Research report

Association of COMT and TPH-2 genes with DSM-5 basedPTSD symptoms

Armen K. Goenjian a,b,n, Ernest P. Noble c, Alan M. Steinberg a, David P. Walling b,Sofia T. Stepanyan d, Sugandha Dandekar e, Julia N. Bailey f

a UCLA/Duke University National Center for Child Traumatic Stress, Department of Psychiatry, Geffen School of Medicine, University of California, Los Angeles(UCLA), CA, USAb Collaborative Neuroscience Network, Garden Grove, CA, USAc Alcohol Research Center, Department of Psychiatry, Geffen School of Medicine, UCLA, CA, USAd Department of Psychology, University of California at Riverside, CA, USAe Sequencing & Genotyping Core, Department of Human Genetics, UCLA, CA, USAf Department of Epidemiology, UCLA Fielding School of Public Health; Epilepsy Genetics/Genomics Laboratories, VA GLAHS, Los Angeles, CA, USA

a r t i c l e i n f o

Article history:Received 12 October 2014Accepted 14 October 2014Available online 22 October 2014

Keywords:Catechol-O-methyltransferase (COMT)GeneticsPTSDTryptophan hydroxylase 2 (TPH 2)Trauma

a b s t r a c t

Background: Dopaminergic and serotonergic systems have been implicated in PTSD. The present studyevaluated the association of four catechol-O-methyltransferase (COMT) gene loci, and the joint effect ofCOMT and tryptophan hydroxylase 2 (TPH2) genes on PTSD symptoms.Methods: Subjects included 200 Caucasian Armenian adults exposed to the 1988 Spitak earthquake from12 multigenerational (3–5 generations) families. Instruments used included the UCLA PTSD ReactionIndex based on DSM-5 criteria, and the Beck Depression Inventory.Results: The adjusted heritabilitiy of vulnerability to DSM-5 based PTSD symptoms was 0.60 (po10�4).There was a significant association of the COMT allele rs4633C with total PTSD (po0.03), and D category(po0.04) (negative alterations in cognitions and mood) severity scores, but not with C category(avoidance) scores. There was no genetic correlation between C and D category severity scores. COMTallele rs4633C and the TPH-2 allele rs11178997T together accounted for 7% of the variance in PTSDseverity scores (po0.001). None of the COMT alleles were associated with depression.Limitations: The ratings of earthquake exposure and prior trauma may have been subject to recall bias.The findings may not be generalizable to other ethnic/racial populations.Conclusion: COMT allele rs4633C may be causally related and/or is in linkage disequilibriumwith gene(s)that are causally related to PTSD symptoms. Carriers of these COMT and the TPH-2 alleles may be atincreased risk for PTSD. The findings provide biological support for dividing DSM-IV category Csymptoms into DSM-5 categories C and D.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

In a majority of individuals with posttraumatic stress disorder(PTSD), symptoms resolve within a few years (Norris et al., 2002).However, in a minority of individuals, the disorder may becomedisabling for decades after a traumatic event (Morgan et al., 2003;Marshall et al., 2005). Variations in severity and course of thedisorder depend on multiple factors, including sex, age, ethnicity,severity of the traumatic experiences, and pre and post-traumafactors (Norris et al., 2002). Additionally, genetic studies amongtwins (True et al., 1993), and multigenerational family members

(Goenjian et al., 2008) have found that genetic make-up also playsa significant role in vulnerability to PTSD symptoms. In the latterstudy, among survivors of the 1988 Spitak earthquake, theadjusted heritability was 0.41.

One method of identifying genes that are involved in complexdisorders such as PTSD is the hypothesis driven candidate-geneapproach. Genes are selected on the basis of prior knowledgeabout their purported role in the pathogenesis of a phenotype. Theobjective is to determine if a particular gene variant is associatedwith the risk for a phenotype.

Depression is of interest in the study of PTSD-related genes, asthese two phenotypes frequently co-occur (Breslau et al., 2000;Goenjian et al., 2000), share genes (Goenjian et al., 2008; Koenenet al., 2008), and respond to similar medications such as the SSRIs.Studies suggest that the dopaminergic system may be relevant tothe pathogenesis of various psychiatric disorders, including PTSD

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/jad

Journal of Affective Disorders

http://dx.doi.org/10.1016/j.jad.2014.10.0340165-0327/& 2014 Elsevier B.V. All rights reserved.

n Correspondence to: Alcohol Research Center, Room 58-242, UCLA Neuropsy-chiatric Institute, 760 Westwood, CA 90024-1759, USA.

E-mail address: agoenjia@ucla.edu (A.K. Goenjian).

Journal of Affective Disorders 172 (2015) 472–478

(Boscarino et al., 2012; Comings et al., 1996; Kolassa et al., 2010;Segman et al., 2002; Young et al., 2002), depression (Baekkenet al., 2008; Massat et al., 2005; Ohara et al., 1998) and anxietydisorders (Domschke et al., 2004; Hamilton et al., 2002; Hettemaet al., 2008; McGrath et al., 2004; Rothe et al., 2006).

Dopamine is metabolized by catechol-O-methyltransferase(COMT). This enzyme has broad biological functions, includingregulation of catecholamines and enkephalins. The COMT gene islocated at the 22q11.2 interval. It encodes two proteins: solublecytoplasmic COMT (S-COMT) and membrane bound COMT (MB-COMT). The former isoform is predominantly expressed in periph-eral tissues, while the latter is expressed in the brain and has beenof interest in psychiatric disorders (Boscarino et al., 2012;Domschke et al., 2004; Kolassa et al., 2010; Massat et al., 2005).The COMT gene has six exons with the first two being non-coding(Diagram 1). The expression of the COMT gene is controlled by twopromoters where there are two distinct ATG start codons for thepromoters P1 and P2 that drive the transcription of S-COMT andMB-COMT respectively (Lachman et al., 1996; Shield et al., 2004;Tenhunen et al., 1993, 1994). SNP rs6269 is located in the promoterregion of exon 3, rs4633 is within exon 3, and rs4818 and rs4680are within exon 4. SNPs rs4633 and rs4818 are synonymous, i.e. donot produce a change in amino acid composition. In contrast,polymorphism of rs4680 is non-synonymous whereby it changesthe amino acid sequence of the protein. It encodes for a substitu-tion of valine (Val) to methionine (Met) at codons 108 and 158 ofS-COMT and MB-COMT respectively. A Val to Met substitution atcodon 158 of MB-COMT has been considered a main source ofindividual variation in COMT activity resulting in a less thermo-stable COMT enzyme that exhibits multiple-fold reduction inactivity (Lachman et al., 1996; Lotta et al., 1995; Shield et al., 2004).

Most association studies of psychiatric disorders with COMThave focused on the functional non-synonymous SNP rs4680(Val158Met polymorphism). The findings of these studies havebeen modest and inconsistent in the direction of effect (Baekkenet al., 2008; Craddock et al., 2006; Domschke et al., 2004; Frischet al., 1999; Massat et al., 2005; Ohara et al., 1998; Rothe et al.,2006). Two studies of PTSD have reported an association with theCOMT rs4680 Met allele (Boscarino et al., 2012; Kolassa et al., 2010).In the first study (Kolassa et al., 2010) the investigators found no-main effect of the genotype on lifetime PTSD. However, they founda gene–environment interaction. Individuals homozygotic for theMet allele exhibited higher risk for PTSD compared to Val allelecarriers. In the second study (Boscarino et al., 2012) the Met allelewas more common among PTSD cases compared to controls.

The purported role of dopamine in psychiatric disordersextends beyond the COMT gene. Studies have implicated the genethat encodes for dopamine receptor D2 (DRD2) (Comings et al.,1996; Young et al., 2002) and dopamine transporter (DAT)(Segman et al., 2002) in the pathogenesis of PTSD. However, thesefindings have not been replicated in other studies (Gelernter et al.,1999; Bailey et al., 2010).

The serotonergic system also plays a role in PTSD and depres-sion (Goenjian et al., 2012; Grabe et al., 2009; Koenen et al., 2009;Kolassa et al., 2010; Thakur et al., 2009). Most of these studies havefocused on the promoter region of the serotonin transporter gene(5HTTLPR). A recent study among the same cohort as in this studyfound an association between two genes that encode for trypto-phan hydroxylase (TPH), the rate limiting enzyme for the synthesisof serotonin, and PTSD (Goenjian et al., 2012).

With regard to symptom criteria for PTSD, the most recentedition of the Diagnostic and Statistical Manual of Mental Dis-orders (DSM-5) (American Psychiatric Association, 2013) hasmodified the diagnostic symptom criteria for PTSD from DSM IV.Many of the original DSM IV symptoms were retained. However,DSM IV C category symptoms (numbing/avoidance) were dividedinto DSM-5 categories of C (avoidance) and D (negative alterationsin cognition and mood). The latter included items from DSM IVand new items relating to extreme negative beliefs. With theaddition of reckless or self-destructive behavior and no othersubstantial changes, the DSM IV D category was classified inDSM-5 as E category (alterations in arousal and reactivity).

Almost all candidate gene studies of PTSD have been case-controlled studies utilizing subjects recruited from clinics orepidemiological studies among subjects exposed to trauma. Incontrast, the present study is a multigenerational study where theentire family was exposed to trauma. Thus, there is increasedlikelihood of affected family members having the same causalgenetic variants. The objective of this study was to investigate theassociation of four polymorphic loci of the COMT gene with PTSDsymptoms based on both DSM-IV and DSM-5 diagnostic symptomcriteria, and their association with depressive symptoms. In addi-tion, the joint effect of the COMT and TPH-2 gene was examined inrelation to the risk for PTSD symptoms.

2. Methods

2.1. Subjects

Subject recruitment for this study has been previouslydescribed (Goenjian et al., 2008). Briefly, a total of 212 subjectswere recruited from Gumri (a city in Armenia nearby the epicenterof the catastrophic 1988 Spitak earthquake) where there wasextensive destruction, morbidity and mortality. Seven of thesubjects were not in the city during the earthquake, while fivewere unable to complete the testing. Thus, a total of 200 adultsexposed (121 females, 79 males) from 12 multigenerational (threeto five generations) families participated and completed the studyprocedures approximately 14 years post-earthquake. Both themean and modal number of subjects per family was 16; the rangewas between 13 and 19. The types of relatives included: 108parent–offspring, 137 full siblings, 48 grandparent–grandchildren,306 avuncular, 64 grand avuncular, 306 first cousins, 225

Diagram 1. COMT gene with exons and SNPS located on chromosome 22.

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first-cousins-once- removed and 111 second cousins. Subjectsbelonged to the same racial/ethnic (Caucasian/Armenian) groupand had similar socioeconomic backgrounds. After obtaining IRBapproval, subjects were given a full description of the study,including potential risks and benefits. Written informed consentwas obtained from all participants.

Subjects had been exposed to extreme threat to life, wide-spread destruction, and extensive morbidity and mortality duringthe earthquake. All the subjects saw destroyed buildings through-out the city, 90% saw dead bodies lying in the streets, and 92% sawpeople who were severely injured.

The mean Posttraumatic Stress Disorder-Reaction Index (PTSD-RI) score based on DSM-IV symptom criteria was 33.9713.2, and33.1712.5 based on DSM-5 criteria. The mean Beck DepressionInventory (BDI) score was 24.476.7. Adjusted heritability ofvulnerability to PTSD symptoms and depressive symptoms in thisgroup has been reported previously as 0.41 and 0.66 respectively(Goenjian et al., 2008). Bailey et al. (2010) reported the heritabilityof DSM IV PTSD B category symptoms to be 0.75; C categorysymptoms 0.39; while D category symptoms were not heritable.

2.2. Instruments

An earthquake exposure profile was completed for each parti-cipant using a modified version of a DSM IV-based exposurequestionnaire previously utilized in studies after disasters(Goenjian et al., 2001; Roussos et al., 2005). This instrumentincluded information on gender, age, location at the time of theearthquake, objective experiences during the earthquake, includingdestruction of residence, death of relatives, seeing dead bodies,being injured, and seeing someone else who was injured. Partici-pants were also evaluated for subjective experiences during theearthquake. These items included fear during the earthquake, fearof getting badly injured or dying, and fear that someone elsewould be badly hurt or killed. Additionally, the questionnaireincluded items relating to pre- and post-earthquake exposure totraumatic experiences; post-earthquake medical illness/treatmentby subject or family member; and post-earthquake adversities.These items were rated on a five point Likert scale ranging from0¼not at all, to 4¼a whole lot.

Posttraumatic stress symptoms were evaluated using the UCLAPTSD Reaction Index (PTSD-RI), a 22-item self-report scalebased on DSM-IV criteria (Steinberg et al., 2004, 2013) comprisedof category B (re-experiencing), C (avoidance/numbing), and D(arousal) symptom subscales. Frequency of symptom occurrenceduring the previous month is rated on a 5-point Likert scale,ranging from 0¼not at all, to 4¼most of the time. Psychometricproperties of this scale have been reported, with high internalconsistency, reliability, and discriminant validity (Steinberg et al.,2004, 2013). Additional analyses were performed using a modifiedversion of the PTSD-RI based on DSM-5 symptom criteria. Thestudy questionnaire incorporated all but one of the DSM-5diagnostic symptom items. The missing item was a D categorysymptom: persistent and exaggerated negative belief or expectationsabout oneself, others or the world. The reason for the missing itemwas that, at the time of the study, the DSM-5 version of thePTSD-RI did not include a tested formulation for evaluating thissymptom. Depressive symptoms were evaluated using the BeckDepression Inventory (BDI). Psychometric properties for the BDIhave been reported elsewhere (Beck and Steer, 1984).

2.3. Genomic processing

Buccal cell samples were taken from each participant in Armeniaand transported to the University of California, Los Angeles (UCLA).Genomic DNA was extracted employing standard techniques per

manufacturer DNA Genotek's protocol. COMT SNPs assessed in thisstudy included rs6269, rs4633, rs4818, and rs4680. Previously theseSNPs were shown to be associated with psychiatric and medicaldisorders (Diatchenko et al., 2005; Hirata et al., 2008; Kocabas et al.,2010, Michaelovsky et al., 2008; Nackley et al., 2006; Schosser et al.,2012; Shifman et al., 2002).

The COMT SNPs rs6269, rs4633, rs4818, rs4680 were genotypedusing a 50 nuclease assay to discriminate between the two alleles.Taqman SNP Genotyping Assays were ordered from Life Technol-ogies formerly known as Applied Biosystems Inc. Polymerasechain reactions were performed using 5-μL reaction volumes in384-well plates with 5 ng of DNA. The standard protocol providedwith the kit Taqman Genotyping Master Mix was followed. Endpoint reads of fluorescence levels were obtained with an ABI7900HT Sequence Detection System.

2.4. Statistical analyses

Statistical genetic modeling was performed using a pedigree-based association method (Almasy and Blangero, 1998; Amos,1994; Almasy et al., 2005) as implemented in SOLAR (Blangeroand Almasy, 1996). This method has been used to analyze data innumerous psychiatric behavioral genetic family studies (Almasyet al., 2008; Carless et al., 2011; Goenjian et al., 2008; Gur et al.,2007). This quantitative genetic method decomposes the pheno-typic variance of a trait into its additive genetic and non-geneticcomponents. The analysis includes a component for the candidategene polymorphism in the general model for genetic and environ-mental effects individually on the quantitative PTSD and depres-sion traits. The expected genetic DNA sharing between eachrelative pair is incorporated into the model via a kinship matrixtimes a variance component. Estimations of all the parameters areobtained simultaneously using maximum likelihood methods.Nested models are then tested to determine significance ofparameters. For example, the likelihood of the models with andwithout the candidate gene component is compared to determineif the candidate gene polymorphism explains a significant portionof the expression of PTSD or depressive symptoms.

In the model, COMT rs4633 and TPH-2 rs11178997 wereincluded for the analyses. TPH-2 was included because it isprimarily involved in the synthesis of serotonin in the centralnervous system, as opposed to TPH-1 which is mainly associatedwith peripheral synthesis of serotonin. Association of COMT andTPH-2 polymorphisms with both DSM-IV and DSM-5 based sub-category scores were assessed separately and jointly. These ana-lyses included the significantly associated COMT SNP (with PTSDsymptoms) found in this study (rs4633), and TPH-2 SNP(rs11178997), previously shown to be significantly associated withDSM-IV based PTSD-RI score in this cohort (Goenjian et al., 2012).For the joint analysis, the contributions of risk alleles of both geneswere studied by comparing individuals with none, 1 and 2 of therisk alleles. The subcategories analyzed included all but the arousalsymptoms, i.e. category D of DSM IV, and the category E of DSM-5,as they had not been found to be heritable in this study andpreviously (Bailey et al., 2010).

We estimated that in this sample we had 80% power to detectvariants that account for 4.6% of the trait variance and are instrong linkage disequilibrium (LD) with one of the genotypedmarkers. This calculation was based on a p-value of 0.025, which isequivalent to correcting the standard marginal p-value of 0.05 fortwo tests. For a variant that has an r-squared (the amount oflinkage in the disequilibrium) of at least 0.85 with a genotypedmarker, we reach 80% power accounting for 5.4% of the traitvariance. On the basis of these calculations, genetic associationsthat account for less of the trait variations may not be detected

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because of the sample size. Simulations were performed usingSOLAR (Almasy and Blangero, 1998).

3. Results

3.1. Heritabilities of PTSD symptoms based on DSM-IV and DSM-5criteria after adjusting for covariates

After adjusting for covariates, the heritability of vulnerability toPTSD symptoms based on DSM IV and DSM-5 criteria were 0.41(po0.001) and 0.60 (po10�4) respectively (Table 1). The Pearsoncorrelation between DSM IV and DSM-5 based PTSD severityscores was 0.98 (r¼0.96; po0.0001). The covariates with sig-nificant association to the variance in PTSD scores included:gender, pre-earthquake traumatic experiences, post-earthquakeserious illnesses (Table 1). These covariates contributed 19% of thevariance in PTSD severity scores measured by both instruments.

3.2. Heritability of subcategories and genetic correlation of DSM-5C and D category symptoms

The heritability of vulnerability to DSM-5 based category B (re-experiencing) severity scores was 0.75 (po0.0001); category C(avoidance) severity scores was 0.64 (p¼3.9�10�10); category D(negative alterations in cognitions and mood) scores was 0.58(po0.0001), while category E (arousal symptoms) symptoms,formerly categorized as DSM-IV category D symptoms, were notheritable.

No significant genetic correlation was found (Ho: ρg40)between DSM-5 categories C and D symptoms (ρg¼0.32, SE¼0.21; p¼0.17), indicating that these phenotypes did not sharegenes. After taking genetic relatedness into account, the derivedestimate of the phenotypic correlation was 0.29.

3.3. Association of Single Nucleotide Polymorphism (SNP) alleleswith PTSD and depressive symptoms

The COMT SNP allele rs4633C was significantly associated withboth DSM-IV and DSM-5 total PTSD severity scores (both withpo0.03) (Table 2). Regarding PTSD subcategories, there was nosignificant association between rs4633C and B category score forboth scales (p¼0.28). There was a significant association ofrs4633C with DSM IV based category C (avoidance/numbing)scores (p¼0.03), although not with DSM-5 category C (avoidance)scores (p¼0.46). However, there was a significant association

between rs4633C and category D scores (negative alterations incognitions and mood) (po0.04). Finally, no significant geneticcorrelation was found between DSM-5 C and D category scores,indicating that they did not share genes.

Regarding the non-synonymous SNP rs4680 (Val158Met), therewas a trend toward significance for the association of the G allele(Val) and total PTSD severity scores (p¼0.07) on both DSM IV andDSM-5 based measures. There was no significant associationbetween rs4680G and subcategory scores, and either of the othertwo COMT SNPs (rs6269 and rs4818) with total PTSD and sub-category scores. None of the four COMT SNPs were associated withdepressive symptoms.

Table 1Heritabilites of PTSD symptoms based on DSM-IV and DSM-5 criteria, after adjusting for covariates, among multigenerational family members exposed to the 1988 Spitakearthquake.

DSM-IV based PTSD severity scores DSM-5 based PTSD severity score

Heritability (h2) after adjusting for significant covariates 0.4170.17 0.6070.17po0.001 po10�4

Covariates with p valuesSex 0.01 0.05Age 0.11 0.23Home destroyed 0.76 0.66Death of family member 0.73 1.0Witnessed death 0.01 0.005Witnessed harm to others 0.82 0.99Was hurt during the earthquake 0.72 0.86Pre-earthquake traumatic experience 0.002 0.002Post-earthquake traumatic experience 0.15 0.58

Post-trauma illness o0.04 0.004Proportion of variance due to all covariates (%) 19 19

Table 2The association of COMT SNPs with DSM-IV and DSM-5 based total PosttraumaticStress Disorder-Reaction Index (PTSD-RI) and subcategory severity scores amongmultigenerational family members exposed to the 1988 Spitak earthquake.

COMTSNPs

Trait DSM-IV based p-value Trait DSM-5 based p-value

Total PTSD sx.severity score

Total PTSD symptomseverity score

Rs6269 c 0.45 c 0.62Rs4633C c o0.03 c o0.03Rs4818 c 0.52 c 0.67Rs4680G c 0.07 c 0.07

B category sx. score B category severity score

Rs6269 c 0.87 c 0.87Rs4633C c 0.28 c 0.28Rs4818 c 0.38 c 0.38Rs4680G c 0.34 c 0.34

C category sx. score C category severity score

Rs6269 c 0.13 c 0.52Rs4633C c 0.03 c 0.46Rs4818 c 0.27 c 0.50Rs4680G c 0.09 c 0.36

D category severity score

Rs6269 c 0.53Rs4633C c o0.04Rs4818 c 0.43Rs4680G c 0.15

sx.¼symptom.N.B.: DSM-5 E category (arousal and reactivity) symptoms were not included asthey were not shown to be heritable. Category C symptoms of DSM-5 diagnosticcriteria included only the avoidance items from DSM-IV Category C symptomswhich included avoidance and numbing items.

A.K. Goenjian et al. / Journal of Affective Disorders 172 (2015) 472–478 475

3.4. Multilocus analyses of the association of COMT and TPH-2 SNPsand PTSD symptoms

The TPH-2 rs11178997T allele was significantly associated withDSM-5 based PTSD severity scores (p¼0.02). With regard to theeffect of both TPH-2 rs11178997T and COMT rs4633C, Table 3shows the contributions of both, assessed separately and jointly(by comparing individuals with none, one, and two risk alleles).For the separate analysis, the associations of rs4633 andrs11178997 with DSM-5 based total PTSD severity scores weresignificant (po0.02 and o0.01 respectively). In combination, theyexplained 7% of the variance in PTSD severity scores. For the jointassessment, the association was also significant (po0.001),explaining 7% of the variance of the severity scores.

3.5. Combined effect of all the significant genetic and environmentalvariables on the variance of PTSD

The combined effect of all of the significant variables examined,including gender, environmental variables (e.g., traumatic experi-ences prior and during the earthquake, severe illness after theearthquake) along with the effect of COMT rs4633C and TPH-2rs11178997T alleles was significant (po0.01) explaining 27% of thevariance of total PTSD severity scores.

4. Discussion

The first finding of this study was the high heritability ofvulnerability to PTSD symptoms based on DSM-5 criteria afteradjusting for covariates (0.60). This was higher than previouslyreported for DSM IV based PTSD severity scores (0.41) (Goenjian etal., 2008). The finding suggests that DSM-5 based assessments, asopposed to DSM-IV, may increase the prospects of finding geneticmarkers among traumatized individuals. The other relevant resultwas the association of synonymous COMT allele rs4633C withDSM-5 based total PTSD symptom score (po0.03) and D categorysymptom score (po0.04) but not C category score.

Emerging studies of psychiatric and medical disorders haveshown that mutations of synonymous SNPs, such as rs4633 andrs4818, can effect protein expression and enzyme activity resultingin phenotypic alterations, including pathology. Even thoughsynonymous changes do not produce a change in the amino acidcomposition of a protein, they can cause disruption of splicing(Cartegni et al., 2002), secondary structural changes and instabilityof mRNA (Bartoszewski et al., 2010; Duan et al., 2003; Nackleyet al., 2006; Shen et al., 1999), and asynchrony of co-translationalfolding (Kimchi-Sarfaty et al., 2007; Sauna et al., 2007), therebyeffecting protein expression and enzyme activity.

With regard specifically to synonymous SNPs of COMT, in astudy of pain sensitivity, the results indicated that synonymouschanges in the coding region of COMT affected secondary mRNA

structure and thereby protein expression (Nackley et al., 2006).The present finding of an association between the synonymousSNP rs4633 and PTSD symptoms (Table 2) may be explained on thebasis of a similar mechanism.

The heritability of DSM-5 based B category symptoms was high(0.75). This finding was expected as DSM-5 B category symptomsremained essentially as in DSM-IV. The DSM-5 category C (avoidance)and D (negative alterations in cognitions and emotions) symptomswere highly heritable, 0.64 and 0.58 respectively, while E categorysymptoms (arousal) formerly described as DSM-IV category D symp-toms, were not. We found a significant association between SNPrs4633 and DSM-IV based category C (avoidance/numbing) symptomscores (p¼0.03). There was no significant association between rs4633and DSM-5 category C symptoms, but there was an associationbetween rs4633 and DSM-5 Category D symptoms (po0.04)(Table 1). Further, there was no significant genetic correlation betweenDSM-5 C and D category symptoms, suggesting that they did not sharegenes. These findings provide support for dividing DSM-IV category Csymptoms into DSM-5 C and D categories.

In this study there was a trend towards significance for theassociation between the non-synonymous SNP rs4680G (Val)allele and PTSD symptoms. The Val allele is known to be associatedwith higher enzyme activity and protein level than the Met variant(Lachman et al., 1996; Lotta et al., 1995; Shield et al., 2004).

Numerous studies have found an association between the Valallele and various anxiety disorders (Domschke et al., 2004;Hamilton et al., 2002; Hettema et al., 2008; McGrath et al.,2004; Rothe et al., 2006), and depression (Massat et al., 2005;Baekken et al., 2008). Based on previous findings of geneticcorrelations among these phenotypes, i.e. PTSD, depression andanxiety (Goenjian et al., 2008) it is expected to find some genesthat will be contributing to these phenotypes. Researchers whohave found an association of the Val allele with panic disorder(Rothe et al., 2006) and depression (Baekken et al., 2008; Massatet al., 2005) have conceptualized the mechanism of action for thepathologies to be the removal of catecholamines from the trans-synaptic space at a higher rate due to high (Val) enzyme activity.The trend found in the present study of the association betweenthe Val allele and PTSD symptoms suggests that a similar mechan-ism may be involved in PTSD.

In contrast to these findings, in a study among survivors of theRwandan Genocide, there was no main-effect of the Val158Metpolymorphism, but there was a gene–environment interaction,whereby Met/Met homozygotes exhibited higher risk for PTSDcompared to Val allele carriers (Kolassa et al., 2010). Also, in astudy of pain patients, Met allele was more common among thosewith PTSD compared to controls (Boscarino et al., 2012). Incon-sistent genetic findings in psychiatric disorders such as PTSD,anxiety and depression are not uncommon. Future family studieswith larger samples of various ethnic/racial groups with minimalheterogeneity may shed light on the relevance of these COMTSNPs and PTSD.

Table 3Contribution of COMT and TPH2 genes, individually and jointly, to the variance in DSM-IV and DSM-5 based total PTSD-RI scores among family members exposed to the 1988Spitak earthquake.

SNP TraitDSM-IV

p-value Variance explained by genes(SNPs) (%)

TraitDSM-5

p-value Variance explained by genes(SNPs) (%)

COMT rs4633C TPH2 rs11178997T a (separate assessmentsof risk alleles)

PTSD sx. 0.02 7 PTSD sx. o0.02 70.01 o0.01

COMT TPH2) b (joint assessments of both risk alleles) PTSD sx. 0.003 6 PTSD sx. o0.001 7

sx.¼symptom.a For the analysis, the contribution of each allele of the two genes was assessed separately.b For the analysis, the contribution of both risk alleles of the two genes were studied by comparing individuals with 0, 1 and 2 risk alleles.

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The COMT rs4633C and TPH-2 rs11178997T alleles togetherexplained 7% of the variance of DSM-5 based PTSD symptoms(Table 3). TPH-2 is the rate determining enzyme of serotoninsynthesis in the central nervous system. Previously it has beenshown to be associated with PTSD symptoms (Goenjian et al.,2012). The SNP being located in the promoter region may affectlevel, location or timing of gene expression and thereby synthesisof serotonin, a neurotransmitter that has been implicated in manypsychiatric disorders. These two genes, along with environmentalvariables and gender, explained 27% of the variance in PTSDsymptoms. Replicating these finding may ultimately provide abasis for psychopharmacologic strategies to ameliorate symptomsof PTSD.

The lack of an association between the four COMT SNPs anddepression in this family study represents an addition to theliterature of inconclusive findings in case-controlled studies,including no associations of Val/Met polymorphism (Cusin et al.,2002; Frisch et al., 1999; Kocabas et al., 2010), association with thelow-activity Met allele (Ohara et al., 1998), and association withthe high activity Val allele (Baekken et al., 2008; Massat et al.,2005).

The present study was designed to control for the effects ofpotentially confounding factors. A family study design was usedbecause of the availability of multi-generational families who wereexposed to the traumatic events surrounding the earthquake.Family studies reduce genetic heterogeneity because there isincreased likelihood of affected family members having the exactsame causal genetic variants, whereas in case-controlled studiesevery case could potentially have a unique causal genetic variant.Also, between family variance is minimized compared withstudies that include mixed racial/ethnic groups. The populationin northern Armenia belongs to the same ethnic group. Finally,family studies require significantly less subjects than case-controlstudies for comparable power.

The variance due to trauma exposure, another potentiallyconfounding variable, was reduced as participants in this studywere exposed to similar earthquake-related severe traumaticevents contemporaneously, thereby reducing variance related totype, severity or timing of trauma exposure. Reducing the varia-bility in the dose and timing of exposure improves the reliability ofthe heritability measures and increases the likelihood of detectinggenetic associations. Finally, families in this study were from thegeneral population exposed to the earthquake as opposed to aclinic population, making it more likely that the findings aregeneralizable. The use of a general population sample permitteda less biased evaluation of the heritability of vulnerability to bothPTSD and depression independently.

4.1. Limitations

The ratings of earthquake exposure and prior trauma weremade on the basis of retrospective reports. These ratings may havebeen subject to recall bias. Second, the subjects were ethnicArmenians, and thus the findings may not be generalizable toother populations.

In summary, the present study found a higher adjusted herit-ability of vulnerability to PTSD symptoms based on DSM-5 criteriacompared to DSM-IV criteria. The significant association betweenCOMT rs4633 C allele and total PTSD severity scores suggest thatthis SNP is either causally related to the pathogenesis of PTSDsymptoms or is in linkage disequilibrium to genes that are causallyrelated to PTSD. The association of rs4633C with DSM-5 category Dbut not C symptoms, and the lack of significant genetic correlationbetween these two categories provide biological support fordividing DSM-IV category C symptoms into DSM-5 categories Cand D. The dopaminergic COMT allele rs4633C and the serotonergic

TPH-2 allele rs11178997T jointly explained 7% of the variance inPTSD severity scores. Carriers of these alleles may be at risk forPTSD upon exposure to trauma. These findings warrant largermultigenerational family studies of dopaminergic and serotonergicgenes among traumatized families in other ethnic/racial groupsusing DSM-5 diagnostic criteria.

Role of funding sourceNone.

Conflict of interestNone

AcknowledgmentsWe would like to thank the family members who participated in this study and

the mental health staff of the Psychiatric Outreach Program who assisted us in theimplementation of the project.

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