q22-23 and identifies a Taql RFLP. Am J Hum Genet 45: 778-85, 1989. 5. Noble E P. Blum K. Ritchie T, Montgomery A. Sheridan P J. Allelic association of the & dopamine receptor gene with receptor-binding characteristics in alcoholism. Arch Gen Psychiatry 48: 648-54. 1991. 6. Blum K, Noble E P, Sheridan P J et al. AlIeIic association of human dopamine Dz receptor gene in alcoholism. JAMA 263: 2055-60,1990. 7. Gordis E, Tabakoff B, Goldman D. Berg K. Finding the gene(s) for alcoholism. JAMA 263:2094-5, 1990 (editorial). 8. Bolos A, Dean M, Lucas-Derse S. Ramsburg M, Brown G L, Goldman G. Population and pedigree studies reveal a lack of association between the dopamine D, receptor gene and alcoholism. JAMA 264: 315660. 1990. 9. Parsian A, Todd R D. Devor E J, O’MaIIey K L, Suarez B K. Reich T, Claringer C R. Alcoholism and alleles of the human Dz dopamine receptor locus: studies of association and linkage. Arch Gen Psychiatry 48:65543. 1991. 10 Conneally P M. Association between the Dz dopamine receptor gene and alcoholism: a continuing controversy. Arch Gen Psychiatry 48: 644-6, 1991. 11. Peele S. Diseasing of America. Lexington Books, Lexington, Mass. 1989.
A Gene for Alcohol Toxicity, not Drinking Behavior R. M. GOODMAN 1402 Astor Ave, Bronx, NY 10469, USA
Abstract-Apparently contradictory results concerning association of an allele of a human dopamine receptor gene with alcoholism are explained by the gene’s being involved with ethanol toxicity, not drinking behavior.
Introduction ‘Alcoholism’ has two meanings. The internist and neurologist use the ‘-ism’ suffix, similarly to ‘-osis’, to denote pathologic condition. Therefore as with salicylism, cinchonism, etc, alcoholism refers to toxic effects of alcohol overdose. Acute alcoholism is drunkenness. Chronic alcoholism is one or more of hepatitis, cirrhosis of the liver, pancreatitis, gastritis, cardiomyopathy, peripheral neuropathy, and encephalopathy. I will call this ‘alcoholism 1’. The psychiatrist uses ‘-ism’ as in bruxism, communism, Judaism, and athleticism, to indicate belief, tendency, practice, etc. Therefore alcoholism refers to a behavior pattern of excessive alcohol consumption. I will call this ‘alcoholism 2’. Although the two are strongly associated, alcoholisms 1 and 2 are not the same, and trouble arises when their meanings are conflated. For instance, there is the inference that the behavior pattern in alcoholism 2 is a pathology as in alcoholism 1 (1). It is also my hypothesis that a gene contributing to alcoholism 1 has been discovered and wrongly implicated in alcoholism 2, leading to apparently contradictory findings. Bunzow et al used a hamster be&-adrenergic receptor gene coding region to probe a rat genome liDate received 4 November 1991 Date accepted 23 December 1991
brary (2). A low stringency Southern blot detected a clone called RGB-2. This hybridized in Southems after a high stringency wash (0.2X SSC, 0.1% SDS, 65°C) to a 2.5 kb cDNA from rat brain, and in Northems to mRNA from various rat brain tissues wherein the D2 dopamine receptor is known to be expressed. The coding sequence is consistent with the molecular weight of the D2 dopamine receptor. An extra 87 bp of coding sequence are seen in some cDNAs of mature transcripts, suggesting alternative splicing (3). When RGB-2 is put in a eucaryotic expression system, binding of ligands to the cell membrane is similar to that seen in rat tissue containing this receptor (2). Using this rat cDNA as a probe, Grandy et al did a similar analysis to identify a human pituitary cDNA and test ligand binding properties of its expression product (3). On the basis of sequence similarities and product ligand binding properties, this was identified as the human D2 dopamine receptor gene (although great similarities are seen between the sequence of dopaminergic and adrenergic receptor proteins in rodents), named DRD2, and localized by hybridization to a place on chromosome 11 (4). The 87 bp variably present in the rat message appear in a distinct exon of the human gene (3).
203
204 Restriction of DRD2 by TaqI showed fragment length bimorphism, identifying alleles Al and A2 (4). The fragment containing the TaqI site contains coding and non-coding regions corresponding to the 3’ end of the mRNA. The alleles produce different average amounts of receptor sites in human caudate nuclei, but with equal affinity for ligand (5)-results suggesting a difference in a non-coding region, affecting translation efficiency, although an amino acid difference affecting efficiency of post-translational processing or stability is not ruled out. Whether the amounts of mRNA are comparable has not been established. Human cadaver studies by Blum and Noble associated allele Al with retrospective diagnoses of alcoholism (5, 6). This finding was hailed as portending prediction of alcoholism as a phenotype by genotyping (7). Differences were also shown in binding affinity and number of sites in caudate nuclei for a dopaminergic ligand between alcoholic and nonalcoholic cadaver populations, although with great variation between individuals (5). The difference in number of binding sites was consistent with that between cadavers homozygous for Al, heterozygous, and homozygous for A2. However, genotyping of living known alcoholics versus control populations led other authors to conclude that there was no link between DRD2 allele Al and alcoholism (8,9). The discrepancy has led to controversy and guesses at how to reconcile the data (10). Discussion The methods and results in the supposedly contradictory studies suggest that Blum and Noble found a marker for susceptibility to alcoholism 1, and that Bolos and Parsian showed this marker not to be associated with alcoholism 2. In other words, allele A2 confers relative resistance, and Al, relative susceptibility, to death or illness from chronic alcohol overdose, and the gene does not affect alcoholic behavior. Although the retrospective diagnoses were made by psychiatrists, for ‘a large majority of alcoholics’ in Blum’s initial study (a), ‘the cause of death was primarily attributed to the chronic damaging effects of alcohol on their bodily systems’. ‘The causes of death included accidents, gunshot wounds, myocardial infarction, heart failure, cancer, hepatorenal failure, gastrointestinal bleeding, suicide, and pneumonia.’ The selected population was similar or identical in the second paper (5). It would be interesting to separate the accident, gunshot, and suicide cases to reanalyze the remainder.
MEDICAL HYPOTHESES
Bolos’ study used psychiatric selection for alcoholism, but excluded subjects with ‘an acutely active medical disorder’ (8). Behavioral history was therefore determining. Parsian’s study used similar behavioral criteria, but did not exclude patients with medical problems (9). Neither study found association between alcoholism in general and presence of allele Al. But Parsian also separately studied alcoholics ‘classified as severe because of serious medical problems’. These included 8 cases comprising cirrhosis of the liver, pancreatitis, gastritis, organic brain syndrome, and peripheral netuopathy, and 2 cases without those somatic conditions, but with shakes or seizures on withdrawal of alcohol. There were 6 of these cases out of the total of 16 subjects in which the Al allele was present, and 4 of these cases out of the total of 41 without Al. The difference is significant at P = 0.05 in a two tailed fourfold table test. Parsian et al saw in this result ‘the possibility that the Al allele is not associated with increased risk of alcoholism in general,’ meaning alcoholism 2, ‘but only modifies the probability that an alcoholic will develop severe physical complications,’ meaning alcoholism 1 (9). They then reported results of a family study, wherein 8 of 17 of those with Al, but only 4 of 18 without Al, had severe medical problems associated with alcoholism (9). The difference, although suggestive, is not statistically significant in the fourfold table test. However, the sample in this test was smaller than in the former, and the failure to find a significant difference does not discount the previous positive result. Since Parsian did not make clear whether the populations in the 2 studies overlapped, I don’t know whether to combine them for statistical reanalysis. The effort to find a cause for alcoholism 2 has been under way for some time, and the line of inquiry cited here extends the work of others with rodents in attempts to model human behavior. My opinion is that the factors causing persons to practice alcoholism 2 are like those causing the practice of such other ‘isms’ as Judaism. Among these factors are the perception by the actor of personal risks in such behavior (11). The determination of such risks is promised by discoveries such as that hypothesized here of a genetic screen for alcoholism 1. Persons can have similar histories of alcohol consumption, yet one will get sick, and the other not. If we could predict susceptibility better, we could help some heavy drinkers cut back, and allay the fears of others.
205
A GENE FOR ALCOHOL TOXlClTY, NOT DRINKING BEHAVIOR
Conclusion
References
For this point mutation in DRD2 to contribute to alcohol toxicity in organs other than brain, one of two possibilities must exist. A receptor in brain tissue could mediate, via autonomic or hormonal mechanism, damage to distant organs, or differences in ethanol metabolism-unlikely. Or the gene could be expressed outside the brain. Dopaminergic transmission is identified with the central nervous system. However, a systematic search for mRNAs or cDNAs from outside the brain which hybridize to the probes has not been undertaken, but should be. Perhaps the scatter in ligand binding data from human cadavers could be eliminated by studies in the clone expression system used by Grandy et al (3). Such a system could also be used to assay any difference in ethanol or acetaldehyde cytotoxicity conferred by Al versus A2 in some ‘neutral’ host. However, failure to find a difference would not rule out a difference in their native habitat, wherein interference with normal operation of a dopamine receptor may be telling. Epidemiology should henceforth concentrate on retrospective studies of individuals with similar drinking histories. Is there an A2 vs Al edge among those who have drunk heavily for long periods, and escaped morbidity? A large enough sample population would provide separate data on the various target organs.
1. Goodman R M. Addiction? ~~278-81 in The Great Issues of Drug Policy (A S Trebach, K B Zeese. eds) Drug Policy Foundation, Washington, 1990. 2. Bunzow J R, Van To1 H H M, Grandy D K et al. Cloning and expression of a rat 4 dcpamine receptor cDNA. Nature 336: 783-7, 1988. 3. Grandy D K, Marchionni M A, Makam H et al. Cloning of the cDNA and gene for a human & dopamine receptor. Proc Nat Acad Sci USA 86: 97626, 1989. 4. Grandy D K, Litt M. Allen L et al.‘Ihe human D2 dcpamine receptor gene is located on chromosome 11 at q22-23 and identifies a Taql RFLP. Am J Hum Genet 45: 778-85, 1989. 5. Noble E P. Blum K. Ritchie T, Montgomery A. Sheridan P J. Allelic association of the & dopamine receptor gene with receptor-binding characteristics in alcoholism. Arch Gen Psychiatry 48: 648-54. 1991. 6. Blum K, Noble E P, Sheridan P J et al. AlIeIic association of human dopamine Dz receptor gene in alcoholism. JAMA 263: 2055-60,1990. 7. Gordis E, Tabakoff B, Goldman D. Berg K. Finding the gene(s) for alcoholism. JAMA 263:2094-5, 1990 (editorial). 8. Bolos A, Dean M, Lucas-Derse S. Ramsburg M, Brown G L, Goldman G. Population and pedigree studies reveal a lack of association between the dopamine D, receptor gene and alcoholism. JAMA 264: 315660. 1990. 9. Parsian A, Todd R D. Devor E J, O’MaIIey K L, Suarez B K. Reich T, Claringer C R. Alcoholism and alleles of the human Dz dopamine receptor locus: studies of association and linkage. Arch Gen Psychiatry 48:65543. 1991. 10 Conneally P M. Association between the Dz dopamine receptor gene and alcoholism: a continuing controversy. Arch Gen Psychiatry 48: 644-6, 1991. 11. Peele S. Diseasing of America. Lexington Books, Lexington, Mass. 1989.