2012 - possible association between oprm1 genetic variance at the 118 locus and alcohol dependence...

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Possible Association Between OPRM1 Genetic Variance atthe 118 Locus and Alcohol Dependence in a Large

Treatment Sample: Relationship to Alcohol Dependence

SymptomsGabriele Koller, Peter Zill, Dan Rujescu, Monika Ridinger, Oliver Pogarell, Christoph Fehr,

Norbert Wodarz, Brigitta Bondy, Michael Soyka, and Ulrich W. Preuss

Background: Several lines of evidence from previous research indicate that opioid receptors play animportant role in ethanol reinforcement and alcohol dependence (AD) risk. Conflicting results were

reported on the role of the mu-opioid receptor (OPRM1) polymorphism A118G (Asn40Asp,

rs1799971) in the development of alcoholism.

Methods: We investigated a total number of 1,845 alcohol-dependent subjects recruited from inpa-

tient facilities in Germany and 1,863 controls for the mu-opioid receptor (OPRM1) polymorphism

using chi-square statistics.

Results: An association between the OPRM variant and AD was detected (p =

0.022), in recessive(AA vs. GA/GG) and co-dominant (AA vs. GA) models of inheritance. An association between theOPRM variant and the DSM-IV criterion efforts to cut down or could not (p = 0.047) was found,

but this did not remain significant after the correction for multiple testing.

Conclusions: The results indicate that this functional OPRM variant is associated with risk of AD

and these findings apply to more severe AD, although the association is only nominally significant.

Key Words: Alcohol, Genetics, Polymorphism, Opioid Receptor, Dependence.

S EVERAL LINES OF evidence suggest that mu- anddelta-opioid receptors have a great impact in ethanol(EtOH) reinforcement and the development of alcohol

dependence (AD) (Me ndez and Morales-Mulia, 2008). The

use of the opioid antagonist naltrexone as anticraving

medication in alcohol-dependent subjects provided addi-

tional evidence of the important role these receptors have in

the etiology of AD (Ro sner et al., 2010). Furthermore, the

majority of relapse prevention studies also indicate that the

amount of heavy drinking was reduced when naltrexone was

administered (Soyka and Ro sner, 2008). Functional changes

in the opioid system in chronic alcohol-dependent subjects

have also been demonstrated in positron emission tomogra-

phy studies, which indicate a negative correlation between

mu-opioid receptor binding and alcohol craving in recently

abstinent alcohol-dependent patients (Bencherif et al., 2004).

In addition, findings from imaging studies (Heinz et al.,

2005) indicate that an increase in mu-opioid receptors in dif-

ferent regions of the brain, including the nucleus accumbens,

correlate with the severity of alcohol craving.

There are at least 3 types of opioid receptorsmu, kappa,

and deltaeach with a distinct pharmacological profile. The

OPRM1 gene encodes the mu-opioid receptor. The mu-

opioid receptor is a member of the G protein-coupled recep-

tor family (Bond et al., 1998).

For the mu-opioid receptor gene, about 100 variants have

been identified with more than 20 producing amino acid

changes and having polymorphic frequencies of more than

1% (Lo tsch and Geisslinger, 2005; Somogyi et al., 2007).

The most common SNP is A118G, resulting in an amino acid

exchange at position 40 from asparagine to aspartate

(Asn40Asp; Bond et al., 1998).

The Asn40Asp substitution polymorphism of the

human mu-opioid receptor (OPRM1, rs1799971) influ-

ences the opioid binding and signal transduction and

could therefore contribute to the development of AD

(Smolka et al., 1999). Clinical studies do not unequivo-

cally support an association between polymorphisms in

OPRM1 and AD (van der Zwaluw et al., 2007).

To directly establish the potentially causal role of OPRM1

A118G variation, 2 humanized mouse lines carrying the

From the Department of Psychiatry and Psychotherapy (GK, PZ,

DR, OP, BB) Ludwig-Maximilians University, Munich, Germany;Department of Psychiatry, Psychosomatics and Psychotherapy (MR,

NW), Johannes-Gutenberg University, Mainz, Germany; Department of

Psychiatry (CF), Johannes-Gutenberg University, Mainz, Germany;Privatklinik Meiringen (MS), Meiringen, Switzerland; Department of

Psychiatry, Psychotherapy, Psychosomatics (UWP), Martin-Luther-

University, Halle, Germany.

Received for publication March 22, 2011; accepted October 26, 2011.

Reprint requests: Dr. Gabriele Koller, Klinik fur Psychiatrie und Psy-

chotherapie der LMU, Nubaumstrae 7, 80336 Munich, Germany; Tel.:

00498951605741; Fax: 00498951605748; E-mail:[email protected]

muenchen.deCopyright 2012 by the Research Society on Alcoholism.

DOI: 10.1111/j.1530-0277.2011.01714.x

1230 Alcohol Clin Exp Res,Vol 36, No 7, 2012: pp 12301236

ALCOHOLISM: CLINICAL ANDEXPERIMENTALRESEARCH Vol. 36, No. 7July 2012


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respective human sequence variant were generated. Brain

microdialysis showed a fourfold greater peak dopamine

response to an alcohol challenge in h/mOPRM1-118GG

than in h/mOPRM1-118AA mice (Ramchandani et al.,

2011). Of greatest clinical relevance is the finding that allelic

variation at OPRM1 has been associated with differential

response to a mu-receptor antagonist (Anton et al., 2008;

Oslin et al., 2003; Kranzler and Edenberg, 2010), but not allstudies show agreement to this finding (Gelernter et al.,

2007).

Several investigations found no significant association of

OPRM1 A118G genetic variation with the phenotype of AD

(Bergen et al., 1997; Franke et al., 2001; Zhang et al., 2006;

van der Zwaluw et al., 2007). In a recent systematic review, 9

of 10 studies did not find significant differences in the

frequency of the 118G allele (40Asp) between alcohol-

dependent subjects and control subjects (van der Zwaluw

et al., 2007). Another study reported a statistical trend

toward increased frequency of 118G allele in alcohol-depen-

dent subjects (Rommelspacher et al., 2001). The role of thisallele in reward-driven alcoholic phenotypes was supported

in a recent investigation (Ray, 2011) and the OPRM1 A118

allele was found to be significantly associated with increased

risk for AD in a subsequent study (Schinka et al., 2002).

Our study examines the association between AD and

OPRM1 among individuals who were recruited from inpa-

tient treatment programs. As a result, these individuals may

have a more severe alcohol use disorder than evaluated in

previous association studies. Prior research demonstrates

that specific phenotypes associated with AD appear earlier in

the development of an alcohol use disorder, like age of fre-

quent drinking. Some occur later, such as delirium tremens

and seizures (Hasin and Beseler, 2009), each of which may

have a separate but overlapping genetic background. Thus,

in our study, we also focused on the association between

OPRM1 variant and related phenotypes like tolerance, with-

drawal, larger and longer consumption, unsuccessful efforts

to cut down, delirium tremens, alcohol withdrawalrelated

seizures, an earlier age of first consumption, an earlier first

age of frequent drinking, and an earlier AD onset age.

MATERIALS AND METHODS

Subjects

Inpatient alcohol-dependent subjects were recruited from 3addiction treatment wards (offering qualified detoxification) inthe following psychiatric hospitals: Ludwig-Maximilians Universityof Munich, Germany (n = 494), Gutenberg University of Mainz,Germany (n = 250), and University of Regensburg, Germany(n = 1,101). All alcohol-dependent individuals (n = 1,845) weretreatment-seeking and admitted via an outpatient motivationalgroup (Munich), an addiction outpatient unit (Regensburg) or foremergency detoxification (Mainz). While the mode of admissionwas different across hospitals, all 3 centers offer an inpatient quali-fied detoxification treatment program, which includes somaticdetoxification, individual and group psychotherapy, counseling forsocial and financial problems as well as somatic medical care. Thisapproach is in correspondence with inpatient treatment standards

in Germany (Mann et al., 2006). Data on recent DSM-IV with-drawal are available for the Munich and Mainz sample.

All subjects met Diagnostic and Statistical Manual of MentalDisorders, Fourth Edition (DSM-IV TR) criteria (German Version;Sass et al., 2003) for AD and were assessed using the GermanVersions of CIDI/DIA-X (Composite International DiagnosticInterview; Wittchen et al., 1998) in Mainz and Regensburg or Semi-Structured Assessment on Genetics in Alcoholism (SSAGA;Bucholz et al., 1994; Hesselbrock et al., 1999) in Munich. Althoughdifferent assessment instruments were employed, all participatinghospitals used the same DSM-IV diagnostic criteria.

AD-Related Traits

Although each recruiting center enrolled alcohol-dependent sub-jects consecutively, subjects were excluded if they had any currentAxis I disorders other than major depression, nicotine, or marijuanadependence. Alcohol-dependent subjects with current and lifetimemajor depression but without current suicidal behavior wereincluded. Subjects incapable of informed consent were excludedfrom the study.

With the exception of alcohol withdrawal symptoms and compli-cations in the days following admission, all assessments were con-

ducted approximately 2 weeks after alcohol detoxification and priorto discharge. At this time, all subjects were still inpatients and freeof any psychotropic medication. The age of AD onset was definedfrom SSAGA or corresponding CIDI/DIA-X items of first agewhen at least 3 criteria of DSM-IV criteria were met.

Daily alcohol intake prior to admission was asked and calculatedin grams per day (g/d) of absolute EtOH. Structured interviews usedin the study employed a calendar method to obtain information ondrinking habits. Daily intake of several alcoholic beverages includ-ing beer, wine, and booze was reported from the patients. While allthe reported amounts of average daily alcohol intake differ signifi-cantly across sites, they all range between 200 and 300 mg/d.

The duration of AD was computed as the difference between sub-jects current age and the age of AD onset. Finally, history of alco-hol withdrawal, delirium tremens, and alcohol withdrawalrelated

seizures was also taken from study participants. Subjects with anyother current substance use disorder except nicotine and marijuanawere excluded from the study. A median split of age at first alcoholintake, age at frequent drinking, and age of AD onset was per-formed to conduct an association analysis with the OPRM variant.

To cross-validate the information from semi-structured inter-views, data reported from subjects were cross-checked with theirinpatient files including current and previous hospital admissions.

Control Samples

The first group of control persons was recruited from the generalpopulation at different locations (e.g., libraries, road constructionsites, and department stores) intending to represent a range of social

classes from university graduates to unskilled workers (Munich I,n = 346). All subjects underwent comprehensive medical and psy-chiatric assessment to establish general dimensional personalitycharacteristics (MMPI, NEO-FFI, and TCI) so as to exclude per-sons with possible psychiatric Axis I/II disorders, such as schizo-phrenia, depression, personality disorders, and substance usedisorders other than AD. This assessment included the SSAGA(Bucholz et al., 1994; Hesselbrock et al., 1999), various personalityquestionnaires, and a brief structured clinical interview conductedby a psychiatrist together with routine laboratory screening.Furthermore, all individuals with any positive first-degree familialhistory of Axis I disorders (including AD) were excluded. This con-trol sample has been described in more detail previously (Zill et al.,2004a,b). All were of German descent, and none of the subjects wererelated.

RELATIONSHIP TO ALCOHOL DEPENDENCE SYMPTOMS 1231


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A second group of controls (Munich II, n = 1,432) was made upof unrelated volunteers of German descent randomly selected fromthe general population of Munich, Germany, and contacted byemail. To exclude subjects with a personal or family history of neu-ropsychiatric disorders, thorough pre-enrollment screening was per-formed. Initially, subjects who responded (approximately 50%)were screened by phone. Here, detailed medical and psychiatric his-tories were taken for both subjects and their first-degree relativesusing systematic forms. Second, a selection of the responders wasinvited to a comprehensive interview (Structural Clinical InterviewDSM-IV disorders [SCID-I] and SCID-II, German Versions; Witt-chen and Fydrich, 1998) at the respective study centers. Psychiatricdiagnoses among their first-degree relatives were assessed using theFamily History Assessment Module (Rice et al., 1995). Again, sub-

jects with relevant somatic diseases or a lifetime history of any AxisI or II psychiatric disorders or suicidal behavior were excluded. Sub-

jects who had first-degree relatives with a lifetime history of a mentaldisorder or suicidal behavior were also excluded.

The third group of controls from the University of Mainz wascontacted via board notices at several university departments(n = 85). The absence of psychiatric disorder was confirmed by adetailed psychiatric investigation in addition to a standardized diag-nostic interview (CIDI/DIA-X; Wittchen et al., 1998).

The entire study was approved by the ethics committee in agree-ment with the principles laid down in the Helsinki Declaration(1964).

Genotyping

Genomic DNA was isolated from whole blood according to stan-dard procedures. Genotyping of the Asn40Asp polymorphism(A118G; rs1799971) in the OPRM gene was performed by the fluo-rescence resonance energy transfer method using the Light CyclerSystem (Roche Diagnostics, Mannheim, Germany). The followingconditions were applied: forward primer: 5-ACC TCG CAC AGCGGT-3; reverse primer: 5-CCG AAG AGC CCC ACC A-3;acceptor hybridization probe: LC 640 Red-5-ATG CGG TCCGAA CCG CAC CGA CCT GGG CGG GA-3phosphate; donor

hybridization probe: 5-CTT AGA TGG CAA CCT GTC CGA-3Fluorescein.

PCR was performed with 50 ng DNA according to the manu-factures instructions for 35 cycles of denaturation (95C,0 seconds, ramp rate 20C/s), annealing (65%C, 10 seconds,ramp rate 20C/s), and extension (72C, 10 seconds, ramp rate 20C/s). After amplification, a melting curve was generated by hold-ing the reaction at 32C for 4 min and then heating slowly to 70C with a ramp rate of 0.15C/s. The fluorescence signal was plot-ted against temperature to give melting curves at 57C (Asp-allele)and at 65C (Asn-allele). All laboratory procedures were carriedout blind to casecontrol status.

For quality control, genotyping was performed using internalcontrols and blanks. For second analyses, 10% of all probes wererandomly selected and genotyped in independent assays. We found100% agreement between the assays.

Statistical Analysis

All continuous data were tested for normal distribution byKolmogorovSmirnov nonparametrical tests. HardyWeinbergequilibrium (HWE) was computed for the OPRM A118G geneticvariant. As recommended (Wigginton et al., 2005), the HWE was

verified using the exact test (Guo and Thompson, 1992). To analyzethe relationship between the defined clinical characteristics andOPRM A118G, chi-square statistics was employed. A p-value of


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Regensburg). Sample characteristics are presented in

Table 1. From Munich and Mainz, 1,863 controls were

enrolled.

To exclude potential site effects on the association statis-

tics, OPRM genotype frequencies were compared across

study sites. For both alcohol-dependent individuals (v2 value

1.19; degree of freedom (df): 4; p: 0.879) and controls (v2

value 2.82; df: 2; p: 0.431), no significant differences acrosscenters were found. Association analysis was also conducted

for both genders. No gender-specific association was found.

Using 1-way ANOVA to compute statistical differences in

continuous variables (e.g., ages of onset of AD and other

drinking patterns, # of DSM-IV criteria met) across OPRM

genotypes did not show any additional statistically signifi-

cant results.

To exclude an association with smoking (Smoking +/ in

AD and controls) or depression history (AD + Depression,

AD Depression, controls), both phenotypes were associ-

ated with OPRM and showed nonsignificance (smoking: v2

value = 0.6, df: 2,p = 0.74; depression: v2

value = 8.8, df: 4,p = 0.06). Further, as described in the Materials and Meth-

ods section, no OPRM genotype differences were detected

across centers in either alcohol-dependent individuals or

controls. Regression analyses with either OPRM A- or

G-alleles as dependent variables did not reveal any additional

statistical significance.

The OPRM polymorphism in both alcohol-dependent

individuals and controls did not deviate from HWE, as pre-

sented in Table 2. Chi-square statistics showed a significant

difference of A-allele frequencies between the control and the

alcohol-dependent groups when AA versus AG/GG (reces-

sive model) and AA versus AG genotypes (co-dominant

model) were compared. Comparing A- versus G-alleles

between groups, however, failed to show any statistical sig-

nificance (v2 value 3.69; df: 1;p: 0.055).

A marginally significant association was detected between

unsuccessful efforts to cut down (v2 value: 6.12; df: 2;

p = 0.047) with OPRM A118G (Table 3). This result, how-

ever, did not remain significant after correction for multiple

testing.

A separate analysis for recent withdrawal with the OPRM1

SNP was conducted and yielded a nonsignificant association.

In this analysis, 588 alcohol-dependent subjects were included

of whom n = 447 (76.3%) met DSM-IV criteria of alcohol

withdrawal. More than 50% of the 447 individuals received

pharmacological treatment during withdrawal.

DISCUSSION

The most important finding in this large, multicenter study

of alcohol-dependent individuals and controls is that OPRM

A118G (Asn40Asp) is significantly associated with AD, in

recessive (AA vs. GA/GG) and co-dominant (AA vs. GA)

models of inheritance. Alcohol-dependent subjects in this

sample have a lower rate of heterozygotes (19.1% vs. 22.5%

in controls). Altogether, in our study, the risk allele OPRM

Table

2.Gen

otypeandAlleleFrequenciesforOPRMA1

18G(Asn40Asp)Alcohol-DependentSubje

cts(n=

1,845)andControls(n=

1,863)

SNPORPM

Geno-

type

Alcohol-

dependent

subjects

%,

N

Controls

%,

N

Allele

Alcohol-

dependent

subjects%

Co

ntrols

%

Hardy

Weinberg

equilibrium

(HWE),cases

(exacttest)

HWE,

controls

(exacttest)

Chi-square

valuecases

vs.controls,

modeof

inheritance

(df:1)

p(exact

test)

cases

vs.

controls

Oddsratio

95%CI

OPRMA118G

(Asn40Asp)

rs1799971

A/AA/GG/G

79.2(1,461

)

19.1(353)

1.7(31)

76.1(1,417)

22.5(419)

1.4(27)

A G

88.8

11.2

87.3

1

2.7

F=

0.041

p=

0.08

F=

0.014

p=

0.60

Allele:

Avs.G

Recessive

models:

5.22(AAvs.

AG/GG)

0.32(GGvs.

AG/AA)

Allele

p:0.06

Recessive

models:

0.022

0.57

Recessive

mode

l:

0.817

0.835

0.861

Recessive

model:

0.697to

0.958

0.715to

0.975

0.512to

1.447

Co-dominant

model:

1.29(GG

vs.GA)

6.18(AA

vs.GA)

Co-dominant

models:

0.2553

0.013

Co-dominant

mode

ls

0.861

0.817

Co-dominant

models:

0.512to

1.447

0.697to

0.958

df,degreeoffreedom

=

1.



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118G (40Asp) occurred less frequently in alcohol-dependent

individuals than controls (Miranda et al., 2010; van den Wil-

denberg et al., 2007), although other previous studies

reported OPRM 118G occurring more frequently in alcohol-

dependent individuals (Bart et al., 2005; Rommelspacheret al., 2001).

We chose to investigate the association between the

broader phenotype of AD and tested in subsequent analyses,

whether individual, physiologically related DSM-IV criteria

of AD are related to the OPRM variant. The results indicate

that there is a relationship to the broader phenotype rather

than individual criteria.

Of course, the association of opposite alleles at the same

bi-allelic locus in different samples with the same or similar

phenotype can be considered a spurious finding. A further

limitation of our study is that a comparison of A versus

G-alleles across groups showed only a significant trend.

Recent publications consider contradictory findings for

the same SNP and the same phenotype as so-called flip-flop

associations (Lin et al., 2007; Zaykin and Shibata, 2008).

These flip-flop associations may indicate heterogeneous

effects of the same variants that are influenced by multilocus

effects and interlocus correlations. All these influences are

likely in AD, known to have a complex genetic background

with various genetic loci contributing to the phenotype.

In a subsequent analysis, the DSM-IV criterion unsuc-

cessful efforts to cut down was also significantly associated

with the OPRM variant. However, because altogether 9

related phenotypes were tested for association, this initially

statistical significance did not remain after Bonferroni cor-

rection for multiple testing.

Opioid agonists like naltrexone were demonstrated to

influence drinking behavior in alcohol-dependent subjects

(Johnson, 2010; OMalley et al., 2008). Naltrexone was

reported to elevate GABAergic neuroactive steroid levels in

heavy drinkers with the ASP40 allele of the OPRM1 gene

(Ray et al., 2010). The OPRM1 Asn40Asp was shown to

predict response to naltrexone treatment (Oroszi et al.,

2009). On the other hand, polymorphic variation in the opi-

oid receptor did not alter therapeutic effect of nalmefene

(Arias et al., 2008). Also, no association between OPRM1

and naltrexone treatment was found in another investigation

(Gelernter et al., 2007). Differential responses to naltrexone

with regard to variations in the OPRM1 receptor may

explain conflicting results (McGeary et al., 2006). Function-

ally significant OPRM1 Asp40 allele predicting naltrexonetreatment response in alcoholic individuals was reported

from a large treatment trial (Anton et al., 2008). Conflicting

results in alcohol sensitivity, craving, and drinking behavior

may be due to different settings (out of or inside a labora-

tory) (Ray et al., 2007).

As for our sample of inpatient alcohol-dependent individ-

uals, the admission procedure is described in the methods

section. Indication for admission (which is common in Ger-

many but not in other countries) is acute alcohol intoxication

or high risk of alcohol withdrawal symptoms and their com-

plications, which may need pharmacological treatment or

medical monitoring. Certainly, this sample may reflect alco-

hol-dependent subjects with a more severe course of disease

and a higher rate of withdrawal symptoms compared to

family (COGA sample; Schuckit et al., 2001; over 50%) or

epidemiological (rates of single withdrawal symptoms

between 30% and 70%; Brower and Perron, 2010) samples.

The more severe AD characteristics of our sample may

explain in part the differences between the results obtained in

this study and prior studies.

There are several limitations to our study. The AD charac-

teristics were obtained retrospectively with the aid of inter-

views and are subject to recall biases of study participants.

Moreover, these characteristics were quite heterogeneous

and differed significantly across centers. These differences

may be due to the use of 2 different interviews (CIDI in

Regensburg and Mainz and SSAGA in Munich) while using

the same AD criteria according to DSM-IV for study inclu-

sion. The interviews of inpatients were obtained or super-

vised by at least 1 experienced psychiatrist in each study site

(Munich: UWP, GK, MS; Mainz: CF; Regensburg: NW,

MR). All interviewers participated in an initial training using

the CIDI/DIA-X and the SSAGA.

Another issue may be the differences in admission proce-

dures across study sites. For instance, while all alcohol-

dependent subjects were treatment-seeking, they were

Table 3. Association Between Alcoholism-Related Phenotypes and OPRM Alleles and Genotypes (rs1799971 A118G)

AD-related phenotype

AD + phenotype; N, (%) AD phenotype; N(%)v2 valuedf = 2

p(corrected p)AA AG GG AA AG GG

Tolerance 129 (83.8) 22 (14.3) 3 (1.9) 1,097 (79.1) 267 (19.3) 23 (1.7) 2.27 0.32Ever withdrawal 204 (77.0) 56 (21.1) 5 (1.9) 1,022 (79.6) 289 (18.8) 21 (1.6) 1.31 0.20Larger and longer 55 (80.9) 11 (16.2) 2 (2.9) 1,171 (79.5) 278 (18.9) 24 (1.6) 0.93 0.63Efforts t o cut d own o r could n ot 64 ( 80.0) 12 ( 15.0) 4 (5.0) 1,162 ( 79.5) 277 ( 19.0) 22 (1.5) 6.12 0.047 ( 0.42)Delirium tremens 182 (84.7) 32 (14.9) 1 (0.5) 1,044 (78.7) 257 (19.4) 25 (1.9) 5.02 0.08Seizures 221 (78.6) 55 (19.6) 5 (1.8) 1,005 (79.8) 234 (18.6) 21 (1.7) 0.17 0.13Earlier age at first consumption ( MS) 348 ( 77.3) 95 (21.1) 7 (1.6) 737 ( 80.5) 164 (17.9) 15 (1.6) 2.02 0.37Earlier first age at frequent drinking (MS) 130 (75.6) 39 (22.7) 3 (1.7) 558 (80.1) 127 (18.2) 12 (1.7) 1.78 0.41Earlier alc. depend. onset age (MS) 129 ( 79.0) 22 (19.4) 3 (1.6) 737 ( 80.5) 164 (17.9) 15 (1.6) 3.91 0.14

df, degrees of freedom; MS, median split; AD, alcohol dependence; +/ phenotype, phenotype present or not present; (), p-value after Bonferronicorrection for multiple testing, 9 phenotypes.

1234 KOLLER ET AL.


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admitted by different modes of admission. Apart from that,

all 3 centers offer a comparable qualified detoxification

program. Although alcohol-dependent individuals across

study sites differed significantly in most of their characteris-

tics, the OPRM variant was still significantly associated with

the risk of AD and 1 related phenotypes. Furthermore, no

subjects with dependence from illicit drugs were included

into the study. This could be a limitation when compared toother studies in which subjects dependent on alcohol and illi-

cit substances were enlisted. Another limitation is the lack of

a genomic control technique to confirm the study results.

Mu-opioid mRNA expression was not investigated. As

reported earlier (i.e., Kim et al., 2011; Kraus et al., 2010),

mu-opioid receptor expression seems to be significantly influ-

enced by several mechanisms distinct from genetics.

The interpretation of results may also be limited as

patients suffering from major depression were included in the

alcohol-dependent sample, whereas other psychiatric disor-

ders were an exclusion criterion for the control sample.

Although the OPRM1 variant was associated with depres-sion, this was not statistically significant. Smoking as a phe-

notype was clearly not associated with the OPRM1 variant

in this study.

The results were also limited by the analytic approach,

which is based on chi-square analysis. Because the case sam-

ples are drawn from different sites within Germany, there is

considerable phenotypic (both demographic and clinical)

heterogeneity among alcohol-dependent individuals, as well

as between them and controls.

While all the subjects enrolled into the study were carefully

examined regarding their ethnic background, stratification

bias cannot be excluded. All individuals were of German des-

cent (ascertained by place of birth of their grandparents),

and all were interviewed to obtain information on their

parental heritage to exclude ethnic stratification.

However, the Hap-Map database reported an allele fre-

quency of rs1799971 minor G-allele of 15.5% for Utah resi-

dents with Northern and Western European ancestry. This

frequency is similar to the rate found for alcohol-dependent

individuals and controls in this study.

In conclusion, our results indicate that this functional

OPRM variant is associated with severe AD risk but it needs

further investigation regarding its relevance for AD-related

phenotypes and pharmacogenomics (Ryan et al., 2006).

ACKNOWLEDGMENT

We thank Dr. med. JWM Wong for native English lan-

guage editing.

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