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Supplementary Materials and Methods
Methods
Participants
The present study was part of the Survey of Adolescent Life in Västmanland, which is a medium-sized
county in Sweden situated approximately 100 km west of Stockholm. Data were collectedin 2006. All
students in the second year of high school (age, 17–18 years) were asked to complete a questionnaire
during class. Questionnaires were given to teachers for students who did not attend class on the day of
the study. In addition, participants were asked to provide a saliva sample for DNA extraction.
Participation was anonymous and voluntary. A total of 2263 students, 77.3% of the target population,
completed the questionnaire, 183 of whom returned their questionnaires and saliva samples by mail.
Among the 183 late responders, 60 individuals had complete data and did not differ from the other
participants regardingsex, genotype frequencies, delinquency, family relations, sexual abuse, and child–
parent relationship. A saliva sample was provided by 2131 participants. DNA was successfully extracted
for 1414 participants. The genotype analysis failed in 717 samples because of the DNA extraction
method. However, these 717 participants did not differ from those with usable DNA regarding the scores
on delinquency, family conflict, and child–parent relationship,although they had lower scores on sexual
abuse (p=0.042). Other adolescents did not complete the questionnaire. In total, 1337 participants were
included in the study: 661 boys and 676 girls (Aslund et al., 2009).
Measures
Genotyping
For MAOA-uVNTR, the reaction volume was 25 L and contained 100 ng of DNA, 1PCR buffer, 400
M dNTP, 1.5 mMMgCl, 2 pmol of each primer, and 2.5 U of Taq-DNA polymerase Gold (Applied
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Biosystems). The sequence ofthe forward primer was 5–Fam-ACA GCC TGA CCG TGG AGA AG–3, and
that of the reverse primer was 5–GAA CGG ACG CTC CAT TCG GA–3. The amplification was performed
as described above, with an initial denaturation temperature of 95°C for 10 min, followed by 35 cycles of
95°C for 15 s, 57°C for 30 s, and 72°C for 30 s, and a final elongation at 72°C for 10 min. The gel was run
at 120 V for 45 min, and the DNA fragments were visualized under UV light by ethidiumbromide staining.
The forward primer was labeled with Fam at the 5 end, and the PCR products were analyzed by capillary
electrophoresis on an ABI PRISM® 3700 DNA Analyzer with ABI GS-500 bp molecular weight size marker
(Applied Biosystems, USA) and theGeneScan Analysis Software (Sabol et al., 1998a)(Gokturk et al., 2008).
As there are some uncertainties regarding the classification of the 5-repeat allele of the MAOAuVNTR(Sabol et al., 1998b; Deckert et al., 1999), we chose to classify the 5-repeat allele according to
Deckert et al.(Deckert et al., 1999). Thus, for the analyses, genotypes were divided into two groups:
presence of short alleles with 2 or 3 copies of the MAOA-uVNTRS (boys)/SS and LS (girls), and long alleles
with 3.5, 4, or 5 copies of the MAOA-uVNTRL (boys)/LL (girls).
The solute carrier family 6 (SLC6A4, neurotransmitter transporter) serotonin transporter-linked
promoter region (5-HTTLPR) polymorphism, which consists of an insertion/deletion that creates a long
(L) or a short (S) allele, and the brain-derived neurotrophic factor (BDNF) polymorphism, which consists
of a single-nucleotide polymorphism (SNP) G/A substitution at codon 66 (Val66Met), were analyzed
using DNA isolated from saliva. The saliva samples were analyzed for the 5-HTTLPR and BDNF Val66Met
genotypes. The BDNF Val66Met (rs6265) polymorphism was analyzed using the TaqManPre-Designed
SNP Genotyping Assay (Applied Biosystems Inc., Foster City, California, USA). Polymerase chain reaction
(PCR) was performed in a 5 L reaction mixture containing 2.5 LTaqMan Universal PCR Master Mix
(Applied Biosystems), 0.125 L of 40 Custom TaqMan® SNP Genotyping Assays Mix (Applied
Biosystems), and 3–20 ng of genomic deoxyribonucleic acid (DNA). The allele-discrimination PCR reaction
was performed on an ABI PRISM 7900HT Sequence Detection System using the following thermal cycler
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conditions: initial step of 10 min at 95°C, followed by 40 cycles of 15 s at 92°C and 60 s at 60°C.
Genotypes were analyzed using SDS 2.3 (Applied Biosystems).
The 5-HTTLPR polymorphism was amplified from 3–20 ng of genomic DNA using the following primer
sequences: forward, 5–AAC ATG CTC ATT TAA GAA GTG GAA C–3; and reverse, 5–XCT AGA GGG ACT
GAG CTG GAC AAC–3. The reverse primer was labeled with the fluorescent dye 5-hex. PCR was
performed in a 10 L reaction mixture containing 1 mM PCR Buffer 109 with 1.5 mM MgCl2, 0.2 M
dNTPs, 0.8 M of two primers, and 0.5 U FastStartTaq DNA polymerase (Roche Diagnostics GmbH,
Mannheim, Germany). The PCRs were performed on a GeneAmp 9700 (Applied Biosystems) using the
following conditions: start at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 45 s,
annealing at 61°C for 1 min, and elongation at 72°C for 90 s, with a final extension at 72°C for 7 min. The
PCR products were analyzed by capillary electrophoresis using an ABI PRISM® 3700 DNA Analyzer
(Applied Biosystems), and allele sizes were determined manually on chromatograms using the Gene
Marker® 1.5 AFLP/Genotyping software (SoftGenetics LLC, State College, PA, USA), as described
previously (Aslund et al., 2009; Comasco et al., 2011).
Family Conflict
Six items assessed the adolescent’s perception of his/her family: Have you ever run away from home?
No (0) and Yes (1); Does anyone in your family have a problem with alcohol or narcotics? No (0) and Yes
(1); Have there ever been any serious, heart-rending quarrels between your parents? Never, or less than
once a month (0) and Yes, at least once a month (1); Has either of your parents ever pushed, beaten, or
used any other kind of violence against the other? Never or seldom (0) and At least once a year (1);Has
either of your parents ever pushed or beaten you, or used any other kind of violence against you? Never
or seldom (0) and At least once a year (1);and Have you ever been treated badly psychologically (for
example, taunted, or scorned) by either of your parents? Never or seldom (0) and At least once a year
(1). Scores were summed to create a family-conflict score that ranged from 0 to 6. Internal consistency
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was estimated withCronbach’s alpha of 0.567. Among the 1,337 participants, 4% reported running away,
14.4% reported parents with substance misuse, 16.7% reported serious quarrels between their parents,
4% had observed physical violence between their parents, 4.6% had experienced physical abuse, and
9.4% had experienced psychological abuse.
Sexual Abuse
Sexual abuse was assessed by three items: Has anyone ever touched you or forced you to touch them
sexually against your will? No, never (0); Yes, once (1); Yes, 2–4 times (2); and Yes, five or more times
(3);Has anyone ever attempted to have intercourse with you (oral, vaginal, or anal) against your will? No,
never (0); Yes, once (1); Yes, 2–4 times (2); and Yes, five or more times (3);and Has anyone ever had
intercourse with you (oral, vaginal, or anal) against your will? No, never (0); Yes, once (1); Yes, 2–4 times
(2); and Yes, 5 or more times (3). Scores were summed to create a sexual-abuse score that ranged from 0
to 9(Nilsson et al., 2011) with a Cronbach’s alphaof 0.668. About a fifth of the of the participants (18.8%;
8.5% of the boys and 27.6% of the girls) reported having been touched against their will, 12.4% (6.3% of
the boys and 18.2% of the girls) experienced an attempted rape, and 5.7% (3.7% of theboys and 7.7% of
the girls) reported having been raped.
Child–Parent Relationship
The participant’s perception of his/her relationship with his/her parents was assessed by four items: I
can talk to my parents about almost everything, I enjoy hanging out with my parents, I can trust my
parents when it really matters, My parents give me many opportunities to do fun things with them. The
answers were: Strongly agree (1), Agree (2), Neither agree nor disagree (3), Disagree (4), and Strongly
disagree (5). Answers were reverse coded and summed to create a child–parent-relationship score that
ranged from 4 (indicating a poor relationship) to 20 (indicating a positive relationship). A total
Cronbach’s alpha of 0.830 estimated internal consistency. Among the adolescents, the following
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percentages of individuals reported that they strongly agreed with these statements: 28.9%,I can talk to
my parents about almost everything; 34.4%, I enjoy hanging out with my parents; 66.3%, I trust my
parents when it really matters; 23.4%,my parents give me many opportunities to do fun things with
them.
Delinquency
Delinquency was measured by 14 questions pertaining to how often had the participant: 1. Broken
into a house, shop, store, kiosk, or other building with the intention to steal things? 2. Sold or bought
something you knew was stolen? 3. Threatened or forced someone to give you money, cigarettes, or
anything else? 4. Been involved in a fight during your leisure time (not at school)? 5. Carried a weapon
(brass knuckles, baseball bat, knife, switchblade, or similar) at school or during your leisure time? 6.
Stolen a car? 7. Stolen a moped, motorbike, or motor scooter? 8. Hit/kicked someone so hard that
he/she needed medical attention? 9. Deliberately hurt someone with a knife, switchblade, brass
knuckles, or similar? 10. Been involved in threatening another person to do something he/she did not
want to do? 11. By yourself threatened another person to do something he/she did not want to do? 12.
Been involved in breaking into and stealing something from a car? 13. By yourself broken into and stolen
something from a car? 14. Had sex with a person who was so drunk/high that he/she did not really
understand what was going on? 15. Had sex with a person against his/her will? Response alternatives
were: never (0), once (1), 2–4 times (2), 5–10 times (3), and more than 10 times (4). These were summed
to create a total delinquency score that ranged from 0 to 90, with a Cronbach’salpha of 0.960.
Among the participants, 9.1% had broken into a house, etc.; 14.6% had sold or bought something
stolen; 5.7% had threatened or forced someone to hand over goods; 18.2% had been involved in fights;
11% had carried weapons; 7.8% had stolen a car; 15.9% had stolen a moped, motorbike, or a scooter;
10% had hit/kicked someone so hard he/she needed medical attention; 13.4% had deliberately hurt
someone with a weapon; 7.4% had been involved in threatening another person; 5.6% by themselves
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had threatened another person; 6.1% had been involved in breaking into a car; 4.5% by themselves had
broken into a car; 5.4% had had sex with a person who was drunk and/or did not understand what was
going on; and 1.7% had had sex with someone against his/her will.
Adjustment variables
Alcohol consumption
Participants answered the first three questions of the structured Alcohol Use Disorders Identification
Test (AUDIT-C) to measure risk-consumption (Bush et al., 1998). Previous studies support the use of the
AUDIT in samples of secondary school adolescents (Fleming et al., 1991; Kokotailo et al., 2004). The three
items, and possible answers were: 1. How often do you have a drink containing alcohol? Never (0) [pass
over question 2 and 3]; every other month or more seldom (1); about once a month (2); 2-4 times a
month (3); 2-3 times a week (4); 4 or more times a week (5).
2. How many drinks containing alcohol do you have on a typical day when you are drinking? 1-2 glasses
(0); 3-4 glasses (1); 5-6 glasses (2); 7-9 glasses (3); 10 or more glasses (4).
3. How often do you have six or more drinks on one occasion? Never (0); every other month or more
seldom (1); about once a month (2); 2-4 times a month (3); 2-3 times a week (4). Due to the young
population, we modified the scale by adding answer alternatives (as shown above) in comparison with
the original AUDIT-C scale.
Symptoms of attention deficit hyperactivity disorder and depression
We used the Swedish translation of the short form of the World Health Organisation adult ADHD
Self-Report Scale (ASRS). The short six-question screening questionnaire has been shown to have 68.7 %
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sensitivity and 99.5 % specificity(Kessler et al., 2005). Minor changes in wording were conducted to
adapt the scale for adolescents.
Questions regarding the frequency of a symptom were answered on a scale ranging from 0 to 4
(never (0), rarely (1), sometimes (2), often (3) and very often (4)). We used the standard cut-off of
Kessler et al for ADHD symptoms, where a positive screening of ADHD corresponded to a rating of two or
more on questions 1-3 and to a rating of three or more on questions 4-6 (referred to as symptoms of
ADHD.
An adolescent version of the Depression Self-Rating Scale (DSRS) was used to estimate symptoms of
depression (Aslund et al., 2009).The DSRS is a self-report inventory based on the DSM-IV criteria for
depression. DSRS consists of 16 questions on depressive symptoms occurring during the last two weeks.
The response format is “yes” or “no”. DSRS have a reported sensitivity of 86 % and a specificity of 75 %
in an adult clinical population(Svanborg and Ekselius, 2003). Symptoms of co-morbidity of ADHD and
depression was measured by combining ASRS and DSRS; resulting in no symptoms (0), symptoms of
ADHD (1), symptoms of depression (2), and co-occurring symptoms (3)
Statistical Analyses
The delinquency variable was skewed, and neither a log nor a log–log transformation produced a
symmetric distribution of the data. Moreover, log transformations seriously violate the possibilities of
detecting interaction effects. In studies of cGE, it is difficult to select the appropriate statistical method
when the outcome variable is a skewed ordinal or interval variable. Interactions are efficiently estimated
by a GLM, but the inference is not valid if the assumption of normally distributed residuals is violated.
Therefore, although GLMs may be used to estimate main and interaction effects, the results must be
interpreted with caution. Consequently, in the present study, cGEswere interpreted cautiously,
calculations of observed power were computed, and data were additionally analyzed using a Poisson loglinear regression, which is suitable for ordinal scaled variables and the distribution of the dependent
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variable. The observed power was generally high, and the Poisson log-linear models yielded results that
were similar to those obtained in the GLM.
Continuous variables for family conflict, sexual abuse, and child–parent relationship were used in the
GLM and passion regressions. However, for descriptive and illustrative purposes, family-conflict and
sexual-abuse scores were dichotomized as present or absent, such that any positive endorsement of an
item was defined as “present”. Participants were described as having experienced no family conflict and
no sexual abuse, either family conflict or sexual abuse, or both family conflict and sexual abuse. The
scores for child–parent relationship were divided into quartiles: poor (lowest, 24%), medium (25% to
74%), and good (75% to 100%).
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