Download opioid receptor gene variants: lack of association with

Molecular Psychiatry (1997) 2, 490–494
 1997 Stockton Press All rights reserved 1359–4184/97 $12.00
ORIGINAL RESEARCH ARTICLE
response to endogenous opioids and exogenous opiates can now be investigated.
m opioid receptor gene
variants: lack of
association with alcohol
dependence
Ninety-one percent (1093 base pairs) of the OPRM1
coding sequence and an additional 1479 bases of
intronic and untranslated sequence (Exon 2, IVS 2,
Exon 3 and 3′UTR) were determined in 30 chromosomes and the 5′UTR and Exon 1 alone were determined in an additional 104 chromosomes (Figure 1).
Four nucleotide variants (+17C/T, +118A/G, +440C/G
(relative to the ATG) and IVS2+691C/G) were observed
and these variants result in three amino acid substitutions (Ala6Val, Asn40Asp and Ser147Cys). The Ala6Val and Ser147Cys variants were rare and were
observed in 2/134 chromosomes (0.015%) and in
3/1062 chromosomes (0.0028%), but were not characterized further. However, the Asn40Asp and IVS2+691
polymorphisms were moderately to highly abundant,
about °11% and °60% in Caucasians. In each population, the frequencies of +118A/G and IVS2+691 genotypes did not differ significantly from Hardy–Weinberg
expectation. The Ala6Val, Asn40Asp, Ser147Cys and
IVS2+691G/C sequence variants are the first alleles of
the human m opioid receptor gene to be identified in
population samples. Direct sequencing and PCR-RFLP
genotyping suggest that eight out of the nine sequence
differences observed among the three extant OPRM1
cDNA sequences available in public databases are
sequencing artifacts or rare haplotypes.
The Asp40 allele is a common and non-conservative
substitution of a phylogenetically conserved asparagine, the fourth of five asparagine residues in the amino
terminal, extracellular domain of the m opioid receptor.
While the amino terminus of the rat MOR1 protein has
been shown not to be necessary for ligand binding or
ligand-mediated inhibition of forskolin-stimulated
cAMP accumulation,16,17 substitution of this phylogenetically conserved asparagine might be expected to
affect protein transport and expression level at the cell
surface due to changes in glycosylation.18–20 Increased
m opioid receptor radioligand binding after chronic
naloxone treatment is not associated with an increase
in OPRM1 mRNA,21 suggesting that another regulatory
mechanism is responsible for the increased binding.
On the basis of sequence conservation among human
and rodent opioid receptors,22 effects of the Ser147Cys
variant on m opioid receptor function might be
expected, while no effects of the Ala6Val variant would
be expected.
The opioidergic hypothesis suggested a test of association between alleles, genotypes and haplotypes at the
OPRM1 locus and alcohol dependence categories,
DSM-III-R lifetime alcohol dependence with and without drug abuse and dependence diagnoses (Tables 1
and 2). In the USC sample, alcohol dependence alone
was used in a test for association at the OPRM1 locus
due to small cell numbers (Table 1). In the FC sample,
the larger number of drug disorder diagnoses permitted
two tests, first alcohol dependence vs unaffected and
second, a comparison of alcohol dependence with and
without drug abuse and dependence diagnoses and
unaffected. The category of drug abuse and depen-
AW Bergen1, J Kokoszka1, R Peterson1,
JC Long1, M Virkkunen 2, M Linnoila3 and
D Goldman1
1
Laboratory of Neurogenetics, National Institute on Alcohol
Abuse and Alcoholism, 12501 Washington Avenue, Flow
Bldg, Room 2, Rockville, Maryland 20852, USA;
2
Department of Psychiatry, University of Helsinki,
Lapinlandentie 1, 001810 Helsinki 18, Finland; 3Laboratory
of Clinical Studies, National Institute on Alcohol Abuse and
Alcoholism, Building 10, Room 3C-103, Bethesda,
Maryland 20892, USA
Keywords: OPRM1; m opioid receptor; polymorphism;
alcoholism; substance abuse
The m opioid receptor is implicated in the reward, tolerance and withdrawal effects of alcohol and other drugs
of abuse.1,2 This hypothesis is supported by the effects
of alcohol on beta-endorphin release,3 of m opioid
receptor agonists and antagonists on alcohol consumption,4,5 and by the activation of the dopaminergic reward
system by both alcohol and opiates.6 In addition, the
murine m opioid receptor locus, Oprm, is implicated as
the major quantitative trait locus (QTL) affecting the different levels of morphine consumption between two
inbred mouse strains that also exhibit differences in
alcohol and cocaine consumption. 7,8 Detection of genetic variation affecting OPRM1 expression or m opioid
receptor function would be an important step towards
understanding the origins of inter-individual variation in
response to m opioid receptor ligands and in diseases of
substance dependence.9–12 We directly sequenced the
human m opioid receptor locus, OPRM1,13–15 to detect
natural variation that might affect function and/or be
associated with psychiatric phenotypes related to
opioid function. Four DNA sequence variants were
found: three non-synonymous substitutions (Ala6Val
[rare], Asn40Asp, [0.10–0.16], Ser147Cys [rare]) and one
intronic variant (IVS2+691G/C [0.55–0.63]). OPRM1
alleles, genotypes and haplotypes from three psychiatrically characterized population samples (US Caucasian [USC, n = 100], Finnish Caucasian [FC, n = 324] and
Southwestern American Indian [SAI, n = 367]), were
used to perform association and sib-pair linkage analyses with alcohol and drug dependence diagnoses. No
significant association of OPRM1 genetic variation to
phenotype was observed. This analysis has 80% power
to detect a small to moderate effect of OPRM1 variation
on alcohol dependence and 100% power to detect
effects of the magnitude of the ALDH2*2 variant. While
these data do not support a role of the m opioid receptor
in susceptibility to alcohol dependence, the potential
relationship between OPRM1 genetic variation and
OPRM1 variants and alcohol dependence
AW Bergen et al
491
Figure 1 Direct sequencing screen of the OPRM1 locus. The domains of the m opioid receptor (MOR) protein screened include
the entire amino terminal extracellular domain (ECD1), 80% of the first transmembrane domain (TM1), 50% of first cytoplasmic
loop (CD1), the entire second through seventh transmembrane domains and intervening extracellular and cytoplasmic loops
and 65% of the carboxyterminal cytoplasmic domain (376/400 amino acids). This direct sequencing screen of the OPRM1 locus
also included a portion of the promoter (37 bases), the entire 2nd intron (773 bases) and a portion of the 3′UTR (669 bases).
The base pair (bp) designations are relative to the start codon. PCR and sequencing primers are indicated by horizontal arrows
and sequence variation by vertical arrows and shaded blocks within the locus schema. Hash marks (//) represent unscreened
regions of the OPRM1 locus.
Table 1 OPRM1 genetic variation (genotype and allele frequency) and dependence phenotype in USC, FC and SAI population samples
Population/phenotype
Genotype
Asn40/Asp40
Allele
Asn40/Asp40
691G/C
691G/C
n
1/1
1/2
2/2
1/1
1/2
2/2
1
2
1
2
US Caucasians
Alcohol Dep
Unaffected
Total
χ2=
20
80
100
0.950
0.750
0.790
0.050
0.250
0.210
0.000
0.000
0.000
0.213
0.150
0.138
0.140
0.550
0.450
0.470
0.300
0.413
0.390
0.001
0.975
0.875
0.895
0.025
0.125
0.105
3.405
0.425
0.363
0.375
0.575
0.638
0.625
0.533
Finnish Caucasians
Alcohol Dep
Unaffected
Total
χ2=
140
184
324
0.743
0.783
0.765
0.243
0.212
0.225
0.014
0.005
0.009
1.174
0.164
0.152
0.157
0.457
0.533
0.500
0.379
0.315
0.343
1.911
0.864
0.889
0.878
0.136
0.111
0.122
0.877
0.393
0.418
0.407
0.607
0.582
0.593
0.432
Indians
238
0.681
129
0.744
367
0.703
0.286
0.233
0.267
0.034
0.023
0.030
2.731
0.185
0.209
0.193
0.529
0.512
0.523
0.286
0.279
0.283
0.792
0.824
0.860
0.837
0.176
0.140
0.163
1.669
0.450
0.465
0.455
0.550
0.535
0.545
0.163
52
88
182
322
0.827
0.693
0.780
0.764
0.173
0.284
0.214
0.227
0.000
0.023
0.005
0.009
5.440
0.154
0.170
0.154
0.158
0.481
0.443
0.533
0.500
0.365
0.386
0.313
0.342
2.146
0.913
0.835
0.887
0.877
0.087
0.165
0.113
0.123
6.132
0.394
0.392
0.420
0.408
0.606
0.608
0.580
0.592
0.496
Indians
122
116
21
108
367
0.656
0.707
0.905
0.713
0.703
0.311
0.259
0.048
0.269
0.267
0.033
0.034
0.048
0.019
0.030
7.143
0.180
0.190
0.095
0.231
0.193
0.525
0.534
0.619
0.491
0.523
0.295
0.276
0.286
0.278
0.283
2.681
0.811
0.836
0.929
0.847
0.837
0.189
0.164
0.071
0.153
0.163
3.902
0.443
0.457
0.405
0.477
0.455
0.557
0.543
0.595
0.523
0.545
0.997
Southwest American
Alcohol Dep
Unaffected
Total
χ2=
Finnish Caucasians
Alc Dep + Drug
Alc Dep − Drug
Unaffected
Total
χ2=
Southwest American
Alc Dep + Drug
Alc Dep − Drug
Drug alone
Unaffected
Total
χ2=
*
All P values nonsignificant (P.0.05).
OPRM1 variants and alcohol dependence
AW Bergen et al
492
Table 2 Sib-pair linkage and regression of OPRM1 Asn40/Asp40 and IVS2+691C/G alleles to alcohol dependence and drug
abuse and dependence in the SAI population sample
Alcohol dependence
Drug dependence
Sib-pair linkage
P-value
Pairs
Sib-pair type
Pairs
Alleles
i.b.d.
Std Err
Asn40/Asp40
Unaffected
Discordant
Affected
37
87
97
0.510
0.495
0.500
0.024
0.016
0.016
0.332
0.380
0.498
IVS2+691C/G
Unaffected
Discordant
Affected
36
88
94
0.485
0.485
0.510
0.032
0.020
0.020
1.000
0.229
0.308
Asn40/Asp40
IVS2+691G/C
D.F.
136
136
Mean p
0.500
0.496
T-value
−0.379
−0.678
dence (drug disorder) alone without alcohol dependence was discarded in this sample because the n was
2. In the SAI sample, the same two tests were used, and
a sufficient number of drug disorder patients without
alcohol dependence was available (n = 21) to include
this category in the second analysis. Also in the SAI
sample, sib-pair linkage analysis was used to test for
association between genotype and alcohol dependence
and drug disorder diagnoses (Table 2). No significant
association or linkage between OPRM1 alleles, OPRM1
genotype or OPRM1 haplotype (data not shown) to any
disorder category was observed in the three population samples.
Human and animal studies implicate the opioid system in both initial sensitivity or response to alcohol
and in the reinforcing effects of alcohol, which have
been attributed to activation of the opioid and dopamine systems.1,2,5 The lack of an alcohol preference or
sensitivity QTL near the Oprm locus on proximal chromosome 10q25 and the presence of QTLs at other
loci23–25 suggest that differences in consumption of
alcohol in the C57BL and DBA mouse strains may be
affected by loci other than Oprm. The results of this
study suggest that a similar conclusion could be drawn
for humans, that is, that the OPRM1 locus does not
affect alcohol consumption, where a diagnosis of alcohol dependence is a measure of increased intake of
alcohol.
The diseases of alcohol and drug dependence are
complex disorders involving genes, the effects of drugs
of abuse on normal physiology and variable host
response to drug exposure,2 suggesting that the effect of
any single gene may be small. To detect subtle genetic
effects on such complex diseases, large samples of
either unrelated individuals or sib-pairs may need to
be analyzed using non-parametric approaches.26 This
Alleles
i.b.d.
Std Err
P-value
89
96
36
0.480
0.518
0.506
0.016
0.015
0.013
1.000
1.000
0.397
87
97
34
0.487
0.513
0.462
0.019
0.019
0.040
1.000
1.000
1.000
Mean p
0.500
0.496
T-value
1.561
1.155
P-value
0.940
0.875
Regression
P-value
D.F.
0.353
136
0.249
136
analysis of three population samples suggests, however
that the negative result may not be due to a lack of
power. The three samples are large enough to have a
power of 80% to detect small (0.1) to medium (0.3)
effect sizes at a nominal significance level of P = 0.05,27
where the effect size of the variant ALDH2*2 allele is
0.32 for DSM-III alcohol dependence.28 At this effect
size, all our samples have complete power to detect
association of OPRM1 variation to alcohol dependence.
These results do not rule out a relationship between
OPRM1 alleles and alcohol dependence, but do suggest
that the effect on alcohol dependence, if it exists, is
less than 10% of the liability.
Other phenotypes related to the normal and diseaseassociated functions of the opioid system could be
tested for association with the OPRM1 genetic variation
discovered and characterized in this study. The lack of
morphine-mediated analgesia, reward, physical dependence and withdrawal symptoms in Oprm knock-out
mice which show no accompanying changes in d or k
opioid receptor number or distribution suggests that it
is the m opioid receptor which is of primary importance in the pharmacology of opiate addiction.29,30
Methadone31 and naltrexone32 have been used to treat
opiate addiction and alcoholism33,34 and patients who
exhibit differential response to opiate pharmacotherapy constitute groups within which to look for association of OPRM1 to behavior. The recent discovery of
endomorphins as powerful m opioid-specific ligands35
suggests that there are multiple endogenous m opioid
ligand-receptor systems regulating opioid system tone.
Thus the function of neuroendocrine systems regulated
by the opioid system, such as the HPA axis and nociception, may be affected by genetic varation at
OPRM1. Association analysis with such phenotypes
may contribute towards understanding the role of the
OPRM1 variants and alcohol dependence
AW Bergen et al
m opioid receptor in vulnerability to addiction,
response to m opioid receptor pharmacotherapy and to
endogenous ligands.
Methods
Subject ascertainment and diagnostic assessment
The US Caucasian population sample is composed of
100 unrelated individuals (42 males, 58 females, mean
age = 43.6 ± 14.0 years) recruited through newspaper
advertisements for an EEG study as described.36 This
sample included five individuals with alcohol dependence alone, three with drug abuse or dependence
(drug abuse) alone, 15 with both alcohol dependence
and drug abuse, and 77 with no alcoholism or drug
abuse diagnoses. The Finnish sample was made up of
324 males, composed of 165 male criminal offenders
(mean age = 31.6 ± 8.1 years) undergoing forensic psychiatric examination in the Psychiatry Department of
the University of Helsinki and 159 male volunteers
(mean age = 31.7 ± 9.7 years) recruited through newspaper advertisements as described. 37,38 This sample
included 88 individuals with alcohol dependence
alone, two with drug abuse alone, 52 individuals with
both alcohol dependence and drug abuse, and 182 with
no drug abuse or dependence diagnoses. The American
Indian population sample is composed of 367 individuals (155 male and 212 female, mean age = 36.5 6 13.2
years) from a single Southwestern tribe. Individuals
were identified from three very large genealogies for a
family-based study on alcoholism and psychiatric disorders as described.39 This sample included 116 with
alcohol dependence diagnoses alone, 21 individuals
with drug abuse alone, 122 individuals with both alcohol dependence and drug abuse, and 108 with no alcohol or drug abuse diagnoses. All subjects provided
informed consent and all studies were approved by the
appropriate IRBs as described.36–39 All subjects were
interviewed using SADS-L or SCID and DSM-III-R diagnoses were determined from interview data by two psychiatrists,36 by a clinical social worker under the supervision of a research psychiatrist37,38 or by a clinical
social worker and a clinical psychologist.39
Genotyping
PCR reactions and direct sequencing and restriction of
PCR products was performed on DNA extracted from
lymphoblastoid cell lines using sequencing primers
derived from cDNA sequence and intronic sequence as
depicted in Figure 1 and as described.40 The +17 C/T
(Ala6Val) transition was observed in the direct sequencing screen and was not genotyped in other individuals. The +118A/G (Asn40Asp) polymorphism
was
genotyped
using
the
forward
primer,
5′CCTTGGCGTACTCAAGTTGCTC3′, bases +45→+66,
and reverse primer, 5′TTCGGACCGCATGGGACG
GAC3′, bases +139→ +119, where an A was substituted
for a T at position −6 to create a DrdI site, with an
annealing temperature of 66°C, to amplify a 95-bp PCR
product, yielding 22 and 73 bp fragments upon DrdI
digestion of the variant PCR product. The +440C/G
(Ser147Cys) transversion was genotyped using forward
primer, 5′CTCCTAGATACACCAAGATG3′, composed
of the last seven bases of IVS141 and bases +285→+297
and reverse primer, 5′TGAACATGTTATAGTAA
TCTCTG3′, bases +462→ 441, where a C was substituted for an A at position −3 to create a PstI site, with
an annealing temperature of 54°C, to amplify a 185-bp
PCR product, yielding 24 and 161 bp fragments upon
PstI digestion of the variant PCR product. The
IVS2+691C/G transversion was genotyped using forward primer, 5′GCTCTGGTCAAGGCTAAGAAT3′,
bases IVS2+670→IVS2+690 where a G was substituted
for an A at position −4 to create a HinfI site, and reverse
primer, 5′GATCATCAGTCCATAGCACACGG3′, bases
+774→+751, with an annealing temperature of 64°C, to
amplify a 235-bp fragment, yielding 215 and 20 bp fragments upon HinfI digestion of the variant PCR product.
Haplotype information derived from direct sequencing
and PCR-RFLP genotyping of bases at positions +151,
+183, +621, +699, +700, +1313, +1655 and +1700 suggests that these sequencing discrepancies observed
among the three OPRM1 cDNA sequences found in
Genbank13–15 and the sequence obtained in this study
are sequencing artifacts or rare haplotypes not
observed in our sample, as noted in Genbank
Accessions AF024515, AF024516 and AF024517.
Statistical analysis
Association analyses between OPRM1 alleles, genotypes and haplotypes and DSM-III-R diagnoses of alcohol dependence and drug abuse and/or dependence
were performed using contingency table analysis. Sibpair linkage analyses in the Southwest American
Indian population between OPRM1 genotype and
DSM-III-R diagnoses of alcohol dependence and drug
abuse and/or dependence diagnoses were performed
using the SAGE program.42
Acknowledgements
The authors thank L Akhtar, R Aragon and M Radel for
technical assistance, MA Enoch, M Eggert and R Robin
for assistance with ascertainment of clinical populations, A Malhotra for advice on power analysis and
the editor and two referees for suggestions on a earlier
version of this manuscript.
References
1 Herz A. Endogenous opioid systems and alcohol addiction. Psychopharmacology 1997; 129: 99–111.
2 Kreek MJ. Opiates, opioids and addiction. Mol Psychiatry 1996; 1:
232–254.
3 Gianoulakis C, Barcomb A. Effect of acute ethanol in vivo and in
vitro on the B-endorphin system in the rat. Life Sci 1987; 40: 19–28.
4 Reid LD, Hunter GA. Morphine and naloxone modulate intake of
ethanol. Alcohol 1984; 1: 33–37.
5 Altschuler HL, Phillips PE, Feinhandler DA. Alteration of ethanol
self-administration by naltrexone. Life Sci 1980; 26: 679–688.
6 Di Chiara G, Imperato A. Drugs abused by humans preferentially
increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci USA 1988; 85:
5274–5278.
493
OPRM1 variants and alcohol dependence
AW Bergen et al
494
7 Berrettini W, Ferraro T, Alexander R, Buchberg A, Vogel W. Quantitative trait loci study of morphine preference in inbred mouse
strains. Nature Genet 1994; 17: 54–58.
8 Alexander RC, Heydt D, Ferraro TN, Vogel W, Berrettini WH.
Further evidence for a quantitative trait locus on murine chromosome 10 controlling morphine preference in inbred mice. Psych
Genet 1996; 6: 29–31.
9 Volpicelli JR. Uncontrollable events and alcohol drinking. Br J
Addict 1987; 82: 381–392.
10 Gianoulakis C, Beliveau D, Angelogianni P, Meaney M, Thavundayil J, Tawar V et al. Different pituitary B-endorphin and adrenal
cortisol response to ethanol in individuals with high and low risk
for future development of alcoholism. Life Sci 1989; 45: 1097–1109.
11 Peterson JB, Pihl RO, Gianoulakis C, Conrod P, Finn PR, Stewart
SH, LeMarquand DG et al. Ethanol-induced change in cardiac and
endogenous opiate function and risk for alcoholism. Alcoholism:
Clin Exper Res 1996; 20: 1542–1552.
12 King AC, Volpicelli JR, Frazer A, O’Brien CP. Effect of naltrexone
on subjective alcohol response in subjects at high and low risk for
future alcohol dependence. Psychopharmacology 1997; 129: 15–22.
13 Wang JB, Johnson PS, Persico A, Hawkins AL, Griffin CA, Uhl GR.
Human mu opiate receptor: cDNA and genomic clones, pharmacological characterization and chromosomal assignment. FEBS Lett
1994; 338: 217–222.
14 Bare LA, Mansson E, Yang D. Expression of two variants of the
human mu opioid receptor mRNA in SK-N-SH cells and human
brain. FEBS Lett 1994; 354: 213–216.
15 Mestek A, Hurley JH, Bye LS, Campbell AD, Chen Y, Tian M et al.
The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C. J Neurosci 1995; 15: 2396–2406.
16 Wang JB, Imai Y, Eppler CM, Gregor P, Spivak CE, Uhl GR. mu
opiate receptor: cDNA cloning and expression. Proc Natl Acad Sci
USA 1993; 90: 10230–10234.
17 Surratt CK, Johnson PS, Moriwaki A, Seidleck BK, Blaschak CJ,
Wang JB et al. m opiate receptor: charged transmembrane domain
amino acids are critical for agonist recognition and intrinsic
activity. J Biol Chem 1994; 269: 20548–20553.
18 Green SA, Turki J, Innis M, Liggett SB. Amino-terminal polymorphisms of the human beta2-adrenergic receptor impart distinct
agonist-promoted regulatory properties. Biochemistry 1994; 33:
9414–9419.
19 Green SA, Turki J, Bejarano P, Hall IP, Liggett SB. Influence of betaadrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am J Respir Cell Mol Biol 1995; 13:
25–33.
20 Liles WC, Nathanson NM. Regulation of neuronal muscarinic
receptor number by protein glycosylation. J Neurochem 1986; 46:
85–95.
21 Unterwald EM, Rubenfeld JM, Imai Y, Wang JB, Uhl GR, Kreek MJ.
Chronic opioid antagonist administration upregulates mu opioid
receptor binding without altering mu opioid receptor mRNA levels.
Mol Brain Res 1995; 33: 351–355.
22 Kieffer BL. Recent advances in molecular recognition and signal
transduction of active peptides: receptors for opioid peptides. Cell
Mol Neurobio 1995; 15: 615–635.
23 Melo JA, Shendure J, Pociask K, Silver LM. Identification of sexspecific quantitative trait loci controlling alcohol preference in
C57BL/6 mice. Nature Genet 1996; 13: 147–153.
24 Market PD, Bennett B, Beeson M, Gordon L, Johnson TE. Confirmation of quantitative trait loci for ethanol sensitivity in long-sleep
and short-sleep mice. Genome Res 1997; 7: 92–99.
25 Kozak CA, Filie J, Adamson MC, Chen Y, Yu L. 1994. Murine
chromosomal location of the m and k opioid receptor genes. Genomics 1994; 21: 659–661.
26 Risch N, Merikangas K. The future of genetic studies of complex
human diseases. Science 1996; 273: 1516–1517.
27 Cohen J. Statistical Power Analysis for the Behavioral Sciences.
Lawrence Erlbaum Associates: Hillsdale, New Jersey, 1988.
28 Thomasson HR, Edenberg HJ, Cragg DW, Mai X-L, Jerome RE, Li
T-K et al. Alcohol and aldehyde dehydrogenase genotypes and
alcoholism in Chinese men. Am J Hum Genet 1991; 48: 677–681.
29 Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S,
Kitchen I et al. Loss of morphine-induced analgesia, reward effect
and withdrawal symptoms in mice lacking the mu-opioid receptor
gene. Nature 1996; 383: 819–823.
30 Sora I, Takahashi N, Funada M, Ujike H, Revay RS, Donovan DM
et al. Opiate receptor knockout mice define mu receptor roles in
endogenous nociceptive responses and morphine-induced analgesia. Proc Natl Acad Sci USA 1997; 94: 1544–1549.
31 Dole VP, Nyswander M. A medical treatment for diacetylmorphine
(heroin) addiction. J Am Med Assoc 1965; 193: 80–84.
32 Martin WR, Jasinski DR, Mansky PA. Naltrexone, an antagonist for
the treatment of heroin dependence. Arch Gen Psych 1973; 28:
784–791.
33 O’Malley SS, Jaffe A, Chang G, Schottenfeld RS, Meyer RE, Rounsaville BJ. Naltrexone and coping skills therapy for alcohol dependence: a controlled study. Arch Gen Psych 1992; 49: 881–887.
34 Volpicelli JR, Alterman AI, Hayashida M, O’Brien CP. Naltrezone
in the treatment of alcohol dependence. Arch Gen Psych 1992; 49:
876–880.
35 Zadina JE, Hackler L, Ge L-J, Kastin AJ. A potent and selective
endogenous agonist for the m-opiate receptor. Nature 1997; 386:
499–502.
36 Enoch M-A, Rohrbaugh JW, Davis EZ, Harris CR, Ellingson RJ, Andreason P et al. Relationship of genetically transmitted alpha EEG
traits to anxiety disorders and alcoholism. Am J Med Gen
(Neuropsych Gen) 1995; 60: 400–408.
37 Virkkunen M, Rawlings R, Tokola R, Pland RE, Guidotti A, Nereroff
C et al. CSF biochemistries, glucose metabolism, and diurnal
activity rhythms in alcoholic, violent offenders, fire setters, and
healthy volunteers. Arch Gen Psych 1994; 51: 20–27.
38 Virkkunen M, Kallio E, Rawlings R, Tokola R, Pland RE, Guidotti
A et al. Personality profiles and state aggressiveness in Finnish
alcoholic, violent offenders, fire setters, and healthy volunteers.
Arch Gen Psych 1994; 51: 28–33.
39 Goldman D, Urbanek M, Guenther D, Robin R, Long JC. Linkage
and association of a functional DRD2 variant [Ser311Cys] and
DRD2 markers to alcoholism, substance abuse and schizophrenia
in Southwestern American Indians. Am J Med Gen (Neuropsych
Gen) 1997; 74: 386–394.
40 Bergen A, Wang C-Y, Nakai B, Goldman D. Mass allele detection
(MAD) of rare 5-HT1A structural variants with allele-specific amplification and electrochemiluminescent detection. Hum Mut 1996; 7:
135–143.
41 Simonin F, Gaveriaux-Ruff C, Befort K, Matthes H, Lannes B, Micheletti G et al. k-Opioid receptor in humans: cDNA and genomic
cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system. Proc
Natl Acad Sci USA 1995; 92: 7006–7010.
42 Statistical Analysis for Genetic Epidemiology, Release 22. Computer program package available from the Department of Biometry
and Genetics, LSU Medical Center, New Orleans, supported by a
USPHS Resource Grant (1 P41 RR03655) from the Division of
Research Resources.
Correspondence: Dr AW Bergen, Laboratory of Neurogenetics,
National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Avenue, Flow Bldg, Room 2, Rockville, Maryland 20852, USA.
E-mail: awbKdicbr.niaaa.nih.gov
Received 9 April 1997; revised 1 August 1997; accepted 4 August
1997