Journal of Affective Disorders 172 (2015) 472–478
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Research report
Association of COMT and TPH-2 genes with DSM-5 based PTSD 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, USA b Collaborative Neuroscience Network, Garden Grove, CA, USA c Alcohol Research Center, Department of Psychiatry, Geffen School of Medicine, UCLA, CA, USA d Department of Psychology, University of California at Riverside, CA, USA e Sequencing & Genotyping Core, Department of Human Genetics, UCLA, CA, USA f Department of Epidemiology, UCLA Fielding School of Public Health; Epilepsy Genetics/Genomics Laboratories, VA GLAHS, Los Angeles, CA, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 12 October 2014 Accepted 14 October 2014 Available online 22 October 2014
Background: Dopaminergic and serotonergic systems have been implicated in PTSD. The present study evaluated the association of four catechol-O-methyltransferase (COMT) gene loci, and the joint effect of COMT and tryptophan hydroxylase 2 (TPH2) genes on PTSD symptoms. Methods: Subjects included 200 Caucasian Armenian adults exposed to the 1988 Spitak earthquake from 12 multigenerational (3–5 generations) families. Instruments used included the UCLA PTSD Reaction Index 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 (p o10 4). There was a significant association of the COMT allele rs4633C with total PTSD (po 0.03), and D category (p o0.04) (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. COMT allele rs4633C and the TPH-2 allele rs11178997T together accounted for 7% of the variance in PTSD severity scores (po 0.001). 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 disequilibrium with gene(s) that are causally related to PTSD symptoms. Carriers of these COMT and the TPH-2 alleles may be at increased risk for PTSD. The findings provide biological support for dividing DSM-IV category C symptoms into DSM-5 categories C and D. & 2014 Elsevier B.V. All rights reserved.
Keywords: Catechol-O-methyltransferase (COMT) Genetics PTSD Tryptophan hydroxylase 2 (TPH 2) Trauma
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 become disabling for decades after a traumatic event (Morgan et al., 2003; Marshall et al., 2005). Variations in severity and course of the disorder depend on multiple factors, including sex, age, ethnicity, severity of the traumatic experiences, and pre and post-trauma factors (Norris et al., 2002). Additionally, genetic studies among twins (True et al., 1993), and multigenerational family members
n Correspondence to: Alcohol Research Center, Room 58-242, UCLA Neuropsychiatric Institute, 760 Westwood, CA 90024-1759, USA. E-mail address:
[email protected] (A.K. Goenjian).
http://dx.doi.org/10.1016/j.jad.2014.10.034 0165-0327/& 2014 Elsevier B.V. All rights reserved.
(Goenjian et al., 2008) have found that genetic make-up also plays a significant role in vulnerability to PTSD symptoms. In the latter study, among survivors of the 1988 Spitak earthquake, the adjusted heritability was 0.41. One method of identifying genes that are involved in complex disorders such as PTSD is the hypothesis driven candidate-gene approach. Genes are selected on the basis of prior knowledge about their purported role in the pathogenesis of a phenotype. The objective is to determine if a particular gene variant is associated with the risk for a phenotype. Depression is of interest in the study of PTSD-related genes, as these two phenotypes frequently co-occur (Breslau et al., 2000; Goenjian et al., 2000), share genes (Goenjian et al., 2008; Koenen et al., 2008), and respond to similar medications such as the SSRIs. Studies suggest that the dopaminergic system may be relevant to the pathogenesis of various psychiatric disorders, including PTSD
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(Boscarino et al., 2012; Comings et al., 1996; Kolassa et al., 2010; Segman et al., 2002; Young et al., 2002), depression (Baekken et al., 2008; Massat et al., 2005; Ohara et al., 1998) and anxiety disorders (Domschke et al., 2004; Hamilton et al., 2002; Hettema et al., 2008; McGrath et al., 2004; Rothe et al., 2006). Dopamine is metabolized by catechol-O-methyltransferase (COMT). This enzyme has broad biological functions, including regulation of catecholamines and enkephalins. The COMT gene is located at the 22q11.2 interval. It encodes two proteins: soluble cytoplasmic COMT (S-COMT) and membrane bound COMT (MBCOMT). The former isoform is predominantly expressed in peripheral tissues, while the latter is expressed in the brain and has been of 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 two promoters where there are two distinct ATG start codons for the promoters P1 and P2 that drive the transcription of S-COMT and MB-COMT respectively (Lachman et al., 1996; Shield et al., 2004; Tenhunen et al., 1993, 1994). SNP rs6269 is located in the promoter region of exon 3, rs4633 is within exon 3, and rs4818 and rs4680 are within exon 4. SNPs rs4633 and rs4818 are synonymous, i.e. do not produce a change in amino acid composition. In contrast, polymorphism of rs4680 is non-synonymous whereby it changes the amino acid sequence of the protein. It encodes for a substitution of valine (Val) to methionine (Met) at codons 108 and 158 of S-COMT and MB-COMT respectively. A Val to Met substitution at codon 158 of MB-COMT has been considered a main source of individual variation in COMT activity resulting in a less thermostable COMT enzyme that exhibits multiple-fold reduction in activity (Lachman et al., 1996; Lotta et al., 1995; Shield et al., 2004). Most association studies of psychiatric disorders with COMT have focused on the functional non-synonymous SNP rs4680 (Val158Met polymorphism). The findings of these studies have been modest and inconsistent in the direction of effect (Baekken et al., 2008; Craddock et al., 2006; Domschke et al., 2004; Frisch et al., 1999; Massat et al., 2005; Ohara et al., 1998; Rothe et al., 2006). Two studies of PTSD have reported an association with the COMT rs4680 Met allele (Boscarino et al., 2012; Kolassa et al., 2010). In the first study (Kolassa et al., 2010) the investigators found nomain effect of the genotype on lifetime PTSD. However, they found a gene–environment interaction. Individuals homozygotic for the Met allele exhibited higher risk for PTSD compared to Val allele carriers. In the second study (Boscarino et al., 2012) the Met allele was more common among PTSD cases compared to controls. The purported role of dopamine in psychiatric disorders extends beyond the COMT gene. Studies have implicated the gene that 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, these findings have not been replicated in other studies (Gelernter et al., 1999; Bailey et al., 2010).
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The serotonergic system also plays a role in PTSD and depression (Goenjian et al., 2012; Grabe et al., 2009; Koenen et al., 2009; Kolassa et al., 2010; Thakur et al., 2009). Most of these studies have focused on the promoter region of the serotonin transporter gene (5HTTLPR). A recent study among the same cohort as in this study found an association between two genes that encode for tryptophan hydroxylase (TPH), the rate limiting enzyme for the synthesis of serotonin, and PTSD (Goenjian et al., 2012). With regard to symptom criteria for PTSD, the most recent edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) (American Psychiatric Association, 2013) has modified 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 divided into DSM-5 categories of C (avoidance) and D (negative alterations in cognition and mood). The latter included items from DSM IV and new items relating to extreme negative beliefs. With the addition of reckless or self-destructive behavior and no other substantial changes, the DSM IV D category was classified in DSM-5 as E category (alterations in arousal and reactivity). Almost all candidate gene studies of PTSD have been casecontrolled studies utilizing subjects recruited from clinics or epidemiological studies among subjects exposed to trauma. In contrast, the present study is a multigenerational study where the entire family was exposed to trauma. Thus, there is increased likelihood of affected family members having the same causal genetic variants. The objective of this study was to investigate the association of four polymorphic loci of the COMT gene with PTSD symptoms based on both DSM-IV and DSM-5 diagnostic symptom criteria, and their association with depressive symptoms. In addition, the joint effect of the COMT and TPH-2 gene was examined in relation to the risk for PTSD symptoms.
2. Methods 2.1. Subjects Subject recruitment for this study has been previously described (Goenjian et al., 2008). Briefly, a total of 212 subjects were recruited from Gumri (a city in Armenia nearby the epicenter of the catastrophic 1988 Spitak earthquake) where there was extensive destruction, morbidity and mortality. Seven of the subjects were not in the city during the earthquake, while five were unable to complete the testing. Thus, a total of 200 adults exposed (121 females, 79 males) from 12 multigenerational (three to five generations) families participated and completed the study procedures approximately 14 years post-earthquake. Both the mean and modal number of subjects per family was 16; the range was between 13 and 19. The types of relatives included: 108 parent–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. Subjects belonged to the same racial/ethnic (Caucasian/Armenian) group and had similar socioeconomic backgrounds. After obtaining IRB approval, subjects were given a full description of the study, including potential risks and benefits. Written informed consent was obtained from all participants. Subjects had been exposed to extreme threat to life, widespread destruction, and extensive morbidity and mortality during the earthquake. All the subjects saw destroyed buildings throughout the city, 90% saw dead bodies lying in the streets, and 92% saw people who were severely injured. The mean Posttraumatic Stress Disorder-Reaction Index (PTSDRI) score based on DSM-IV symptom criteria was 33.9713.2, and 33.1 712.5 based on DSM-5 criteria. The mean Beck Depression Inventory (BDI) score was 24.47 6.7. Adjusted heritability of vulnerability to PTSD symptoms and depressive symptoms in this group has been reported previously as 0.41 and 0.66 respectively (Goenjian et al., 2008). Bailey et al. (2010) reported the heritability of DSM IV PTSD B category symptoms to be 0.75; C category symptoms 0.39; while D category symptoms were not heritable. 2.2. Instruments An earthquake exposure profile was completed for each participant using a modified version of a DSM IV-based exposure questionnaire previously utilized in studies after disasters (Goenjian et al., 2001; Roussos et al., 2005). This instrument included information on gender, age, location at the time of the earthquake, objective experiences during the earthquake, including destruction of residence, death of relatives, seeing dead bodies, being injured, and seeing someone else who was injured. Participants were also evaluated for subjective experiences during the earthquake. These items included fear during the earthquake, fear of getting badly injured or dying, and fear that someone else would be badly hurt or killed. Additionally, the questionnaire included items relating to pre- and post-earthquake exposure to traumatic experiences; post-earthquake medical illness/treatment by subject or family member; and post-earthquake adversities. These items were rated on a five point Likert scale ranging from 0 ¼not at all, to 4¼ a whole lot. Posttraumatic stress symptoms were evaluated using the UCLA PTSD Reaction Index (PTSD-RI), a 22-item self-report scale based on DSM-IV criteria (Steinberg et al., 2004, 2013) comprised of category B (re-experiencing), C (avoidance/numbing), and D (arousal) symptom subscales. Frequency of symptom occurrence during the previous month is rated on a 5-point Likert scale, ranging from 0¼ not at all, to 4 ¼most of the time. Psychometric properties of this scale have been reported, with high internal consistency, reliability, and discriminant validity (Steinberg et al., 2004, 2013). Additional analyses were performed using a modified version of the PTSD-RI based on DSM-5 symptom criteria. The study questionnaire incorporated all but one of the DSM-5 diagnostic symptom items. The missing item was a D category symptom: persistent and exaggerated negative belief or expectations about oneself, others or the world. The reason for the missing item was that, at the time of the study, the DSM-5 version of the PTSD-RI did not include a tested formulation for evaluating this symptom. Depressive symptoms were evaluated using the Beck Depression Inventory (BDI). Psychometric properties for the BDI have been reported elsewhere (Beck and Steer, 1984). 2.3. Genomic processing Buccal cell samples were taken from each participant in Armenia and 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 this study included rs6269, rs4633, rs4818, and rs4680. Previously these SNPs were shown to be associated with psychiatric and medical disorders (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 genotyped using a 50 nuclease assay to discriminate between the two alleles. Taqman SNP Genotyping Assays were ordered from Life Technologies formerly known as Applied Biosystems Inc. Polymerase chain reactions were performed using 5-μL reaction volumes in 384-well plates with 5 ng of DNA. The standard protocol provided with the kit Taqman Genotyping Master Mix was followed. End point reads of fluorescence levels were obtained with an ABI 7900HT Sequence Detection System.
2.4. Statistical analyses Statistical genetic modeling was performed using a pedigreebased association method (Almasy and Blangero, 1998; Amos, 1994; Almasy et al., 2005) as implemented in SOLAR (Blangero and Almasy, 1996). This method has been used to analyze data in numerous psychiatric behavioral genetic family studies (Almasy et al., 2008; Carless et al., 2011; Goenjian et al., 2008; Gur et al., 2007). This quantitative genetic method decomposes the phenotypic variance of a trait into its additive genetic and non-genetic components. The analysis includes a component for the candidate gene polymorphism in the general model for genetic and environmental effects individually on the quantitative PTSD and depression traits. The expected genetic DNA sharing between each relative pair is incorporated into the model via a kinship matrix times a variance component. Estimations of all the parameters are obtained simultaneously using maximum likelihood methods. Nested models are then tested to determine significance of parameters. For example, the likelihood of the models with and without the candidate gene component is compared to determine if the candidate gene polymorphism explains a significant portion of the expression of PTSD or depressive symptoms. In the model, COMT rs4633 and TPH-2 rs11178997 were included for the analyses. TPH-2 was included because it is primarily involved in the synthesis of serotonin in the central nervous system, as opposed to TPH-1 which is mainly associated with peripheral synthesis of serotonin. Association of COMT and TPH-2 polymorphisms with both DSM-IV and DSM-5 based subcategory scores were assessed separately and jointly. These analyses included the significantly associated COMT SNP (with PTSD symptoms) found in this study (rs4633), and TPH-2 SNP (rs11178997), previously shown to be significantly associated with DSM-IV based PTSD-RI score in this cohort (Goenjian et al., 2012). For the joint analysis, the contributions of risk alleles of both genes were studied by comparing individuals with none, 1 and 2 of the risk alleles. The subcategories analyzed included all but the arousal symptoms, 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 and previously (Bailey et al., 2010). We estimated that in this sample we had 80% power to detect variants that account for 4.6% of the trait variance and are in strong linkage disequilibrium (LD) with one of the genotyped markers. This calculation was based on a p-value of 0.025, which is equivalent to correcting the standard marginal p-value of 0.05 for two tests. For a variant that has an r-squared (the amount of linkage in the disequilibrium) of at least 0.85 with a genotyped marker, we reach 80% power accounting for 5.4% of the trait variance. On the basis of these calculations, genetic associations that account for less of the trait variations may not be detected
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because of the sample size. Simulations were performed using SOLAR (Almasy and Blangero, 1998).
3. Results 3.1. Heritabilities of PTSD symptoms based on DSM-IV and DSM-5 criteria after adjusting for covariates After adjusting for covariates, the heritability of vulnerability to PTSD symptoms based on DSM IV and DSM-5 criteria were 0.41 (p o0.001) and 0.60 (po 10 4) respectively (Table 1). The Pearson correlation between DSM IV and DSM-5 based PTSD severity scores was 0.98 (r ¼0.96; po 0.0001). The covariates with significant association to the variance in PTSD scores included: gender, pre-earthquake traumatic experiences, post-earthquake serious illnesses (Table 1). These covariates contributed 19% of the variance in PTSD severity scores measured by both instruments. 3.2. Heritability of subcategories and genetic correlation of DSM-5 C and D category symptoms The heritability of vulnerability to DSM-5 based category B (reexperiencing) severity scores was 0.75 (p o0.0001); category C (avoidance) severity scores was 0.64 (p¼ 3.9 10 10); category D (negative alterations in cognitions and mood) scores was 0.58 (p o0.0001), while category E (arousal symptoms) symptoms, formerly categorized as DSM-IV category D symptoms, were not heritable. No significant genetic correlation was found (Ho: ρg 40) between DSM-5 categories C and D symptoms (ρg ¼0.32, SE¼ 0.21; p ¼0.17), indicating that these phenotypes did not share genes. After taking genetic relatedness into account, the derived estimate of the phenotypic correlation was 0.29. 3.3. Association of Single Nucleotide Polymorphism (SNP) alleles with PTSD and depressive symptoms The COMT SNP allele rs4633C was significantly associated with both DSM-IV and DSM-5 total PTSD severity scores (both with p o0.03) (Table 2). Regarding PTSD subcategories, there was no significant association between rs4633C and B category score for both scales (p¼ 0.28). There was a significant association of rs4633C with DSM IV based category C (avoidance/numbing) scores (p ¼0.03), although not with DSM-5 category C (avoidance) scores (p ¼ 0.46). However, there was a significant association
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between rs4633C and category D scores (negative alterations in cognitions and mood) (p o0.04). Finally, no significant genetic correlation was found between DSM-5 C and D category scores, indicating that they did not share genes. Regarding the non-synonymous SNP rs4680 (Val158Met), there was a trend toward significance for the association of the G allele (Val) and total PTSD severity scores (p ¼0.07) on both DSM IV and DSM-5 based measures. There was no significant association between rs4680G and subcategory scores, and either of the other two COMT SNPs (rs6269 and rs4818) with total PTSD and subcategory scores. None of the four COMT SNPs were associated with depressive symptoms.
Table 2 The association of COMT SNPs with DSM-IV and DSM-5 based total Posttraumatic Stress Disorder-Reaction Index (PTSD-RI) and subcategory severity scores among multigenerational family members exposed to the 1988 Spitak earthquake. COMT SNPs
Trait DSM-IV based
p-value
Total PTSD sx. severity score
Trait DSM-5 based
p-value
Total PTSD symptom severity score
Rs6269 Rs4633C Rs4818 Rs4680G
c c c c B category sx. score
0.45 o 0.03 0.52 0.07
c c c c B category severity score
0.62 o0.03 0.67 0.07
Rs6269 Rs4633C Rs4818 Rs4680G
c c c c C category sx. score
0.87 0.28 0.38 0.34
c c c c C category severity score
0.87 0.28 0.38 0.34
Rs6269 Rs4633C Rs4818 Rs4680G
c c c c
0.13 0.03 0.27 0.09
c c c c D category severity score
0.52 0.46 0.50 0.36
c c c c
0.53 o0.04 0.43 0.15
Rs6269 Rs4633C Rs4818 Rs4680G
sx.¼ symptom. N.B.: DSM-5 E category (arousal and reactivity) symptoms were not included as they were not shown to be heritable. Category C symptoms of DSM-5 diagnostic criteria included only the avoidance items from DSM-IV Category C symptoms which included avoidance and numbing items.
Table 1 Heritabilites of PTSD symptoms based on DSM-IV and DSM-5 criteria, after adjusting for covariates, among multigenerational family members exposed to the 1988 Spitak earthquake. DSM-IV based PTSD severity scores
DSM-5 based PTSD severity score
0.417 0.17 p o 0.001
0.60 70.17 p o 10 4
Covariates with p values Sex Age Home destroyed Death of family member Witnessed death Witnessed harm to others Was hurt during the earthquake Pre-earthquake traumatic experience Post-earthquake traumatic experience
0.01 0.11 0.76 0.73 0.01 0.82 0.72 0.002 0.15
0.05 0.23 0.66 1.0 0.005 0.99 0.86 0.002 0.58
Post-trauma illness Proportion of variance due to all covariates (%)
o 0.04 19
0.004 19
2
Heritability (h ) after adjusting for significant covariates
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structure and thereby protein expression (Nackley et al., 2006). The present finding of an association between the synonymous SNP rs4633 and PTSD symptoms (Table 2) may be explained on the basis 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 symptoms remained essentially as in DSM-IV. The DSM-5 category C (avoidance) and D (negative alterations in cognitions and emotions) symptoms were highly heritable, 0.64 and 0.58 respectively, while E category symptoms (arousal) formerly described as DSM-IV category D symptoms, were not. We found a significant association between SNP rs4633 and DSM-IV based category C (avoidance/numbing) symptom scores (p¼0.03). There was no significant association between rs4633 and DSM-5 category C symptoms, but there was an association between rs4633 and DSM-5 Category D symptoms (po0.04) (Table 1). Further, there was no significant genetic correlation between DSM-5 C and D category symptoms, suggesting that they did not share genes. These findings provide support for dividing DSM-IV category C symptoms into DSM-5 C and D categories. In this study there was a trend towards significance for the association between the non-synonymous SNP rs4680G (Val) allele and PTSD symptoms. The Val allele is known to be associated with 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 Val allele 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 genetic correlations among these phenotypes, i.e. PTSD, depression and anxiety (Goenjian et al., 2008) it is expected to find some genes that will be contributing to these phenotypes. Researchers who have found an association of the Val allele with panic disorder (Rothe et al., 2006) and depression (Baekken et al., 2008; Massat et al., 2005) have conceptualized the mechanism of action for the pathologies to be the removal of catecholamines from the transsynaptic space at a higher rate due to high (Val) enzyme activity. The trend found in the present study of the association between the Val allele and PTSD symptoms suggests that a similar mechanism may be involved in PTSD. In contrast to these findings, in a study among survivors of the Rwandan Genocide, there was no main-effect of the Val158Met polymorphism, but there was a gene–environment interaction, whereby Met/Met homozygotes exhibited higher risk for PTSD compared to Val allele carriers (Kolassa et al., 2010). Also, in a study of pain patients, Met allele was more common among those with PTSD compared to controls (Boscarino et al., 2012). Inconsistent genetic findings in psychiatric disorders such as PTSD, anxiety and depression are not uncommon. Future family studies with larger samples of various ethnic/racial groups with minimal heterogeneity may shed light on the relevance of these COMT SNPs and PTSD.
3.4. Multilocus analyses of the association of COMT and TPH-2 SNPs and PTSD symptoms The TPH-2 rs11178997T allele was significantly associated with DSM-5 based PTSD severity scores (p ¼ 0.02). With regard to the effect of both TPH-2 rs11178997T and COMT rs4633C, Table 3 shows 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 and rs11178997 with DSM-5 based total PTSD severity scores were significant (po 0.02 and o0.01 respectively). In combination, they explained 7% of the variance in PTSD severity scores. For the joint assessment, the association was also significant (po 0.001), explaining 7% of the variance of the severity scores. 3.5. Combined effect of all the significant genetic and environmental variables on the variance of PTSD The combined effect of all of the significant variables examined, including gender, environmental variables (e.g., traumatic experiences prior and during the earthquake, severe illness after the earthquake) along with the effect of COMT rs4633C and TPH-2 rs11178997T alleles was significant (p o0.01) explaining 27% of the variance of total PTSD severity scores.
4. Discussion The first finding of this study was the high heritability of vulnerability to PTSD symptoms based on DSM-5 criteria after adjusting for covariates (0.60). This was higher than previously reported for DSM IV based PTSD severity scores (0.41) (Goenjian et al., 2008). The finding suggests that DSM-5 based assessments, as opposed to DSM-IV, may increase the prospects of finding genetic markers among traumatized individuals. The other relevant result was the association of synonymous COMT allele rs4633C with DSM-5 based total PTSD symptom score (p o0.03) and D category symptom score (p o0.04) but not C category score. Emerging studies of psychiatric and medical disorders have shown that mutations of synonymous SNPs, such as rs4633 and rs4818, can effect protein expression and enzyme activity resulting in phenotypic alterations, including pathology. Even though synonymous changes do not produce a change in the amino acid composition of a protein, they can cause disruption of splicing (Cartegni et al., 2002), secondary structural changes and instability of mRNA (Bartoszewski et al., 2010; Duan et al., 2003; Nackley et al., 2006; Shen et al., 1999), and asynchrony of co-translational folding (Kimchi-Sarfaty et al., 2007; Sauna et al., 2007), thereby effecting protein expression and enzyme activity. With regard specifically to synonymous SNPs of COMT, in a study of pain sensitivity, the results indicated that synonymous changes in the coding region of COMT affected secondary mRNA
Table 3 Contribution 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 1988 Spitak earthquake. SNP
Trait DSM-IV
COMT rs4633C TPH2 rs11178997T a (separate assessments PTSD sx. of risk alleles) COMT TPH2) b (joint assessments of both risk alleles) PTSD sx.
p-value
Variance explained by genes (SNPs) (%)
Trait DSM-5
p-value
Variance explained by genes (SNPs) (%)
0.02 0.01 0.003
7
PTSD sx.
7
6
PTSD sx.
o 0.02 o 0.01 o 0.001
7
sx. ¼symptom. a b
For the analysis, the contribution of each allele of the two genes was assessed separately. 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 together explained 7% of the variance of DSM-5 based PTSD symptoms (Table 3). TPH-2 is the rate determining enzyme of serotonin synthesis in the central nervous system. Previously it has been shown to be associated with PTSD symptoms (Goenjian et al., 2012). The SNP being located in the promoter region may affect level, location or timing of gene expression and thereby synthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric disorders. These two genes, along with environmental variables and gender, explained 27% of the variance in PTSD symptoms. Replicating these finding may ultimately provide a basis for psychopharmacologic strategies to ameliorate symptoms of PTSD. The lack of an association between the four COMT SNPs and depression in this family study represents an addition to the literature 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 the low-activity Met allele (Ohara et al., 1998), and association with the high activity Val allele (Baekken et al., 2008; Massat et al., 2005). The present study was designed to control for the effects of potentially confounding factors. A family study design was used because of the availability of multi-generational families who were exposed to the traumatic events surrounding the earthquake. Family studies reduce genetic heterogeneity because there is increased likelihood of affected family members having the exact same causal genetic variants, whereas in case-controlled studies every case could potentially have a unique causal genetic variant. Also, between family variance is minimized compared with studies that include mixed racial/ethnic groups. The population in northern Armenia belongs to the same ethnic group. Finally, family studies require significantly less subjects than case-control studies for comparable power. The variance due to trauma exposure, another potentially confounding variable, was reduced as participants in this study were exposed to similar earthquake-related severe traumatic events contemporaneously, thereby reducing variance related to type, severity or timing of trauma exposure. Reducing the variability in the dose and timing of exposure improves the reliability of the heritability measures and increases the likelihood of detecting genetic associations. Finally, families in this study were from the general population exposed to the earthquake as opposed to a clinic population, making it more likely that the findings are generalizable. The use of a general population sample permitted a less biased evaluation of the heritability of vulnerability to both PTSD and depression independently. 4.1. Limitations The ratings of earthquake exposure and prior trauma were made on the basis of retrospective reports. These ratings may have been subject to recall bias. Second, the subjects were ethnic Armenians, and thus the findings may not be generalizable to other populations. In summary, the present study found a higher adjusted heritability of vulnerability to PTSD symptoms based on DSM-5 criteria compared to DSM-IV criteria. The significant association between COMT rs4633 C allele and total PTSD severity scores suggest that this SNP is either causally related to the pathogenesis of PTSD symptoms or is in linkage disequilibrium to genes that are causally related to PTSD. The association of rs4633C with DSM-5 category D but not C symptoms, and the lack of significant genetic correlation between these two categories provide biological support for dividing DSM-IV category C symptoms into DSM-5 categories C and D. The dopaminergic COMT allele rs4633C and the serotonergic
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TPH-2 allele rs11178997T jointly explained 7% of the variance in PTSD severity scores. Carriers of these alleles may be at risk for PTSD upon exposure to trauma. These findings warrant larger multigenerational family studies of dopaminergic and serotonergic genes among traumatized families in other ethnic/racial groups using DSM-5 diagnostic criteria.
Role of funding source None.
Conflict of interest None
Acknowledgments We 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 the implementation of the project.
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