Behavioral genetic research designs have often been attacked because they rely on comparing monozygotic twins (MZ) to dizygotic twins (DZ). Critics of twin-based research maintain that MZ twins look m

European Journal of Human Genetics(2008)16,626 – 634; doi:10.1038/sj.ejhg.5201999; published online 23 January 2008 Keywords:delinquency; crime; violence; MAOA; genotype; antisocial behavior Introduction Studies that investigate the connections between genetic variants and delinquent and criminal behavior in humans have been rare in spite of the wide recognition that individuals may differ in the propensity to commit serious delinquent and criminal acts. 1–3 TheMAOAgene has been a focus in the investigation of aggression in animals and violent behavior in humans.

Monoamine oxidase A (MAOA) is one major enzyme thatcatalyzes the oxidative deamination of a number of biogenic amines in the brain, including dopamine. Because of its ability to catabolize neurotransmitters,MAOAis frequently a candidate gene in the study of psychiatric diseases and behavioral traits. Evidence that implicates the MAOAgene in aggressive behavior has come from knock- out mouse models and human data. Caseset al 4and Shih and Thompson 5developed a line of mice with a targeted disruption of theMAOAgene. They observed an increase in the brain levels of dopamine, serotonin, and norepinephr- ine, and an increase in manifested aggression among men.

Brunneret al 6reported mental retardation and impulsive aggression among eight men in an extended Dutch family with an uncommon sex-specific point mutation in the MAOAgene.

TheMAOAgenomic sequences and promoters 7–9 were studied extensively in search of polymorphisms that might Received 9 June 2007; revised 10 December 2007; accepted 11 December 2007; published online 23 January 2008 *Correspondence: Professor G Guo, Department of Sociology, Carolina Center for Genome Sciences, Carolina Population Center, University of North Carolina – Chapel Hill, CB no. 3210, Chapel Hill, NC 27599-3210, USA.

Tel:þ919 962 1246; Fax: 919 962 7568; E-mail: [email protected] European Journal of Human Genetics (2008) 16,626 – 634 &2008 Nature Publishing Group All rights reserved 1018-4813/08 $30.00 www.nature.com/ejhg be potentially associated with psychiatric disorders and behavioral traits. These basic studies led to a discovery of a 30-bp promoter region VNTR inMAOA, affecting level of transcriptional activity by Sabolet al. 10 The PCR product usually consists of five possible fragment sizes that include 2, 3, 3.5, 4, and 5 copies of the repeat sequence; the 3 and 4 repeats are much more common than the 2, 3.5, and 5 repeats in human populations. Sabolet al 10 showed that alleles with 3.5 (3.5R) or 4 (4R) copies of the repeat sequence are transcripted more efficiently than alleles with 3 (3R) or 5 (5R) copies of the repeat. Sabol and colleagues did not examine theMAOA2R.

Caspiet al 11did not find a main effect ofMAOAvariants, but reported that maltreated male children in New Zealand with the 3R or 5R of the VNTR inMAOAwere more likely to engage in violent behavior than maltreated children with the 3.5R or 4R of the VNTR. Widom and Brzustowicz 12 reported a replication of these results. Habersticket al 13 failed to replicate the gene – environment interaction findings in this same Add Health data set used in this study. The present study focuses on the main effects of MAOAvariants. Meyer-Lindenberget al 14 studied the impact of theMAOAVNTR on brain structure and function with MRI in a large sample of healthy human volunteers.

The study showed that the low expression variants predict differences in the size of limbic structures such as the amygdala and that men with the low expression variants exhibited increased reactivity of the left amygdale and hippocampus during the recall of aversive information.

The objective of this study is twofold. The First, to investigate the association between the self-reported serious and violent delinquency and the 30-bp VNTR in theMAOAgene in a cohort of 2524 adolescents and young adults in the United States in the National Longitudinal Study of Adolescent Health (Add Health). The specific hypothesis is that theMAOA2R is associated with higher levels of delinquency. Second, to perform a functional analysis that evaluates the promoter activity in anMAOA 1.3-kb promoter-luciferase construct containing 2-, 3-, and 4-repeat sequences of the 30-bp VNTR, using two human brain-derived cell lines: neuroblastoma SH-SY5Y and hu- man glioblastoma 1242-MG. Materials and methodsSubject The data source for our analysis is the sibling subsample of 2524 participants in the National Longitudinal Study of Adolescent Health (Add Health), which started as a nationally representative sample of more than 20 000 adolescents in grades 7 – 12 in 1994 – 1995 (Wave I) in the United States. 15 Add Health is longitudinal; the respon- dents have been followed by two additional in-home interviews in 1995 – 1996 (Wave II) and 2001 – 2002 (Wave III). Add Health was stratified by region, ethnic mix, size,urbanicity (urban/suburban/rural), and school type (pub- lic/private/parochial). Our analysis uses the sibling sample of Add Health because DNA measures collected at Wave III in 2002 are available only for this subset of the Add Health respondents. Table 1 provides the mean (SD), proportion, and frequency of the sample characteristics. Measures We constructed a serious delinquency scale and a violent delinquency scale using the 12 questions asked to all the Add Health respondents at Waves I – III. The questions and scaling weights used to create the scales are given in Appendix 1. These two scales are variations of a widely used type of scales in contemporary research on delin- quency and criminal behavior. 16 Our scales are closely related to the scales used by, for example, Hagan and Foster 17and Haynie 18,19 in the analysis of Add Health data and by Hannon 20in the analysis of data from the National Longitudinal Study of Youth. Our serious delinquency scale overlaps with the delinquency scale of Hagan and Foster 17 to a substantial extent. As the descriptor suggests, our violent delinquency scale focuses on an array of violent delinquent behavior that could potentially be classified as violent offenses by the criminal justice system.

Table 1 Mean (SD), proportion, or frequency of sample characteristics Males Females Serious delinquency Wave I 2.42 (4.31) 1.14 (2.60) Wave II 1.64 (3.42) 0.75 (1.85) Wave III 1.20 (2.39) 0.32 (1.15) Violent delinquency Wave I 1.67 (3.11) 0.70 (1.75) Wave II 1.05 (2.35) 0.43 (1.75) Wave III 0.71 (1.66) 0.15 (0.64) Genotype Any2R 0.92% 2.39% Only 3R or 5R 42.4% 17.3% 3.5R or 4R 56.7% 80.35% Ethnicity Caucasian 56.7% 57.6% Non-Caucasian 43.2% 42.3% Age (mean; in years) Wave 1 15.6 15.5 Wave II 16.57 16.42 Wave III 21.34 21.29 Sibling type MZ pairs 92 94 DZ pairs (same sex) 184 164 Full sib pairs (same sex) 97 102 Singletons 554 504 Sample size 1200 1324 VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 627 European Journal of Human Genetics Following the delinquency literature, 17 – 19 we divided the 12 items into the nonviolent and violent types. The nonviolent delinquency includes stealing amounts larger or smaller than $50, breaking and entering, and drug selling. Violent delinquency includes serious physical fighting that resulted in injuries needing medical treat- ment, use of weapons to get something from someone, involvement of physical fighting between groups, shooting or stabbing someone, deliberately damaging property, and pulling a knife or gun on someone. The serious delin- quency scale (nonviolent and violent) is based on the entire 12 items and the violent scale is based on a subset (8) of the 12 items. The Cronbach’sa-values for the serious delinquency scale for Waves I, II, and III are 0.81, 0.79, and 0.73, respectively. For Waves I, II, and III, the Cronbach’sa- values for the violent delinquency scale are 0.75, 0.74, and 0.66, respectively.

Measuring delinquency and crime is challenging. Offi- cial measures based on the police reports and the justice system have been long known to substantially under- estimate delinquency and crime, 16,21 – 23 because official measures reflect not only the behavior of offenders, but also the decisions made by the justice system. For these reasons, many criminologists have turned to self-reports in recent decades. 24,25 Self-reports are now a fundamental method of measuring criminality and seem capable of yielding reliable and valid data. 16 As with any survey of sensitive private information, reporting accuracy is a concern. To protect confidentiality, reduce nonresponses, and increase reporting accuracy, this section of the interview in Add Health was self-adminis- tered by audio-CASI (computer-assisted self-interview). The sensitive question was read to respondents by means of audio headphones. Respondents were given instructions by the computer on how to complete their answers. Self- reported rates of illegal and embarrassing behavior are higher when computer-assisted techniques, particularly self-administered techniques, are used. 26,27 DNA preparation and genotyping At Wave III, in collaboration with the Institute for Behavioral Genetics in Boulder (CO, USA), Add Health collected, extracted, and quantified DNA samples from the sibling subsample. Genomic DNA was isolated from buccal cells using a modification of published methods. 28 – 31 All of the methods employed Applied Biosystems instruments and reagents. Microsatellite and VNTR polymorphisms were performed using fluorescent primers that were analyzed on an ABI capillary electrophoresis instrument.

To reduce errors, two individuals independently scored all genotyping. The additional details on DNA collection and genotyping can be found at Add Health website (Smolen and Hewitt, http://www.cpc.unc.edu/projects/addhealth/).

The MAOA-uVNTR polymorphism was assayed by a modified method. 10,13 The primer sequences for the30-bp VNTR in the promoter region of theMAOAopen reading frame were: forward, 5 0ACAGCCTGACCGTGG AGAAG-3 0(fluorescently labeled); and reverse, 5 0-GAACGTG ACGCTCCATTCGGA-3 0.10 The reaction yielded five frag- ment sizes that included 291, 321, 336, 351, and 381 bp (2, 3, 3.5, 4, and 5 repeats, respectively). This analysis focuses on the effect of 2R (291)vsall the other alleles. Only 11 men possess a 2R. These 11 individuals are from 11 families with six from six pairs of DZ twins and five from another five families of full siblings. Of 31 women who possess one or two 2R alleles, six are from three pairs of MZ twins, three from three pairs of DZ twins, and 22 from 19 families of full siblings. We performed a Hardy – Weinberg equilibrium test for the 2R allele among women and obtained aw 2-value of 0.012 for one degree of freedom indicating that the equilibrium is not violated. Analytical strategies To test the associations of theMAOA30-bp VNTR polymorphisms with serious and violent delinquency, we followed a three-step analytical strategy. The first step is a contingency table analysis in which the mean scores of serious delinquency and violent delinquency across geno- types were compared within each Add Health Wave and gender (Add Health Waves refer to the initial Add Health study in 1994 and two follow-up studies in 1995 – 1996 and 2002).

The second step is regression analysis. Our sample consists of twins and siblings as well as the repeated observations of the same individual over different Add Health Waves; these observations are not independent. The mixed model has long been established in the statistical literature for the analysis of data that are not indepen- dent. 32,33 The following equation describes the basic structure of the mixed models used in our analysis Delinquency jitðsÞ ¼b 0þb 1genotype jiþb 2age jitþb 3age 2 jitþb 4gender ji þb 5ethnicity jiþu j0ðsÞ þv jiþe jitðsÞ ð1Þ wherej,i, andtindex sibling pair or cluster, individual, and Add Health Waves, respectively;s¼m,d,orfindicates whether the sibling cluster or pair are MZ twins, DZ twins, or full biological siblings. The basic trajectory of serious and violent delinquency is described by age and age 2, and their parameters. The model allows the random effect at the sibling cluster level and the level of observations to vary by type of sibling cluster because the strength of the correlation in these types of sibling clusters varies con- siderably. Conditional on the three random intercepts at the level of sibling clusters and one random intercept at the individual level, the siblings and repeated measures are assumed to be independent. The models are estimated by SAS. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 628 European Journal of Human Genetics In our third step, we used two strategies to address the potential impact of population structure. First, we adjusted for self-reported race/ethnicity in all regression analysis.

Tanget al 34showed a near-perfect correspondence between the four self-reported ethnic categories (European Amer- icans, African Americans, East Asians, and Hispanics) and the categories determined by 326 microsatellite markers.

As a second strategy, we applied the procedure by Allison et al 35 to test for possible population stratification.

Following the idea used in the development of sibship tests of linkage and association, 36 – 38 Allisonet alreasoned that the probabilities of genotypes of siblings depended entirely on parental genotypes and that controlling for the effects of sibship would be equivalent to controlling for parental genotypes. Indexing sibships byj, individuals by k, and genotypes byi, they proposed a procedure that can be written as a mixed model Y ijk¼mþa iþb jþðabÞ ijþe ijk ð2Þ wherea i, or the effect of genotypei, is assumed to be fixed; b j, or the effect of sibshipj, is assumed to be random; and (ab) ijis an interaction term specifying the dependence of the random effect of sibship on genotype. This model is a special case of the mixed model. 32,33 Functional analysis of promoter activity The analysis of promoter activity was based upon two human brain-derived cell lines: neuroblastoma SH-SY5Y and human glioblastoma 1242-MG. TheMAOA1.3-kb promoter-luciferase construct was generated by PCR using anMAOA2-kb luciferase reporter gene construct (contain- ing 4.5 repeat) as a template. The PCR product (MluI/ HindIII)ofMAOApromoter fragment ( 1336/ 64 bp) was cloned into the polylinker site (MluI/HindIII) upstream of the luciferase gene in the pGL2-Basic vector. Site-directed mutagenesis was utilized to generate 4, 3, and 2 repeats, respectively, using theMAOA1.3-kb promoter-luciferase construct as a template (Figure 1). The three primers used for mutagenesis of VNTR sites were the following (deletednucleotides are underlined and in lowercase): 5 0- GCACCA GTACCCGCACCAGT accggcaccggcaccGAGCGCAAGGCGG AGGGCCCGCC-3 0( 1117 to 1113; for generating 4- repeat sequence; deleted 15-bp nucleotides usingMAOA 1.3-kb promoter-luciferase construct containing 4.5-repeat sequence as a template); 5 0-GCACCAGTACCCGCACCAG T accggcaccggcaccagtacccgcaccagtGAGCGCAAGGCGGAGG GCCCGCC-3 0( 1199 to 1113 bp; for generating 3-repeat sequence; deleted a 30-bp nucleotides usingMAOA1.3-kb promoter-luciferase construct containing 4-repeat se- quence as a template); and 5 0-GCACCAGTACCCGCACCA GT accggcaccggcaccagtacccgcaccagtGAGCGCAAGGCGGAG GGCCCGCC-3 0( 1231 to 1113 bp; for generating 2- repeat sequence; deleted another 30-bp nucleotides using MAOA1.3-kb promoter-luciferase construct containing 3- repeat sequence as a template).

The mutated nucleotide sequences of all mutant con- structs were confirmed by DNA sequencing. Transfections in SH-SY5Y and 1242-MG cells were performed using Lipofectamine 2000 (Invitrogen). Cells were plated at a density of 5 10 5 cells/well in 6-well plates. In the following day, 0.5mgMAOApromoter-luciferase construct (for one well) was co-transfected with 20 ng of plasmid pRL-TK (the herpes simplex virus thymidine kinase promoter fused upstream to theRenillaluciferase gene, which is used as an internal control; Promega) into the cells as described previously. Controls were the pGL2-basic luciferase reporter gene vector instead ofMAOApromoter- luciferase construct. After 24 h, cells were harvested with luciferase assay lysis buffer (Promega). The cell lysates were assayed for luciferase activity using the Promega Dual Luciferase Assay system. ResultsContingency table analysis Table 2 compares the mean score of serious and violent delinquency across genotypes within each gender and each of the three Add Health Waves. The number of observa- tions and the standard deviation are also given for each mean score in parentheses. The declining trend of both serious and violent delinquency over the Waves is a reflection of the well-known age pattern of delinquent and criminal behavior. For men, the genetic variants on the single X chromosome are grouped into three categories based on the finding by Sabolet al(1998): 2R; 3R or 5R; and 3.5R or 4R. The most striking result for men is the much higher scores of those with a 2R allele than those in the other two categories. The much higher score for 2R holds for both serious and violent delinquency and is present at all Waves of Add Health. In contrast, the delinquency scores of those with 3R or 5R do not seem to differ systematically from individuals with the 3.5R or 4R.

The delinquency score for 2R tends to be twice as high as those in the other two genotype categories. The basic -2072 bp -1336 bpVNTR VNTR 4.5-repeats PCR & site-directed mutagenesis 4-repeats 3-repeats 2-re peatsSpl sites Spl sites Luciferase Luciferase Figure 1 The human MAOA promoter-luciferase construct. The MAOA 1.3-kb promoter containing 2, 3, and 4 repeats were generated by PCR and site-directed mutagenesis using MAOA 2-kb promoter luciferase reporter gene vector as a template. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 629 European Journal of Human Genetics findings remain the same whether 3.5R and 5R are included or not, because 3.5R and 5R each account for only about 1% of the samples in the data of Sabolet alas well as ours.

Grouping women into genotype categories is less straightforward because each woman possesses two X chromosomes and it is unknown which of the two alleles is inactivated. The female participants were classified into three genotype categories: Any2R, those with only 3R or 5R, and those with 3.5R or 4R. For women in Table 2, the 2R stands out being associated with much higher scores for both serious and violent delinquency at Waves II and III.

The serious and violent delinquency scores for the other two genotype categories do not seem to differ from one another.Because of the sibling clustering in the data, standard significance tests are not valid for these comparisons. The next section presents significance tests for the genotype effects obtained from the mixed regression models that take the correlations into consideration. These exploratory results suggest that the genotype effects may be relatively constant over ages in adolescence and young adulthood, or the trajectories of delinquency across genotypes appear to be parallel over the age range. Regression analysis The regression analysis compares serious and violent delinquency scores between the 2R genotype and all the other genotypes within each gender after adjusting for the effects of age and self-reported race/ethnicity (Table 3). The Table 2 Mean score of serious and violent delinquency scales, number of observations, SD byMAOAgenotype, Add Health Wave (age), and gender Genotype Serious delinquency (n; SD) Violent delinquency (n; SD) Wave I Wave II Wave III Wave I Wave II Wave III Age (in years) 12 – 18 13 – 19 19 – 23 12 – 18 13 – 19 19 – 23 Males 2R 5.63 (11; 9.05) 3.18 (10; 6.08) 1.59 (10; 2.65) 3.95 (11; 7.04) 2.45 (10; 5.03) 1.36 (10; 2.26) 3R or 5R 2.33 (508; 4.27) 1.61 (478; 3.24) 1.55 (379; 2.45) 1.13 (508; 3.15) 1.18 (478; 2.25) 0.74 (397; 1.84) 3.5R or 4R 2.41 (681; 4.21) 1.66 (625; 3.49) 1.19 (580; 2.33) 1.64 (681; 2.95) 1.04 (625; 2.36) 0.66 (520; 1.51) Females Any2R 1.16 (31; 2.18) 1.96 (28; 3.56) 1.03 (27; 3.76) 0.77 (31; 1.75) 1.46 (28; 3.12) 0.68 (27; 2.05) Only 3R or 5R 1.17 (227; 3.10) 0.75 (212; 1.62) 0.43 (189; 1.10) 0.78 (227; 2.16) 0.50 (212; 1.17) 0.16 (189; 0.55) Any3.5R and Any4R 1.17 (1066; 2.50) 0.75 (988; 1.82) 0.33 (853; 0.96) 0.68 (1066; 1.65) 0.42 (988; 1.13) 0.15 (853; 0.56) Table 3 Estimated association of the genetic variants in the VNTR ofMAOAwith serious and violent delinquency among adolescents and young adults a Serious delinquency Violent delinquency Males Females Males Females Model 1 Model 2 Model 3 Model 4 b a 95% CIb95% CIb95% CIb95% CI Intercept 3.65 ( 7.81, 0.51) 5.88 (3.59, 8.19) 1.35 ( 4.30, 1.59) 4.65 (3.14, 6.18) Age (in years) 0.73 (0.26, 1.20) 0.45 ( 0.714, 0.201) 0.37 (0.048, 0.70) 0.40 ( 0.570, 0.229) Age squared (in years) 0.024 ( 0.035, 0.012) 0.009 (0.002, 0.016) 0.013 ( 0.022, 0.003) 0.008 (0.004, 0.014) European AmericanFFFFFFFF African American 0.38 ( 0.054, 0.82) 0.072 ( 0.15, 0.29) 0.34 (0.038, 0.65) 0.24 (0.099, 0.383) Hispanic 0.49 (0.031, 0.96) 0.16 ( 0.080, 0.41) 0.38 (0.055, 0.71) 0.17 (0.013, 0.322) Asian 0.26 ( 0.33, 0.85) 0.12 ( 0.46, 0.21) 0.12 ( 0.52, 0.29) 0.050 ( 0.259, 0.160) Others b FFFFFFFF 2R 1.72 (0.21, 3.24)FF1.46 (0.37, 2.5)FF Others c FFFFFFFF Any2RFF0.48 ( 0.087, 1.04)FF0.29 ( 0.065, 0.65) 2 logL16 844.0 14 893.5 14 626.6 11 896.4 No. of persons 1198 1314 1198 1314 No. of measures 3221 3592 3221 3592 aEstimated regression coefficient.bReference category for 2R for men: 3.5R, 4R, 3R, or 5R.cReference category for Any2R for women: Any3.5R, Any4R, Any3R, or Any5R.

Random parameters are not presented in Table 3. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 630 European Journal of Human Genetics regression analysis has yielded findings that are consistent with those from the contingency table analysis (Table 2).

The regression coefficients in mixed models can be interpreted exactly as those in the ordinary least square linear regression. For men, the 2R genotype scored 1.72 (P¼0.025) points higher than the other genotypes on the serious delinquency scale. For the violent delinquency scale, men with the 2R genotype scored 1.45 (P¼0.008) points higher than the other genotypes. Figure 2 plots the model-predicted serious delinquency over age for those with the 2R and those with 3R, 3.5R, 4R, or 5R. The 2Rs on average scored about twice as high as the non-2Rs. The effects of 2R for violent delinquency are similar to those in Figure 1 and the data not presented. Using 3.5R or 4R as the reference category in a reestimated regression model shows that the 3R or 5R genotype does not differ from the 3.5R or 4R genotype.The female effect of Any2R genotype is similar to the male effect. Women with the Any2R genotype scored 0.47 (P¼0.097) and 0.29 (P¼0.108) points higher than the other genotypes on the serious and violent delinquency scales, respectively. These regression coefficients represent a large increase in the serious and violent delinquency scores among women.

To address potential bias from population stratification, we controlled for self-reported race/ethnicity in all regres- sion models presented in Table 3. In addition, we performed the procedure of Allisonet al 4(Equation (2)) The model includes one random effect (b j) at the sibling- cluster level, a second random effect at the individual level (e ijk), and the key interaction term [(ab) ij] between genotype and the random effect at the sibling-cluster level.

In all Allison’s models we have estimated, the random interaction term is not significant indicating that within- family effects may not be sufficiently influential to generate population stratification. Promoter activity To test whether the statistical link between theMAOA VNTR 2R and delinquency has a biochemical basis, we carried out an analysis ofMAOApromoter activity by genotype. As shown in Figure 3, the transient transfection and luciferase assay reveals three levels of promoter activity for the three 30-bp nucleotide repeat sequences inMAOA in SH-SY5Y and 1242-MG cells. The 4R sequence exhibited a higher level of promoter activity than the 3R sequence.

Alleles with the 3.5R or 4R sequence had been previously shown to transcribe more efficiently than those with the 3R or 5R sequence. 10Our finding is that the 2R sequence of theMAOApromoter displayed the lowest level of promoter + pGL2 Basic + MAO A promoter 1242-MG SH-SY5Y MAO A Promoter 1.3 kb 2 repeats 3 repeats 4 repeats Luc Luc Luc 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Figure 3 Relative luciferase activity. The effect of the 30-bp nucleotide repeat sequence on the human MAOA promoter activity in SH-SY5Y and 1242-MG cells. The MAOA promoter 1.3-kb luciferase constructs were transfected into either SH-SY5Y or 1242-MG cells for 24 h. Then cells were harvested and luciferase activity was determined. Controls were pGL2-Basic vector as indicated. Please note that 2-repeat sequence of MAOA promoter shows the lowest activity and 4-repeat sequence of MAOA promoter shows the highest activity. Data were the mean± SD from three independent experiments with triplicates for each experiment. The sixt-tests (three for each cell) between 2 and 3 repeats, 2 and 4 repeats, and 3 and 4 repeats are all highly significant (t46). 4 3.5 3 2.5 2 1.5 1 0.5 0 Serious Deliquency 12 13 14 15 16 17 18 19 20 21 22 23 Age No 2R 2R Figure 2 ProjectedMAOA2R and non-2R serious delinquency by age. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 631 European Journal of Human Genetics activity. In both SH-SY5Y and 1242-MG cells, the level of promoter activity for the 2R sequence is substantially lower than that of the 3R and only a fraction of that for 4R. The extremely low level of promoter activity in the 2R sequence corresponds to the much heightened level of serious and violent delinquency for the 2R genotype.

We performed a series oft-tests between the 2R, 3R, 4R, and the controls (pGL2-Basic) and among 2R, 3R, and 4R (3Rvs2R, 4Rvs2R, and 3Rvs4R). All thet-tests are highly significant, witht-ratios reaching 10 or above in most cases. The smallestt-ratio is 6.

Discussion Genetic studies of serious delinquent and criminal beha- vior in non-patient or general human populations are rare.

Caspiet al 11reported an interaction between maltreatment in childhood and level ofMAOAactivity for violent behavioral problems; they did not find a main effect of theMAOAgene. Chenet al 39 hypothesized the association of aggressive behavior in adolescents with both the dopamine D2 receptor gene and the dopamine transporter gene. They provided suggestive evidence from a small study of 11 adolescents diagnosed to have impulsive – aggressive violent behavior. Guoet al 40 reported the main effects of theTa qI polymorphism in theDRD2gene and the 40-bp VNTR in theDAT1gene on serious and violent delinquency among men in the same Add Health data.

The present study demonstrated a link between the 2 repeat of the 30-bp VNTR in theMAOAgene and much higher levels of self-reported serious and violent delin- quency. The finding is supported by a statistical association analysis and a functional analysis ofMAOApromoter activity. The association analysis showed that men with a 2R reported a level of serious delinquency in adolescence and young adulthood that were at least twice as high as that for those with the other variants in the VNTR. A very similar finding was obtained for violent delinquency for men. Women with Any2R also reported much higher levels of serious and violent delinquency than those with other repeats, although theP-values associated with these estimates are 0.097 and 0.108, respectively.

In the functional analysis, the 2 repeat exhibited the lowest level of promoter activity, that is, 25 – 30% of the promoter activity exhibited by the 4 repeat. The level of promoter activity for the 3 repeat is located between the 2 and 4 repeats, which is consistent with the previous report. 10 The excessively low promoter activity of the 2R suggests a biochemical basis for the excessively high levels of serious and violent delinquency.

Although the promoter activity differs significantly between 3R and 4R, we did not find a significant difference in serious and violent delinquency between those posses- sing 3R and those possessing 4R. This result is notinconsistent with the findings reported by Caspiet al, 11 who found a higher level of violent behavior for 3R (and 5R) than 4R (and 3.5R) only among men who were maltreated in childhood. Kim-Cohenet al 41 reported a main effect of 3R against 4R as well as an interaction effect in a sample of 7-year old Caucasian boys born in England and Wales.

Although our sample is large, the 2 repeat is rare.

Though the findings concerning 2R for men have passed the standard tests of significance in spite of the small category of 2R, it is possible that some of our findings could be attributable to chance. For this reason, it is important that these findings are replicated in a much larger population-based study. Future replications may prove the importance of the 2R allele, but the allele cannot possibly be involved in most delinquent behavior because of its rarity, just like the rare mutation in the MAOAgene the Dutch family 6cannot explain most of the delinquency.

The weaker results for women could be due to the ambiguity in defining the X-linkedMAOAVNTR genotypes for women, which is equivalent to measurement errors.

The Any2R category used in the present study may include those whose 2 repeat is not fully active. A test of this hypothesis requires a much larger sample that contains a sufficient number of participants homozygous for the 2 repeat.

Our measures of serious and violent delinquency are constructed in the tradition of research on delinquency and crime. Serious delinquency measures the overall delinquency including violent delinquency, but it does not include acts more typically viewed as common adolescent deviance such as lying to parents/guardians about where they had been, minor vandalism, being loud in a public place, and driving a car without its owner’s permission. Violent delinquency measures violent behavior that is typically treated as violent offense by the criminal justice system. Our findings suggest that the MAOA*2R may be more predictive of violent delinquency than nonviolent delinquency. Future work should test this hypothesis explicitly using nonviolent and violent delinquency measures.

Acknowledgements This research uses data from Add Health, a program project designed by J Richard Udry, Peter S Bearman, and Kathleen Mullan Harris, and funded by the Grant P01-HD31921 from the National Institute of Child Health and Human Development, with cooperative funding from 17 other agencies (http://www.cpc.unc.edu/addhealth/con- tract.html). Special acknowledgment is due to Andrew Smolen and John K Hewitt of the Institute for Behavior Genetics, University of Colorado for DNA isolation and genotyping. We gratefully acknowl- edge grant supports from NIH, P01-HD31921 to Add Health; R03 HD042490-02 and R03 HD053385-01 to Guang Guo; and from NSF, SES-0210389 to Guang Guo. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 632 European Journal of Human Genetics References1 Gottfredson MR, Hirschi T:A General Theory of Crime. Stanford, CA: Stanford University Press, 1990.

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41 Kim-Cohen J, Caspi A, Taylor Aet al: MAOA, maltreatment, and gene – environment interaction predicting children’s mental health: new evidence and a meta-analysis.Mol Psychiatry2006; 11: 903 – 913. Appendix 1 The serious and the violent delinquency scales 1. In the past 12 months, how often did you hurt someone badly enough to need bandages or care from a doctor or nurse? a 2. In the past 12 months, how often did someone hurt you badly enough to need bandages or care from a doctor or nurse? a 3. In the past 12 months, how often did you use or threaten to use a weapon to get something from someone? a VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 633 European Journal of Human Genetics 4. In the past 12 months, how often did you take part in a fight where a group of your friends was against another group? a 5. In the last 12 months, how often did you deliberately damage property that did not belong to you? a 6. In the past 12 months, how often did you carry a handgun to school or work? a 7. In the past 12 months, how often did you steal something worth more than $50? a 8. In the past 12 months, how often did you steal something worth less than $50? a 9. In the past 12 months, how often did you go into a house or building to steal something? a 10. In the past 12 months, how often did you sell marijuana or other drugs? a 11. In the past 12 months, have you shot or stabbed someone? b 12. In the past 12 months, have you pulled a knife or gun on someone? b aFor this question, the score value on the scale is determined in the following manner: the score is coded as zero if the event did not occur in the past 12 months; the score is coded as one if the event occurred once or twice in the past 12 months; the score is coded as two if the event occurred three or four times in the past 12 months; the score is coded as three if the event occurred five or more times in the past 12 months.

bFor this question, the score value on the scale is determined in the following manner: the score is coded as zero if the event did not occur in the past 12 months; the score is coded as three if the event did occur once or more during the past 12 months.

In the construction of the serious delinquency scale, individuals with more than two missing responses were excluded from analysis. In the construction of the violent delinquency scale, individuals with more than one missing response were excluded from analysis.

The violent scale is based upon 8 of the 12 items and they are items 1 – 6, 11, and 12. VNTR 2 repeat inMAOAand delinquent behavior G Guoet al 634 European Journal of Human Genetics