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JAD-05478; No of Pages 9 Journal of Affective Disorders xxx (2012) xxx–xxx

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Journal of Affective Disorders

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Research report Association of TPH1, TPH2, and 5HTTLPR with PTSD and depressive symptoms

Armen K. Goenjian a,b,e,⁎, Julia N. Bailey c,d, David P. Walling b, Alan M. Steinberg a, Devon Schmidt b, Uma Dandekar d, Ernest P. Noble e a UCLA/Duke University National Center for Child Traumatic Stress, Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles (UCLA), United States b Collaborative Neuroscience Network, Garden Grove, CA 92845, United States c Department of Epidemiology, UCLA School of Public Health, Los Angeles, CA, United States d Epilepsy Genetics/Genomics Laboratories, VA GLAHS, Los Angeles, CA, United States e Alcohol Research Center, Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, United States article info abstract

Article history: Objective: To examine the potential contribution of the serotonin hydroxylase (TPH1 and Received 29 January 2012 TPH2) genes, and the serotonin transporter promoter polymorphism (5HTTLPR) to the unique Accepted 5 February 2012 and pleiotropic risk of PTSD symptoms and depressive symptoms. Available online xxxx Methods: Participants included 200 adults exposed to the 1988 Spitak earthquake from 12 multigenerational families (3 to 5 generations). Severity of trauma exposure, PTSD, and de- Keywords: pressive symptoms were assessed using standard psychometric instruments. Pedigree-based Genetics variance component analysis was used to assess the association between select genes and PTSD the phenotypes. Depression Results: After adjusting for age, sex, exposure and environmental variables, there was a signif- Tryptophan hydroxylase icant association of PTSD symptoms with the ‘t’ allele of TPH1 SNP rs2108977 (pb0.004), Serotonin transporter explaining 3% of the phenotypic variance. This allele also showed a non-significant trend for an association with depressive symptoms (p=0.08). Also, there was a significant association of PTSD symptoms and the ‘t’ allele of TPH2 SNP rs11178997 (p=0.03), explaining 4% of the variance. Depressive symptoms were significantly associated with the ‘s’ allele of 5HTTLPR (p=0.03), explaining 4% of the variance. Limitations: Retrospective rating of exposure may have been subject to memory failure leading to misestimation of symptom severities. Second, findings may not be generalizable to other ethnic/racial populations. Conclusion: To our knowledge, this is the first published report showing that variants in TPH1 and TPH2 genes constitute risk factors for PTSD symptoms. Additionally, the TPH1 gene may be associated pleiotropically with PTSD and depressive symptoms. The association of the ‘s’ allele of 5HTTLPR polymorphism with depression adds to similar findings from case/case–control studies. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

1.1. Comorbidity of PTSD and depression

⁎ Corresponding author at: Alcohol Research Center, Rm. 58-242, UCLA Comorbidity of PTSD and depression has been documented Neuropsychiatric Institute, 760 Westwood, CA 90024, United States. Tel.: +1 310 523 4200; fax: +1 310 373 4039. in numerous psychiatric epidemiologic studies (for example, E-mail address: [email protected] (A.K. Goenjian). see Breslau et al., 1991; Kessler et al., 1995). In the National

Comorbidity Survey (Kessler et al., 1995), affective disorders in 5HT synthesis (Arrango et al., 2003; Harvey et al., 2004). were two to three times more likely to occur among those Tryptophan is converted into 5hydroxytyptohan (5HTP) by with PTSD compared to those without PTSD. Longitudinal stud- the enzyme TPH, which then is converted to 5HT. The two ies after the Spitak earthquake in Armenia have shown signifi- isomorphisms of this compound are TPH1 (gene located on cant comorbidity of PTSD and depressive symptoms among chromosome 11) and TPH2 (gene located on chromosome survivors (Goenjian et al., 2000; 2005). And in a family study 12). Both are expressed in several brain regions, including in the same population, Goenjian et al. (2008) found a moder- the hippocampus, amygdala, frontal cortex, and hypothala- ate heritability of PTSD and depressive symptoms and a high mus. TPH2 expression is absent in the peripheral tissues, genetic correlation (0.71) between the two phenotypes. Like- whereas TPH 1 controls most of the peripheral 5HT synthesis wise, Koenen et al. (2008) in a study among Vietnam War vet- (Walther and Bader, 2003; Zill et al., 2007). erans, found a high genetic correlation (0.77) between PTSD To our knowledge, there are no published reports of the and major depression. In the study among the Spitak earth- association of TPH and PTSD. With regard to depression, quake survivors the genetic correlation was statistically signif- some studies have shown the TPH gene to be associated icantly different from 0, indicating pleiotropy (shared genes with depression (Table 1). However, these findings have between two phenotypes), and significantly less than 1, imply- not been replicated in other studies (Cusin et al., 2001; ing the presence of unique genes for each phenotype. Serretti et al., 2002). A gene that may be related to both PTSD and depression 1.2. Psychiatric genetics and the serotonergic system in the serotonergic system is the 5HTT gene. This gene has been of interest because depression has been associated Association studies of candidate genes with a phenotype with reduced serotonin transporter sites in platelets and var- are performed to determine if a particular gene variant has ious brain regions, including the amygdala, prefrontal cortex, a direct effect on risk for the phenotype or if it is tightly hypothalamus, and brainstem (Malison et al., 1998). linked in linkage disequilibrium to the gene which has a di- 5HTTLPR is a 44-bp insertion/deletion functional polymor- rect effect. The serotonergic system has been shown to play phism of the 5HTT gene located in the promoter region. The a role in controlling, arousal, sleep, anxiety, and depression two main alleles are the short ‘s’ allele and the long ‘l’ allele. (Jacobs, 1991; Lesch et al., 1996; Owens and Nemeroff, It is thought that the ‘s’ allele impairs transcriptional activity 1998). Serotonin (5HT) has been the target of various types of 5-HTT and lowers biological activity of the transporter of antidepressants, including tricyclics and serotonin reup- (Lesch et al., 1996) thereby increasing the risk for depression. take inhibitors. These medications have been effectively With regard to the association of 5HTTLPR with PTSD, find- used to treat both depression and PTSD symptoms (Blier ings have been mixed (Table 2). Likewise, results of studies and de Montigny, 1994; Davidson, 2000). Tryptophan hy- that have evaluated the association between 5HTTLPR and droxylase (TPH) gene and serotonin transporter (5HTT) depression have been equivocal (see Table 3). gene (SLC64A), have been the focus of many mood disorder In order to assess for pleiotropy, a study should include studies (Gizatullin et al., 2006; Karg et al., 2011; Munafo subjects with an adequate degree of co-morbid PTSD and de- et al., 2009; Risch et al., 2009; Serretti et al., 2002; Sun pressive symptoms. Prior studies in this population have et al., 2004; Tsai et al., 2009; Van Den Bogaert et al., 2006). shown a significant genetic correlation of both phenotypes The TPH gene encodes tryptophan, the rate limiting enzyme (Goenjian et al., 2008) making the present sample appropri-

Table 1 List of published association studies of TPH1 and TPH2 SNPs with depression.

Author Year Sample TPH1 TPH1 TPH1 TPH2 TPH2 TPH2 TPH2 TPH2

rs1799913 rs1800532 rs2108977 rs11178997 rs1386494 rs4570625 rs4290270 rs17110747

Sun et al. 2004 Case–control Asso. w. comorbid (females) MD+AD Taiwan Zill et al. 2004 Case–control Asso. w. MD Germany Gizatullin et al. 2006 Case–control Asso. w. Sweden MD Van Den Bogaert 2006 Case–control Asso. w. UP et al. Sweden Jokela et al. 2007 Cohort No main effect. G/E Finland effect w. dep. symptoms (BDI) Nobile et al. 2009 Cohort Asso. w. Aff. Prob. Italy Ages 10–14 Tsai et al. 2009 Case–control Asso. w. MD Taiwan

Abbreviation: Asso. w. = association with; AD = anxiety disorders; dep. = depression; G/E = gene/environment; UP = unipolar depression; MD = major depression; Aff. Prob. = affective problems: MDD, dysthymia, anxiety, ADHD. Blank cells indicate that the SNPs were not reported.

Table 2 List of published association studies of 5HTTLPR polymorphism (s and l alleles) with PTSD.

Author Year Sample Finding

Lee et al. 2005 Case/control, clinic population Korea Main effect (s allele ↑ risk) Kilpatrick et al. 2007 Population based cohort, hurricane exposed, USA No main effect; G/E 3 way interaction, s allele ↑ risk Xie et al. 2009 Case–control, substance dependent PTSD subjects, USA No main effect; G/E effect (s allele ↑ risk) Koenen et al. 2009 Population based cohort, hurricane exposed, USA No main effect; G/E effect (s allele can ↑ or ↓ risk) Grabe et al. 2009 Population based cohort, Germany Main effect (l allele ↑ risk) Thakur et al. 2009 Cohort, ER accident cases, Canada Main effect (l allele ↑ risk) Kolassa et al. 2010 Cohort, Refugees, Rwanda Main effect (s allele ↑ risk)

Abbreviations: G/E = gene environment. ate to test for candidate genes. The purpose of the present experiences during the earthquake. These items included study was to assess the potential contribution of serotonergic fear during the earthquake, fear of getting badly injured or candidate genes TPH1, TPH2, and 5HTTLPR to the genetic risk dying, and fear that someone else would be badly hurt or for these phenotypes, uniquely and pleiotropically. killed. Additionally, the questionnaire included items relating to pre- and post-earthquake exposure to traumatic experi- 2. Methods ences, pre- and post-earthquake psychiatric treatment, post-earthquake medical illness/treatment by subject or 2.1. Subjects family member, treatment for such illness, post-earthquake psychiatric illness/treatment by family members, and adver- Subject recruitment for this study has been previously de- sities including lack of heat, electricity, food and financial scribed (Goenjian et al., 2008). A total of 200 adults (121 fe- difficulties. These items were rated on a five-point Likert males, 79 males) from 12 multigenerational families scale, ranging from 0 = not at all to 4 = a whole lot. exposed to the Spitak earthquake in Armenia participated in Posttraumatic stress symptoms were evaluated using the the study which was conducted approximately 14 years UCLA Posttraumatic Stress Disorder Reaction Index (PTSD-RI), post-earthquake. Families consisted of three to five genera- a 22-item self-report scale based on DSM-IV PTSD criteria tions. Both the mean and modal number of subjects per fam- (Steinberg et al., 2004). This instrument assesses categories ily were 16, and the range was between 13 and 19. Subjects A (Exposure); B (Re-experiencing); C (Avoidance); and D belonged to the same ethnic/racial (Caucasian Armenian) (Hyper-arousal) symptoms. Frequency of symptom occurrence and religious group and had similar socioeconomic back- during the previous month is rated on a 5-point Likert scale, grounds. After obtaining IRB approval, subjects were given a ranging from 0 = not at all, to 4 = most of the time. Psycho- full description of the study, including potential risks and metric properties of this scale have been reported, with high in- benefits. Written informed consent was obtained for all par- ternal consistency reliability and validity (Steinberg et al., ticipating subjects. 2004). Depressive symptoms were evaluated using the Beck Depression Inventory (BDI). Psychometric properties for the 2.2. Instruments BDI have been reported previously (Beck and Steer, 1984).

An earthquake exposure profile was completed for each 2.3. Genomic processing participant using a modified version of a DSM IV-based exposure questionnaire previously utilized in studies after disas- Buccal cell samples were taken from each participant in ters (Roussos et al., 2005). This instrument included Armenia and transported to the University of California at information on objective experiences during the earthquake Los Angeles (UCLA). Genomic DNA was extracted employing including destruction of residence, deaths of relatives, seeing standard techniques. Three TPH1 and five TPH2 SNPs were dead bodies, being injured, and seeing someone else who was investigated in this study. The TPH1 SNPs, rs2108977, injured. Participants were also evaluated for subjective rs1799913 and 1800532, were chosen due to prior evidence showing association with: comorbid major depression and anxiety (Sun et al., 2004), major depression (Gizatullin et al., 2006) and depressive symptoms (Jokela et al., 2007). Table 3 The TPH2 SNPs, rs11178997, rs1386494, rs4570625, Meta-analyses of 5HTTLPR (‘s’ and ‘l’ allele) and depression. rs4290270, and rs17110747, were included due to prior find- Author Year Method Finding ings indicating association between the various SNPs and Lotrich and Pollock 2004 Meta-analysis OR 1.16, s allele major depression or affective problems (Nobile et al., 2009; risk factor Tsai et al., 2009; Van Den Bogaert et al., 2006; Zill et al., Lasky-Su et al. 2005 Meta-analysis No association 2004). Lopez-Leon et al. 2008 Meta-analysis OR 1.11, s allele These SNPs were genotyped using a 5′ nuclease assay to dis- risk factor Risch et al. 2009 Meta-analysis No association criminate between the two alleles. Applied Biosystems Inc Taq- Munafo et al. 2009 Meta-analysis Negligible findings; man SNP Genotyping Assays were used for genotyping of: attributable to chance TPH1 — rs1799913, rs1800532, rs2108977, and TPH2 — Karg et al. 2011 Meta-analysis s allele risk factor rs11178997, rs1386494, rs4570625, rs4290270, and Abbreviation: OR = odds ratio. rs17110747. Polymerase chain reactions (PCR) were

Please cite this article as: Goenjian, A.K., et al., Association of TPH1, TPH2, and 5HTTLPR with PTSD and depressive symptoms, J. Affect. Disord. (2012), doi:10.1016/j.jad.2012.02.015 4 A.K. Goenjian et al. / Journal of Affective Disorders xxx (2012) xxx–xxx performed using 5-μL reaction volumes in 384-well plates with incorporated into the model to test for co-segregation of the 5 ng of DNA. The standard protocol provided with the kit using marker and trait or linkage. Taqman Genotyping Master mix was followed. End point reads We estimated that in this sample we have 80% power to of fluorescence levels were obtained with an ABI 7900HT Se- detect variants that account for 4.6% of the trait variance quence Detection System. and are in strong linkage disequilibrium (LD) with one of The short 5-HTTLPR was identified using a protocol mod- the genotyped markers. This calculation used a p-value of ified from Lesch et al. (1996). Briefly, the forward primer was 0.025, which is equivalent to correcting the standard mar- 5′-ATGCCAGCACCTAACCCCTAATGT-3′ (labeled with 6- ginal p-value of 0.05 for two tests. For a variant that has an carboxyfluorescein fluorophore), and the reverse primer r-squared (the amount of linkage in the disequilibrium) of was 5′GGACCGCAAGGTGGGCGGGA-3′, which yielded ampli- at least 0.85 with a genotyped marker, we reach 80% power cons of 419 (l) or 376 (s) bp. The assay we used for the accounting for 5.4% of the trait variance. On the basis of 5HTTLPR (Anchordoquy et al., 2003) is a modification of the these calculations, genetic associations that account for less method of Lesch et al. (1996). The primer sequences are of the trait variations may not be detected because of the from Gelernter et al. (1999). PCR was performed in a total sample size. volume of 20 μL, containing 100 ng of DNA; 10 nM of each Power of our current sample to detect a gene through primer; 1× buffer; 2 mM MgCl2; 10% DMSO (v/v); 2 U Ampli- linkage can be estimated by simulating genetic markers, as- taq Gold DNA polymerase (Applied Biosystems, Foster City, suming that the phenotype is influenced by a single bi- California); 200 μM of dATP, dCTP, and dTTP; 100 μMof allelic quantitative trait locus (QTL) with a residual additive dGTP; and 7-deaza-2′-dGTP. Cycling conditions consisted of effect due to polygenes and that fully informative marker ge- 1) an initial 12 min denaturation at 94 °C; 2) touchdown notype data will be available. Simulations were performed from 65 °C to 55 °C decreasing by 0.5 °C per cycle with dena- using SOLAR (Almasy et al., 1998). We had 80% power to de- turation for 30 s at 94 °C, varied annealing temperatures con- tect a lod score of 1 with a QTL single gene heritability over sisting of 30 s at 66 °C (2 cycles), then 65 °C (3 cycles), then 0.3, and a lod score of 3 with a QTL heritability over 0.4. 64 °C (3 cycles), followed by hybridization for 1 min at This means that we could detect a gene for PTSD in our cur- 72 °C; 3) 35 cycles with an annealing temperature of 63 °C rent sample if one gene explained most of the 42% and the same denaturation and hybridization parameters; heritability. and 4) a final extension for 20 min at 72 °C. The PCR products were electrophoresed on an ABI 3730 DNA analyzer (Applied Biosystems) with GeneScan LIZ500 size standard (Applied 3. Results Biosystems). Data collection and analysis used GeneScan and Genemapper software (Applied Biosystems). Ambiguous Subjects in this study were exposed to the severe destruc- genotypes were retyped. The call rates of genotypes were sat- tion, morbidity, and mortality of the Spitak earthquake, with isfactory, ranging between 88 and 100%. All PCR and geno- a mean exposure score for the subjective experiences of 3.6 typing were done at UCLA GenoSeq Core. (severe). With regard to their objective experiences during the earthquake, all subjects experienced the tremor and witnessed the destruction 90% saw dead bodies lying in the 2.4. Statistical analyses streets, and 92% saw people who were severely injured. The mean PTSD-RI score was 33.9±13.2, with a range of 4–65. Statistical genetic modeling was performed using a The mean BDI score was 24.4±6.7 with a range of 12–44. pedigree-based association method (Almasy and Blangero, Covariates significantly associated with severity of PTSD 1998; Almasy et al., 2005; Amos, 1994) as implemented in symptoms included the following: sex (females had higher SOLAR (Blangero and Almasy, 1996). This method has been scores); witnessing death during the earthquake or its after- used to analyze data in numerous psychiatric behavioral ge- math; and exposure to pre-earthquake traumatic experi- netic family studies, e.g. Gur et al. (2007); Almasy et al. ences. Covariates that were significantly associated with (2008); Carless et al. (2011). This quantitative genetic meth- severity of depressive symptoms included: sex (females had od decomposes the phenotypic variance of a trait into its ad- higher scores); age (older subjects had higher scores); pre- ditive genetic and non-genetic components. This analysis earthquake traumatic experiences; and death of a family includes a component for the candidate gene polymorphism member. For the pleiotropic analyses, we adjusted each indi- in the general model for genetic and environmental effects vidual outcome for their significant covariates prior to per- individually on the quantitative PTSD and depression traits. forming the bi-variate analyses. The expected genetic DNA sharing between each relative After adjustment for sex, age, exposure and environmen- pair is incorporated into the model via a kinship matrix tal variables including adversities, the heritability rate for times a variance component. Estimations of all the parame- PTSD and depressive symptoms was respectively 0.41 and ters are obtained simultaneously using maximum likelihood 0.73 (Table 4). The genetic correlation (ρg) of PTSD symp- methods. Nested models are then tested to determine signif- toms with depressive symptoms was 0.71 (Table 4). This icance of parameters. For example, the likelihood of the being significantly greater than 0 (ρg >0; pb0.0006) indi- models with and without the candidate gene component is cates that some genes are shared, and being significantly compared to determine if the candidate gene polymorphism less than 1 (ρg b1; pb0.0001) indicates that not all genes explains a significant portion of the expression of PTSD or de- are shared. Fig. 1 shows the scatter-gram of PTSD-RI and pressive symptoms. Likewise, a component that includes the BDI scores. The Pearson correlation of PTSD-RI and BDI scores identical by descent (ibd) sharing of the marker can be was r=0.55. Since these were related individuals, their

Table 4 Ratesa of heritabilities and bi-variate genetic correlations for PTSD and depressive symptoms in 12 multi-generational families from Armenia exposed to the 1988 Spitak earthquake.

Genetic heritability Bi-variate genetic correlation

2 h (se) p ρg (se) p PTSD-RI (PTSD symptoms) 0.41 (0.18) b0.0001 0.71 (0.11) p (RhoG=0)=0.0006 BDI (depressive symptoms) 0.73 (0.21) b0.0001 p (RhoG=1)=0.0001

2 Abbreviations: h =heritability; ρg (Rho G)=Genetic correlation. a Adjusted for age, sex, measured exposure and environmental variables. phenotypic correlation does not account for the genetic the two phenotypes among subjects exposed to the 1988 Spi- relatedness. tak earthquake in Armenia was due to separate or similar TPH1 (Table 5): After adjusting for age, sex, exposure and candidate genes of the serotonergic system. The study dif- environmental variables, the family based variance compo- fered from prior candidate gene studies that have used case nent analyses revealed a significant association of PTSD-RI or case–control designs in that the subjects for the present with the ‘t’ allele of TPH1 SNP rs2108977 (χ2 =8.44;df1; study were members of multigenerational (3 to 5 genera- pb0.004). This gene explained 3% of the variance in PTSD-RI tions) families. The two main findings were the association scores. Linkage analysis of PTSD-RI scores of TPH1 of PTSD symptoms with TPH1 SNP rs2108977 (pb0.004), rs2108977 revealed a lod score of 1.05. With regard to BDI and TPH2 SNP rs11178997 (p=0.03). To our knowledge, and rs2108977, there was a trend (p=0.08) indicating this is the first published report showing the association of more severe depressive symptoms among those with the ‘t’ TPH genes with PTSD symptoms. The family study design allele. allowed determination of the proportion of variance of TPH2 (Table 5): After adjusting for age, sex, exposure and PTSD symptoms due to the TPH1 gene (3%) and the TPH2 environmental variables, analyses indicated a significant as- gene (4%). Scheuch et al. (2007) found that rs11178997 re- sociation of PTSD-RI and the ‘t’ allele of TPH2 SNP duced TPH2 transcriptional activity in neurons from rat rs11178997 (χ2 =3.87; df=1; p=0.03). This gene raphe nuclei and in human small cell lung carcinoma cells. explained 4% of the variance of PTSD-RI scores. There was A possible mechanism underlying the present findings is no association between rs11178997 and BDI scores. The re- that these alleles reduce the transcriptional activity of the mainder of the TPH1and TPH2 SNPs tested did not show a two TPH enzymes, thereby reducing the production of intra- significant association with both PTSD-RI and BDI scores; cellular serotonin resulting in vulnerability to PTSD symp- and the linkage scores were below 1.0. toms. Indirect support for this proposal comes from clinical 5HTTLPR (Table 5): After adjusting for age, sex, and expo- studies that have shown reduction of PTSD symptoms with sure and environmental variables, analyses indicated that BDI serotonin re-uptake inhibitors. A third finding of the study scores were significantly associated with the ‘s’ allele of was the association between depressive symptoms and the 5HTTLPR (χ2 =4.54; df=1; p=0.03). This allele explained ‘s’ allele of the 5HTTLPR gene. This finding adds to prior sim- 4% of the variance of BDI scores. There was no significant as- ilar findings in case/case–control studies. The proportion of sociation of either ‘s’ or ‘l’ allele and PTSD-RI scores. the variance due to this gene was 4%. With regard to the association of the TPH genes with de- 4. Discussion pression, studies have shown that some TPH1 and TPH2 polymorphisms are associated with depression (Table 1). In the The aim of the present study was to determine the extent present study we did not replicate these findings. However, to which the observed comorbidity of PTSD and depressive the TPH1 gene that was associated with PTSD (‘t’ allele of symptoms and high genetic correlation (ρg =0.71) between rs2108977) in this study, showed a trend for depression

Phenotypic Correlation of PTSD Symptoms and Depression Symptoms 45

PTSD-Reaction Index Scores 10 0 102030405060 Beck Depression Inventory Scores

Fig. 1. Scattergram of PTSD-RI and BDI scores among the participants exposed to the 1988 Spitak earthquake in Armenia.

Table 5 Association (p values) and linkage (lod scores) of polymorphisms of TPH1, TPH2 and 5HTTLPR with PTSD symptoms and depressive symptoms, adjusted for age, sex, exposure and measured environmental variables.

Genes SLC6A4 TPH1 TPH1 TPH2 TPH2 TPH2 TPH2 TPH2 TPH2

Polymorphism 5HTTLPR, `s' rs1799913 rs2108977 rs11178997 rs2129575 rs1386494 rs4570625 rs4290270 rs17110747 and `l' alleles

PTSD-RI Association 0.96 0.69 0.004 0.03 0.89 0.94 0.42 0.13 0.45 (PTSD symptoms) (p value) Linkage 0 0.56 1.05 0.02 0.46 0 0.32 0 0 (lod score) BDI Association 0.03 0.85 0.08 0.77 0.82 0.87 0.72 0.35 0.80 (Depressive symptoms) (p value) Linkage 0.01 0.58 0.34 0 0.20 0.09 0 0.03 0 (lod score)

(p=0.08), and linkage analyses showed a lod score of 1.05. case–control study by Lee et al. (2005), may misclassify sub- In a case–control study among Taiwanese women, Sun et al. jects as controls. Some of those subjects who may have been (2004) found an association of this gene in group of patients carrying vulnerability genes for PTSD and were not exposed with co-morbid depression and anxiety. With regard to the to trauma may have been inaccurately classified as controls' TPH2 gene, Van Den Bogaert et al. (2006) found the TPH2 thereby reducing the detectable effect-size. In the study by rs11178997, that is associated with PTSD in this study, was Lee et al. (2005) the result indicating that the ‘s’ allele is a associated with unipolar depression among patients from risk factor for PTSD may have been more robust had non- northern Sweden. These two genes may be appropriate can- exposed subjects been excluded from the control group. didates to study the genetics of both phenotypes, uniquely Similarly, adjusting for depression in the analyses for the and pleiotropically, in larger samples of traumatized subjects identification of PTSD genes is essential, as both phenotypes of different ethnic/racial cohorts for several reasons: First, have unique and shared genes. For example, in the study by there is a high genetic correlation between PTSD symptoms Lee et al. (2005) subjects who had major depression prior and depressive symptoms in this population. Second, there to the trauma but not those who had it after the trauma is a significant association of rs2108977 and rs11178997 were excluded. Elimination of depressed individuals who with PTSD symptoms, and lod score of rs2108977 for PTSD may have been at greater risk for developing PTSD may is 1.05, and there is a trend for an association of rs2108977 have skewed the sample and led to missing important with depression. Lastly, there is an association of the two genes that are pleiotropic for depression and PTSD. In the genes with depressive symptoms in the studies by Sun et al. study by Kilpatrick et al. (2007) among subjects exposed to (2004) and Van Den Bogaert et al. (2006). hurricanes in Florida, the results showed no main effect for The lack of association of these genes with depression in 5HTTLPR and PTSD. However, these authors found a main ef- this study may be due to lack of adequate sample size to de- fect for trauma (hurricane exposure) and depression. They tect the association and low effect-size of the gene in this also found a three way gene–environment interaction ethnic/racial group population. Additionally, cohorts with (5HTTLPR by high exposure by low social support) whereby different proportions of co-morbid individuals (not necessar- the ‘s’ allele was found to be a risk factor for PTSD and ily due to genetic differences) may not produce comparable major depression in the group with high exposure and low findings (stratification bias). Finally, the positive findings in social support. It remains unclear whether the significance prior studies may have been false positives. A priori expecta- of the ‘s’ allele with PTSD would have emerged after account- tion in such studies is that 5% of the results have type 1 er- ing for depression and exposure. Since PTSD and depression rors. Possible publication bias against negative findings may are often comorbid conditions and pleiotropic, future genetic have excluded other true negatives from being published, studies should utilize a design that includes assessment of thereby magnifying the importance of rare positive findings. the independent effects of exposure, PTSD symptoms and de- Regarding the association of 5HTTLPR and PTSD, results pressive symptoms. have varied and have been inconsistent (Table 2). In two With regard to the relation of 5HTTLPR gene and depres- studies the ‘s’ allele was reported to be causal (Kolassa sion, Table 3 lists six meta-analyses. Three of the studies et al., 2010; Lee et al., 2005), in two others protective reported a positive relation between the ‘s’ allele and depres- (Grabe et al., 2009; Thakur et al., 2009), in one not associated sion, and three did not find such an association. The most re- (Mellman et al., 2009), and in another three only associated cent and largest meta-analysis by Karg et al. (2011) included under specific environmental conditions (Kilpatrick et al., many of the studies reviewed in the other meta-analysis. The 2007; Koenen et al., 2009; Xie et al., 2009). In addition to positive findings in three of the studies indicate that the ‘s’ al- the possible reasons noted above as to why findings in the lit- lele was a risk factor for depression, but had a small effect erature, including this study, may be dissimilar (e.g. insuffi- size. cient sample size, low effect-size of the gene, heterogeneity The results in the present study, using a family study de- of the sample), the misclassification of controls and the co- sign also indicated a positive association between the ‘s’ al- existence of depression (which may be independently associ- lele and depressive symptoms thus extending prior positive ated with 5HTTLPR) may be confounding factors. For exam- findings in studies that have used case/case–control designs. ple, not screening for exposure among controls, as in the The phenotypic variance in depression due to the 5HTTLPR

Please cite this article as: Goenjian, A.K., et al., Association of TPH1, TPH2, and 5HTTLPR with PTSD and depressive symptoms, J. Affect. Disord. (2012), doi:10.1016/j.jad.2012.02.015 A.K. Goenjian et al. / Journal of Affective Disorders xxx (2012) xxx–xxx 7 gene was 4%. This small effect-size may constitute one reason non for the diagnosis. Reducing the variability in the dose why the positive finding of the ‘s’ allele is not often replicat- and timing of exposure improves the reliability of the herita- ed. Additionally, publication bias toward negative findings bility measures and increases the likelihood of detecting ge- may have excluded true negative results from being pub- netic associations. lished, thereby skewing the outcome of meta-analyses in Limitations of the present study include the following. favor of positive results. First, retrospective rating of exposure to the earthquake The present study differs from the other candidate gene may have been subject to memory failure leading to misesti- studies of 5HTTLPR and TPH among subjects with co- mation of severities of phenotypes and heritability measures. occurring PTSD and depression in several significant ways. Second, the subjects were ethnic Armenians thus the findings First, as opposed to case and case–control studies, the present may not be generalizable. Third, the sample size may have study was a multigenerational family study. Family studies been insufficient to detect genes with relatively very small control for genetic heterogeneity within a family where the effects. likelihood of affected family members having the same causal In summary, the findings of an association between TPH1 variants is high. In case–control studies each case may be af- SNP rs2108977 and TPH2 rs11187997 and PTSD symptoms fected due to unique variants, making it difficult to establish a suggest that the serotonergic system may be causally impli- genetic association. Also, in the present study between family cated in the onset and/or persistence of PTSD. Also, the asso- variances were reduced because the population was relative- ciation between depression and the ‘s’ allele of HTTLPR ly homogenous. extends prior similar findings in studies that have used case Second, the families in this study were unascertained and case–control designs. Even though the results did not which permitted us to evaluate the heritability of both PTSD demonstrate definite pleiotropy for the two phenotypes, the and depression independently. In case or case–control stud- significant association of TPH1 gene (SNP rs2108977) with ies cases are typically ascertained. If a sample is ascertained PTSD and the non-significant trend with depressive symp- for PTSD, such as when subjects with a known diagnosis are toms is suggestive that the gene may be pleiotropic for both recruited from a clinic, independent analysis for depression phenotypes. Finding unique and shared genes for the two may not be valid. This would be especially problematic if phenotypes may be helpful in establishing biologically de- the diseases being investigated are related and share genes, fined illnesses and developing specific treatments. The pre- as in the present study. Our study focused on unascertained sent findings justify further family studies in a larger family members to represent the general population in the sample of traumatized individuals in other racial/ethnic region. Only two of these subjects were seeking treatment. groups to replicate the results. Families were recruited based on family size and exposure to the Spitak earthquake and not based on any phenotypic Role of funding source symptom. This design allowed us to assess the association Nothing declared. of both PTSD and depressive symptoms uniquely and pleiotropically. Another difference between ascertained and unascertained samples may be the severity of symptoms. Usually, Conﬂict of interest a case is assumed to be more severe and that is why subjects No conflict declared. may be seeking treatment. It is possible that genes involved for different severity level of symptoms vary from those Acknowledgments who have less severe symptoms. We would like to thank the family members who participated in this Third, in this study symptoms were measured on a con- study and the mental health staff of the Psychiatric Outreach Program who tinuous scale which allows for greater statistical power than assisted us in the implementation of the project. symptoms measured dichotomously with a yes/no item response option (Bailey and Almasy, 1995). Decrease in References power occurs when information is lost in the dichotomization of phenotypes. For example, individuals with many Almasy, L., Blangero, J., 1998. Multipoint quantitative-trait linkage analysis in symptoms (but not enough for diagnosis) are considered as general pedigrees. The American Journal of Human Genetics 62 (5), – ‘ ’ 1198 1211. unaffected and classified together with individuals with no Almasy, L., Rainwater, D.L., Cole, S., Mahaney, M.C., Vandeberg, J.L., Hixson, symptoms. 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Please cite this article as: Goenjian, A.K., et al., Association of TPH1, TPH2, and 5HTTLPR with PTSD and depressive symptoms, J. Affect. Disord. (2012), doi:10.1016/j.jad.2012.02.015