Problematic alcohol use associates with sodium channel and clathrin linker 1 (SCLT1) in trauma-exposed populations.

Excessive alcohol use is extremely prevalent in the United States, particularly among trauma-exposed individuals. While several studies have examined genetic influences on alcohol use and related problems, this has not been studied in the context of trauma-exposed populations. We report results from a genome-wide association study of alcohol consumption and associated problems as measured by the alcohol use disorders identification test (AUDIT) in a trauma-exposed cohort. Results indicate a genome-wide significant association between total AUDIT score and rs1433375 [N = 1036, P = 2.61 × 10-8 (dominant model), P = 7.76 × 10-8 (additive model)], an intergenic single-nucleotide polymorphism located 323 kb upstream of the sodium channel and clathrin linker 1 (SCLT1) at 4q28. rs1433375 was also significant in a meta-analysis of two similar, but independent, cohorts (N = 1394, P = 0.0004), the Marine Resiliency Study and Systems Biology PTSD Biomarkers Consortium. Functional analysis indicated that rs1433375 was associated with SCLT1 gene expression and cortical-cerebellar functional connectivity measured via resting state functional magnetic resonance imaging. Together, findings suggest a role for sodium channel regulation and cerebellar functioning in alcohol use behavior. Identifying mechanisms underlying risk for problematic alcohol use in trauma-exposed populations is critical for future treatment and prevention efforts.

Post-traumatic stress disorder is associated with PACAP and the PAC1 receptor.

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 post-traumatic stress disorder (PTSD). Here we find, in heavily traumatized subjects, a sex-specific association of PACAP blood levels with fear physiology, PTSD diagnosis and symptoms in females. We examined 44 single nucleotide polymorphisms (SNPs) spanning the PACAP (encoded by ADCYAP1) and PAC1 (encoded by ADCYAP1R1) genes, demonstrating a sex-specific association with PTSD. A single SNP in a putative oestrogen response element within ADCYAP1R1, rs2267735, predicts PTSD diagnosis and symptoms in females only. This SNP also associates with fear discrimination and with ADCYAP1R1 messenger RNA expression in human brain. Methylation of ADCYAP1R1 in peripheral blood is also associated with PTSD. Complementing these human data, ADCYAP1R1 mRNA is induced with fear conditioning or oestrogen 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 oestrogen regulation of ADCYAP1R1. PACAP levels and ADCYAP1R1 SNPs may serve as useful biomarkers to further our mechanistic understanding of PTSD.

Childhood maltreatment is associated with distinct genomic and epigenetic profiles in posttraumatic stress disorder.

Childhood maltreatment is likely to influence fundamental biological processes and engrave long-lasting epigenetic marks, leading to adverse health outcomes in adulthood. We aimed to elucidate the impact of different early environment on disease-related genome-wide gene expression and DNA methylation in peripheral blood cells in patients with posttraumatic stress disorder (PTSD). Compared with the same trauma-exposed controls (n = 108), gene-expression profiles of PTSD patients with similar clinical symptoms and matched adult trauma exposure but different childhood adverse events (n = 32 and 29) were almost completely nonoverlapping (98%). These differences on the level of individual transcripts were paralleled by the enrichment of several distinct biological networks between the groups. Moreover, these gene-expression changes were accompanied and likely mediated by changes in DNA methylation in the same loci to a much larger proportion in the childhood abuse (69%) vs. the non-child abuse-only group (34%). This study is unique in providing genome-wide evidence of distinct biological modifications in PTSD in the presence or absence of exposure to childhood abuse. The findings that nonoverlapping biological pathways seem to be affected in the two PTSD groups and that changes in DNA methylation appear to have a much greater impact in the childhood-abuse group might reflect differences in the pathophysiology of PTSD, in dependence of exposure to childhood maltreatment. These results contribute to a better understanding of the extent of influence of differences in trauma exposure on pathophysiological processes in stress-related psychiatric disorders and may have implications for personalized medicine.

Accounting for population stratification in DNA methylation studies.

DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable.

Cross-cultural gene- environment interactions in depression, post-traumatic stress disorder, and the cortisol awakening response: FKBP5 polymorphisms and childhood trauma in South Asia.

Despite increased attention to global mental health, psychiatric genetic research has been dominated by studies in high-income countries, especially with populations of European descent. The objective of this study was to assess single nucleotide polymorphisms (SNPs) in the FKBP5 gene in a population living in South Asia. Among adults in Nepal, depression was assessed with the Beck Depression Inventory (BDI), post-traumatic stress disorder (PTSD) with the PTSD Checklist-Civilian Version (PCL-C), and childhood maltreatment with the Childhood Trauma Questionnaire (CTQ). FKBP5 SNPs were genotyped for 682 participants. Cortisol awakening response (CAR) was assessed in a subsample of 118 participants over 3 days. The FKBP5 tag-SNP rs9296158 showed a main effect on depressive symptoms (p = 0.03). Interaction of rs9296158 and childhood maltreatment predicted adult depressive symptoms (p = 0.02) but not PTSD. Childhood maltreatment associated with endocrine response in individuals homozygous for the A allele, demonstrated by a negative CAR and overall hypocortisolaemia in the rs9296158 AA genotype and childhood maltreatment group (p < 0.001). This study replicated findings related to FKBP5 and depression but not PTSD. Gene-environment studies should take differences in prevalence and cultural significance of phenotypes and exposures into account when interpreting cross-cultural findings.

DNA extracted from saliva for methylation studies of psychiatric traits: evidence tissue specificity and relatedness to brain.

DNA methylation has become increasingly recognized in the etiology of psychiatric disorders. Because brain tissue is not accessible in living humans, epigenetic studies are most often conducted in blood. Saliva is often collected for genotyping studies but is rarely used to examine DNA methylation because the proportion of epithelial cells and leukocytes varies extensively between individuals. The goal of this study was to evaluate whether saliva DNA is informative for studies of psychiatric disorders. DNA methylation (HumanMethylation450 BeadChip) was assessed in saliva and blood samples from 64 adult African Americans. Analyses were conducted using linear regression adjusted for appropriate covariates, including estimated cellular proportions. DNA methylation from brain tissues (cerebellum, frontal cortex, entorhinal cortex, and superior temporal gyrus) was obtained from a publically available dataset. Saliva and blood methylation was clearly distinguishable though there was positive correlation overall. There was little correlation in CpG sites within relevant candidate genes. Correlated CpG sites were more likely to occur in areas of low CpG density (i.e., CpG shores and open seas). There was more variability in CpG sites from saliva than blood, which may reflect its heterogeneity. Finally, DNA methylation in saliva appeared more similar to patterns from each of the brain regions examined overall than methylation in blood. Thus, this study provides a framework for using DNA methylation from saliva and suggests that DNA methylation of saliva may offer distinct opportunities for epidemiological and longitudinal studies of psychiatric traits.

A genome-wide identified risk variant for PTSD is a methylation quantitative trait locus and confers decreased cortical activation to fearful faces.

Genetic factors appear to be highly relevant to predicting differential risk for the development of post-traumatic stress disorder (PTSD). In a discovery sample, we conducted a genome-wide association study (GWAS) for PTSD using a small military cohort (Systems Biology PTSD Biomarkers Consortium; SBPBC, N = 147) that was designed as a case-controlled sample of highly exposed, recently returning veterans with and without combat-related PTSD. A genome-wide significant single nucleotide polymorphism (SNP), rs717947, at chromosome 4p15 (N = 147, ß = 31.34, P = 1.28 × 10(-8) ) was found to associate with the gold-standard diagnostic measure for PTSD (the Clinician Administered PTSD Scale). We conducted replication and follow-up studies in an external sample, a larger urban community cohort (Grady Trauma Project, GTP, N = 2006), to determine the robustness and putative functionality of this risk variant. In the GTP replication sample, SNP rs717947 associated with PTSD diagnosis in females (N = 2006, P = 0.005), but not males. SNP rs717947 was also found to be a methylation quantitative trait locus (meQTL) in the GTP replication sample (N = 157, P = 0.002). Further, the risk allele of rs717947 was associated with decreased medial and dorsolateral cortical activation to fearful faces (N = 53, P < 0.05) in the GTP replication sample. These data identify a genome-wide significant polymorphism conferring risk for PTSD, which was associated with differential epigenetic regulation and with differential cortical responses to fear in a replication sample. These results may provide new insight into understanding genetic and epigenetic regulation of PTSD and intermediate phenotypes that contribute to this disorder.

Methylation quantitative trait loci (meQTLs) are consistently detected across ancestry, developmental stage, and tissue type.

BACKGROUND: Individual genotypes at specific loci can result in different patterns of DNA methylation. These methylation quantitative trait loci (meQTLs) influence methylation across extended genomic regions and may underlie direct SNP associations or gene-environment interactions. We hypothesized that the detection of meQTLs varies with ancestral population, developmental stage, and tissue type. We explored this by analyzing seven datasets that varied by ancestry (African American vs. Caucasian), developmental stage (neonate vs. adult), and tissue type (blood vs. four regions of postmortem brain) with genome-wide DNA methylation and SNP data. We tested for meQTLs by constructing linear regression models of methylation levels at each CpG site on SNP genotypes within 50 kb under an additive model controlling for multiple tests. RESULTS: Most meQTLs mapped to intronic regions, although a limited number appeared to occur in synonymous or nonsynonymous coding SNPs. We saw significant overlap of meQTLs between ancestral groups, developmental stages, and tissue types, with the highest rates of overlap within the four brain regions. Compared with a random group of SNPs with comparable frequencies, meQTLs were more likely to be 1) represented among the most associated SNPs in the WTCCC bipolar disorder results and 2) located in microRNA binding sites. CONCLUSIONS: These data give us insight into how SNPs impact gene regulation and support the notion that peripheral blood may be a reliable correlate of physiological processes in other tissues.

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 fearpotentiated 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.

Adcyap1r1 genotype, posttraumatic stress disorder, and depression among women exposed to childhood maltreatment.

BACKGROUND: A growing literature indicates that genetic variation, in combination with adverse early life experiences, shapes risk for later mental illness. Recent work also suggests that molecular variation at the ADCYAP1R1 locus is associated with posttraumatic stress disorder (PTSD) in women. We sought to test whether childhood maltreatment (CM) interacts with ADCYAP1R1 genotype to predict PTSD in women. METHODS: Data were obtained from 495 adult female participants from the Detroit Neighborhood Health Study. Genotyping of rs2267735, an ADCYAP1R1 variant, was conducted via TaqMan assay. PTSD, depression, and CM exposure were assessed via structured interviews. Main and interacting effects of ADCYAP1R1 and CM levels on past month PTSD and posttraumatic stress (PTS) severity were examined using logistic regression and a general linear model, respectively. As a secondary analysis, we also assessed main and interacting effects of ADCYAP1R1 and CM variation on risk of past-month depression diagnosis and symptom severity. RESULTS: No significant main effects were observed for ADCYAP1R1 genotype on either PTSD/PTS severity. In contrast, a significant ADCYAP1R1 × CM interaction was observed for both past month PTSD and PTS severity, with carriers of the "C" allele showing enhanced risk for these outcomes among women exposed to CM. No significant main or interaction effects were observed for past month depression/depression severity. CONCLUSIONS: Genetic variation at the ADCYAP1R1 locus interacts with CM to shape risk of later PTSD, but not depression, among women. The molecular mechanisms contributing to this interaction require further investigation.

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).

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.

Linkage analysis of plasma dopamine ß-hydroxylase activity in families of patients with schizophrenia.

Dopamine ß-hydroxylase (DßH) catalyzes the conversion of dopamine to norepinephrine. DßH enters the plasma after vesicular release from sympathetic neurons and the adrenal medulla. Plasma DßH activity (pDßH) varies widely among individuals, and genetic inheritance regulates that variation. Linkage studies suggested strong linkage of pDßH 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 pDßH. Prior studies have suggested that variation in pDßH, or genetic variants at DßH, 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 pDßH in patients with schizophrenia and their relatives. The results strongly confirm linkage of markers at DBH to pDßH 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 pDßH, suggest that rs1611115, rs1611122, rs6271 and additional unidentified variants at or near DBH contribute to the genetic regulation of pDßH, and suggest that a locus near 20p12 also influences pDßH.

UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle.

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 beta-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.

Association between childhood maltreatment and adult emotional dysregulation in a low-income, urban, African American sample: moderation by oxytocin receptor gene.

The ability to effectively regulate emotions and a secure attachment style are critical for maintaining mental health across the life span. The experience of childhood maltreatment interferes with normal development of emotional regulation and dramatically increases risk for a wide range of psychiatric disorders in adulthood. The central nervous system oxytocin systems are critically involved in mediating social attachment and buffering psychophysiological responses to stress. We therefore investigated the impact of childhood maltreatment and an oxytocin receptor (OXTR) single nucleotide polymorphism (rs53576) and their interaction on emotional dysregulation and attachment style in adulthood in a sample of low-income, African American men and women recruited from primary care clinics of an urban, public hospital. Consistent with prior research, we found that the severity of childhood maltreatment was associated with increased levels of emotional dysregulation in adulthood. Childhood maltreatment was also positively associated with ratings of disorganized/unresolved adult attachment style and negatively associated with ratings of secure adult attachment style. There was no direct association between rs53576 and emotional dysregulation or ratings of adult attachment style. However, there were significant interactions between rs53576 and childhood maltreatment in predicting level of adult emotional dysregulation and attachment style. Specifically, G/G genotype carriers were at risk for increased emotional dysregulation when exposed to three or more categories of childhood abuse. In addition, G/G genotype carriers exhibited enhanced disorganized adult attachment style when exposed to severe childhood abuse compared to A/A and A/G carriers. Our findings suggest that A allele carriers of OXTR rs53576 are resilient against the effects of severe childhood adversity, by protection against emotional dysregulation and disorganized attachment.

Differential immune system DNA methylation and cytokine regulation in post-traumatic stress disorder.

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.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay.

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Rapid development of neutralizing and diagnostic SARS-COV-2 mouse monoclonal antibodies.

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~ 300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nM-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.

Two LIM domain proteins and UNC-96 link UNC-97/pinch to myosin thick filaments in Caenorhabditis elegans muscle.

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.

Polymorphisms in CRHR1 and the serotonin transporter loci: gene x gene x environment interactions on depressive symptoms.

Gene x environment (G x 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 x gene (G x G) interactions between CRHR1 and 5-HTTLPR polymorphisms. We used an association study examining G x G x 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 x 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 x G interaction.