Patent Application: US-88945004-A

Abstract:
the present invention relates to diagnostic methods for assessing predisposition of a subject to a mental disorder phenotype having an association with an at - risk allele of a brain - functional gene having a plurality of alleles , the association being conditioned by a pathogenic environmental risk factor status condition . additionally , the invention relates to methods for discovering a conditional association between a mental disorder phenotype and an at - risk allele of a brain - functional gene having a plurality of alleles , the association being conditioned by a pathogenic environmental risk factor status condition .

Description:
we studied a large sample of male children from birth to adulthood to determine why some children who are maltreated grow up to develop antisocial behavior whereas others do not . a functional polymorphism in the gene encoding the neurotransmitter metabolizing enzyme monoamine oxidase a ( maoa ) was found to moderate the effect of maltreatment . maltreated children with a genotype conferring high levels of maoa expression were less likely to develop antisocial problems . these findings may partly explain why not all victims of maltreatment grow up to victimize others , and they provide epidemiological evidence that genotypes can moderate children &# 39 ; s sensitivity to environmental insults . in this study , individual differences at a functional polymorphism in the promoter of the monoamine oxidase a ( maoa ) gene were used to characterize genetic susceptibility to maltreatment and to test whether the maoa gene modifies the influence of maltreatment on children &# 39 ; s development of antisocial behavior . based on the hypothesis that maoa genotype can moderate the influence of childhood maltreatment on neural systems implicated in antisocial behavior , we tested whether antisocial behavior would be predicted by an interaction between a gene ( maoa ) and an environment ( maltreatment ). a well - characterized variable number tandem repeat ( vntr ) polymorphism exists at the promoter of the maoa gene , which is known to affect expression . we genotyped this polymorphism in members of the dunedin multidisciplinary health and development study , a sample without population stratification confounds . the history of the study is described in p . a . silva , w . stanton , eds . from child to adult : the dunedin study ( oxford university press , 1996 ). this birth cohort of 1 , 037 children ( 52 % male ) has been assessed at ages 3 , 5 , 7 , 9 , 11 , 13 , 15 , 18 , 21 , and was virtually intact ( 96 %, n = 499 males ) at age 26 years . research sample . the dunedin longitudinal study was constituted at age 3 when the investigators enrolled 91 % of the consecutive births between april 1972 and march 1973 in dunedin , new zealand . cohort families represent the full range of socioeconomic status in the general population of new zealand &# 39 ; s south island . follow - ups have been carried out at ages 3 , 5 , 7 , 9 , 11 , 13 , 15 , 18 , 21 , and most recently at age 26 , when we assessed 96 % of the living cohort members ( n = 499 males ). at each age , participants are brought back to the research unit within 60 days of their birthday for a full day of individual tests and interviews . these data are supplemented by questionnaires completed by persons who know the study members well and by official record searches . dna extraction and genotyping . at age 26 , dna was obtained from 953 study members ( 97 % of those assessed at that age ; 51 % male ); 93 % of dna samples were obtained via blood and 7 % via buccal swabs for those not wishing to undergo phlebotomy . dna was extracted from blood samples using standard procedures . a modified procedure was used to extract dna from buccal cells . primer sequences are described by sabol et al ., namely mao apt1 ( 5 ′- acagcctgaccgtggagaag - 3 ′; seq id no : 1 ) and mao apb1 ( 5 ′- gaacggacgctccattcgga - 3 ′; seq id no : 2 ), although here mao apt1 was 5 ′- labelled with the tet fluorophore . pcr was carried out on a ptc - 225 dna engine ( mj research ), using the following cycling conditions : initial 2 - min denaturing step at 95 ° c ., followed by 35 cycles of 94 ° c . for 1 min , 58 . 2 ° c . for 1 min and 72 ° c . for 1 min 30 secs , and a final extension phase of 72 ° c . for 5 min . reactions were performed in 25 μl geneamp pcr buffer i ( pe applied biosystems ), 1 . 5 mm mgcl 2 , 50 ng of genomic dna , 10 pmols of each primer , 0 . 33 mm dntps and 1 . 5 units of native taq ( promega ). pcr products were assayed on an applied biosystems 377 genetic analyzer ( pe applied biosystems ), set up in genotyping mode , using 4 . 25 % w / v polyacrylamide gel ( amresco ) and tamra - labelled gs500 ( pe applied biosystems ) size standard . results were analyzed using genescan v2 . 1 and genotyper v1 . 1 software ( applied biosystems ). table 1 shows the allele frequencies observed among non - maori members of our study . the genotypes were classified according to previous results showing that an optimum sequence length of 3 . 5 or 4 repeats results in high expression levels . in terms of expression , all studies agree on the functional classification of the two most common alleles , i . e . 3 repeats ( low activity ) and 4 repeats ( high activity ). these two alleles account for 95 . 7 % of our sample . of rare alleles , both sabol et al . and deckert et al . assayed the 3 . 5 repeat with the same result ( high activity ), whereas a discrepancy arises for the 5 repeat . we chose the classification of sabol et al . as they assayed 3 cell lines as opposed to one . however , we carried out analyses using both classifications and observed the same effects . the rare 2 repeat , of which only 1 exists in our sample , was classified as low activity due to its short length . population stratification can probably be ruled out as a confounding factor in this study . first , cohort members reporting maori ethnicity ( 7 %) were not included in our analysis . second , caucasian study members reported the ethnicity of all four grandparents , and only 4 % reported 1 or 2 non - european grandparents . third , allele frequencies among caucasian study members matched closely frequencies reported in caucasian samples . as a final check for stratification we adopted a genomic control approach based on latent class analysis . one hundred individuals were selected at random from the sample and typed for 40 unlinked microsatellite markers . in a stratified sample one would expect to observe hardy - weinberg disequilibrium and linkage disequilibrium across the unlinked markers : our genomic control approach aimed to identify subpopulations ( latent classes ) such that within each there is hardy - weinberg and linkage equilibrium . in the current sample , however , there was no support for having more than one latent class , which is consistent with the sample being homogeneous . childhood maltreatment . evidence of childhood maltreatment during the first decade of life ( ages 3 to 11 years ) was ascertained using behavioral observations , parental reports , and retrospective reports by study members once they reached adulthood . first , mother - child interactions were observed during the child &# 39 ; s age - 3 assessment . the mother was rated by an observer on eight categories : mother &# 39 ; s affect toward the child was consistently negative ; harshness toward the child ; rough , awkward handling of the child ; no effort to help child ; unaware or unresponsive to child &# 39 ; s needs ; indifferent to child &# 39 ; s performance ; demanding of child &# 39 ; s attention ; soiled , unkempt appearance of child ). mothers engaging in 2 or more such behaviors were classified as rejecting ( 16 %), based on evidence that such maternal behavior is associated with increased risk of children &# 39 ; s later antisocial behavior . second , harsh discipline was measured at ages 7 and 9 using a checklist on which parents indicated if they engaged in ten disciplinary behaviors such as “ smack him or hit him with something .” parents scoring in the top decile of the sample - wide distribution were classified as unusually harsh , relative to the culture in which this cohort grew up ( 10 %), based on evidence that such parenting styles are associated with subsequent antisocial behavior of children . third , changes in the person occupying the role of the child &# 39 ; s primary caregiver were ascertained at each assessment . children who experienced 2 or more such changes during the first decade of life were classified as having suffered disruptive caregiver changes ( 6 %), based on evidence that such family changes are predictive of later antisocial behavior . fourth , exposure to child physical abuse was assessed retrospectively at age 26 as part of an interview about victimization . study members were classified as physically abused if they reported multiple episodes of severe physical punishment ( e . g ., strapping leaving welts ; whipping with electric cords ) resulting in lasting bruising or injury before age 11 ( 3 %). fifth , unwanted sexual contact was assessed retrospectively at age 26 as part of an interview about reproductive health . study members were classified as sexually abused if they reported having their genitals touched , touching another &# 39 ; s genitals , or attempted / completed sexual intercourse before age 11 ( 5 %). the percentages of males experiencing physical and sexual abuse are consistent with rates reported elsewhere . we examined these maltreatment experiences based on evidence that they too are linked to antisocial behavior . we derived a cumulative exposure index for each child by counting the number of maltreatment experiences during the first decade of life ; 64 % of the children experienced no maltreatment , 28 % experienced 1 indicator of maltreatment ( hereafter referred to as “ probable maltreatment ”), and 8 % experienced 2 or more indicators of maltreatment ( hereafter “ severe maltreatment ”). antisocial behavior outcomes in adolescence and in adulthood . we examined four different outcome measures of antisocial behavior , using information from independent data sources that were appropriate at different stages of development . conduct disorder was measured according to the criteria of the diagnostic and statistical manual of mental disorders ( dsm ), which identify adolescents displaying a persistent pattern of behavior that violates the rights of others , including physical harm . a diagnosis of conduct disorder ( using a 12 - month reporting period for symptoms ) was made in our longitudinal study when we assessed the research participants at each of four ages : ages 11 , 13 , 15 , and 18 . a ‘ lifetime ’ diagnosis was arrived at by establishing whether a study member received the diagnosis at one or more of the four ages ( according to the dsm , conduct disorder is not normally diagnosed after age 18 ). court records of violent convictions in adulthood were searched via the australian and new zealand police for 97 % of male study members . among study males , 11 % received 174 convictions for violent crimes ( e . g ., common assault , aggravated assault with intent to injure with weapon , domestic violence , manslaughter , rape ). a disposition toward violence was ascertained at age 26 as part of the multidimensional personality questionnaire ( mpq ) aggression scale ( e . g ., “ when i get angry i am ready to hit someone ,” “ i admit that i sometimes enjoy hurting someone physically ”). α reliability of the summed scale was 0 . 71 . symptoms of antisocial personality disorder were ascertained at age 26 , when informant reports about 95 % of male study members were collected by mailing a questionnaire to persons they nominated as “ someone who knows you well ”. informants were friends , partners , or family members . informants described the study members on seven cardinal symptoms : “ has problems controlling anger ,” “ blames others for own problems , “ does not show guilt after doing something bad ,” “ impulsive , rushes into things without thinking ,” “ good citizen ( reversed ),” “ does things against the law ,” and “ gets into fights .” response options were “ not a problem , “ a bit of a problem ,”, and “ yes , a problem .” α reliability of the summed scale was 0 . 84 . intercorrelations between the four outcomes ranged from 0 . 32 to 0 . 46 . we fitted a common factor model to the four measures of antisocial behavior , using methods appropriate to the mixture of categorical and continuous measures . according to multiple fit indices , the model fit well ( χ 2 ( 2 )= 2 . 56 , p = 0 . 28 , cfi = 0 . 99 , rmsea = 0 . 02 ), with factor loadings ranging from 0 . 64 to 0 . 74 , showing that all four measures index liability to antisocial behavior . on the basis of the factor analysis , we created a composite index of antisocial behavior by counting the number of antisocial outcomes observed for each study member . this summary index counts whether they ( a ) met diagnostic criteria for adolescent conduct disorder , ( b ) were convicted for a violent crime , ( c ) scored in the top quartile of the distribution on a self - reported disposition toward violence , and ( d ) scored in the top quartile of the distribution on informant - reported antisocial personality disorder symptoms . we created this composite because the most reliable way to measure antisocial behavior is to aggregate multiple sources of information . we also report separate analyses of each of the four measures of antisocial behavior , in order to test whether the observed findings were robust or sensitive to the four different ways in which the antisocial phenotype was measured . a robust finding is one whose pattern should be observed irrespective of how antisocial behavior is measured ( 20 ). the effects of maoa activity , maltreatment , and their interaction on antisocial behavior were estimated in a moderated regression framework , using logistic regression for categorical outcomes ( e . g ., conduct disorder ) and ordinary least squares ( ols ) for continuous measures ( e . g ., personality disposition toward violence ). the full results are contained in table 2 . the interaction effect was consistent with the hypothesis that maoa activity moderated the effect of maltreatment on antisocial outcomes . as shown in fig1 , the dose - response association between maltreatment and antisocial behavior was significantly weaker in the high - maoa activity group than in the low - maoa activity group . we probed the gene × environment interaction further ( 21 ) and found that the difference in antisocial behavior between the high - and low - maoa groups became larger at increasing levels of maltreatment . t - tests for these differences are as follows : t =− 1 . 48 , p = 0 . 14 at no maltreatment , t = 1 . 62 , p = 0 . 11 at probable maltreatment , and t = 2 . 31 , p = 0 . 02 at severe maltreatment . we further considered the possibility that the observed protective effect of high - maoa activity could have been brought about because of individual differences in iq . we considered this alternative hypothesis because complete and selective deficiency of enzymatic activity of maoa was associated with mild mental retardation in the dutch kindred ( 22 ), and low iq is linked to high levels of antisocial behavior in the general population ( 23 ), including in this sample ( r =− 0 . 28 , p & lt ; 0 . 001 ). therefore , the observed protective effect of high - maoa activity could have been an epiphenomenon of higher iq among males with this genotype . however , we found no iq differences between males with low - and high - maoa activity ( m = 107 ( sd = 14 ) vs . m = 108 ( sd = 13 ), t ( 430 )=− 0 . 70 , p = 0 . 48 ), and no significant linear association between maltreatment and iq in either the low - maoa activity group , t ( 157 )=− 0 . 87 , p = 0 . 38 , or the high - maoa activity group , t ( 269 )= 0 . 93 , p = 0 . 34 . we repeated the regression analysis shown in supplementary table 2 ( first row ), with the addition of iq as a covariate . the interaction effect between maoa and maltreatment remained statistically significant and of equivalent magnitude after controlling for iq ( b =− 0 . 34 , se = 0 . 14 , t = 2 . 43 , p = 0 . 015 ). finally , we considered the possibility that the observed protective effect of high - maoa activity could be brought about if children with this genotype were likely to be reared in favorable environments . as such , we introduced into our analyses a further environmental covariate , social class , that is associated with antisocial behavior ( 24 ), including in this sample ( r =− 0 . 46 , p & lt ; 0 . 001 ). the childhood social class variable used in our analyses is the average of the highest social class level of either parent , assessed repeatedly at the study member &# 39 ; s birth and ages 3 , 5 , 7 , 9 , 11 , 13 , and 15 . this variable reflects the socioeconomic conditions experienced by the study members while they grew up ( 25 ). there were no social class differences between males with low - and high - maoa activity , t ( 439 )= 0 . 90 , p = 0 . 37 . we repeated the regression analysis shown in supplementary table 2 ( first row ), with the addition of social class as a covariate . the interaction effect between maoa and maltreatment remained statistically significant and of equivalent magnitude after controlling for childhood social class origins ( b =− 0 . 33 , se = 0 . 14 , t = 2 . 36 , p = 0 . 019 ). the study offers three advantages for testing gene - environment ( g × e ) interactions . first , in contrast to studies of adjudicated or clinical samples , the representative general population sample avoids potential distortions in association between variables . second , the sample has well - characterized environmental adversity histories . between ages 3 - 11 years , 8 % of the study children experienced “ severe ” maltreatment , 28 % experienced “ probable ” maltreatment , and 64 % experienced no maltreatment . ( maltreatment groups did not differ on maoa activity , χ 2 ( 2 )= 0 . 38 , p = 0 . 82 , suggesting that genotype did not influence exposure to maltreatment .) third , the study has ascertained antisocial outcomes rigorously . antisocial behavior is a complicated phenotype , and each method and data source used to measure it ( e . g ., clinical diagnoses , personality checklists , official conviction records ) is characterized by different strengths and limitations . using information from independent sources appropriate to different stages of development , we examined four outcome measures . adolescent conduct disorder was assessed according to criteria of the diagnostic and statistical manual of mental disorders ( dsm ); convictions for violent crimes were identified via the australian and new zealand police ; a personality disposition toward violence was measured as part of a psychological assessment at age 26 ; symptoms of antisocial personality disorder were ascertained at age 26 by collecting information about the study members from people they nominated as “ someone who knows you well .” a common - factor model fit the four measures of antisocial behavior well , with factor loadings ranging from 0 . 64 to 0 . 74 , showing that all four measures index liability to antisocial behavior . using moderated regression analysis , we predicted scores on a composite antisocial index comprising the four measures of antisocial behavior ( fig1 ). the main effect of maoa activity on the composite index of antisocial behavior was not significant ( b = 0 . 01 , se = 0 . 09 , t = 0 . 13 , p = 0 . 89 ) while the main effect of maltreatment was significant ( b = 0 . 35 , se = 0 . 07 , t = 4 . 82 , p & lt ; 0 . 001 ). the hypothesized interaction between maoa activity and maltreatment revealed a significant g × e interaction ( b =− 0 . 36 , se = 0 . 14 , t = 2 . 53 , p = 0 . 01 ). probing the interaction within each genotype group showed that the effect of childhood maltreatment on antisocial behavior was significantly weaker among males with high - maoa activity ( b = 0 . 24 , se = 0 . 11 , t = 2 . 15 , p = 0 . 03 ) than among males with low - maoa activity ( b = 0 . 68 , se = 0 . 12 , t = 5 . 54 , p & lt ; 0 . 001 ). we conducted further analyses to test if the g × e interaction was robust across each of the four measures of antisocial behavior that made up the composite index . for all four antisocial outcomes , the pattern of findings was consistent with the hypothesis that the association between maltreatment and antisocial behavior is conditional , depending on the child &# 39 ; s maoa genotype ( g × e interaction p = 0 . 06 , 0 . 05 , 0 . 10 , and 0 . 04 , respectively ). for adolescent conduct disorder ( fig2 a ), maltreated males ( including probable and severe cases ) with the low - maoa activity genotype were more likely than nonmaltreated males to develop conduct disorder by a significant odds ratio of 2 . 8 ( 95 % ci : 1 . 42 - 5 . 74 ). in contrast , among males with high - maoa activity , maltreatment did not confer significant risk for conduct disorder ( or = 1 . 54 , 95 % ci : 0 . 89 - 2 . 68 ). for adult violent conviction ( fig2 b ), maltreated males with the low - maoa activity genotype were more likely than nonmaltreated males to be convicted of a violent crime by a significant odds ratio of 9 . 8 ( 95 % ci : 3 . 10 - 31 . 15 ). in contrast , among males with high - maoa activity , maltreatment did not confer significant risk for violent conviction ( or = 1 . 63 , 95 % ci = 0 . 72 - 3 . 68 ). for self - reported disposition toward violence ( fig2 c ) and informant - reports of antisocial personality disorder symptoms ( fig2 d ), males with the low - maoa activity genotype who were maltreated in childhood had significantly elevated antisocial scores relative to their low - maoa counterparts who were not maltreated . in contrast , males with high - maoa activity did not have elevated antisocial scores , even when they had experienced childhood maltreatment . these findings provide initial evidence that a functional polymorphism in the maoa gene moderates the impact of early childhood maltreatment on the development of antisocial behavior in males . this study focused on males because their single x chromosome yields two straightforwardly characterized maoa genotypes : high - activity ( 63 % in this sample ) and low - activity ( 37 %). females , having two copies of the x chromosome , fall into two homozygous groups , high - high ( 42 % in this sample ), low - low ( 12 %), and a third heterozygous group , low - high ( 46 %), that cannot be characterized with certainty because it is not possible to determine which of the two alleles is inactivated for each female participant . given the rarity in females of both the low - low genotype ( 12 %) and severe antisocial outcomes , such as violent conviction ( 2 %), our cohort of 481 females , 11 % of whom were severely maltreated , was too small to support all of the analyses reported here for males . however , adolescent conduct disorder could be analyzed , revealing that girls with the low - maoa activity genotype were more likely to develop conduct disorder by a significant odds ratio of 5 . 5 ( 95 % ci : 1 . 0 - 32 . 0 ) if they were maltreated . in contrast , among girls with high - maoa activity , maltreatment did not confer significant risk for conduct disorder ( or = 1 . 7 , 95 % ci : 0 . 75 - 4 . 2 ). this suggests that high - maoa activity exerts a protective influence against maltreatment for girls as well as boys , and raises the possibility that further research into x - linked genotypes may help to explain one of the least understood facts about serious antisocial behavior : the sex difference . the findings have implications for research and clinical practice . with regard to research in psychiatric genetics , knowledge about environmental context might help gene - hunters refine their phenotypes . genetic effects in the population may be diluted across all individuals in a given sample , if the effect is apparent only among individuals exposed to specific environmental risks . with regard to research on child health , knowledge about specific genetic risks may help to clarify risk processes . numerous biological and psychological processes have been put forward to explain why and how experiences of maltreatment are converted into antisocial behavior toward others , but there is no conclusive evidence that any of these processes can account for the progression from childhood maltreatment to later criminal violence . moreover , some youngsters make the progression , but others do not , and researchers have sought to understand why . the search has focused on social experiences that may protect some children , overlooking a potential protective role of genes . genes are assumed to create vulnerability to disease , but from an evolutionary perspective they are equally likely to protect against environmental insult . maltreatment studies may benefit from ascertaining genotypes associated with sensitivity to stress , and the known functional properties of maoa may point toward hypotheses , based on neurotransmitter system development , about how stressful experiences are converted into antisocial behavior toward others in some , but not all , victims of maltreatment . although individuals having the combination of low - activity maoa genotype and maltreatment were only 12 % of the male birth cohort , they were 22 % of those with multiple antisocial outcomes , yielding an attributable risk fraction ( 11 %) comparable to that of the major risk factors associated with cardiovascular disease . moreover , 85 % of cohort males having a low - activity maoa genotype who were severely maltreated developed some form of antisocial behavior . both attributable risk and predictive sensitivity indicate that these findings could inform the development of future pharmacological treatments . members of the dunedin multidisciplinary health and development study were also tested in connection with a hypothesis relating to conditional association between the short allele of human 5 - htt and depression , where the association is conditioned on experience of stressful life events . research sample . participants were members of the dunedin multidisciplinary health and development study . the birth cohort of 1 , 037 children ( 52 % male ) was established at age 3 when the investigators enrolled 91 % of the consecutive births between april 1972 and march 1973 in dunedin , new zealand . cohort families represent the full range of socioeconomic status in the general population of new zealand &# 39 ; s south island . follow - ups have been carried out at ages 3 , 5 , 7 , 9 , 11 , 13 , 15 , 18 , 21 , and most recently at age 26 , when we assessed 96 % of the living cohort members . the sample and its history are described in detail elsewhere . serotonin transporter genetic variation . we selected to study the 5 - htt gene based on two criteria . ( a ) evidence of functionality and ( b ) evidence that it may moderate response to stress . the promoter activity of the 5 - htt gene , located on 17q11 . 2 , is modified by sequence elements within the proximal 5 ′ regulatory region , designated the serotonin transporter gene - linked polymorphic region ( 5 - httlpr ). a 20 - 23 base pair repeat motif within this region occurs as 2 prevalent alleles : one consisting of 14 repeats ( the short allele ‘ s ’) and another of 16 repeats ( the long allele ‘ l ’). this polymorphic region has functional significance ; ‘ l / l ’ homozygote lymphoblast cells produce 1 . 4 - 1 . 7 times the concentration of 5 - htt mrna than ‘ s / l ’ and ‘ s / s ’ cells , uptake of labeled serotonin in ‘ l / l ’ homozygote lymphoblast cells is 2 times greater than in ‘ s / l ’ or ‘ s / s ’ cells , and the protein produced from ‘ l / l ’ cells binds 30 - 40 % more serotonin than cells with the short variant . although the short promoter variant has not been conclusively linked to depression , experimental paradigms , including studies of 5 - htt knockout mice , stress - reared macaques , and human functional neuroimaging have shown that the 5 - htt gene can interact with environmental conditions to shape reactions to stressful experiences , suggesting to us the hypothesis that variations in the 5 - htt gene may explain why stress leads to depression in some people but not in others . dna extraction and genotyping . when the study members were age 26 years , we obtained dna from 953 participants ( 97 % of those assessed at that age ; 51 % male ); 93 % of the dna samples were obtained via blood and 7 % via buccal swabs for those not wishing to undergo phlebotomy . dna was extracted from blood samples using standard procedures . a modified procedure was used to extract dna from buccal cells . primer sequences for 5 - httlpr are described by gelernter et al ., the forward primer having the sequence ( 5 ′- atgccagcacctaacccctaatgt - 3 ′, seq id no : 3 ) and the reverse ( 5 ′- ggaccgcaaggtgggcggga - 3 ′, seq id no : 4 ). this amplifies a 419 base pair product for the 16 repeat (‘ l ’) allele and a 375 base pair product for the 14 repeat (‘ s ’) allele . pcr was carried out on a ptc - 225 dna engine ( mj research ), using the following cycling conditions : initial 15 - min denaturing step at 95 ° c ., followed by 35 cycles of 94 ° c . for 30 sec , 66 ° c . for 30 sec and 72 ° c . for 40 sec , and a final extension phase of 72 ° c . for 15 min . reactions were performed in 10 × reaction buffer iv ( abgene ), 1 . 5 mm mgcl 2 , 50 ng of genomic dna , 5 pmols of each primer , 0 . 3 mm dntps and 1 unit of native taq ( promega ). pcr products were separated on a 2 . 5 % agarose gel ( multiabgarose , abgene ) supplemented with ethidium bromide ( 0 . 03 %, bdh ) and visualised by ultraviolet transillumination . population stratification can probably be ruled out as a confounding factor in this study . first , cohort members reporting maori ethnicity ( 7 %) were not included in our analysis . second , a genomic control approach based on latent class analysis was adopted , which suggested that the caucasian sample was genetically homogeneous . third , allele frequencies among the non - maori members of our study were consistent with previously reported allele frequencies in caucasian populations : 57 % for the 16 repeat (‘ l ’) allele and 43 % for the 14 repeat (‘ s ’) allele . no other alleles were detected . we followed the well - documented functional classification described by lesch et al . the sample was split into three groups on the basis of genotype , s / s ( n = 147 , 17 % of sample , 51 % male ), s / l ( n = 435 , 51 % of sample , 51 % male ) and l / l ( n = 265 , 31 % of sample , 51 % male ). the three groups were in hardy - weinberg equilibrium ( χ 2 ( 2 )= 1 . 91 , p = 0 . 41 ), and there was no significant difference in genotype frequencies between the sexes ( χ 2 ( 2 )= 0 . 02 , p = 0 . 99 ). stressful life events were assessed at age 26 with the aid of a life history calendar , a highly - reliable method for ascertaining life - event histories . the 5 - year reporting period covered events occurring after the 21 st birthday and before the 26 th birthday . events included employment problems ( long - term unemployment ; being made redundant ; losing a job because the company moved ; being fired ); financial problems ( problems with debt , such as having items repossessed ; not having enough money to pay for food or household expenses ; lacking money for medical expenses ; difficulty paying bills ); housing problems ( homelessness ; multiple residential changes ); health problems ( a disabling physical illness lasting a month or more ; a disabling injury ); and relationship problems ( being involved in a physically violent relationship ; a break - up of a cohabiting , intimate relationship ). to ensure that the collection of information on life events was not influenced by knowledge of psychiatric outcomes , this information was gathered from study members by a different interviewer in a separate session . 30 % of the study members experienced no stressful life events , 25 % experienced 1 event , 20 % 2 events , 11 % 3 events , and 15 % 4 or more events . males experienced more stressful life events than females , x 2 ( 4 )= 10 . 6 , p = 0 . 03 . there were no significant differences between the three genotype groups in the number of life events they experienced , f ( 2 , 846 )= 0 . 56 , p = 0 . 59 , suggesting that 5 - httlpr genotype did not influence exposure to stressful life events in adulthood . childhood maltreatment . to assess children &# 39 ; s experience of stressful life events , we measured their experience of maltreatment between ages 3 to 11 years , as previously described by caspi et al . evidence of childhood maltreatment was ascertained using behavioral observations , parental reports , and retrospective reports by the study members . first , mother - child interactions were observed during the child &# 39 ; s age - 3 assessment . the mother was rated by an observer on eight categories : mother &# 39 ; s affect toward the child was consistently negative ; harshness toward the child ; rough , awkward handling of the child ; no effort to help child ; unaware or unresponsive to child &# 39 ; s needs ; indifferent to child &# 39 ; s performance ; demanding of child &# 39 ; s attention ; soiled , unkempt appearance of child . mothers engaging in 2 or more such behaviors were classified as rejecting . second , harsh discipline was measured at ages 7 and 9 using a checklist on which parents indicated if they engaged in ten disciplinary behaviors such as “ smack him or hit him with something .” parents scoring in the top decile of the sample - wide distribution were classified as unusually harsh , relative to the culture in which this cohort grew up . third , changes in the person occupying the role of the child &# 39 ; s primary caregiver were ascertained at each assessment . children who experienced 2 or more such changes during the first decade of life were classified as having suffered disruptive caregiver changes . fourth , exposure to child physical abuse was assessed retrospectively at age 26 as part of an interview about victimization . study members were classified as physically abused if they reported multiple episodes of severe physical punishment ( e . g ., strapping leaving welts ; whipping with electric cords ) resulting in lasting bruising or injury before age 11 . fifth , unwanted sexual contact was assessed retrospectively at age 26 as part of an interview about reproductive health . study members were classified as sexually abused if they reported having their genitals touched , touching another &# 39 ; s genitals , or attempted / completed sexual intercourse before age 11 . we derived a cumulative exposure index for each child by counting the number of maltreatment experiences during the first decade of life ; in the full sample , 64 % of the children experienced no maltreatment , 27 % experienced 1 indicator of maltreatment , and 9 % experienced 2 or more indicators of maltreatment . there was no significant association between the three genotype groups and maltreatment ( x 2 ( 4 )= 1 . 67 , p = 0 . 80 ), suggesting that 5 - httlpr genotype did not influence exposure to maltreatment in childhood . depression outcomes at age 26 . depression was assessed at age 26 using the diagnostic interview schedule , administered by clinicians with a medical or clinical psychology degree . the reporting period was 12 months prior to interview , which occurred within 60 days of the 26 th birthday . this structured interview yields a continuous measure of depressive symptoms ( m = 5 . 2 , sd = 10 . 5 ; cronbach &# 39 ; s alpha = 0 . 95 ) as well as a diagnosis of a major depressive episode according to dsm - iv criteria . the essential feature of a major depressive episode is a period of at least two weeks during which there is either depressed mood or the loss of interest or pleasure in all activities . one must also experience four of the following additional symptoms : changes in weight or appetite , sleep , or psychomotor activity ; decreased energy ; feelings of worthlessness or guilt ; difficulty thinking or concentrating ; or recurrent thoughts of death or suicidal ideation . lastly , the episode must be accompanied by clinically significant distress or impairment in social , occupational or other important areas of functioning . 17 % of study members ( 58 % female vs . 42 % male ; or = 1 . 6 , 95 % ci : 1 . 1 - 2 . 2 ) met criteria for a past - year major depressive episode , which is comparable to age and sex prevalence rates observed in u . s . epidemiological studies . in addition to analyzing the diagnostic outcome of depression , we also examined specific evidence of suicide ideation / attempt ; 3 % of the study members reported suicide attempts or recurrent thoughts about suicide in the context of a depressive episode . we also collected informant reports about symptoms of depression for 96 % of study members at age 26 , by mailing a brief questionnaire to persons nominated by each study member as “ someone who knows you well .” informants were best friends , partners , or other family members . using a 3 - point scale ( 0 = no , doesn &# 39 ; t apply ; 1 = applies somewhat ; 2 = certainly applies ), informants rated the study member on 4 different symptoms : “ feels depressed , miserable , sad , or unhappy ,” “ feels that no one loves them ,” “ seems lonely ,” and “ talks about suicide ” ( m = 1 . 0 ; sd = 1 . 2 ; cronbach &# 39 ; s alpha = 0 . 80 ). measures of depression at ages 18 and 21 . depression symptoms and diagnoses were derived in the same way at ages 18 and 21 as at age 26 ( described above ). study members were interviewed with the diagnostic interview schedule at ages 18 and 21 years . at those assessments , the interviews covered the 12 - month periods prior to the 18 th ( age 17 years ) and 21 st ( age 20 years ) birthdays . statistical analysis . we used a moderated regression framework to estimate the association between depression and ( a ) 5 - httlpr genotype , ( b ) stressful life events , and ( c ) their interaction . sex was entered into the regressions as a covariate . the equation for the models is as follows b0 is the intercept , b1 is the regression coefficient associated with the effect of sex , which is coded as : b2 is the regression coefficient associated with the effect of variations in the serotonin transporter gene promoter , which is here coded to reflect the number of long (‘ l ’) alleles , such that : b3 is the coefficient associated with the effect of stressful life events , coded to reflect the number of life events , such that : b4 is the coefficient associated with the interaction effect , which is the product of the two variables ( 5 - httlpr and stressful life events ). for continuous measures ( self - reports and informant reports of depression symptoms ), we used ordinary least squares ( ols ) regression ; for categorical measures ( diagnosis of major depression and suicide ideation / attempt ), we used logistic regression . the full results of these regression analyses are provided in tables 3 through 5 . the coefficients ( labeled as b ) in tables 3 - 7 are the model parameters for each type of model ( e . g ., ols , logistic ) before any transformation ( e . g ., exponentiation to obtains odds ratios ). predicted values can be plotted using variable values . in additional analyses we examined the moderating effect of 5 - httlpr on the association between stress and depression , as a function of maoa genotype . genotyping details about maoa are provided in caspi et al . study members were grouped as “ low ” maoa activity ( carrying the 2 , 3 or 5 repeat variants ; 61 % male ) and “ high ” maoa activity ( carrying the 3 . 5 or 4 repeat variants ; 75 % male ). as the gene is situated on the x chromosome , only females are heterozygous ( 23 % of the sample ). we observed that the influence of life stress on depression was moderated by variation in the 5 - htt gene , regardless of individuals &# 39 ; maoa genotype . among carriers of an ‘ s ’ allele , the effect of stressful life events on depression was consistently significant , whether they had low - or high - maoa activity status ( table 6 ). in contrast , among l / l homozygotes , the effect of stressful life events on depression was nonsignificant , regardless of maoa status ( table 7 ). assessing the robustness of the g × e effect . we incorporated five analytic features into this study to test the robustness of the g × e effect . first , we tested that the g × e interaction on depression obtained whether stress occurred in childhood or in adulthood . second , we tested that the g × e interaction predicted within - individual increases in depression from a baseline measured before life events occurred . third , we tested that the g × e interaction was not an artefact of genetic vulnerability evoking life events . fourth , we used informant reports of depression to rule out the possibility of self - report biases . fifth , we examined multiple outcome measures , which is of particular importance in the behavioral sciences because different measurements have different sources of error associated with them . conducting multiple tests is problematic in the following situation : when ( a ) several tests are conducted , ( b ) only a small subset of the tests attain significance , and ( c ) the small number that attain significance can be explained by chance . this situation is even more problematic if ( d ) no hypothesis was stated in advance , or ( e ) researchers selectively report only the test that attained significance . in contrast , as in the present study , multiple statistical tests can provide evidence that a finding is robust in the following situation : ( a ) several tests are conducted using different methods of measurement and analysis , ( b ) all findings are in the same direction and all of the tests attain significance ( or very near - significance ), and ( c ) this number of significant tests exceeds the proportion that could be explained by chance . this situation provides even better evidence of a sturdy finding if , as in the present study , ( d ) a clear hypothesis was stated in advance , and ( e ) the researchers collect multiple outcome measures and report all of them to document that the finding is not an artefact of one measurement approach . 847 caucasian non - maori study members , without stratification confounds , were divided into three groups on the basis of their 5 - httlpr genotype : those with two copies of the ‘ s ’ allele ( s / s homozygotes ; n = 147 , 17 %), those with one copy of the ‘ s ’ allele ( s / l heterozygotes ; n = 435 , 51 %), and those with two copies of the ‘ l ’ allele ( l / l homozygotes ; n = 265 , 31 %). there was no difference in genotype frequencies between the sexes ( χ 2 ( 2 )= 0 . 02 , p = 0 . 99 ). stressful life events occurring after the 21 st birthday and before the 26 th birthday were assessed with the aid of a life history calendar ( 17 ), a highly reliable method for ascertaining life - event histories ( 18 ). the 14 events included employment , financial , housing , health , and relationship stressors . 30 % of the study members experienced no stressful life events , 25 % experienced 1 event , 20 % 2 events , 11 % 3 events , and 15 % 4 or more events . there were no significant differences between the three genotype groups in the number of life events they experienced , f ( 2 , 846 )= 0 . 56 , p = 0 . 59 , suggesting that 5 - httlpr genotype did not influence exposure to stressful life events . study members were assessed for past - year depression at age 26 using the diagnostic interview schedule , which yields a quantitative measure of depressive symptoms and a categorical diagnosis of a major depressive episode according to dsm - iv criteria . 17 % of study members ( 58 % female vs . 42 % male ; odds ratio = 1 . 6 , 95 % ci : 1 . 1 - 2 . 2 ) met criteria for a past - year major depressive episode , which is comparable to age and sex prevalence rates observed in u . s . epidemiological studies . in addition , 3 % of the study members reported past - year suicide attempts or recurrent thoughts about suicide in the context of a depressive episode . we also collected informant reports about symptoms of depression for 96 % of study members at age 26 by mailing a brief questionnaire to persons nominated by each study member as “ someone who knows you well .” we used a moderated regression framework , with sex as a covariate , to test the association between depression and ( a ) 5 - httlpr genotype , ( b ) stressful life events , and ( c ) their interaction ( table 3 ). the interaction between 5 - httlpr and life events showed that the effect of life events on self - reports of depression symptoms at age 26 was significantly stronger ( p = 0 . 02 ) among individuals carrying an ‘ s ’ allele than among l / l homozygotes ( fig3 a ). we further tested whether life events could predict within - individual increases in depression symptoms over time among individuals with an ‘ s ’ allele , by statistically controlling for the baseline number of depressive symptoms they had before the life events occurred ( table 3 ). the significant interaction ( p = 0 . 05 ) showed that individuals carrying an ‘ s ’ allele whose life events occurred after their 21 st birthday experienced increases in depressive symptoms from age 21 to 26 years ( b = 1 . 55 , se = 0 . 66 , t = 2 . 35 , p = 0 . 02 among s / s homozygotes , and b = 1 . 25 , se = 0 . 34 , t = 3 . 66 , p & lt ; 0 . 001 among s / l heterozygotes ) whereas l / l homozygotes did not ( b = 0 . 17 , se = 0 . 41 , t = 0 . 41 , p = 0 . 68 ). the g × e interaction also showed that stressful life events predicted a diagnosis of major depression among carriers of an ‘ s ’ allele , but not among l / l homozygotes ( p = 0 . 056 , fig3 b ). we further tested whether life events could predict the onset of new diagnosed depression among carriers of an ‘ s ’ allele ( table 3 ). we excluded from analysis study members who were diagnosed with depression prior to age 21 . the significant interaction ( p = 0 . 02 ) showed that life events occurring after their 21 st birthdays predicted depression at age 26 among carriers of an ‘ s ’ allele who did not have a prior history of depression ( b = 0 . 79 , se = 0 . 25 , z = 3 . 16 , p = 0 . 002 among s / s homozygotes , and b = 0 . 41 , se = 0 . 12 , z = 3 . 29 , p = 0 . 001 among s / l heterozygotes ), but did not predict onset of new depression among l / l homozygotes ( b = 0 . 08 , se = 0 . 20 , z = 0 . 42 , p = 0 . 67 ). further analyses showed that stressful life events predicted suicide ideation / attempt among individuals carrying an ‘ s ’ allele , but not among l / l homozygotes ( p = 0 . 05 , fig3 c ). the hypothesized g × e interaction was also significant when we predicted informant - reports of age - 26 depression ( p & lt ; 0 . 01 ); an analysis that ruled out the possibility of self - report bias ( fig3 d ). the interaction showed that the effect of life events on informant reports of depression was stronger among individuals carrying an ‘ s ’ allele than among l / l homozygotes . these analyses attest that the 5 - htt gene interacts with life events to predict depression symptoms , an increase in symptoms , depression diagnoses , new - onset diagnoses , suicidality , and an informant &# 39 ; s report of depressed behavior . this evidence that 5 - httlpr variation moderates the effect of life events on depression does not constitute unambiguous evidence of a g × e interaction because exposure to life events may be influenced by genetic factors ; if individuals have a heritable tendency to enter situations where they encounter stressful life events , these events may simply be a genetically - saturated marker ( 23 , 24 ). thus , what we have identified as a gene × environment interaction predicting depression could actually reflect a gene ×“ gene ” interaction between the 5 - httlpr and other genes we did not measure . we reasoned that if our measure of life events represents merely genetic risk , then life events would interact with 5 - httlpr even if they occurred after the the depression episode . however , if our measure of life events represents environmental stress , then the timing of life events relative to depression must follow cause - effect order and life events that occur after depression should not interact with 5 - httlpr to postdict depression . we tested this hypothesis by substituting the age - 26 measure of depression with depression assessed in this longitudinal study when study members were 21 and 18 years old , prior to the occurrence of the measured life events between ages 21 - 26 years . whereas the 5 - httlpr × life events interaction predicted depression at age 26 years , this same interaction did not postdict depression reported at age 21 nor at age 18 years ( table 4 ), indicating our finding is a true g × e interaction . if 5 - htt genotype moderates the depressogenic influence of stressful life events , it should moderate the effect of life events that occurred not just in adulthood , but also of stressful experiences that occurred in earlier developmental periods . based on this hypothesis , we tested whether adult depression was predicted by the interaction between 5 - httlpr and childhood maltreatment that occurred during the first decade of life . consistent with the g × e hypothesis , the longitudinal prediction from childhood maltreatment to adult depression was significantly moderated by 5 - httlpr ( table 5 ). the interaction showed ( p = 0 . 05 ) that childhood maltreatment predicted adult depression only among individuals carrying an ‘ s ’ allele , but not among l / l homozygotes ( fig4 ). we previously showed that variations in the gene encoding the neurotransmitter - metabolizing enzyme monoamine oxidase a ( maoa ) moderate children &# 39 ; s sensitivity to maltreatment ( 25 ). maoa has high affinity for 5 - ht , raising the possibility that the protective effect of the l / l allele on psychiatric morbidity is further augmented by the presence of a genotype conferring high maoa activity ( 13 , 26 ). however , we found that the moderation of life stress on depression was specific to a polymorphism in the 5 - htt gene , as this effect was observed regardless of individuals &# 39 ; maoa gene status ( tables 6 , 7 ). although carriers of an ‘ s ’ 5 - httlpr allele who experienced 4 or more life events constituted only 10 % of the birth cohort , they accounted for almost one - quarter ( 23 %) of the 133 cases of diagnosed depression . moreover , among cohort members suffering 4 or more life events , 33 % of individuals with an ‘ s ’ allele became depressed , whereas only 17 % of the l / l homozygotes developed depression ( fig5 ). thus , the g × e &# 39 ; s attributable risk and predictive sensitivity indicate that more knowledge about the functional properties of the 5 - htt gene may lead to better pharmacological treatments for those already depressed . although the short 5 - httlpr variant is too prevalent for discriminatory screening ( over half of the caucasian population has an ‘ s ’ allele ), a microarray of genes might eventually identify those needing prophylaxis against life &# 39 ; s stressful events ( 27 ). evidence of a direct relation between the 5 - httlpr and depression has been inconsistent ( 12 ), perhaps because prior studies have not considered participants &# 39 ; stress histories . in this study , no direct association between the 5 - htt gene and depression was observed . previous experimental paradigms including 5 - htt knockout mice ( 13 ), stress - reared rhesus macaques ( 14 ), and human functional neuroimaging ( 15 ) have shown that the 5 - htt gene can interact with environmental conditions , although these experiments did not address depression . our study demonstrates that this g × e interaction extends to the natural development of depression in a representative sample of humans . however , we could not test hypotheses about brain endophenotypes ( 28 ) intermediate between the 5 - htt gene and depression because of the difficulty of taking csf or fmri measures in an epidemiological cohort . much genetic research has been guided by the assumption that genes cause diseases , but the expectation that direct paths will be found from gene to disease has not proven fruitful for complex psychiatric disorders ( 29 ). our findings of g × e interaction for the 5 - htt gene , and another candidate gene maoa ( 25 ), point to a different , evolutionary model . this model assumes that genetic variants maintained at high prevalence in the population probably act to promote organisms &# 39 ; resistance to environmental pathogens ( 30 ). we extend the concept of environmental pathogens to include traumatic , stressful life experiences , and propose that the effects of genes may be uncovered when such pathogens are measured ( in naturalistic studies ) or manipulated ( in experimental studies ). to date , few linkage studies detect genes , many candidate gene studies fail consistent replication , and genes that replicate account for little variation in the phenotype ( 29 ). our g × e findings have implications for improving research in psychiatric genetics . incomplete gene penetrance , a major source of error in linkage pedigrees , can be explained if a gene &# 39 ; s effects are expressed only among family members exposed to environmental risk . if risk exposure differs between samples , candidate genes may fail replication . if risk exposure differs among participants within a sample , genes may account for little variation in the phenotype . we speculate that some multifactorial disorders , instead of resulting from variations in many genes of small effect , may result from variations in fewer genes whose effects are conditional on exposure to environmental risks . the present invention is not intended to be limited to the foregoing examples but to encompass all such variations and modifications as fall within the scope of the appended claims . references , each incorporated by reference as if set forth herein in its entirety l . s . aiken , s . g . west , multiple regression : testing and interpreting interactions ( sage , thousand oaks , calif ., 1991 ). m . de bellis , develop . psychopathol . 13 , 539 ( 2001 ). a . j . bennet et al ., molec . psychiatry 7 , 188 ( 2002 ). m . e . berman , r . j . kavoussi , e . f . coccaro , in handbook of antisocial behavior , d . m . stoff , d . bowtell , anal . biochem . 162 , 463 ( 1987 ). j . breiling , j . d . maser , eds . 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