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Corticotrophin-releasing hormone type 1 receptor gene (CRHR1) variants predict posttraumatic stress

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NIH Public AccessAuthor ManuscriptDis Markers. Author manuscript; available in PMC 2013 July 25.Published in final edited form as:Dis Markers. 2011 ; 30(0): –99. doi:10.3233/DMA-2011-0761.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptCorticotrophin-releasing hormone type 1 receptor gene (CRHR1)variants predict posttraumatic stress disorder onset and coursein pediatric injury patientsAnanda B. Amstadtera,*, Nicole R. Nugentb, Bao-Zhu Yangc, Alisa Millerd, Richie Siburiane,Priya Moorjanie, Stephen Haddade, Aditi Basue, Jesen Fagernesse, Glenn Saxed,f, JordanW. Smollere,f, and Karestan C. KoenengaDepartment of Psychiatry, Virginia Institute for Psychiatric and Behavioral Genetics, VirginiaCommonwealth University, Richmond, VA, USAbDepartment of Psychiatry, Brown Medical School and Rhode Island Hospital, Providence, RI,USAcDepartment of Psychiatry, Division of Human Genetics, Yale University School of Medicine, NewHaven, Connecticut, CT, USAdDepartment of Psychiatry, Boston Children’s Hospital, Boston, MA, USAePsychiatric Genetics Program in Mood and Anxiety Disorders and Psychiatric andNeurodevelopmental Genetics Unit, Center for Human Genetic Research, MassachusettsGeneral Hospital, Boston, MA, USAfHarvard Medical School, Boston, MA, USAgDepartments of Society, Human Development and Health and Epidemiology, Harvard School ofPublic Health, Boston, MA, USA and the Harvard Center on the Developing Child, Cambridge,MA, USAAbstractPosttraumatic stress disorder (PTSD) is a common and disabling anxiety disorder that may occurin the aftermath of exposure to potentially traumatic life events. PTSD is moderately heritable, butfew specific molecular variants accounting for this heritability have been identified. Genesregulating the hypothalamic-pituitary-adrenal (HPA) axis, such as corticotrophin-releasinghormone type 1 receptor gene (CRHR1), have been implicated in traumatic-stress relatedphenotypes but have yet to be studied in relation to PTSD. The present study sought to examinethe relation between 9 single nucleotide polymorphisms (SNPs) in the CRHR1 gene andposttraumatic stress symptoms in a prospective study of pediatric injury patients (n = 103) whowere first assessed in the acute aftermath of their injury at the hospital. Results indicated thatmultiple SNPs were associated with acute symptoms at a univariate level, and after correction formultiple testing, rs12944712 was significantly related to acute PTSD symptoms. Longitudinallatent growth curve analyses suggest that rs12944712 is also related to both acute symptom leveland trajectory of symptoms over time. The present study adds support for the role of CRHR1 inthe stress response following potentially traumatic event exposure in youth. It should be noted thatthe sample size in this study was small, and therefore statistical power was low; following, results© 2011 – IOS Press and the authors. All rights reserved*Corresponding author: Ananda B. Amstadter, PhD; Virginia Institute for Psychiatric and Behavioral Genetics, VirginiaCommonwealth University, Department of Psychiatry, 800 E. Leigh Street, PO Box 980126, Richmond, VA 23298-0126, USA. Tel.:+1 804 828 8129; Fax: +1 804 828 8801; abamstadter@vcu.edu.Amstadter et al.Page 2

from this study should be considered preliminary. Although results are not definitive, the findingsfrom this study warrant future replication studies on how variation in this gene relates to responseto traumatic event exposure in youth.Keywords

Posttraumatic stress disorder; CRHR1; hypothalamic-pituitary-adrenal axis; genetic; injury

NIH-PA Author Manuscript1. Introduction

NIH-PA Author ManuscriptNIH-PA Author ManuscriptStressful experiences involve the recruitment of the body’s major stress systems, includingthe hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis, activated by corticotrophin-releasing hormone (CRH), regulates the release of stress hormones such as cortisol [1]. Inmany individuals, this normative stress response is followed by a return to baseline once thestressor has passed. However, a subset of individuals evidence alterations in this initial stressresponse and followed by the chronic dysregulation of the HPA axis characteristic oftraumatic stress related phenotypes, including post-traumatic stress disorder (PTSD) [2].There are a number of key findings suggesting HPA axis dysregulation that are exhibited inthose with PTSD (e.g., elevated cerebral spinal fluid corticotrophin releasing hormone,enhanced suppression of cortisol, lower glucocorticoid receptors in lymphocytes), all ofwhich are consistent with the notion of a sensitized HPA axis in individuals with PTSD [forreview see 2]. Therefore, there is reason to hypothesize that genes regulating the HPA axiswould be relevant to study in relation to posttraumatic stress symptom trajectory. Notably,however, these findings suggest that the HPA axis may be dysregulated in individuals withPTSD, but they are not specific to PTSD. Many phenotypes are common following exposureto traumatic events, such as depression and other anxiety disorders, and many of thesedisorders also evidence HPA axis dysregulation.

Although there are numerous candidate genes with potential influence on the HPA axis [3],CRH system genes play a critical role in modulation of HPA stress reactivity [4–7]. As such,these genes may influence acute and/or chronic alterations in HPA axis functioning.

Although a total of thirty investigations have examined candidate genes and PTSD [8], onlytwo studies have examined whether polymorphisms believed to influence HPA axis

functioning are related to PTSD [9,10], and no prior studies of PTSD have examined CRHgenes. The present investigation examines the association between nine genetic markersspanning the CRH receptor 1 (CRHR1) gene (rs11657992, rs12936181, rs12944712,

rs17690314, rs17763658, rs242942, rs4074461, rs4458044, rs11657992) and longitudinaltrajectories of PTSD symptoms in pediatric injury patients.

To date, only one study of candidate genes and PTSD has focused on children [11]. Animaland human studies support the developmental sensitivity of stress effects on the HPA axis[12,13]. Indeed, age and developmental stage at the time of the trauma and at the time oflater assessment have been linked to different patterns of both acute and chronic HPA axisalterations [14,15]. For example, whereas low levels of peritrauma cortisol predict laterdevelopment of PTSD in adults [16], high peritrauma cortisol levels predict subsequentdevelopment of PTSD in children [17–19]. Interestingly, longitudinal assessmentscomparing trauma-exposed youth who did versus did not develop PTSD found that,although acute measures of acute salivary cortisol were predictive of subsequent

development of PTSD (measured at 1 and 6 months), salivary cortisol levels and rhythmnormalized by 6 months [19]. This pattern of changing direction in cortisol-PTSDassociations was also found in an investigation of youth assessed within a year of theirtrauma compared with youth assessed more than one year post-trauma [20]; whereas PTSD

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symptoms were linearly and positively associated with cortisol in recently traumatizedyouth, symptoms were negatively associated with cortisol in youth whose experiencesoccurred more than a year prior. These investigations highlight key considerations for

researchers interested in understanding the relationship between the HPA axis (as indicatedby cortisol) and PTSD: (1) the association between cortisol and subsequent PTSD differs inyouth and adults and (2) the direction of the association between cortisol and PTSD in youthchanges as time since trauma elapses. Thus, while the genetic predictors of associationsbetween HPA axis functioning and PTSD are unknown, it is clear that both age at time oftrauma and duration of time since trauma are important considerations in traumatic stressresearch.

Recently, variation in the CRHR1 gene (CRHR1 rs110402 and rs242924) was found tointeract with a history of childhood maltreatment to predict adult cortisol response to thedexamethasone/CRH test [7]; in both markers, the minor (i.e., less common) alleles werefound to protect against the effects of childhood maltreatment, with maltreated participantshomozygous (GG) for the major allele found to evidence increased stress reactivity relativeto maltreated participants who were carriers of the minor alleles. This finding was partiallyreplicated in a second investigation of one of the variants (rs110402), in which the minorallele was associated with decreased cortisol response to the dexamethasone/CRH test inmales, but not females, reporting childhood trauma [21]. Notably, these effects wereobserved in participants with adverse early life experiences.

Numerous studies of depression provide support for the CRHR1 gene as a moderator ofpsychopathology following exposure to early life stressors. For example, Bradley andcolleagues (2008) reported an interaction between genetic markers spanning CRHR1 andchildhood abuse in prediction of adult depression [22]. Seven out of ten markers spanningthe gene showed significant interactions with childhood abuse in the prediction of

depression, with rs110402 and rs7209436 significant even after correction for multiple tests.Consistent with prior research, participants experiencing childhood abuse and possessing theGG rs110402 genotype were at greatest risk for depression. Examination of commonhaplotypes of CRHR1 revealed a protective effect of a TAT haplotype formed by threeCRHR1 variants (rs7209436, rs110402, rs242924). Providing even further support for thesefindings, the results were replicated with an independent sample that was ethnically distinctfrom the original sample. Polanczyk and colleagues [23] attempted to replicate and extendthe Bradley et al. investigation using data from two longitudinal cohort studies. Althoughfindings from one cohort replicated the protective nature of the TAT haplotype (rs7209436,rs110402, and rs242924) in the prediction of depression, results of the second cohort did notsupport the expected effects. However both of these investigations assessed both early lifestress and the psychiatric outcome many years after these experiences, introducing thepotential for recall bias and limiting researchers’ ability to prospectively examine

psychobiological developmental processes in response to trauma. Additionally, althoughboth depression and PTSD are stress-sensitive conditions characterized by alterations of theHPA axis, the patterns of HPA activity and reactivity found in PTSD diverge markedly fromthe patterns found in depression [24].

Although there is considerable theoretical support for the importance of CRHR1 in thedevelopment of psychopathology following exposure to significant early life stress, noextant studies have examined CRHR1 markers in the prediction of PTSD following traumaexposure during childhood. Indeed, no investigations to date have examined CRHR1 asrelated to PTSD in any sample. Another limitation of extant research is the absence of

studies that examine the influence of genes on both onset of symptoms and symptom course.As we have argued elsewhere [25], genes implicated in the development or onset ofsymptoms of PTSD may differ from genes implicated in the maintenance or course of

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symptoms of PTSD. Unlike previous cross-sectional investigations of PTSD candidategenes, the present investigation involves recruitment and assessment of participants withinhours of trauma exposure followed by subsequent assessments of symptoms of PTSD at 3months, 12 months, and 18 months. Thus, the present investigation can model not only theeffects of genotype at a single time point but also influence of genotype over time.Consistent with cross-sectional research, we expected that the minor allele would beprotective at the intercept (within hours of injury). However, as no prior studies haveassessed the influence of candidate genes on PTSD symptom course, we did not make apriori hypotheses about the effects of CRHR1 on symptom course.

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2. Methods and materials

2.1. Participants

Participants were 103 children who were sequentially admitted to an inner-city hospital forinjuries between April 2002 and January 2004 [26,27]. All children aged 7–18 admitted tothe hospital with an injury were eligible to participate unless they or their parents did notspeak sufficient English to complete the study instruments, had a Glasgow Coma Scaleequal to or less than 7 at the time of their admission, or lived more than 2 hours away fromthe hospital (complicating follow up interviews). Participant characteristics are displayed inTable 1.

2.2. Enrollment and acute assessment procedure

The families of children who were admitted to the hospital with an injury were introduced tothe study by a master’s level research associate, once the child was deemed medically stableby the attending surgeon (e.g., they did not have a delirium, an active infection and were notreceiving mechanical ventilation). Families were told that the study was investigating achild’s coping following injury. All families were informed that participation in the studywas voluntary and to decline participation would not affect their quality of care. Writteninformed consent was obtained from the parents and child after the researcher gave a

comprehensive description of the study. The study was approved by the Institutional ReviewBoard and treatment of human subjects adhered to established guidelines. The researchassociate interviewed the child and the primary caregiver during the initial hospital stayusing the psychometric assessment package described below. Every effort was made tointerview the child and caregiver separately, however the sensitive and complicated natureof the hospital stay and setting (e.g., grief, anxiety, doctor consultations, nurses visits, vitalsbeing taken, etc.) precluded this in some cases. Each participant provided a saliva sampleusing a standard mouthwash protocol for DNA extraction [28]. Participants were paid $50for participation in the acute assessment, and they were also paid $25 (and transportationcosts) to complete follow-up assessments.

2.3. DNA isolation

Buccal DNA samples were obtained from each subject via mouthwash. DNA was isolatedvia standard procedures using the Gentra DNA isolation kit (Gentra Systems, MinneapolisMN).

2.4. SNP selection and genotyping

SNP selection was determined using the Phase II HAPMAP [29]. We used the aggressivetagging option (2- or 3-haplotype tagging of SNPs) of Tagger implemented in the programHaploview [30,31]. We further enriched our SNP selection by including SNPs implicated inanxiety phenotypes from the published literature. Genotyping of SNP markers was

performed by mass spectrometry through use of the iPlex assay (Sequenom). The majorsteps in this process included the following: primer design using SpectroDE-SIGNER

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software; DNA amplification by PCR; post-amplification removal of the phosphate groupsfrom the unincorporated dNTPs using shrimp alkaline phosphotase; primer extension

reactions for allele differentiation, salt removal using ion-exchange resin; and SpectroCHIPplating/analysis by mass spectrometry.

2.5. Outcome measure

Posttraumatic stress symptoms were assessed during the acute hospitalization, and at 3-, 12-,and 18-month follow-ups using the University of California at Los Angeles Child Post-traumatic Stress Disorder Reaction Index (UCLA PTSD-RI; [32–34]). The UCLA PTSD-RIis a 20-item semi-structured interview that assesses posttraumatic symptoms in children.Children are asked to rate the frequency of their posttraumatic symptoms on a 5-point Likertscale from 0 = “never” to 4 = “ most of the time”. The measure is most often usedcontinuously, but a clinical cutoff of 38 can also be used [34]. The UCLA PTSD RI isinternally consistent, with Cronbach’s alpha in a disaster sample was reported to be 0.92,and the measure has demonstrated excellent test-retest reliability (0.84) [35]. The measurehas convergent validity coefficients ranging from 0.70 (in comparison with the PTSDModule of the Schedule for Affective Disorders and Schizophrenia for School-AgeChildren) to 0.83 (in comparison to the Child and Adolescent Version of the Clinician-administered PTSD Scale). Using a cutoff score of 38, sensitivity has been found to be 0.93and specificity to be 0.87 in detecting accurate PTSD diagnoses [33]. The UCLA PTSD-RIis one of the most widely used measures of PTSD symptoms in children [34].

2.6. Covariates

Race/ethnicity information was gathered via self-report. For analytic purposes, participantswere characterized as Non-Hispanic white, non-Hispanic black, or other. Sex was defined asmale or female, and age at admission was measured in years. Injury Severity Score (ISS)[36] is a well-validated index of the injury severity. The ISS is related to the likelihood ofsurvival after injury and is determined by rating the severity of injury for six body areas (i.e.,head, neck, face, chest, abdomen, extremity and external) on a five-point scale known as theabbreviated injury scale (AIS). The AIS ranges from 1 (minor injury) to 5 (critical injury).The numerical score represents the degree of life threat associated with the anatomicalinjury. The ISS is derived from the sum of the squares of the AIS score with a range of 0–75. For patients with multiple traumas the three most severe injures are squared and

summed. A trained trauma nurse coordinator assigned the ISS score of the participants inthis study. Number of days in the hospital and whether the participant had experienced aviolent (e.g., was shot, stabbed or physically assaulted) versus non-violent injury (e.g., caraccident) were extracted from medical records.

2.7. Statistical analyses

Given the low number of children with probable PTSD at baseline (n = 18, 17.5%) and at 3-month follow-up (n = 7, 6.8%) the UCLA PTSD-RI was used continuously to maximizepower. First, single SNP analyses were conducted in PLINK [37] to determine if any of thenine SNPs genotyped within CRHR1 were associated at a bivariate level to UCLA PTSD-RItotal score. Also using PLINK, we derived empirical p-values using permutation tests(10,000 permutations [38,39]). We adjusted for multiple testing using a Bonferronicorrection.

Finally, the longitudinal influence of CRHR1 rs1294 4712 genotype was modeled usinglatent growth modeling (LGM) in MPlus. Analyses explored linear growth as well a

quadratic slope factor [40]. Parameter estimates were conducted using maximum likelihoodestimation with robust standard errors (MLR), which permits application of the missingnessoption, allowing retention of all participants on endogenous variables. Minimum

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missingness in the present study was 0.33, suggesting sufficient covariance coverage for areliable model [41]. Trajectories of linear time were parameterized using months, with theintercept indicated at the acute in-hospital assessment to permit examination of the influenceof rs12944712 on initial symptom onset. On the basis of prior research supporting additiveprotective effects of the minor allele of rs12944712, the effects of rs12944712 were modeledin assumption of an additive genetic effect, with the alleles coded for symptom risk

conferred (i.e., major alleles (G) were coded to indicate greater “dose,” AA = 0, AG = 1, GG= 2). To control for potential population stratification self-reported racial/ethnic status wasincluded as a covariate. All analyses also covaried for key individual-level influences (i.e.,age, sex) as well as injury-related factors (i.e., injury severity, days in hospital, whetherinjury was sustained due to violence).

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript3. Results

3.1. Descriptive analyses

Sample characteristics are presented in Table 1. Participants’ racial/ethnic status wasreported as: non-Hispanic white (n = 42, 40.8%), non-Hispanic black (n = 47, 45.6%), orother (n = 14, 13.6%). In regard to sex, 73.8% (n = 76) of the participants were male, andthe remaining 26.2% (n = 27) were female. The average age of participants was 14.63 years(S.D. = 3.18). With regard to injury characteristics, the average number of days spent in thehospital was 5.90 (S.D. = 5.78). The majority of the injuries were non-violent in nature

(71.8%, n = 74), with the remaining 28.3% (n = 29) being violent in nature. The average ISSscore was 8.91 (S.D. = 6.91). With regard to UCLA PTSD-RI scores, the average score was24.09 (S.D. = 12.21) at the hospital, 21.68 (S.D. = 12.59) at 3-month follow-up, 20.20(S.D.= 11.60) at 12 month follow-up, and 16.85 (S.D. = 10.26) at the 18-month follow-up.Racial/ethnic status was not related to UCLA PTSD-RI scores at any of the assessment timepoints, suggesting that even if we did not control for racial/ethnic status, populationstratification was not a possible cause of bias in results. Female sex was related to PTSDsymptoms at the acute assessment, 3-month follow-up, and female sex was marginallyrelated to PTSD symptoms at the other follow-up assessments. Age of participant was notrelated to UCLA PTSD-RI total score at any time point. In regard to injury characteristics,violent assaults were related to PTSD-RI score at 12-and 18-month follow-up assessments.No other injury characteristics were correlated with UCLA PTSD-RI score as any timepoint.

3.2. Genetic analyses

One marker, rs11657992 was excluded from analyses due to its low minor allele frequency(0.0115). The remaining SNP identifications, their locations, the Hardy-WeinbergEquilibrium test P – values, minor/major alleles, minor allele frequencies, and call ratepercentages are shown in Table 2. Call rates for SNPs ranged from 91 to 95% which

included samples that failed all assays and for which DNA quality was inferior. All SNPswere in Hardy-Weinberg Equilibrium.

The gene encoding CRHR1 is located on chromosome 17q21.31 and contains 13 exons

spanning 51 kb. To capture genetic variation across the CRHR1 locus, we selected a set of 9SNP located in a 71-kb region with an average intermarker distance of 9.6-kb (note that oneSNP was removed from analyses). We used Haploview [30] to determine the LD structureof the SNPs within the CRHR1 gene which is presented in Fig. 1. These 8 SNPs capture 46of the 79 (58%) alleles at an R2 > 0.8 using a pair-wise tagging approach based on the

HAPMAP [29] CEPH sample set, and the SNPs capture 62% of the CRHR1 alleles at an r2≥ 0.80 using an aggressive 2- and 3-haplotype. We identified two different pairs of blocks

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using the Four Gamete Rule and the Solid Spine of LD definitions in Haploview. Of the 103children from whom we received DNA, 4 were removed for low genotyping (< 90%).

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript3.3. Association analyses

We first conducted single SNP analyses to determine which markers were associated withUCLA PTSD-RI score at the acute hospital assessment. As displayed in Table 3, three SNPswere significantly related to PTSD symptom frequency (rs4074461, rs12944712,

rs17763104) using empirical p-values. With Bonferroni correction, one of the eight SNPs(rs12944712) remained significantly associated with PTSD symptom frequency (see Table3) and was therefore examined in the latent growth analysis below.

3.4. Latent growth analyses

The influence of rs12944712 in CRHR1 on the trajectory of PTSD symptoms was examinedusing latent growth curve analysis, controlling for empirically- and theoretically-determinedcovariates including individual-level influences (i.e., age, sex, race) as well as injury-relatedfactors (i.e., injury severity, days in hospital, whether injury was sustained due to violence);all participants with complete data on exogenous variables were retained in this analysis (N= 87). Using random coefficients regression (Raudenbush and Bryk, 2002) and maximumlikelihood estimation procedures, a trend for each participant’s symptoms of PTSD overeach time point (in hospital, 3 months, 12 months, and 18 months) was modeled. Participantintercept (acute symptoms of PTSD) and both linear and quadratic slope (symptoms of

PTSD over time) were predicted by covariates and rs12944712. Estimation difficulties wereencountered in attempts to add nonlinear terms. Findings revealed significant effects ofrs12944712 on both the intercept of acute PTSD symptoms as well as the linear slope ofPTSD symptoms over time. Specifically, having an increasing number of G alleles was

significantly associated with more acute symptoms of PTSD, Estimate = 5.77, SE (Estimate)= 1.43, z-statistic = 3.35, p-value < 0.001. Additionally, having an increasing number of Galleles was associated with a significantly greater decline in PTSD symptoms over time,Estimate = −0.42, SE (Estimate) = 0.11, z-statistic = −3.74, p-value < 0.001. As shown inTable 4, female gender was associated with greater levels of acute PTSD symptoms andhaving sustained a violent injury was associated with greater symptoms of PTSD over time.Unadjusted mean values for these data are shown in Fig. 2.

4. Discussion

Data reported in this study yielded two main findings. First, results suggest that

polymorphisms in the CRHR1 gene, which is involved in activation of the HPA pathway,are related to acute PTSD symptoms in pediatric injury patients. Second, latent growth curveanalyses modeling the trajectory of PTSD symptoms over time suggest that CRHR1variation is associated with both onset and course of PTSD symptoms. This study is uniquein numerous ways; it is not only the first study to examine CRHR1 variation in relation toPTSD symptoms, but it is also the first longitudinal examination of genetic variation onPTSD symptom trajectory. Furthermore, of the 30 candidate gene studies of PTSD [8], thisis only the second study in which genetic influences on PTSD were examined in childrenand adolescents. Specifically, three SNPs (rs4074461, rs12944712, rs17763104) wererelated to PTSD symptoms on a univariate level, and after correction for multiple testing,rs12944712 remained significant. For rs1294412, the minor allele (A) was protective withregard to level of PTSD symptoms reported at the acute hospital visit, and given that self-reported race/ethnicity was not related to PTSD symptoms at any time point, it is unlikelythat this was due to issues related to population stratification.

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In contrast to the univariate analysis, the longitudinal analysis controlled for potentialindividual level influences (i.e., age, sex, self-reported race/ethnicity) and injury

characteristics (i.e., injury severity, days in the hospital, whether the injury was violent innature). With regard to covariates, elevations in initial PTSD symptom levels was predictedonly by female gender, which was unrelated to subsequent course of symptoms. The onlycovariate found to significantly predict subsequent symptom course was having sustained aviolent injury; youth sustaining injuries secondary to violence evidenced significantly moresymptoms over time than youth who sustained nonviolent injuries. Interestingly, growthanalyses revealed significant effects of rs1294412 on both onset and course of PTSDsymptoms. Consistent with univariate analyses, the minor allele of rs1294412 was

associated with lower acute PTSD symptoms, again suggesting a protective effect. However,interestingly, the major allele (G) was associated with a significantly greater decline of

symptoms over time. It is possible that those with the major allele (G) had higher symptomsacutely and therefore had more opportunity for a sharper decline over the course of thestudy. Alternatively, it is possible that children exhibiting the most acute distress were alsomore likely to receive attention from family and medical personnel and that this supportmitigated symptoms over time. This is a question we hope to explore in future studies.Although this is the first examination of CRHR1 genetic variation in relation to PTSD, ourfindings related to the degree of PTSD symptoms are generally consistent with previousstudies of examining depression in which protective effects of certain CRHR1 SNPs (e.g.,rs110402, TAT haplotype) in individuals with a child abuse history have been reported[42,43]. Our rs12944712 SNP is in strong LD with three of the four SNPs found to be

protective these in prior reports (rs7209436, rs110402, rs242924). Given that rs12944712 isan intronic SNP with no known function, future research is needed to determine if this SNPis causally related, or if it is just in high LD with a functional SNP. In other words, thespecific biological mechanism via which the minor allele of rs12944712 in CRHR1 may beassociated with decreased risk of PTSD symptoms acutely, but be associated with a slowerdecline in symptoms over time remains to be elucidated.

Although our sample size was too small to fully examine specific sex effects, previousreports of CRHR1 variation suggest that the genes’ protective effect of buffering againstdepression in adulthood in those with a history of child abuse may be specific to men [21],however this finding is not universally replicated [43]. The relatively consistent findingacross these CRHR1 investigations with our results is rather remarkable, given the majormethodologic differences (e.g., different SNPs genotyped, all previous papers examinedadults, the majority of traumatic events experienced in the previous studies were physical orsexual assault). Taken together, these findings point towards relevant polymorphisms forstress-related psychopathology in the CRHR1 gene.

Although the specific functional variants in the CRHR1 gene and their downstream effectsare yet to be discovered, the biologic relevance of this gene as a whole to stress-relatedpsychopathology such as PTSD is clear in light of the key role that CRH has on activatingthe HPA axis. Given evidence that PTSD patients hypersuppress cortisol in response to low-dose dexamethasone treatment [44], models of PTSD development have been expanded toincorporate altered posttrauma cortisol response. Yehuda and colleagues proposed thatexaggerated catecholamine increases during traumatic stress without the regulatory

influence of accompanying cortisol increases could lead to inappropriate memory formation(either over-salient or fragmented memories) and result in the intrusion symptoms thatcharacterize PTSD [45]. It is possible that variation in CRHR1 plays a role in this process,although that is an empirical question not yet answered. Nonetheless, the importance ofCRHR1 in stress-related pathology is supported by preclinical and clinical studies [4,5].

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4.1. Limitations and future directions

The present data, although unique, are not without limitations. First, the study was limitedby its small sample size and attrition over the course of follow-up assessments. Due to thesmall sample size we were under-powered to detect small effects, and therefore resultsshould be considered preliminary. This also limited our ability to fully examine GxEinteractions, possible developmental specific effects, and possible sex effects. Further,estimates derived from small samples may not be stable. Clearly, replication in a largersample is warranted. Second, this investigation examined PTSD symptoms, not a clinicaldiagnosis of PTSD; therefore generalization to the PTSD diagnosis may be limited.

Additionally, as noted above, HPA-axis dysregulation is not specific to PTSD, which wasthe only phenotype examined in the present paper. Future research should also examineCRHR1 variation in relation to other mood and anxiety disorders. Third, the results may beconfounded by population stratification. However, genotype distribution did not differ byracial group and self-reported racial/ethnic status was not related to PTSD symptoms, so thisis unlikely. Fourth, although 8 SNPs were analyzed, only 58–62% of the variation in theCRHR1 gene was accounted for. Future studies of this gene would be strengthened by finemapping and sequencing, thereby allowing for identification of functional variants. Lastly,although a stress-exposed cohort study has advantages, generalizability of the results may belimited as the only traumatic event category examined in the present study was injuries.Future studies should examine a range of traumatic event experiences to determine if thefindings in the present study hold.

4.2. Conclusions

Our results suggest that CRHR1 variation is related to PTSD symptoms in pediatric injurypatients, both acutely, and longitudinally. This is the first longitudinal genetic associationstudy of PTSD symptoms in children that we are aware of that examines gene-symptomrelations within a sample that was recently exposed to a significant stressor. Furthermore,this is the first examination of CRHR1 SNPs in association with PTSD in a trauma-exposedsample, and it is also the second study to examine PTSD in children and adolescents. Theprospective nature of our study makes our findings less vulnerable to ascertainment or recallbiases than for studies that retrospectively assess adults regarding childhood experiences.Results from this study are not definitive and additional research is needed to replicate ourmethodology and findings, but our results suggest that studying an acutely-exposedhospitalized sample is both feasible and may improve power to find gene-disorder

associations. Moreover, our findings in conjunction with data from preclinical models [4]and the literature on CRHR1 and stress-related pathology in humans [42,43,46] suggest thatthis gene is indeed implicated in the post-trauma trajectory.

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcknowledgments

Dr. Amstadter is supported by US-NICHD HD0558 85. Dr. Nugent is supported by US-NIMH K01 MH 087240.Dr. Yang is supported by US-NIDA K01 DA 024758. Dr. Koenen is supported by US-NIMH K08 MH070627 anda Junior Faculty Sabbatical from the Harvard School of Public Health. This work was also supported by US-NIHgrants MH0728, MH063247, MH086309 and the Robert Wood Johnson Foundation.

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Amstadter et al.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 12

Fig. 1.

The position of the CRHR1 gene and its exons (filled rectangles) on chromosome 17 as wellas a linkage disequilibrium (LD) plot of all tested SNPs using r2 as the measure of LD.

Dis Markers. Author manuscript; available in PMC 2013 July 25.

Amstadter et al.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 13

Fig. 2.

Mean PTSD-RI symptoms by rs12944712 genotype.

Dis Markers. Author manuscript; available in PMC 2013 July 25.

Amstadter et al.Page 14

Table 1

Participant characteristics

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptN (%)/M (S.D.)

Sex Male FemaleAge 7–8 9–10 11–12 13–14 15–16 17–18

Self-Reported Race Non-Hispanic White Non-Hispanic Black OtherInjury type Violent Non-ViolentInjury Severity Score

Number of Days in the Hospital

Acute UCLA Posttraumatic Stress Disorder Reaction Index3-month UCLA Posttraumatic Stress Disorder Reaction Index12-month UCLA Posttraumatic Stress Disorder Reaction Index18-month UCLA Posttraumatic Stress Disorder Reaction Index

29 (28.2)74 (71.8)8.91 (6.91)5.90 (5.78)24.09 (12.21)21.68 (12.59)20.20 (11.60)16.85 (10.26)42 (40.8)47 (45.6)14 (13.6)8 (7.8)7 (6.8)9 (8.7)11 (10.7)31 (30.1)37 (35.9)76 (73.8)27 (26.2)

Dis Markers. Author manuscript; available in PMC 2013 July 25.

r/major alleles, minor alleleAmstadter et al.

NIH-PA Author ManuscriptPage 15

NIH-PA Author Manuscript2 elbaTNIH-PA Author Manuscript%eta33273333........r44314444 l99999999laCycneuqerf 83326227e43121110l........e00000000lla roniMselella rojTGTTa//G/GG/GmGA/CC//TAG/A/roniMeulav-p greb00770159n00330633........i11001000eW-ydraHnoitisop288030 l21951762a994245m06927158o12234677es22222222to11111111am44444444ro lrlhacC dnreak rya21448cnm114801567412136 eP440303uN4483969679592767qS02424777eb41412111rdssssssssrrrrrrrrfDis Markers. Author manuscript; available in PMC 2013 July 25.

List of tested CRHR1 SNPS. Their positions on human chromosome 17, hardy-weinberg equilibrium test p value, minoNIH-PA Author ManuscriptNIH-PA Author Manuscript3 elbaTNIH-PA Author ManuscriptAmstadter et al.

Page 16

eulav-p lacir.)i*p55m200282862000470026E........0010000000.0 42067=0948 4..9...98/.209.0211.5t2−−0−−−10.0 42=68010742 p200000000(R........ 00000000noit.cE13804197er.78414782........rS11222223oc i63061n03570or1..0...0r.51.1659.eβ4−−2−−−3fnorB erketrfa21448am1148015 t67412136n P440303aN44839696c79592767ifS02424777ib41412111ndssssssssgrrrrrrrris*Dis Markers. Author manuscript; available in PMC 2013 July 25.

Single SNP analyses in relation to acute UCLA PTSD-RI scoreAmstadter et al.Page 17

Table 4

Latent growth curve analysis results

NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript**B

Intercept

African American Other NonWhite Age

Days in Hospital Injury Severity Score Violent Injury Female rs12944712Slope

African American Other NonWhite Age

Days in Hospital Injury Severity Score Violent Injury Female rs12944712

0.220.180.020.03−0.030.910.13−0.420.261.59−0.01−0.180.26−1.996.835.77

S.E.(B)B/S.E.(B)StdYX

2.593.750.410.190.242.172.811.72

0.100.42−0.03−0.931.10−0.2.43*3.35*0.020.07−0.01−0.130.22−0.150.390.52

0.170.200.020.020.020.270.230.11

1.30.901.121.75−1.403.33*0.56−3.74*0.230.130.150.36−0.310.860.86−0.58

Note. B = Estimate; SE(B) = standard error of estimate; B/SE(B) = z-statistic;

p < 0.01; StdXY = standardized increase in Y given a standard deviation increase in Xp < 0.01.

Dis Markers. Author manuscript; available in PMC 2013 July 25.

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