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Case-control and within-family tests for an association between conduct disorder and 5HTTLPR.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:825 –832 (2006)
Case-Control and Within-Family Tests for an Association
Between Conduct Disorder and 5HTTLPR
Joseph T. Sakai,1* Susan E. Young,2 Michael C. Stallings,2 David Timberlake,2 Andrew Smolen,2
Gary L. Stetler,2 and Thomas J. Crowley1
Division of Substance Dependence, Department of Psychiatry, University of Colorado School of Medicine, Denver, Colorado
Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado
Several lines of research have suggested that
serotonin dysfunction is associated with aggression, impulsivity, and antisocial behavior. A functional polymorphism in the promoter region (s,
short and l, long allele variant) of the serotonin
transporter gene (SLC6A4) that results in
decreased transcription of the serotonin transporter gene has been linked with such serotonin
dysfunction. To test for an association between
5HTTLPR genotype and conduct disorder diagnosis/aggression. Analysis for association between 5HTTLPR and conduct disorder/aggression
using a case-control design and the transmission
disequilibrium test. Conduct-disordered adolescents, who were drawn from admissions to a
program that treats adolescents with serious
substance and behavior problems, and conductdisordered siblings of these patients (n, 297) were
compared with non-conduct-disordered control
adolescents and non-conduct-disordered siblings
of these controls (n, 93). Second, using patient
families where parental DNA was available,
transmission disequilibrium tests were conducted for two phenotypes: (1) conduct disorder
(74 trios), and (2) conduct disorder with at least
one aggressive symptom (57 trios). Case-control
analyses suggested a strong association between
the ss genotype and conduct disorder (x22 ¼ 14.3;
P < 0.01). Within-family analyses for conduct disorder with at least one aggressive symptom
significantly favored greater transmission of the
s-allele to affected offspring (xtdt2 ¼ 4.13; P ¼ 0.04);
for conduct disorder, without aggressive symptoms, however, results were non-significant
(xtdt2 ¼ 1.61; P ¼ 0.20). These data suggest that the
s-allele may confer some risk for aggressive
behavior or may be in linkage disequilibrium
with such an allele.
ß 2006 Wiley-Liss, Inc.
aggression; serotonin; serotonin
Please cite this article as follows: Sakai JT, Young SE,
Stallings MC, Timberlake D, Smolen A, Stetler GL, and
Crowley TJ. 2006. Case-Control and Within-Family
Grant sponsor: NIDA; Grant numbers: DA 09842, DA 11015,
DA 12845, K08DA016314; Grant sponsor: NIMH; Grant numbers:
5T32MH15442, MH01865.
*Correspondence to: Dr. Joseph T. Sakai, 4200 East Ninth Ave.,
Box C268-35, Denver CO 80262. E-mail: joseph.sakai@uchsc.ed
Received 7 April 2005; Accepted 27 October 2005
DOI 10.1002/ajmg.b.30278
ß 2006 Wiley-Liss, Inc.
Tests for an Association Between Conduct Disorder
and 5HTTLPR. Am J Med Genet Part B 141B:825–832.
Conduct disorder is characterized by repetitive violation of
the rights of others and violation of age-appropriate societal
norms. It is common, associated with great morbidity [Crowley
and Riggs, 1995], and better treatments for this disorder are
needed. Because antisocial behavior and aggression are substantially heritable traits (accounting for about 40%–50% of the
population variance) [Miles and Carey, 1997; Rhee and Wladman, 2002], genetic studies may offer one avenue to search for
factors contributing to the liability toward this disorder.
Serotonin dysfunction has long been implicated in impulsivity, aggression, and antisocial behavior. Low CSF (cerebrospinal
fluid) levels of 5-HIAA (5-hydroxyindoleacetic acid), a serotonin
metabolite, have been demonstrated in aggressive patients
[Brown et al., 1979], violent offenders [Linnoila et al., 1983],
oppositional defiant disordered or conduct-disordered adolescents versus controls [Van Goozen et al., 1999], and have
predicted persistence of aggressive behavior in adolescent males
with disruptive behavior disorders (62% had conduct disorder)
[Kruesi et al., 1992]. Tests of serotonin responsiveness by
fenfluramine administration have shown a blunted prolactin
response in offenders with antisocial personality disorder
[O’Keane et al., 1992]; similar results have been demonstrated
in general population samples for males but not females
[Manuck et al., 1998]. Measures of platelet serotonin binding
offers one other peripheral measure of serotonin function;
adolescents with conduct disorder have been shown to have
significantly lower 3-H imipramine maximal binding sites on
platelets compared with control children [Stoff et al., 1987].
The serotonin transporter is a high affinity transporter
that removes serotonin from the presynaptic space, thus
terminating serotonin-mediated neurotransmission. In humans a functional polymorphism in the promoter region of the
serotonin transporter gene (SLC6A4) has been described
(5HTTLPR) with a long (l) and short (s) variant. The short
promoter variant is transcriptionally less active than the long
allele resulting in a 30%–40% reduction of the serotonin
transporter and a two-fold reduction in serotonin transport. In
addition, imaging studies suggest that this promoter deletion
alters brain activity in vivo [Heinz et al., 2000; Hariri et al.,
Importantly, the s-allele has been associated with serotonin
dysfunction, similar to that seen in impulsive-aggressive
persons. The ss genotype has been associated with lower CSF
5-HIAA in Caucasians and males but not other groups
[Williams et al., 2003]. The s-allele has been associated with
blunted serotonin function as measured by prolactin release
following fenfluramine administration among males [Reist
et al., 2001] and with lower platelet serotonin binding
availability [Stoltenberg et al., 2002].
Several case-control studies have tested for an association
between 5HTTLPR genotype and aggressive behavior. Positive
Sakai et al.
associations between the l-allele and violence or aggression
have been reported [Twitchell et al., 2001; Zalsman et al.,
2001]. In contrast, some studies have found an excess of
the s-allele among alcoholic, incarcerated, violent offenders
[Hallikainen et al., 1999], Chinese males convicted of violent
crime [Liao et al., 2004], violent heroin dependent males in
Italy [Gerra et al., 2004], and physically violent males referred
for forensic evaluations in Germany [Retz et al., 2004]. One
study found an association between the s-allele and externalizing behaviors in males; but the opposite result was found for
females [Cadoret et al., 2003]. Three studies found no
association between 5HTTLPR genotype and aggression [Reist
et al., 2001; Beitchman et al., 2003; Davidge et al., 2004].
These seemingly contradictory findings may have been due
to confounding factors such as population stratification or
small sample sizes in the studies reporting null results. In
order to examine the relationship between 5HTTLPR and
aggression, using larger samples than previous studies, we
conducted case-control and within-family analyses of
5HTTLPR genotypes and measures of aggression (conduct
disorder diagnosis (CD), and CD with at least one aggressive
The research protocol was approved by the Colorado Multiple Institutional Review Board, and all participants gave
informed written consent.
discriminative validity for conduct disorder diagnosis [Crowley
et al., 2001]. Patients also completed the CARI (Colorado
Adolescent Rearing Inventory), which provides quantitative
information about severity of physical/sexual abuse and
neglect and has shown discriminative validity and clinical
utility in this population [Crowley et al., 2003].
Pre-determined phenotypes were (1) conduct disorder
(defined as at least three lifetime DSM-IV conduct disorder
symptoms), and (2) conduct disorder with at least one
aggressive symptom.
The second phenotype was included because adolescents
may meet criteria for conduct disorder by committing rule
violations and not aggressive acts. Aggressive symptoms were
defined by the DSM-IV’s first seven criteria for conduct
disorder, which are grouped under the heading ‘‘aggression
to people and animals.’’
Genotype Determination
A DNA sample was obtained by cheek swab buccal cell
collection. 5HTTLPR genotypes were determined by modification of the protocols [Gelernter et al., 1999; Anchordoquy et al.,
2003]. The availability of genotypic data on nuclear families
allowed for the confirmation of allele calls via Mendelian
inheritance verification; all samples were genotyped twice. The
recently described single nucleotide polymorphism in the lallele [Hu et al., 2005], which is present at low frequency, was
not evaluated in this population.
Data Analysis
Patient and control families. These patient and control
samples have been described previously [Miles et al., 1998].
Briefly, patients 13–19 years of age were recruited from a
University-based treatment program; most were referred by
the criminal justice system or social services. Patients and
siblings were included in the case-control analyses if they met
lifetime criteria for conduct disorder, defined here as meeting
three lifetime conduct disorder criteria (though the impairment criteria was not required only 3 of the 297 cases did not
meet this criteria). Cases consisted of 297 individuals with
conduct disorder; 261 patients and 36 siblings of patients. The
within-patient-family analyses included those with at least
one affected offspring and at least one participating parent
(74 offspring-mother-father trios and 101 proband-parent
duos). Control adolescents were recruited to be similar on
average for age, gender, ethnicity, and zip code of residence to
adolescent patients [Miles et al., 1998]. For inclusion in these
case-control analyses, controls, or control siblings were
required to have never met criteria for conduct disorder (at
least three lifetime conduct disorder symptoms) (n, 93; 48
controls and 45 siblings of controls). Patients, controls, and
siblings were administered the same study instruments and
DNA samples were collected from consenting patients, siblings, and parents.
Adolescents from patient families meeting lifetime diagnostic criteria for conduct disorder were compared with nonconduct-disordered adolescents from control families. Differences in demographic characteristics and selected diagnoses
were compared using w2 test for categorical variables and
independent t-tests for continuous, approximately normal
variables. Adolescents from patient and control families were
compared by genotype frequencies, using the w2 test. Withinpatient-family analyses were carried out using the transmission disequilibrium test (TDT) [Spielman et al., 1993; Sham,
1998] for our two pre-determined phenotypes. Because both
parental genotypes were not available for all 297 adolescents,
these analyses included 74 trios (offspring-mother-father). To
avoid a potential bias, trios with heterozygous offspring and
parents were included in all TDT tests [Sham et al., 2000]. TDT
tests were also completed including informative duos and all
TDT tests were rerun excluding siblings.
Although not part of our pre-hoc hypotheses, because 95% of
conduct disordered sample met lifetime criteria for at least one
lifetime substance use disorder, we conducted post-hoc
analyses to test for a possible association between 5HTTLPR
genotype and measures of conduct disorder and substance
dependence severity. We utilized two phenotypes: (1) conduct
disorder symptom count and (2) dependence vulnerability
(DV), defined as the lifetime substance dependence symptom
count summed across 10 drug categories, divided by the
number of substances ever used (using the CIDI-SAM minimum threshold for use definition, which generally requires
that a substance has been tried at least five times). Both
phenotypes were standardized for age trends and sex differences using large community samples from Colorado as has
been done previously [Stallings et al., 2005]. We tested whether
these phenotypes varied by genotype within patients-only (n,
261) using ANOVA (ss vs. sl vs. ll) and t-tests (ss/sl vs. ll).
To test for a possible gene-environment interaction, we used
general linear modeling to test whether the slopes of the
association between standardized, age-and-sex corrected conduct disorder symptom count and physical/sexual abuse and
Assessments and Phenotype Selection
Trained interviewers administered structured diagnostic
instruments to all consenting subjects. Patients, siblings of
patients, control adolescents, and their siblings were administered the CIDI-SAM (Composite International Diagnostic
Interview–Substance Abuse Module) [Cottler et al., 1989]
and the DISC (Diagnostic Interview Schedule for Children).
The CIDI-SAM provides DSM-IV [American Psychiatric
Association, 1994] diagnoses of substance abuse and dependence for 10 drug categories: alcohol, nicotine, hallucinogens,
cocaine, amphetamines, opioids, PCP, sedatives, cannabis, and
inhalants. Adolescent self-reporting on the DISC has shown
Conduct Disorder and 5HTTLPR
neglect differed across the three 5HTTLPR genotypes. If a
5HTTLPR by maltreatment interaction were present we would
expect the slopes to differ significantly.
cents from control families (examining probands and siblings,
and probands only) were in HWE; adolescents from patient
families (examining probands and siblings, and probands only)
were not.
Relative Risk Required for About 80% Power
We estimated the relative risk for disease conferred by
possession of one or two s-alleles, assuming about 80% power to
detect an association [Purcell et al., 2003]. Relative risks were
estimated (see Table I) assuming an s-allele frequency of 0.40,
disease prevalence 0.10, setting the linkage disequilibrium
parameter to one, alpha to 0.05, a power of approximately 80%,
and using 74 trios.
Demographic and Diagnostic Comparisons
Table II shows females and Caucasians were over-represented in the adolescent controls. As would be expected from
studies of comorbidity with conduct disorder [Crowley and
Riggs, 1995], adolescents from patient families were significantly more likely to have met lifetime criteria for major
depression, attention-deficit hyperactivity disorder, and oppositional defiant disorder. Interestingly, the two groups did not
differ in the lifetime prevalence of generalized anxiety
disorder. About 95% of conduct-disordered adolescents and
13% of controls met lifetime criteria for at least one substance
use disorder.
Case Control Analyses
Table III shows a significant association of the ss genotype in
patients with conduct disorder and conduct disorder with
aggression. This association was found to be significant for the
entire sample (w2 ¼ 14.3, 2df, P < 0.01), or when analyzed
separately for Caucasians (w2 ¼ 7.0, 2df, P ¼ 0.03), Hispanics
(w2 ¼ 8.3, 2df, P ¼ 0.02), and males only (w2 ¼ 10.5, 2df,
P < 0.01). For females the association of the ss genotypes was
found only in those with the aggressive form of conduct
disorder (w2 ¼ 6.1, 2df, P ¼ 0.05). No gender by genotype
frequency differences were seen in cases (w2 ¼ 0.22, 2df,
P ¼ 0.89) or controls (w2 ¼ 0.19, 2df, P ¼ 0.91). A significant
association was also seen within Caucasian-males for conduct
disorder (124 cases, 39 controls; w2 ¼ 6.5, 1df, P ¼ 0.04) and
conduct disorder with at least one aggressive symptom (106
cases; w2 ¼ 7.5, 1df, P ¼ 0.02). Excluding siblings, ss genotype
frequency differences were similar to those seen in the overall
sample but the difference was non-significant (w2 ¼ 4.1, 2df,
P ¼ 0.13).
Adolescent genotype frequencies were tested for Hardy–
Weinberg equilibrium (HWE) within control and patient
families. Regarding 5HTTLPR genotype frequencies, adolesTABLE I. Power Versus Relative Risk
Dominant versus additive
versus recessive
s-allele dominant
s-allele additive
s-allele recessive
Relative risk
sl ¼ 6
ss ¼ 6
sl ¼ 2
ss ¼ 4
sl ¼ 1
ss ¼ 3.4
Assumptions include: s-allele frequency of 0.40, disease prevalence 0.10,
linkage disequilibrium parameter of one, alpha to 0.05. Using 74 trios, the
table presents the relative risk for disease given genotype with approximately 80% power.
Within Family Analyses
Table IV shows transmission disequilibrium tests for
5HTTLPR and conduct disorder phenotypes. Although the
s-allele was more often transmitted from heterozygous parents, the difference was non-significant (w2 ¼ 1.61, 1 df,
P ¼ 0.20) for the 74 conduct disorder trios; however, for the
57 aggressive-conduct-disorder trios, the s-allele was significantly more likely to be transmitted to offspring from
heterozygous parents (w2 ¼ 4.1, 1df, P ¼ 0.04).
Examination of Informative Duos
(Parent-Offspring) and Sibling Effects
Because of a large number of missing fathers, both TDT
analyses were also calculated using informative duos (parentoffspring). For all comparisons (Table IV, secondary analyses)
examining conduct disorder with at least one aggressive
symptom, the s-allele was found to be significantly more likely
to be transmitted to affected adolescents. There is a potential
bias from including several children from a single family in
these analyses. However, TDT analyses excluding siblings also
showed significantly greater transmission of the s-allele to
cases from heterozygous parents (Table IV).
Because we also had genotypic information on parents of
controls we undertook secondary analyses utilizing these
families (Table IV). We conducted TDT analyses for control
adolescents (controls and their siblings) who had never met
criteria for conduct disorder. Using the 63 available probandfather-mother trios, we found that the l-allele was significantly
more likely to be transmitted from heterozygous parents to
offspring; including informative duos did not affect the level of
significance. Excluding siblings of controls we had small
samples (28 trios and 11 duos) and within-family tests were
Post Hoc Analyses—Conduct Disorder Symptom
Count and Dependence Vulnerability
Means for standardized, age-and-sex corrected conduct
disorder symptom count were calculated within each
5HTTLPR genotype for patients (n, 261). The means for ss,
sl, ss/sl, and ll genotypes were 3.49, 3.40, 3.44, and 3.01,
respectively. Comparisons across three genotypes (ss vs. sl vs.
ll) were non-significant (F ¼ 1.70; P ¼ 0.18) as were comparisons between the ss/sl and ll genotypes (t259 ¼ 1.82; P ¼ 0.07).
Means for standardized, age-and-sex corrected dependence
vulnerability (DV) was calculated within each 5HTTLPR
genotype for patients (n, 261). The means for ss, sl, ss/sl, and
ll genotypes were 2.20, 2.00, 2.08, and 2.35, respectively.
Comparisons across three genotypes were non-significant
(F ¼ 1.74; P ¼ 0.18) as were comparisons between the ss/sl
and ll genotypes (t259 ¼ 1.56; P ¼ 0.12).
Gene-Environment Interaction
Using the subsample of patients with available CARI scores
(n, 197), we used general linear modeling to model standardized age-and-sex corrected conduct disorder symptom count
by the total number of abuse and neglect items endorsed on the
CARI within 5HTTLPR genotype. Slopes of the individual lines
(within ss, sl, and ll genotypes) did not significantly differ
(F ¼ 0.37; P ¼ 0.69); 5HTTLPR genotype was not associated
with differences in abuse and neglect (F ¼ 0.08; P ¼ 0.93).
Sakai et al.
TABLE II. Adolescents From Patient and Control Families: Demographic Variables and
Psychiatric Diagnoses
Patients and
with CD (n, 297)
Age (standard deviation)
Gender (male)
Generalized anxietya disorder
Major Depressiona
Conduct disorder symptom count
Sum of aggressive CD symptoms
Any substance use disorder
Substance dependencea
Any substance dependence
Controls and their
without CD (n, 93)
15.69 (1.35)
252 (84.8%)
15.24 (1.85)
64 (68.8%)
143 (48.1%)
118 (39.7%)
36 (12.1%)
18 (6.1%)
46 (15.5%)
59 (19.9%)
6.3 (2.48)
2.1 (1.54)
281 (94.6%)
61 (65.6%)
23 (24.7%)
9 (9.7%)
3 (3.2%)
6 (6.5%)
0 (0%)
0.7 (0.74)
0.08 (0.28)
12 (12.9%)
216 (72.7%)
162 (54.5%)
134 (45.1%)
83 (27.9%)
40 (13.5%)
40 (13.5%)
26 (8.8%)
2 (2.2%)
1 (1.1%)
1 (1.1%)
1 (1.1%)
0 (0)
0 (0)
0 (0)
t124.3 ¼ 2.2*
w2 ¼ 11.8*
w2 ¼ 8.8*
w2 ¼ 1.1
w2 ¼ 5.0*
w2 ¼ 21.8*
t387.6 ¼ 34.2*
t349.7 ¼ 21.2*
w2 ¼ 253.1*
w2 ¼ 143.1*
w2 ¼ 83.3*
w2 ¼ 60.7*
w2 ¼ 30.3*
w2 ¼ 14.0*
w2 ¼ 14.0*
w2 ¼ 8.7*
DSM-IV diagnosis (lifetime).
*P < 0.05; , CD, at least three whole life conduct disorder symptoms.
This study using two methods (case-control and withinfamily analyses) suggests that 5HTTLPR (s-allele) contributes
to the liability to conduct disorder and aggressive behavior.
This work is consistent with other studies indicating that this
variant in the promoter region is a functional variant that
alters serotonin-mediated behavior. Although our case-control
analyses (even with ethnic subgroup analyses) leave open the
possibility of bias from population stratification, the withinfamily analyses, which eliminate the possibility of this bias,
support the case-control results. Interestingly, some of our
TABLE III. Case Versus Control Association: 5HTTLPR Genotype
Primary analyses
Control (no CD)
Case (CD)
Case (CD þ 1)
Secondary analyses
Control (no CD) (n ¼ 61)
Case (CD) (n ¼ 143)
Case (CD þ 1) (n ¼ 114)
Control (no CD) (n ¼ 23)
Case (CD) (n ¼ 118)
Case (CD þ 1) (n ¼ 102)
Control (no CD) (n ¼ 29)
Case (CD) (n ¼ 45)
Case (CD þ 1) (n ¼ 31)
Control (no CD) (n ¼ 64)
Case (CD) (n ¼ 252)
Case (CD þ 1) (n ¼ 215)
Control (no CD) (n ¼ 48)
Case (CD) (n ¼ 261)
Case (CD þ 1) (n ¼ 219)
8 (8.6%)
72 (24.2%)
62 (25.2%)
37 (39.8%)
124 (41.8%)
104 (42.3%)
48 (51.6%)
101 (34.0%)
80 (32.5%)
w22 ¼ 14.3; P < 0.01
w22 ¼ 15.6; P < 0.01
w22 ¼ 7.0; P ¼ 0.03
w22 ¼ 8.7; P ¼ 0.01
w22 ¼ 8.3; P ¼ 0.02
w22 ¼ 8.5; P ¼ 0.01
w22 ¼ 3.9; P ¼ 0.14
w22 ¼ 6.1; P ¼ 0.05
w22 ¼ 10.5; P < 0.01
w22 ¼ 11.0; P < 0.01
w22 ¼ 4.1; P ¼ 0.13
w22 ¼ 4.2; P ¼ 0.12
Control (no CD), controls and their siblings never meeting criteria for conduct disorder (n, 93); Case (CD),
adolescent patients and their siblings who met at least three lifetime criteria for conduct disorder (n, 297); Case
(CD þ 1), adolescent patients and their siblings who met at least three lifetime criteria for conduct disorder and
endorsed at least one aggressive symptom (n, 246); s, short variant of 5HTTLPR (484); l, long variant of the
5HTTLPR (528).
Conduct Disorder and 5HTTLPR
TABLE IV. Transmission Disequilibrium Tests For 5HTTLPR
Adolescents with conduct disorder from patient families and both parents (74 trios)a
Not transmitted
TDT ¼ (43–32)2/(43 þ 32)
wtdt2 ¼ 1.61; P ¼ 0.20
Adolescents with whole-life conduct disorder with at least one aggressive symptom from patient families and both parents (57
TDT ¼ (39–23)2/(39 þ 23)
wtdt2 ¼ 4.13; P ¼ 0.04
Secondary analyses including informative duosc and/or excluding siblings, for both conduct disorder and conduct disorder with
at least one aggressive symptom
Patient families
Patients and sibs
Case (CD)
wtdt ¼ 3.06; P ¼ 0.08
Patients and sibs
Case (CD þ 1)
wtdt2 ¼ 5.94; P ¼ 0.02
Patients only
Case (CD)
wtdt2 ¼ 4.74; P ¼ 0.03
Patients only
Case (CD þ 1)
wtdt2 ¼ 5.33; P ¼ 0.02
Patients only
Case (CD)
wtdt2 ¼ 8.24; P < 0.01
Patients only
Case (CD þ 1)
wtdt2 ¼ 9.39; P < 0.01
Control families
Controls and sibs
Control (no CD)
wtdt2 ¼ 4.57*; P ¼ 0.03
Controls and sibs
Control (no CD)
wtdt2 ¼ 6.25*; P ¼ 0.01
One trio is two parents and one offspring.
Each parent has two alleles (i.e., ss, sl, or ll), one of which is transmitted to an offspring. Cells in the table indicate which allele was and was not transmitted
from parent to offspring, for example, in the upper right cell, there were 43 parents with the sl genotype who transmitted their s-allele to an offspring but not
their l-allele.
One duo is one parent and one offspring.
*Favoring greater transmission of the l-allele; Control (no CD), controls and their siblings never meeting criteria for conduct disorder (defined here as
meeting at least three lifetime conduct disorder criteria); Case (CD), adolescent patients and their siblings who met at least three criteria for conduct
disorder; Case (CD þ 1), adolescent patients and their siblings who met at least three lifetime criteria for conduct disorder and endorsed at least one
aggressive symptom; s, short variant of 5HTTLPR (484); l, long variant of the 5HTTLPR (528).
findings also are consistent with recently published animal
work on an analogous polymorphism found in non-human
primates [Suomi, 2003].
Case-Control Analyses
Our case-control analyses showed significant 5HTTLPR
genotype differences between cases and controls but the casecontrol method has certain limitations that may make the
results difficult to interpret. Family structure, ethnicity, and
gender can be confounding variables in these comparisons. For
example, the control sample included more siblings than did
the cases, which could be a source of bias. Compared to the
overall analysis, an analysis of probands only was nonsignificant even though the ss genotype frequencies were
similar to those seen in the overall sample. The ethnic makeup
of the cases and controls can be a source of bias since significant
ethnic differences in 5HTTLPR allele frequencies have been
reported [Gelernter et al., 1999]. The ethnic makeup in this
study did differ between conduct-disordered and non-conductdisordered adolescents. Despite these differences, case-control
analyses within our two largest ethnic subgroups were
consistent with our overall results. It has been suggested that
the link between serotonin dysfunction and aggression may
differ by gender [Manuck et al., 1998]. We found no gender-bygenotype differences within cases or controls. Although gender
differences are not likely to have contributed to our results,
they cannot be ruled out completely since our adolescent
sample from patient families was mostly male. One concern is
that the control ss genotype frequencies are lower than might
be expected from some studies of general population Cauca-
sians [Munafo et al., 2003]. It is important to note that these
control adolescents were not a homogeneous ethnic group, and
this was not a general population sample; instead controls were
selected from similar neighborhoods to patients and then were
further selected for never having met criteria for conduct
disorder. It is somewhat reassuring that our ss-genotype
frequencies are similar to a non-violent control group from a
previous study [Retz et al., 2004], but potential confounds to
our case-control results cannot be ruled out.
Within-Family Analyses
We conducted TDT analyses, which control for population
stratification by using within family tests. Our analyses for
conduct disorder were non-significant. The analyses of conduct
disorder with at least one aggressive symptom demonstrated
significantly greater transmission of the s-allele in these
families. Additional tests that excluded siblings (restriction
to simplex families allows a test of association/linkage disequilibrium) were significant, again favoring greater transmission
of the s-allele from heterozygous parents.
Many patient families had only one parent available for
interview (fathers with antisocial personality disorder often
are absent) [Sakai et al., 2004]. To control for this potential bias
TDT analyses were repeated using informative parent-offspring duos. Analyses using duos may introduce a bias since on
average, transmission of the common allele from a known
heterozygous parent is more likely to be included as informative, because the missing parent is most likely to have also
contributed a common allele [Curtis and Sham, 1995]. In our
sample, the opposite was true; when duos were included in the
Sakai et al.
analyses, support for the s-allele (usually the less common
allele) [Munafo et al., 2003] being preferentially transmitted in
patient families increased. Although this may have occurred
simply by chance, it is likely that the s-allele was more
commonly transmitted to the offspring from missing parents. If
true, this result adds support to the observed association
between 5HTTLPR and conduct disorder with at least one
aggressive symptom. The TDT was also conducted for control
adolescents who had never met criteria for conduct disorder. In
contrast to the cases, the l-allele was preferentially transmitted from heterozygous parents to unaffected offspring. This
result is entirely consistent with our overall results.
Hardy–Weinberg Equilibrium
The lack of HWE in the clinical sample at first glance is
somewhat concerning. However, this population is a selected
one in which non-random mating occurs for the phenotype of
interest [Sakai et al., 2004]. We can examine a simple model of
selection, where the s-allele acts recessively, the ss genotype
confers a relative risk of 3.4 (Table I), disease prevalence is
0.10, and the s-allele frequency is 0.40. The expected ss, sl, and
ll genotypes in the general population would be 16%, 48%, and
36%, respectively. Because of differences in relative risk,
prevalence of conduct disorder among those with the ss
genotype would be about 24.6%, and 7.2% among those with
the sl or ll genotype. Within those with conduct disorder we
would expect the ss, sl, and ll genotype frequencies to be 39%,
35%, and 26%, respectively; with a sample the size examined in
this report (n, 297), such genotype frequencies would be out of
HWE as we have found. Alternatively, those never meeting
criteria for conduct disorder would be less affected by such
selection and would have ss, sl, and ll genotype frequencies of
13%, 50%, and 37%, respectively; with a sample the size
examined in this report (n, 93), such genotype frequencies
would be in HWE as in our control sample. Consistent with this
simple model and our results, two studies of violent offenders
have been published with 5HTTLPR genotype frequencies out
of HWE [Hallikainen et al., 1999; Liao et al., 2004].
Comparison of Results With the Existing Literature
The observed association of the s-allele with conduct
disorder with at least one aggressive symptom is consistent
with expectations from the literature. The s-allele has been
associated with serotonin dysfunction (low CSF 5-HIAA,
blunted prolactin secretion, and low platelet binding) and such
serotonin dysfunction has been associated with aggressive
behavior. An analogous polymorphism in the regulatory region
of the 5HTT gene in monkeys has been described and the short
allele has been associated in vivo with reductions in serotonin (reduced CSF 5-HIAA). This association in non-human
primates between genotype, biochemical changes (measures of
serotonin), and behaviors (aggression) appears to be moderated by environmental factors [Suomi, 2003]. Monkeys with
the s-allele who experience adverse rearing environments
(peer-reared) have significantly reduced CSF 5-HIAA and
exhibit greater aggression when compared with monkeys with
the ll genotype reared in the same environment; in less
stressful rearing environments the sl and ll monkeys do not
differ in CSF 5-HIAA or aggression [Bennett et al., 2002;
Suomi, 2003]. Our results differ in that they suggest an
association between the s-allele and conduct disorder with
aggression, one that is not mediated by physical/sexual abuse
and neglect.
Two previous studies have demonstrated an association of
the ll genotype with violence or aggression [Twitchell et al.,
2001; Zalsman et al., 2001], and three found no association
[Reist et al., 2001; Beitchman et al., 2003; Davidge et al., 2004].
However, none of these studies employed a within-family study
design, all utilized small samples relative to the current study,
and none used conduct disorder with aggression as their
phenotype. Finally, we utilized two tests (case-control and
TDT) and found consistent results. Thus, there are clear
differences between the current study and those of previous
samples perhaps explaining the discrepancies in results.
Comment on Comorbity
Antisocial behavior and substance use disorders are highly
comorbid. For example, in one national epidemiological sample
84% of those with antisocial personality disorder also met
criteria for a lifetime substance use disorder [Regier et al.,
1990] and in another alcohol dependence (odds ratio 7.1) and
drug dependence (odds ratio 18.5) were strongly associated
with antisocial personality disorder [Grant et al., 2004]. Our
sample is consistent with these studies. Although with great
effort it may be possible to collect samples of conduct disordered
adolescents who have never met criteria for a substance use
disorder, this would, in fact, be a very atypical, non-representative sample, one which would likely also be less severely
affected [Krueger, 1999].
This high rate of comorbidity raises the concern about
whether co-occurring conduct and substance problems in most
cases represent independent disorders or alternatively,
whether the conduct symptoms are more likely substanceinduced. Although use of substances can lead to aggressive
antisocial behavior, we argue that it was highly unlikely that
many subjects in our study had substance-induced conduct
disorder. First, examination of an available dataset [NESARC
(National Epidemiologic Survey on Alcohol and Related
Conditions), 2001–2002, 2005] suggests that among those
with conduct disorder, most report that none of their antisocial
symptoms before age 15 were associated with substance use.
Second, conduct disorder symptoms tend to predate the use of
drugs and alcohol by many years [Kuperman et al., 2001],
again supporting that for most adolescents conduct disorder
symptoms are not simply induced during periods of intoxication. Third, our patients while substance-free in treatment
often continue to exhibit impulsive, aggressive behavior and
show continued risk-taking behavior on psychological testing
when substance free for at least 7 days [Crowley et al., 2005,
under review]. Thus, we do not believe that many of our
subjects suffered from substance-induced conduct disorder.
We did undertake analyses in an attempt to clarify whether
the association was with conduct disorder or substance
dependence. Unfortunately, our post-hoc analyses could not
differentiate which disorder was driving our results (conduct
disorder and substance dependence symptom counts did not
differ significantly by genotype within patients). Therefore,
this must simply remain an important limitation in our
analyses. This distinction may, however, be of relatively
limited value as several investigators have shown that there
are substantial common genetic influences on both disorders
[Hicks et al., 2004]. Thus it is conceivable that 5HTTLPR may
ultimately be associated with both disorders, perhaps through
an intermediate phenotype such as behavioral dysinhibition
[Young et al., 2000].
Comment on Power
Power estimates for the sample size used for within-family
analyses in this report vary based on the mode of action of the
risk increasing allele and other assumptions. Some studies
suggest that the s-allele operates in a dominant fashion [Heils
et al., 1996] but others do not [Williams et al., 2003]. If the sallele operates in a dominant fashion, to have had about 80%
power to detect the association in our TDT analyses would
require the s-allele to confer a relative risk of about six (Table I).
Even assuming an additive or recessive mode of action, the
Conduct Disorder and 5HTTLPR
relative risk conferred to those possessing the s-allele is
higher than expected given the assumptions that most
complex traits are influenced by a number of genes of small
to moderate effect, and that, at most, such genes would
double the liability for such a trait [Munafo et al., 2003]. Our
results suggest that within the population of study, that we
have found an allelic association of at least moderate
influence, though we cannot rule out that we have made a
chance finding. Although the findings in this sample are
consistent with recent animal literature and within the
dataset two different methods of analysis show convergent
results, these power calculations emphasize the importance
of replication with larger samples.
Conclusions and Limitations
Our results add support to the evidence that the s-variant of
the SLC6A4 promoter contributes to the liability toward
aggressive behavior in humans. These results require careful
interpretation and further replication across multiple samples. Although cases were selected to meet diagnostic criteria
for conduct disorder, adolescent patients were drawn from a
treatment program for youth with serious substance and
behavior problems. Nearly all patients also meet criteria for at
least one substance dependence diagnosis on standardized
testing [Miles et al., 1998]. Therefore, we cannot determine
whether the association is with conduct disorder, substance
dependence, or some unmeasured but highly correlated latent
factor. Still conduct disorder and substance dependence are
commonly comorbid, in keeping with recent research suggesting strong, common genetic influences on both disorders [Hicks
et al., 2004].
Portions of this study were previously presented at annual
meeting of the College on Problems of Drug Dependence (2005).
American Psychiatric Association. 1994. Diagnostic and statistical manual
of mental disorders, (DSM IV), 4th edn. Washington, DC: American
Psychiatric Association.
Anchordoquy HC, McGeary C, Liu L, Krauter KS, Smolen A. 2003.
Genotyping of three candidate genes after whole-genome preamplification of DNA collected from buccal cells. Behav Genet 33:73–78.
Beitchman JH, Davidge KM, Kennedy JL, Atkinson L, Lee V, Shapiro S,
Douglas L. 2003. The serotonin transporter gene in aggressive children
with and without ADHD and nonaggressive matched controls. Ann NY
Acad Sci 1008:248–251.
Bennett AJ, Lesch KP, Heils A, Long JC, Lorenz JG, Shoaf SE, Champoux M,
Suomi SJ, Linnoila MV, Higley JD. 2002. Early experience and serotonin
transporter gene variation interact to influence primate CNS function.
Mol Psychiatry 7:118–122.
Brown GL, Goodwin FK, Ballenger JC, Goyer PF, Major LF. 1979.
Aggression in humans correlates with cerebrospinal fluid amine
metabolites. Psychiatry Res 1:131–139.
Cadoret RJ, Langbehn D, Caspers K, Troughton EP, Yucuis R, Sandhu HK,
Philibert R. 2003. Associations of the serotonin transporter promoter
polymorphism with aggressivity, attention deficit, and conduct disorder
in an adoptee population. Compr Psychiatry 44:88–101.
Cottler L, Robins L, Helzer J. 1989. The reliability of the CIDI-SAM. Br
J Addictions 84:801–814.
Crowley TJ, Riggs PD. 1995. Adolescent substance use disorder with conduct
disorder and comorbid conditions. NIDA Res Monogr 156:49–111.
Crowley TJ, Mikulich SK, Ehlers KM, Whitmore EA, MacDonald MJ. 2001.
Validity of structured clinical evaluations in adolescents with conduct and
substance problems. J Am Acad Child Adolesc Psychiatry 40:265–273.
Crowley TJ, Mikulich SK, Ehlers KM, Hall SK, Whitmore EA. 2003.
Discriminative validity and clinical utility of an abuse-neglect interview for adolescents with conduct and substance use problems. Am
J Psychiatry 160:1461–1469.
Crowley TJ, Raymond KE, Mikulich-Gilbertson SK, Thompson LL, Lejuez
CW. 2005. A risk-taking ‘‘set’’ in a novel task among adolescents with
serious conduct and substance problems. J Am Acad Child Adolesc
Psychiatry 45:175–183.
Curtis D, Sham PC. 1995. A note on the application of the transmission
disequilibrium test when a parent is missing. Am J Hum Genet 56:811–
Davidge KM, Atkinson L, Douglas L, Lee V, Shapiro S, Kennedy
JL, Beitchman JH. 2004. Association of the serotonin transporter
and 5HT1Dbeta receptor genes with extreme, persistent and pervasive
aggressive behaviour in children. Psychiatry Genet 14:143–146.
Gelernter J, Cubells JF, Kidd JR, Pakstis AJ, Kidd KK. 1999. Population
studies of polymorphisms of the serotonin transporter protein gene. Am
J Med Genet 88:61–66.
Gerra G, Garofano L, Santoro G, Bosari S, Pellegrini C, Zaimovic A, Moi G,
Bussandri M, Moi A, Brambilla F, Donnini C. 2004. Association between
low-activity serotonin transporter genotype and heroin dependence:
Behavioral and personality correlates. Am J Med Genet (Neuropsychiatric Genetics) PartB 126B:37–42.
Grant BF, Stinson FS, Dawson DA, Chou SP, Ruan WJ, Pickering RP. 2004.
Co-occurrence of 12-month alcohol and drug use disorders and
personality disorders: Results from the National Epidemiological
Survey on Alcohol and Related Conditions. Arch Gen Psychiatry
Hallikainen T, Saito T, Lachman HM, Volavka J, Pohjalainen T, Ryynanen
OP, Kauhanen J, Syvalahti E, Hietala J, Tiihonen J. 1999. Association
between low activity serotonin transporter promoter genotype and early
onset alcoholism with habitual impulsive violent behavior. Mol
Psychiatry 4:385–388.
Hariri AR, Mattay VS, Tessitore A, Kolachana B, Fera F, Goldman D, Egan
MF, Weinberger DR. 2002. Serotonin transporter genetic variation and
the response of the human amygdala. Science 297:400–403.
Heils A, Teufel A, Petri S, Stober G, Riederer P, Bengel D, Lesch KP. 1996.
Allelic variation of human serotonin transporter gene expression.
J Neurochem 66:2621–2624.
Heinz A, Jones DW, Mazzanti C, Goldman D, Ragan P, Hommer D, Linnoila
M, Weinberger DR. 2000. A relationship between serotonin transporter
genotype and in vivo protein expression and alcohol neurotoxicity. Biol
Psychiatry 47:643–649.
Hicks BM, Krueger RF, Iacono WG, McGue M, Patrick CJ. 2004. Family
transmission and heritability of externalizing disorders: A twin-family
study. Arch Gen Psychiatry 61:922–928.
Hu X, Oroszi G, Chun J, Smith TL, Goldman D, Schuckit MA. 2005. An
expanded evaluation of the relationship of four alleles to the level
of response to alcohol and the alcoholism risk. Alcohol Clin Exp Res 29:
Krueger RF. 1999. The structure of common mental disorders. Arch Gen
Psychiatry 56:921–926.
Kruesi MJ, Hibbs ED, Zahn TP, Keysor CS, Hamburger SD, Bartko JJ,
Rapoport JL. 1992. A 2-year prospective follow-up study of children and
adolescents with disruptive behavior disorders. Prediction by cerebrospinal fluid 5-hydroxyindoleacetic acid, homovanillic acid, and
autonomic measures? Arch Gen Psychiatry 49:429–435.
Kuperman S, Schlosser SS, Kramer JR, Bucholz K, Hesselbrock V, Reich T,
Reich W. 2001. Developmental sequence from disruptive behavior
diagnosis to adolescent alcohol dependence. Am J Psychaitry 158:
Liao DL, Hong CJ, Shih HL, Tsai SJ. 2004. Possible association between
serotonin transporter promoter region polymorphism and extremely
violent crime in Chinese males. Biological Psychiatry 50:284–287.
Linnoila M, Virkkunen M, Scheinin M, Nuutila A, Rimon R, Goodwin FK.
1983. Low cerebrospinal fluid 5-hydroxyindoleacetic acid concentration
differentiates impulsive from nonimpulsive violent behavior. Life Sci
Manuck SB, Flory JD, McCaffery JM, Matthews KA, Mann JJ, Muldoon MF.
1998. Aggression, impulsivity, and central nervous system serotonergic
responsivity in a nonpatient sample. Neuropsychopharmacology 19:
Miles DR, Carey G. 1997. Genetic and environmental architecture of human
aggression. J Pers Soc Psychol 72:207–217.
Miles DR, Stallings MC, Young SE, Hewitt JK, Crowley TJ, Fulker DW.
1998. A family history and direct interview study of the familial
aggregation of substance abuse: The adolescent substance abuse study.
Drug Alcohol Depend 49:105–114.
Sakai et al.
Munafo MR, Clark TG, Moore LR, Payne E, Walton R, Flint J. 2003. Genetic
polymorphisms and personality in healthy adults: A systematic review
and meta-analysis. Mol Psychiatry 8:471–484.
NESARC (National Epidemiologic Survey on Alcohol and Related Conditions),
2001–2002. 2005. Accessed July 27.
O’Keane V, Moloney E, O’Neill H, O’Connor A, Smith C, Dinan TG. 1992.
Blunted prolactin responses to d-fenfluramine in sociopathy. Evidence
for subsensitivity of central serotonergic function. Br J Psychiatry
Purcell S, Cherny SS, Sham PC. 2003. Genetic power calculator: Design of
linkage and association genetic mapping studies of complex traits.
Bioinformatics 19:149–150.
Regier DA, Farmer ME, Rae DS, Locke BZ, Keith SJ, Judd LL, Goodwin FK.
1990. Comorbidity of mental disorders with alcohol and other drug
abuse: Results from the epidemiologic catchment area (ECA) study.
JAMA 264:2511–2518.
Reist C, Mazzanti C, Vu R, Tran D, Goldman D. 2001. Serotonin transporter
promoter polymorphism is associated with attenuated prolactin
response to fenfluramine. Am J Med Genet 105:363–368.
Retz W, Retz-Junginger P, Supprian T, Thome J, Rosler M. 2004.
Association of serotonin transporter promoter gene polymorphism with
violence: Relation with personality disorders, impulsivity, and childhood
ADHD psychopathology. Behav Sci Law 22:415–425.
Rhee SH, Wladman ID. 2002. Genetic and environmental influences on
antisocial behavior: A meta-analysis of twin and adoption studies.
Pscyhol Bull 128:490–529.
Sakai JT, Stallings MC, Mikulich-Gilbertson SK, Corley RP, Young SE,
Hopfer CJ, Crowley TJ. 2004. Mate similarity for substance dependence
and antisocial personality disorder symptoms among parents of patients
and controls. Drug Alcohol Depend 75:165–175.
Sham P. 1998. Statistics in human genetics. New York: Oxford University
Sham PC, Zhao JH, Waldman I, Curtis D. 2000. Should ambiguous trios for
the TDT be discarded? Ann Hum Genet 64:575–576.
Spielman RS, McGinnis RE, Ewens WJ. 1993. Transmission test for linkage
disequilibrium: The insulin gene region and insulin-dependent diabetes
mellitus (IDDM). Am J Hum Genet 52:506–516.
Stallings MC, Corley RP, Dennehey B, Hewitt JK, Krauter KS, Lessem JM,
Mikulich-Gilbertson SK, Rhee SH, Smolen A, Young SE, Crowley TJ.
2005. A genome-wide search for quantitative trait loci influencing
antisocial drug dependence in adolescence. Arch Gen Psychiatry
Stoff DM, Pollock L, Vitiello B, Behar D, Bridger WH. 1987. Reduction of
(3H)-imipramine binding sites on platelets of conduct-disordered
children. Neuropsychopharmacology 1:55–62.
Stoltenberg SF, Twitchell GR, Hanna GL, Cook EH, Fitzgerald HE,
Zucker RA, Little KY. 2002. Serotonin transporter promoter
polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism. Am J Med Genet 114:230–
Suomi SJ. 2003. Gene-environment interactions and the neurobiology of
social conflict. Ann NY Acad Sci 1008:132–139.
Twitchell GR, Hanna GL, Cook EH, Stoltenberg SF, Fitzgerald HE,
Zucker RA. 2001. Serotonin transporter promoter polymorphism genotype is associated with behavioral disinhibition and
negative affect in children of alcoholics. Alcohol Clin Exp Res 25:953–
Van Goozen SH, Matthys W, Cohen-Kettenis PT, Westenberg H, van
Engeland H. 1999. Plasma monoamine metabolites and aggression: Two
studies of normal and oppositional defiant disorder children. Eur
Neuropsychopharmacol 9:141–147.
Williams RB, Marchuk DA, Gadde KM, Barefoot JC, Grichnik K,
Helms MJ, Kuhn CM, Lewis JG, Schanberg SM, StaffordSmith M, Suarez EC, Clary GL, Svenson IK, Siegler IC. 2003.
Serotoning-related gene polymorphisms and central nervous system
serotonin function. Neuropsychopharmacology 28:533– 541.
Young SE, Stallings MC, Corley RP, Krauter KS, Hewitt JK. 2000. Genetic
and environmental influences on behavioral disinhibition. Am J Med
Genet 96:684–695.
Zalsman G, Frisch A, Bromberg M, Gelernter J, Michaelovsky E,
Campino A, Erlich Z, Tyano S, Apter A, Weizman A. 2001.
Family-based association study of serotonin transporter promoter
in suicidal adolescents: No association with suicidality but
possible role in violence traits. Am J Med Genet 105:239–
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