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1.  FKBP5 and Attention Bias for Threat 
JAMA psychiatry (Chicago, Ill.)  2013;70(4):392-400.
Importance
The FKBP5 gene product regulates glucocorticoid receptor (GR) sensitivity and hypothalamic-pituitary-adrenal axis functioning and has been associated with many stress-related psychiatric disorders. The study of intermediate phenotypes, such as emotion-processing biases and their neural substrates, provides a way to clarify the mechanisms by which FKBP5 dysregulation mediates risk for psychiatric disorders.
Objective
To examine whether allelic variations for a putatively functional single-nucleotide polymorphism associated with FKBP5 gene regulation (rs1360780) would relate differentially to attention bias for threat. this was measured through behavioral response on a dot probe task and hippocampal activation during task performance. Morphologic substrates of differential hippocampal response were also measured.
Design
Cross-sectional study conducted from 2010 to 2012 examining associations between genotype, behavioral response, and neural response (using functional magnetic resonance imaging [fMRI]) on the dot probe; voxel-based morphometry and global and local shape analyses were used to measure structural differences in hippocampi between genotype groups.
Setting
Participants were recruited from primary care clinics of a publicly funded hospital in Atlanta, Georgia.
Participants
An African American cohort of adults (N=103) was separated into 2 groups by genotype: one genotype group included carriers of the rs1360780 T allele, which has been associated with increased risk for posttraumatic stress disorder and affective disorders; the other group did not carry this allele. Behavioral data included both sexes (N=103); the MRI cohort (n=36) included only women.
Main Outcome Measures
Behavioral and fMRI (blood oxygen level–dependent) response, voxel-based morphometry, and shape analyses.
Results
Carriers of the rs1360780 T allele showed an attention bias toward threat compared with individuals with-out this allele (F1,90=5.19, P=.02). Carriers of this allele demonstrated corresponding increases in hippocampal activation and differences in morphology; global and local shape analyses revealed alterations in hippocampal shape for TT/TC compared with CC genotype groups.
Conclusion
Genetic variants of FKBP5 may be associated with risk for stress-related psychiatric disorders via differential effects on hippocampal structure and function, resulting in altered attention response to perceived threat.
doi:10.1001/2013.jamapsychiatry.210
PMCID: PMC3732315  PMID: 23407841
2.  ADCYAP1R1 Genotype Associates With Post-Traumatic Stress Symptoms in Highly Traumatized African-American Females 
Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor (PAC1) play a critical role in biological processes that mediate stress response and have been implicated in psychological outcome following trauma. Our previous work [Ressler et al. (2011); Nature 470:492–497] demonstrated that a variant, rs2267735, in the gene encoding PAC1 (ADCYAP1R1) is associated with post-traumatic stress disorder (PTSD) in a primarily African-American cohort of highly traumatized females. We sought to extend and replicate our previous finding in a similarly trauma-exposed, replicate sample of 1,160 African-American adult male and female patients. Self-reported psychiatric measures were collected, and DNA was obtained for genetic analysis. Using linear regression models to test for association with PTSD symptom severity under an additive (allelic) model, we found a genotype × trauma interaction in females (P< 0.001), but not males (P> 0.1); however, there was no main effect of genotype as in our previous study. The observed interaction suggests a genetic association that increases with the degree of trauma exposure in females only. This interaction remained significant in females, but not males, after controlling for age (P< 0.001), income (P< 0.01), past substance abuse (P< 0.001), depression severity (P= 0.02), or child abuse (P< 0.0005), and all five combined (P= 0.01). No significant effects of genotype (or interactions) were found when modeling depression severity when controlling for comorbid PTSD symptom severity (P> 0.1), demonstrating the relative specificity of this variant for PTSD symptoms. A meta-analysis with the previously reported African-American samples revealed a strong association between PTSD symptom severity and the interaction between trauma and genotype in females (N = 1424, P< 0.0001).
doi:10.1002/ajmg.b.32145
PMCID: PMC3738001  PMID: 23505260
PACAP; PAC1R; ADCYAP1R1; gene; PTSD; gene × environment; sex differences; anxiety
3.  Sex Dependent Influence of a Functional Polymorphism in Steroid 5-α-Reductase Type 2 (SRD5A2) on Post-Traumatic Stress Symptoms 
A non-synonymous, single nucleotide polymorphism (SNP) in the gene coding for steroid 5-α-reductase type 2 (SRD5A2) is associated with reduced conversion of testosterone to dihydrotestosterone (DHT). Because SRD5A2 participates in the regulation of testosterone and cortisol metabolism, hormones shown to be dysregulated in patients with PTSD, we examined whether the V89L variant (rs523349) influences risk for post-traumatic stress disorder (PTSD). Study participants (N = 1,443) were traumatized African-American patients of low socioeconomic status with high rates of lifetime trauma exposure recruited from the primary care clinics of a large, urban hospital. PTSD symptoms were measured with the post-traumatic stress symptom scale (PSS). Subjects were genotyped for the V89L variant (rs523349) of SRD5A2. We initially found a significant sex-dependent effect of genotype in male but not female subjects on symptoms. Associations with PTSD symptoms were confirmed using a separate internal replication sample with identical methods of data analysis, followed by pooled analysis of the combined samples (N = 1,443, sex × genotype interaction P < 0.002; males: n = 536, P < 0.001). These data support the hypothesis that functional variation within SRD5A2 influences, in a sex-specific way, the severity of post-traumatic stress symptoms and risk for diagnosis of PTSD.
doi:10.1002/ajmg.b.32147
PMCID: PMC3770127  PMID: 23505265
trauma; African-American; genetic; testosterone; cortisol; male; civilian; human; PTSD
4.  Family environment and adult resilience: contributions of positive parenting and the oxytocin receptor gene 
European Journal of Psychotraumatology  2013;4:10.3402/ejpt.v4i0.21659.
Background
Abundant research shows that childhood adversity increases the risk for adult psychopathology while research on influences of positive family environment on risk for psychopathology is limited. Similarly, a growing body of research examines genetic and gene by environment predictors of psychopathology, yet such research on predictors of resilience is sparse.
Objectives
We examined the role of positive factors in childhood family environment (CFE) and the OXTR rs53576 genotype in predicting levels of adult resilient coping and positive affect. We also examined whether the relationship between positive factors in the CFEs and adult resilient coping and positive affect varied across OXTR rs53576 genotype.
Methods
We gathered self-report data on childhood environment, trauma history, and adult resilience and positive affect in a sample of 971 African American adults.
Results
We found that positive CFE was positively associated with higher levels of resilient coping and positive affect in adulthood after controlling for childhood maltreatment, other trauma, and symptoms of posttraumatic stress disorder. We did not find a direct effect of OXTR 53576 on a combined resilient coping/positive-affect-dependent variable, but we did find an interaction of OXTR rs53576 with family environment.
Conclusions
Our data suggest that even in the face of adversity, positive aspects of the family environment may contribute to resilience. These results highlight the importance of considering protective developmental experiences and the interaction of such experiences with genetic variants in risk and resilience research.
doi:10.3402/ejpt.v4i0.21659
PMCID: PMC3778209  PMID: 24058725
Childhood maltreatment; abuse; family environment; resilience; positive affect; Connor–Davidson Resilience Scale (CD-RISC); oxytocin; OXTR; rs53576
5.  Inhibition of fear is differentially associated with cycling estrogen levels in women 
Background
Although the prevalence of posttraumatic stress disorder (PTSD) is twice as high in women as it is in men, the role of estrogen in the risk for PTSD is not well understood. Deficits in fear inhibition and impaired safety signal learning may be biomarkers for PTSD. We examined menstrual cycle phase and serum estradiol levels in naturally cycling women while they were undergoing a novel conditioned inhibition procedure that measured their ability to discriminate between cues representing danger versus safety and to inhibit fear in the presence of safety cues.
Methods
Sample 1 included healthy participants in whom we compared inhibition of fear-potentiated startle during the follicular (lower estrogen) and luteal (higher estrogen) phases of the menstrual cycle. We used the same paradigm in a traumatized clinical population (sample 2) in whom we compared low versus high estradiol levels.
Results
In both samples, we found that lower estrogen in cycling women was associated with impaired fear inhibition.
Limitations
In the clinical sample, the low estradiol group was on average older than the high estradiol group owing to the random recruitment approach; we did not exclude participants based on hormonal status or menopause.
Conclusion
Our results suggest that the lower estrogen state during normal menstrual cycling may contribute to risk for anxiety disorders through dysregulated fear responses.
doi:10.1503/jpn.120129
PMCID: PMC3756118  PMID: 23611176
6.  A C. elegans homolog of Huntingtin-Associated Protein 1 is Expressed in Chemosensory Neurons and in a Number of Other Somatic Cell Types 
Huntingtin Associated Protein 1 (HAP1) is a binding partner for huntingtin, the protein responsible for Huntington’s Disease. In mammals, HAP1 is mostly found in brain where it is expressed in neurons. Although several functions have been proposed for HAP1, its role has not yet been clearly established. Here we report on the identification of a HAP1 C.elegans homolog called T27A3.1. T27A3.1 shows conservation with rat and human HAP1 as well as with Milton, a Drosophila HAP1 homolog. To determine the cellular expression of T27A3.1 (multiple isoforms; a-e), we generated several transgenic worm lines expressing a fluorescent reporter protein (GFP and DsRed2) or full length T27A3.1a/c isoforms fused to GFP under the control of the promoter for T27A3.1. We have found that T27A3.1 is expressed in many cell types including a subset of chemosensory neurons in the head and tail. These include the amphid chemosensory neurons ASKL and R, ASIL and R, ADFL and ASEL; the phasmid neurons PHBL and R; and the CAN neurons which are required for worm survival. Furthermore, we show that the subcellular localization of T27A3.1a/c resemble that of mammalian HAP1 and that T27A3.1a/c localize to stigmoid body like structures.
doi:10.1007/s12031-008-9109-z
PMCID: PMC3748950  PMID: 18592415
Huntington; localization; amphid; phasmid; stigmoid body
7.  Acute and Posttraumatic Stress Symptoms in a Prospective Gene × Environment Study of a University Campus Shooting 
Archives of general psychiatry  2011;69(1):89-97.
Context
The serotonin transporter (SLC6A4) has been associated with several stress-related syndromes including posttraumatic stress disorder (PTSD). The ability to detect meaningful associations is largely dependent on reliable measures of preexisting trauma.
Objective
To study the association of genetic variants within SLC6A4 with acute and posttraumatic stress symptoms in a civilian cohort with known levels of preexisting trauma and PTSD symptoms collected prior to a shared index traumatic event.
Design
Ongoing longitudinal study.
Setting
On February 14, 2008, a lone gunman shot multiple people on the campus of Northern Illinois University in DeKalb, Illinois, killing 5 and wounding 21. As part of an ongoing longitudinal study on that campus, a cohort of female undergraduate students, interviewed prior to the shooting, completed follow-up trauma-related measures including PTSD symptom severity (follow-up survey was launched 17 days postshooting; n=691). To obtain DNA, salivary samples were collected from a subset of the original study population based on willingness to participate (n=276).
Participants
Two hundred four undergraduate women.
Main Outcome Measures
SLC6A4 polymorphisms STin2, 5-HTTLPR, and rs25531 were genotyped in 235 individuals.
Results
We found that although the STin2 variant and 5-HTTLPR alone did not associate with increased PTSD symptoms, rs25531 and the 5-HTTLPRmultimarker genotype (combined 5-HTTLPR and rs25531) were associated with significantly increased acute stress disorder symptoms at 2 to 4 weeks postshooting (n = 161; P<.05). This association remained significant when controlling for race and for level of shooting exposure (n = 123; P<.007). The association was most robust with the 5-HTTLPR multimarker genotype and avoidance symptoms (P=.003).
Conclusion
These data suggest that differential function of the serotonin transporter may mediate differential response to a severe trauma. When examined in a relatively homogenous sample with shared trauma and known prior levels of child and adult trauma, the 5-HTTLPR multimarker genotype may serve as a useful predictor of risk for PTSD-related symptoms in the weeks and months following the trauma.
doi:10.1001/archgenpsychiatry.2011.109
PMCID: PMC3738299  PMID: 21893641
8.  Using Polymorphisms in FKBP5 to Define Biologically Distinct Subtypes of Posttraumatic Stress Disorder 
Archives of general psychiatry  2011;68(9):901-910.
Context
Polymorphisms in the gene encoding the glucocorticoid receptor (GR) regulating co-chaperone FKBP5 have been shown to alter GR sensitivity and are associated with an increased risk to develop posttraumatic stress disorder (PTSD).
Objective
To investigate interactions of the FKBP5 single-nucleotide polymorphism rs9296158 and PTSD symptoms on baseline cortisol level, low-dose dexamethasone suppression, and whole-blood gene expression.
Design
Association of FKBP5 genotypes and PTSD symptoms with endocrine measures and genome-wide expression profiles.
Setting
Waiting rooms of general medical and gynecological clinics of an urban hospital at Emory University.
Participants
The 211 participants were primarily African American (90.05%) and of low socioeconomic status and had high rates of trauma and PTSD.
Main Outcome Measures
Baseline and post–dexamethasone suppression cortisol measures and gene expression levels.
Results
In our endocrine study, we found that only risk allele A carriers of rs9296158 showed GR supersensitivity with PTSD; in contrast, baseline cortisol levels were decreased in PTSD only in patients with the GG genotype. Expression of 183 transcripts was significantly correlated with PTSD symptoms after multiple testing corrections. When adding FKBP5 genotype and its interaction with PTSD symptoms, expression levels of an additional 32 genes were significantly regulated by the interaction term. Within these 32 genes, previously reported PTSD candidates were identified, including FKBP5 and the IL18 and STAT pathways. Significant overrepresentation of steroid hormone transcription factor binding sites within these 32 transcripts was observed, highlighting the fact that the earlier-described genotype and PTSD-dependent differences in GR sensitivity could drive the observed gene expression pattern. Results were validated by reverse transcriptase–polymerase chain reaction and replicated in an independent sample (N=98).
Conclusions
These data suggest that the inheritance of GR sensitivity–moderating FKBP5 polymorphisms can determine specific types of hypothalamic-pituitary-adrenal axis dysfunction within PTSD, which are also reflected in gene-expression changes of a subset of GR-responsive genes. Thus, these findings indicate that functional variants in FKBP5 are associated with biologically distinct subtypes of PTSD.
doi:10.1001/archgenpsychiatry.2011.50
PMCID: PMC3686481  PMID: 21536970
9.  Estrogen Levels Are Associated with Extinction Deficits in Women with Posttraumatic Stress Disorder 
Biological psychiatry  2012;72(1):19-24.
Background
Women are twice as likely to develop posttraumatic stress disorder (PTSD) than men. As shown in our previous work, the inability to suppress fear responses in safe conditions may be a biomarker for PTSD. Low estrogen in naturally cycling women is associated with deficits in fear extinction. On the basis of these findings, we have now examined the influence of estrogen levels on fear extinction in women with and without PTSD.
Methods
We measured fear-potentiated startle during fear conditioning and extinction in women. The study sample (N = 81) was recruited from an urban, highly traumatized civilian population at Grady Memorial Hospital in Atlanta, Georgia. We assayed serum estrogen levels and used a median split to divide the sample into high and low estradiol (E2) groups. Seventeen of 41 women (41.5%) in the low E2 group and 15 of 40 women (37.5%) met criteria for PTSD in the high E2 group.
Results
The results showed that all groups had equivalent levels of fear conditioning. However, we found significant interaction effects between high versus low E2 groups and PTSD diagnosis [F(1,71) = 4.55, p < .05] on extinction. Among women with low estrogen levels, fear-potentiated startle was higher during extinction in the PTSD group compared with traumatized control women [F(1,38) = 5.04, p < .05]. This effect was absent in the High E2 group.
Conclusion
This study suggests that low estrogen may be a vulnerability factor for development of PTSD in women with trauma histories. Research on the role of estrogen in fear regulation may provide insight into novel treatment strategies for PTSD.
doi:10.1016/j.biopsych.2012.02.031
PMCID: PMC3675159  PMID: 22502987
Anxiety disorders; estrogen; fear extinction; fear-potentiated startle; PTSD; trauma
10.  Differential Genetic and Epigenetic Regulation of catechol-O-methyltransferase is Associated with Impaired Fear Inhibition in Posttraumatic Stress Disorder 
The catechol-O-methyltransferase (COMT) enzyme is critical for the catabolic regulation of synaptic dopamine, resulting in altered cortical functioning. The COMT Val158Met polymorphism has been implicated in human mental illness, with Met/Met homozygotes associated with increased susceptibility to posttraumatic stress disorder (PTSD). Our primary objective was to examine the intermediate phenotype of fear inhibition in PTSD stratified by COMT genotype (Met/Met, Val/Met, and Val/Val) and differential gene regulation via methylation status at CpG sites in the COMT promoter region. More specifically, we examined the potential interaction of COMT genotype and PTSD diagnosis on fear-potentiated startle during fear conditioning and extinction and COMT DNA methylation levels (as determined using genomic DNA isolated from whole blood). Participants were recruited from medical and gynecological clinics of an urban hospital in Atlanta, GA, USA. We found that individuals with the Met/Met genotype demonstrated higher fear-potentiated startle to the CS− (safety signal) and during extinction of the CS+ (danger signal) compared to Val/Met and Val/Val genotypes. The PTSD+ Met/Met genotype group had the greatest impairment in fear inhibition to the CS− (p = 0.006), compared to Val carriers. In addition, the Met/Met genotype was associated with DNA methylation at four CpG sites, two of which were associated with impaired fear inhibition to the safety signal. These results suggest that multiple differential mechanisms for regulating COMT function – at the level of protein structure via the Val158Met genotype and at the level of gene regulation via differential methylation – are associated with impaired fear inhibition in PTSD.
doi:10.3389/fnbeh.2013.00030
PMCID: PMC3622057  PMID: 23596403
catechol-O-methyltransferase; fear-potentiated startle; posttraumatic stress disorder; epigenetic; methylation; trauma
11.  Linkage analysis of plasma dopamine β-hydroxylase activity in families of patients with schizophrenia 
Human genetics  2011;130(5):635-643.
Dopamine β-hydroxylase (DβH) catalyzes the conversion of dopamine to norepinephrine. DβH enters the plasma after vesicular release from sympathetic neurons and the adrenal medulla. Plasma DβH activity (pDβH) varies widely among individuals, and genetic inheritance regulates that variation. Linkage studies suggested strong linkage of pDβH to ABO on 9q34, and positive evidence for linkage to the complement fixation locus on 19p13.2-13.3. Subsequent association studies strongly supported DBH, which maps adjacent to ABO, as the locus regulating a large proportion of the heritable variation in pDβH. Prior studies have suggested that variation in pDβH, or genetic variants at DβH, associate with differences in expression of psychotic symptoms in patients with schizophrenia and other idiopathic or drug-induced brain disorders, suggesting that DBH might be a genetic modifier of psychotic symptoms. As a first step toward investigating that hypothesis, we performed linkage analysis on pDβH in patients with schizophrenia and their relatives. The results strongly confirm linkage of markers at DBH to pDβH under several models (maximum multipoint LOD score, 6.33), but find no evidence to support linkage anywhere on chromosome 19. Accounting for the contributions to the linkage signal of three SNPs at DBH, rs1611115, rs1611122, and rs6271 reduced but did not eliminate the linkage peak, whereas accounting for all SNPs near DBH eliminated the signal entirely. Analysis of markers genome-wide uncovered positive evidence for linkage between markers at chromosome 20p12 (multi-point LOD = 3.1 at 27.2 cM). The present results provide the first direct evidence for linkage between DBH and pDβH, suggest that rs1611115, rs1611122, rs6271 and additional unidentified variants at or near DBH contribute to the genetic regulation of pDβH, and suggest that a locus near 20p12 also influences pDβH.
doi:10.1007/s00439-011-0989-6
PMCID: PMC3193571  PMID: 21509519
12.  Differential Immune System DNA Methylation and Cytokine Regulation in Post-Traumatic Stress Disorder 
American Journal of Medical Genetics  2011;156B(6):700-708.
DNA methylation may mediate persistent changes in gene function following chronic stress. To examine this hypothesis, we evaluated African American subjects matched by age and sex, and stratified into four groups by post-traumatic stress disorder (PTSD) diagnosis and history of child abuse. Total Life Stress (TLS) was also assessed in all subjects. We evaluated DNA extracted from peripheral blood using the HumanMethylation27 BeadChip and analyzed both global and site-specific methylation. Methylation levels were examined for association with PTSD, child abuse history, and TLS using a linear mixed model adjusted for age, sex, and chip effects. Global methylation was increased in subjects with PTSD. CpG sites in five genes (TPR, CLEC9A, APC5, ANXA2, and TLR8) were differentially methylated in subjects with PTSD. Additionally, a CpG site in NPFFR2 was associated with TLS after adjustment for multiple testing. Notably, many of these genes have been previously associated with inflammation. Given these results and reports of immune dysregulation associated with trauma history, we compared plasma cytokine levels in these subjects and found IL4, IL2, and TNFα levels associated with PTSD, child abuse, and TLS. Together, these results suggest that psychosocial stress may alter global and gene-specific DNA methylation patterns potentially associated with peripheral immune dysregulation. Our results suggest the need for further research on the role of DNA methylation in stress-related illnesses.
doi:10.1002/ajmg.b.31212
PMCID: PMC3292872  PMID: 21714072
PTSD; epigenetic; total life stress; TPR; APC5; TLR8; NPFFR2
13.  Genotype-controlled analysis of serum dopamine β-hydroxylase activity in civilian Post-traumatic Stress Disorder 
Background
Norepinephrine (NE) plays a central role in post-traumatic stress disorder (PTSD). Dopamine β-hydroxylase (DβH) converts dopamine (DA) to NE and its activity varies widely across individuals. Mustapic et al. (2007) reported a PTSD-associated deficit in serum DβH activity (sDβH) in a genotype-controlled analysis of combat veterans. We tested whether such a deficit would occur in a sample of civilians.
Methods
The severity of current adult PTSD symptoms and current DSM-IV diagnosis of PTSD were determined by the PTSD Symptom Scale (PSS). Adulthood trauma exposure was assessed using the Traumatic Experience Inventory (TEI). sDβH was assayed by HPLC with electrochemical detection and genotypes were determined using the Taqman® platform.
Results
Two hundred and twenty seven African American (AA) subjects were enrolled in this study, with a mean age (± SD) of 42.9 (±12.9) years. We found a strong association between rs1611115 genotype and sDβH (p<0.0001). After controlling for adulthood trauma exposure, there were no significant differences of sDβH between subjects who met a PTSD diagnosis and those who did not (p>0.05) in any genotype group. No significant correlations were found between sDβH and PTSD severity, but sDβH significantly associated with the status of comorbid depression based on the cutoff of HAMD (p=0.014) in subjects with PTSD.
Conclusions
We have replicated in this sample the prior finding that DBH rs1611115 genotype strongly associates with sDβH. No associations between sDβH and PTSD diagnosis or symptom severity in this civilian sample.
doi:10.1016/j.pnpbp.2010.07.002
PMCID: PMC2974949  PMID: 20621148
post-traumatic stress disorder; serum dopamine β-hydroxylase; genotype; depression; civilian trauma; association
14.  Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor 
Nature  2011;470(7335):492-497.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to broadly regulate the cellular stress response. In contrast, it is unclear if the PACAP/PAC1 receptor pathway has a role in human psychological stress responses, such as posttraumatic stress disorder (PTSD). In heavily traumatized subjects, we find a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females (N=64, replication N=74, p<0.005). Using a tag-SNP genetic approach (44 single nucleotide polymorphisms, SNPs) spanning the PACAP (ADCYAP1) and PAC1 (ADCYAP1R1) genes, we find a sex-specific association with PTSD. rs2267735, a SNP in a putative estrogen response element within ADCYAP1R1, predicts PTSD diagnosis and symptoms in females only (combined initial and replication samples: N=1237; p<2x10−5). This SNP also associates with fear discrimination and with ADCYAP1R1 mRNA expression. Methylation of ADCYAP1R1 is also associated with PTSD (p < 0.001). Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP/PAC1 pathway are involved in abnormal stress responses underlying PTSD. These sex-specific effects may occur via estrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.
doi:10.1038/nature09856
PMCID: PMC3046811  PMID: 21350482
15.  Polymorphisms in CRHR1 and the serotonin transporter loci: gene x gene x environment interactions on depressive symptoms 
Background
Gene x environment (GxE) interactions mediating depressive symptoms have been separately identified in the stress-sensitive serotonergic (5-HTTLPR) and corticotropin-releasing hormone (CRHR1) systems. Our objective was to examine whether the effects of child abuse are moderated by gene x gene (GxG) interactions between CRHR1 and 5-HTTLPR polymorphisms.
Methods
We used an association study examining GxGxE interactions of CRHR1 and 5-HTTLPR polymorphisms and measures of child abuse on adult depressive symptomatology. The participant population (N=1392) was African-American, of low socioeconomic status (60% with <$1000/month family income), and with high rates of childhood and lifetime trauma. Depressive symptoms were measured with Beck Depression Inventory (BDI) and history of Major Depression by Structure Clinical Interview based on DSM-IV (SCID).
Results
We first replicated an interaction of child abuse and 5-HTTLPR on lifetime SCID diagnosis of major depression in a subsample (N= 236) of the study population – the largest African American 5-HTTLPR cohort reported to date. We then extended our previously reported interaction with both a CRHR1 SNP (rs110402) and TCA haplotype interacting with child abuse to predict current symptoms (N=1059; p = 0.0089). We found that the 5-HTTLPR S allele interacted with CRHR1 haplotypes and child abuse to predict current depressive symptoms (N = 856, p = 0.016).
Conclusions
These data suggest that GxE interactions predictive of depressive symptoms may be differentially sensitive to levels of childhood trauma, and the effects of child abuse are moderated by genetic variation at both the CRHR1 and 5-HTTLPR loci and by their GxG interaction.
doi:10.1002/ajmg.b.31052
PMCID: PMC2924813  PMID: 20029939
Child Abuse; Childhood Maltreatmet; Trauma; Depression; PTSD; Genetic; risk factor
16.  CSN-5, a Component of the COP9 Signalosome Complex, Regulates the Levels of UNC-96 and UNC-98, Two Components of M-lines in Caenorhabditis elegans Muscle 
Molecular Biology of the Cell  2009;20(15):3608-3616.
In Caenorhabditis elegans two M-line proteins, UNC-98 and UNC-96, are involved in myofibril assembly and/or maintenance, especially myosin thick filaments. We found that CSN-5, a component of the COP9 signalosome complex, binds to UNC-98 and -96 using the yeast two-hybrid method. These interactions were confirmed by biochemical methods. The CSN-5 protein contains a Mov34 domain. Although one other COP9 signalosome component, CSN-6, also has a Mov34 domain, CSN-6 did not interact with UNC-98 or -96. Anti-CSN-5 antibody colocalized with paramyosin at A-bands in wild type and colocalized with abnormal accumulations of paramyosin found in unc-98, -96, and -15 (encodes paramyosin) mutants. Double knockdown of csn-5 and -6 could slightly suppress the unc-96 mutant phenotype. In the double knockdown of csn-5 and -6, the levels of UNC-98 protein were increased and the levels of UNC-96 protein levels were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Taken together, we found that CSN-5 functions in muscle cells to regulate UNC-98 and -96, two M-line proteins.
doi:10.1091/mbc.E09-03-0208
PMCID: PMC2719578  PMID: 19535455
17.  unc-94 encodes a tropomodulin in C. elegans 
Journal of molecular biology  2007;374(4):936-950.
unc-94 is one of about 40 genes in C. elegans, that when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size, and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some, abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform-a; sf20, has a stop codon in exon 5, which is shared by both isoform-a and isoform-b. The use of promoter-GFP constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles, and in spermatheca and intestinal epithelial cells. By western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell to cell boundaries.
doi:10.1016/j.jmb.2007.10.005
PMCID: PMC2175264  PMID: 17976644
striated muscle; myofibrils; thin filaments; tropomodulin; C. elegans
18.  A Novel Protein Phosphatase is a Binding Partner for the Protein Kinase Domains of UNC-89 (Obscurin) in Caenorhabditis elegans 
Molecular Biology of the Cell  2008;19(6):2424-2432.
Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.
doi:10.1091/mbc.E08-01-0053
PMCID: PMC2397297  PMID: 18337465
19.  Association of FKBP5 Polymorphisms and Childhood Abuse With Risk of Posttraumatic Stress Disorder Symptoms in Adults 
Context
In addition to trauma exposure, other factors contribute to risk for development of posttraumatic stress disorder (PTSD) in adulthood. Both genetic and environmental factors are contributory, with child abuse providing significant risk liability.
Objective
To increase understanding of genetic and environmental risk factors as well as their interaction in the development of PTSD by gene × environment interactions of child abuse, level of non–child abuse trauma exposure, and genetic polymorphisms at the stress-related gene FKBP5.
Design, Setting, and Participants
A cross-sectional study examining genetic and psychological risk factors in 900 non psychiatric clinic patients (762 included for all genotype studies) with significant levels of childhood abuse as well as non–child abuse trauma using a verbally presented survey combined with single-nucleotide polymorphism (SNP) genotyping. Participants were primarily urban, low-income, black (>95%) men and women seeking care in the general medical care and obstetrics-gynecology clinics of an urban public hospital in Atlanta, Georgia, between 2005 and 2007.
Main Outcome Measures
Severity of adult PTSD symptomatology, measured with the modified PTSD Symptom Scale, non–child abuse (primarily adult) trauma exposure and child abuse measured using the traumatic events inventory and 8 SNPs spanning the FKBP5 locus.
Results
Level of child abuse and non–child abuse trauma each separately predicted level of adult PTSD symptomatology (mean [SD], PTSD Symptom Scale for no child abuse, 8.03 [10.48] vs ≥2 types of abuse, 20.93 [14.32]; and for no non–child abuse trauma, 3.58 [6.27] vs ≥4 types, 16.74 [12.90]; P<.001). Although FKBP5 SNPs did not directly predict PTSD symptom outcome or interact with level of non–child abuse trauma to predict PTSD symptom severity, 4 SNPs in the FKBP5 locus significantly interacted (rs9296158, rs3800373, rs1360780, and rs9470080; minimum P=.0004) with the severity of child abuse to predict level of adult PTSD symptoms after correcting for multiple testing. This gene × environment interaction remained significant when controlling for depression severity scores, age, sex, levels of non–child abuse trauma exposure, and genetic ancestry. This genetic interaction was also paralleled by FKBP5 genotype-dependent and PTSD-dependent effects on glucocorticoid receptor sensitivity, measured by the dexamethasone suppression test.
Conclusions
Four SNPs of the FKBP5 gene interacted with severity of child abuse as a predictor of adult PTSD symptoms. There were no main effects of the SNPs on PTSD symptoms and no significant genetic interactions with level of non–child abuse trauma as predictor of adult PTSD symptoms, suggesting a potential gene-childhood environment interaction for adult PTSD.
doi:10.1001/jama.299.11.1291
PMCID: PMC2441757  PMID: 18349090
20.  UNC-98 and UNC-96 Interact with Paramyosin to Promote Its Incorporation into Thick Filaments of Caenorhabditis elegans 
Molecular Biology of the Cell  2008;19(4):1529-1539.
Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.
doi:10.1091/mbc.E07-07-0723
PMCID: PMC2291434  PMID: 18256289
21.  Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle 
Molecular Biology of the Cell  2007;18(11):4317-4326.
By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.
doi:10.1091/mbc.E07-03-0278
PMCID: PMC2043538  PMID: 17761533
22.  UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle 
The Journal of Cell Biology  2006;175(6):853-859.
Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion–like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of β-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.
doi:10.1083/jcb.200608043
PMCID: PMC2064695  PMID: 17158957
23.  Caenorhabditis elegans UNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments 
Molecular Biology of the Cell  2006;17(9):3832-3847.
To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent “needles.” By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel ∼47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.
doi:10.1091/mbc.E06-02-0144
PMCID: PMC1593161  PMID: 16790495
24.  Caenorhabditis elegans UNC-98, a C2H2 Zn Finger Protein, Is a Novel Partner of UNC-97/PINCH in Muscle Adhesion ComplexesD⃞ 
Molecular Biology of the Cell  2003;14(6):2492-2507.
To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.
doi:10.1091/mbc.E02-10-0676
PMCID: PMC194897  PMID: 12808046
25.  Polymorphisms in CRHR1 and the Serotonin Transporter Loci: Gene × Gene × Environment Interactions on Depressive Symptoms 
American Journal of Medical Genetics  2009;153B(3):812-824.
Gene × environment (G × E) interactions mediating depressive symptoms have been separately identified in the stress-sensitive serotonergic (5-HTTLPR) and corticotropin-releasing hormone (CRHR1) systems. Our objective was to examine whether the effects of child abuse are moderated by gene × gene (G × G) interactions between CRHR1 and 5-HTTLPR polymorphisms. We used an association study examining G × G × E interactions of CRHR1 and 5-HTTLPR polymorphisms and measures of child abuse on adult depressive symptomatology. The participant population (N = 1,392) was African-American, of low socioeconomic status (60% with <$1,000/month family income), and with high rates of childhood and lifetime trauma. Depressive symptoms were measured with Beck Depression Inventory (BDI) and history of Major Depression by Structure Clinical Interview based on DSM-IV (SCID). We first replicated an interaction of child abuse and 5-HTTLPR on lifetime SCID diagnosis of major depression in a subsample (N = 236) of the study population—the largest African-American 5-HTTLPR cohort reported to date. We then extended our previously reported interaction with both a CRHR1 SNP (rs110402) and TCA haplotype interacting with child abuse to predict current symptoms (N = 1,059; P = 0.0089). We found that the 5-HTTLPR S allele interacted with CRHR1 haplotypes and child abuse to predict current depressive symptoms (N = 856, P = 0.016). These data suggest that G × E interactions predictive of depressive symptoms may be differentially sensitive to levels of childhood trauma, and the effects of child abuse are moderated by genetic variation at both the CRHR1 and 5-HTTLPR loci and by their G × G interaction. © 2009 Wiley-Liss, Inc.
doi:10.1002/ajmg.b.31052
PMCID: PMC2924813  PMID: 20029939
child abuse; childhood maltreatment; trauma; depression; PTSD; genetic; risk factor

Results 1-25 (25)