Patent Application: US-61745303-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 the report ( 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 . replications of this g × e interaction are now needed . replication studies should use valid and reliable ascertainments of maltreatment history and obtain multiple measures of antisocial outcomes , in large samples of males and females . 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 - 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