Vol. 16, No. 4 July/August 1992
0 I45-6008/92/1604-0786$3.00/0 AI.COHVI.ISM: CLINICAL A N D E X P F R I M E N T A RESEARCH L
COMMENTARY
Dt or Not D2? Robert W. Karp
DRAMATIZED at a recent workshop (D2 Receptor association of a variant serum vitamin D binding protein and, most recently, assoAlleles in Substance Abuse, September 19-20, I99 1, with susceptibility to at the NIDA Addiction Research Center, Baltimore, MD), ciation of a restriction fragment length polymorphism the controversy surrounding the finding of an association (RFLP) near the porphobilinogen deaminase gene with between alcoholism and the A1 allele of the D2 dopamine schizophrenia.'2 receptor gene shows little sign of abating. The original The finding of an incomplete association (as in the case claim of an association with alcoholism in general' has of the A1 allele of the D2 receptor with medical complibeen moderated to apply only to cases of alcoholism with cations of alcoholism) can be interpreted in any of the medical complications (e.g., liver cirrhosis, pancreatitis, three following ways: (1) the DZreceptor is a factor in the gastritis). Based on these observations, Parsian et a1.2 and vulnerability to complications of alcoholism, but since it Cloninger3 have suggested that the A1 allele, while not is not the only such factor, its association with complicaitself a determinant of alcoholism, might act to increase tions is less than 100%;(2) the A 1 allele of the TaqI RFLP susceptibility to medical complications of alcoholism. In marking the D2 receptor gene is not itself a factor in the two separate studies the laboratories of Goldman4 and vulnerability to complications, but is located near a gene Gelernter5 have failed to find an association of the A1 (either D2 itself or some other gene) which is; because of allele with alcoholism. Other investigators have claimed recombination between the RFLP and this gene, the asassociation of the A 1 allele with polydrug abuse, Tourette sociation between the A I allele and medical complications syndrome, and other neuropsychiatric disorders.6 Disa- is less than 100%;(3) neither (1) nor (2) is true, but for greement among these reports centers around methodo- completely independent (and unknown) reasons the allogical details ( e g , proper choice of a sample of normal coholic population studied happens to have a different individuals for comparison to alcoholics, proper criteria frequency of the A1 allele from the normal population. for diagnosis of alcoholism, exclusion of patients with For a number of reasons, interpretation (3) is most likely medical complications from the alcoholic population), as to be the correct one. It is now known, for example, that well as around insufficient consideration of possible artithe frequency of the A 1 allele differs greatly among differfactual explanations for the association. ent ethnic population^'^'^^ and both Gelernter and GoldThe controversy surrounding this finding reflects the man have suggested that observed differences between A 1 notorious difficulties both of execution and interpretation frequencies in some alcoholic and nonalcoholic populaof allelic association studies. Although such studies have tions may result from a skewing of the ethnic composition occasionally served as a useful prelude to linkage studies, of one population relative to the other. There is little direct their history (both in psychiatric research and elsewhere) evidence in the existing studies to rule out such skewing. is richly endowed with findings that have either been Additional evidence (besides the finding of an allelic impossible to replicate, or even when replicated have association) is needed to distinguish among the above contributed little to our understanding of the etiology of interpretations. A finding of cosegregation of the D2 gene the disorder under study. Examples include associations with alcoholism in a subset of alcoholic pedigrees would of blood group A of the ABO system and the S allele of greatly bolster the significance of the apparent association, complement C3 with alc~holism,~ association of a partic, ~ early attempts to detect such cosegregation have so far ular variant of alpha- 1 antitrypsin with Down s y n d r ~ m e , ~but failed.2,4A further test of the significance of the apparent From the Division of Basic Research, National Institute on Alcohol association has been suggested by Goldman and Gelernter. If the A 1 allele really aggravates susceptibility to the meda4huscand Alcoholism, 5600 Fishers Lane, Rockville, Maryland. Rcci.ivcJd,forpublication November 29, 1991; accepted February 18, ical complications of alcoholism, then within pedigrees 1992 segregating alcoholism, those individuals with medical Rqwint rc7yirc'sf.s:Roberl W. Karp, Ph.D., Division of Basic Research, complications should have a higher frequency of the A1 Room 16C-05, Nuiional Institute on Alcohol Abuse anddlcoholism, 5600 allele than those without such complications; this test li.sher.7 Lane, Rockville, MD 2085 7. circumvents the objection of a skewed ethnic composition CoIIyright 0 1992 by The Research Society on Alcoholism.
AS
706
Alcohol Clin Exp Res, Vol 16, No 4, 1992: pp 786-787
D? OR NOT Dz
of the sampled populations, and can be easily performed on existing data. Since the A 1/A2 polymorphism is at a TaqI restriction site now known to be several thousand base pairs downstream of the protein coding portion of the D2 gene,15it is extremely unlikely that this polymorphism itself results in a functional difference between the two alleles. The notion of a functional difference between the two alleles implies that there must also be either other structural differences between the two alleles (in addition to the A1/A2 polymorphism), or else subtle quantitative differences in tissue distribution of the encoded receptor protein, which are directly responsible for the presumed functional differences. Another critical test of the significance of the apparent association between the A1 allele and medical complications of alcoholism would therefore be the demonstration that the chromosomes bearing the A1 and A2 alleles actually encode D2 receptors which differ structurally, or that there is some difference in the quantity or tissue distribution of the receptor encoded by the two alleles. Early attempts to demonstrate variation in the coding portion of the D2 gene have so far failed.I6 The original notion that defective dopaminergic neurotransmission is etiologically involved in alcoholism remains attractive because of a large body of independent neurobiological evidence that this system mediates many reward, reinforcement, and motivation mechanisms. There seems little a priori reason, however, to suspect an involvement of dopaminergic neurotransmission in the development of medical complications of alcoholism ( e g , liver cirrhosis, gastritis, pancreatitis). An understanding of the true significance of the apparent association of the A 1 allele of the dopamine D2 receptor with medical complications of alcoholism remains thus for the moment elusive, awaiting proof against artifact by some of the more rigorous tests described above. Even with such proof in hand, it is important for researchers to recognize that an allelic association, however often validated and replicated, will provide at best circumstantial evidence for a cause-and-effectrelationship between a variant D2 receptor and medical complications of alcoholism. Definitive proof of a causal relationship will come ultimately from experiments (performed out of ethical necessity in animals) in which deliberate disruption of the D2 gene by genetic means results in medical complications after exposure to ethanol. While this standard of proof may be many years away, alcohol researchers should settle now for no less than the standard commonly applied for genetically well-defined disorders such as cystiC fibrosis or Duchenne muscular dystrophy. According to
787
that standard a causal role for a variant D2 gene should be inferred only after the demonstration of cosegregation of the variant allele with medical complications of alcoholism in a subset of alcoholic pedigrees, and demonstration of either variant function of the encoded receptor protein, or else of a variant tissue distribution of that protein. ACKNOWLEDGMENTS The author thanks Drs. Cathy Barr, Ken Kidd, Bruce O’Kara, and George Uhl for permission to cite their work prior to publication, and Drs. David Goldman and Joel Gelernter for valuable comments on the manuscript.
REFERENCES I . Blum K, Noble EP, Sheridan PJ, et al: Allelic association of human dopamine D2 receptor gene in alcoholism. JAMA 263:20552060, 1990 2. Parsian A, Todd RD, Devor EJ, et al: Alcoholism and alleles of the human Dz dopamine receptor locus: Studies of association and linkage. Arch Gen Psychiatry 48:655-663, 1991 3. Cloninger CR: DZdopamine receptor gene is associated but not linked with alcoholism. JAMA 266:1833-1834, 1991 4. Bolos AM, Dean M, Lucas-Derse S, et al: Population and pedigree studies reveal a lack of association between the doparnine D2 receptor gene and alcoholism. JAMA 264:3156-3160, 1990 5. Gelernter J, OMalley S, Risch N, et al: No association between an allele at the Dz dopamine receptor gene (DRD2) and alcoholism. JAMA 266:1801-1807, 1991 6. Comings DE, Comings BG, Muhlemann D, et al: The dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders. JAMA 266: 1793- 1 800, I99 1 7. Hill SY, Goodwin DW, Cadoret R, et al: Association and linkage between alcoholism and eleven serological markers. J Stud Alcohol 361981-992, 1975 8. Fineman RM, Kidd KK, Johnson AM, Breg WR: Increased frequency of heterozygotes for x 1 antitrypsin variants in individuals with either sex chromosome mosaicism or trisomy 2 I . Nature 260:320, 1976 9. Breg WR, Fineman RM, Johnson AM, Kidd KK: The current status of alpha,-antitrypsin and other factors in Down syndrome, in de la Cruz F, Gerald PS (eds): Trisomy 21 (Down Syndrome): Research Perspectives. Baltimore, University Park Press, 1980, pp 205-2 14 10. Eales LJ, Nye KE, Parkin JM, el al: Association of different allelic forms of group specific component with susceptibility to and clinical manifestation of human immunodeficiency virus infection. Lancet 1(8540):999- 1002, 1987 11. Eales LJ, Nye KE, Pinching AJ: Group-specific component and AIDS: Erroneous data. Lancet 1(8591):936, 1988 12. Sanders AR, Hamilton JD, Fann WE, Patel PI: Association of genetic variation at the porphobilinogen deaminase gene with schizophrenia (abstract). Am J Hum Genet 49(Suppl):358, 199 1 13. Barr C, Kidd KK, submitted for publication 14. OHara B, Uhl G, submitted for publication 15. Hauge XY, Grandy DK, Eubanks JH, et al: Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene. Genomics 10527-530, 1991 16. Sarkar G, Kapelner S, Grandy DK, et al: Direct sequencing of the dopamine Dz receptor ( D R D Z )in schizophrenics reveals 3 polymorphisms but no structural change in the receptor. Genomics 11:8-14, 1991
0 I45-6008/92/1604-0786$3.00/0 AI.COHVI.ISM: CLINICAL A N D E X P F R I M E N T A RESEARCH L
COMMENTARY
Dt or Not D2? Robert W. Karp
DRAMATIZED at a recent workshop (D2 Receptor association of a variant serum vitamin D binding protein and, most recently, assoAlleles in Substance Abuse, September 19-20, I99 1, with susceptibility to at the NIDA Addiction Research Center, Baltimore, MD), ciation of a restriction fragment length polymorphism the controversy surrounding the finding of an association (RFLP) near the porphobilinogen deaminase gene with between alcoholism and the A1 allele of the D2 dopamine schizophrenia.'2 receptor gene shows little sign of abating. The original The finding of an incomplete association (as in the case claim of an association with alcoholism in general' has of the A1 allele of the D2 receptor with medical complibeen moderated to apply only to cases of alcoholism with cations of alcoholism) can be interpreted in any of the medical complications (e.g., liver cirrhosis, pancreatitis, three following ways: (1) the DZreceptor is a factor in the gastritis). Based on these observations, Parsian et a1.2 and vulnerability to complications of alcoholism, but since it Cloninger3 have suggested that the A1 allele, while not is not the only such factor, its association with complicaitself a determinant of alcoholism, might act to increase tions is less than 100%;(2) the A 1 allele of the TaqI RFLP susceptibility to medical complications of alcoholism. In marking the D2 receptor gene is not itself a factor in the two separate studies the laboratories of Goldman4 and vulnerability to complications, but is located near a gene Gelernter5 have failed to find an association of the A1 (either D2 itself or some other gene) which is; because of allele with alcoholism. Other investigators have claimed recombination between the RFLP and this gene, the asassociation of the A 1 allele with polydrug abuse, Tourette sociation between the A I allele and medical complications syndrome, and other neuropsychiatric disorders.6 Disa- is less than 100%;(3) neither (1) nor (2) is true, but for greement among these reports centers around methodo- completely independent (and unknown) reasons the allogical details ( e g , proper choice of a sample of normal coholic population studied happens to have a different individuals for comparison to alcoholics, proper criteria frequency of the A1 allele from the normal population. for diagnosis of alcoholism, exclusion of patients with For a number of reasons, interpretation (3) is most likely medical complications from the alcoholic population), as to be the correct one. It is now known, for example, that well as around insufficient consideration of possible artithe frequency of the A 1 allele differs greatly among differfactual explanations for the association. ent ethnic population^'^'^^ and both Gelernter and GoldThe controversy surrounding this finding reflects the man have suggested that observed differences between A 1 notorious difficulties both of execution and interpretation frequencies in some alcoholic and nonalcoholic populaof allelic association studies. Although such studies have tions may result from a skewing of the ethnic composition occasionally served as a useful prelude to linkage studies, of one population relative to the other. There is little direct their history (both in psychiatric research and elsewhere) evidence in the existing studies to rule out such skewing. is richly endowed with findings that have either been Additional evidence (besides the finding of an allelic impossible to replicate, or even when replicated have association) is needed to distinguish among the above contributed little to our understanding of the etiology of interpretations. A finding of cosegregation of the D2 gene the disorder under study. Examples include associations with alcoholism in a subset of alcoholic pedigrees would of blood group A of the ABO system and the S allele of greatly bolster the significance of the apparent association, complement C3 with alc~holism,~ association of a partic, ~ early attempts to detect such cosegregation have so far ular variant of alpha- 1 antitrypsin with Down s y n d r ~ m e , ~but failed.2,4A further test of the significance of the apparent From the Division of Basic Research, National Institute on Alcohol association has been suggested by Goldman and Gelernter. If the A 1 allele really aggravates susceptibility to the meda4huscand Alcoholism, 5600 Fishers Lane, Rockville, Maryland. Rcci.ivcJd,forpublication November 29, 1991; accepted February 18, ical complications of alcoholism, then within pedigrees 1992 segregating alcoholism, those individuals with medical Rqwint rc7yirc'sf.s:Roberl W. Karp, Ph.D., Division of Basic Research, complications should have a higher frequency of the A1 Room 16C-05, Nuiional Institute on Alcohol Abuse anddlcoholism, 5600 allele than those without such complications; this test li.sher.7 Lane, Rockville, MD 2085 7. circumvents the objection of a skewed ethnic composition CoIIyright 0 1992 by The Research Society on Alcoholism.
AS
706
Alcohol Clin Exp Res, Vol 16, No 4, 1992: pp 786-787
D? OR NOT Dz
of the sampled populations, and can be easily performed on existing data. Since the A 1/A2 polymorphism is at a TaqI restriction site now known to be several thousand base pairs downstream of the protein coding portion of the D2 gene,15it is extremely unlikely that this polymorphism itself results in a functional difference between the two alleles. The notion of a functional difference between the two alleles implies that there must also be either other structural differences between the two alleles (in addition to the A1/A2 polymorphism), or else subtle quantitative differences in tissue distribution of the encoded receptor protein, which are directly responsible for the presumed functional differences. Another critical test of the significance of the apparent association between the A1 allele and medical complications of alcoholism would therefore be the demonstration that the chromosomes bearing the A1 and A2 alleles actually encode D2 receptors which differ structurally, or that there is some difference in the quantity or tissue distribution of the receptor encoded by the two alleles. Early attempts to demonstrate variation in the coding portion of the D2 gene have so far failed.I6 The original notion that defective dopaminergic neurotransmission is etiologically involved in alcoholism remains attractive because of a large body of independent neurobiological evidence that this system mediates many reward, reinforcement, and motivation mechanisms. There seems little a priori reason, however, to suspect an involvement of dopaminergic neurotransmission in the development of medical complications of alcoholism ( e g , liver cirrhosis, gastritis, pancreatitis). An understanding of the true significance of the apparent association of the A 1 allele of the dopamine D2 receptor with medical complications of alcoholism remains thus for the moment elusive, awaiting proof against artifact by some of the more rigorous tests described above. Even with such proof in hand, it is important for researchers to recognize that an allelic association, however often validated and replicated, will provide at best circumstantial evidence for a cause-and-effectrelationship between a variant D2 receptor and medical complications of alcoholism. Definitive proof of a causal relationship will come ultimately from experiments (performed out of ethical necessity in animals) in which deliberate disruption of the D2 gene by genetic means results in medical complications after exposure to ethanol. While this standard of proof may be many years away, alcohol researchers should settle now for no less than the standard commonly applied for genetically well-defined disorders such as cystiC fibrosis or Duchenne muscular dystrophy. According to
787
that standard a causal role for a variant D2 gene should be inferred only after the demonstration of cosegregation of the variant allele with medical complications of alcoholism in a subset of alcoholic pedigrees, and demonstration of either variant function of the encoded receptor protein, or else of a variant tissue distribution of that protein. ACKNOWLEDGMENTS The author thanks Drs. Cathy Barr, Ken Kidd, Bruce O’Kara, and George Uhl for permission to cite their work prior to publication, and Drs. David Goldman and Joel Gelernter for valuable comments on the manuscript.
REFERENCES I . Blum K, Noble EP, Sheridan PJ, et al: Allelic association of human dopamine D2 receptor gene in alcoholism. JAMA 263:20552060, 1990 2. Parsian A, Todd RD, Devor EJ, et al: Alcoholism and alleles of the human Dz dopamine receptor locus: Studies of association and linkage. Arch Gen Psychiatry 48:655-663, 1991 3. Cloninger CR: DZdopamine receptor gene is associated but not linked with alcoholism. JAMA 266:1833-1834, 1991 4. Bolos AM, Dean M, Lucas-Derse S, et al: Population and pedigree studies reveal a lack of association between the doparnine D2 receptor gene and alcoholism. JAMA 264:3156-3160, 1990 5. Gelernter J, OMalley S, Risch N, et al: No association between an allele at the Dz dopamine receptor gene (DRD2) and alcoholism. JAMA 266:1801-1807, 1991 6. Comings DE, Comings BG, Muhlemann D, et al: The dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders. JAMA 266: 1793- 1 800, I99 1 7. Hill SY, Goodwin DW, Cadoret R, et al: Association and linkage between alcoholism and eleven serological markers. J Stud Alcohol 361981-992, 1975 8. Fineman RM, Kidd KK, Johnson AM, Breg WR: Increased frequency of heterozygotes for x 1 antitrypsin variants in individuals with either sex chromosome mosaicism or trisomy 2 I . Nature 260:320, 1976 9. Breg WR, Fineman RM, Johnson AM, Kidd KK: The current status of alpha,-antitrypsin and other factors in Down syndrome, in de la Cruz F, Gerald PS (eds): Trisomy 21 (Down Syndrome): Research Perspectives. Baltimore, University Park Press, 1980, pp 205-2 14 10. Eales LJ, Nye KE, Parkin JM, el al: Association of different allelic forms of group specific component with susceptibility to and clinical manifestation of human immunodeficiency virus infection. Lancet 1(8540):999- 1002, 1987 11. Eales LJ, Nye KE, Pinching AJ: Group-specific component and AIDS: Erroneous data. Lancet 1(8591):936, 1988 12. Sanders AR, Hamilton JD, Fann WE, Patel PI: Association of genetic variation at the porphobilinogen deaminase gene with schizophrenia (abstract). Am J Hum Genet 49(Suppl):358, 199 1 13. Barr C, Kidd KK, submitted for publication 14. OHara B, Uhl G, submitted for publication 15. Hauge XY, Grandy DK, Eubanks JH, et al: Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene. Genomics 10527-530, 1991 16. Sarkar G, Kapelner S, Grandy DK, et al: Direct sequencing of the dopamine Dz receptor ( D R D Z )in schizophrenics reveals 3 polymorphisms but no structural change in the receptor. Genomics 11:8-14, 1991
