Andreas Menke

Peripheral blood gene expression: it all boils down to the RNA collection tubes

BackgroundGene expression profiling from peripheral blood is a valuable tool for biomarker discovery in clinical studies. Different whole blood RNA collection and processing methods are highly variable and might confound comparisons of results across studies. The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgene™ and Tempus™ tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals.ResultsRNA quality, yield and numbers of detected transcripts from the two RNA collection systems was comparable, with no significant differences between the tube types. Globin reduction resulted in a significant increase in present call rates (p = 8.17 × 10-5 and p = 1.95 × 10-3 in PAXgene™ and Tempus™ tubes respectively) and significant decrease in gene expression variance in both RNA collection tubes (p = 0.0025 and p = 0.041 in PAXgene™ and Tempus™ tubes respectively). Comparisons of glucocorticoid receptor-stimulated gene expression profiles between the two collection tube systems revealed an overlap of only 17 to 54%, depending on the stringency level of the statistical thresholds. This overlap increased by 1-8% when the RNA samples were processed to remove the globin mRNA.ConclusionRNA obtained from PAXgene™ and Tempus™ tubes was comparable in terms of quality and yield, however, detectable gene expression changes after glucocorticoid receptor stimulation were distinct, with an overlap of only up to 46% between the two collection systems. This overlap increased to 54% when the samples were depleted of globin mRNA and drastically reduced to 17-18% when only gene expression differences with a fold change greater than 2.0 were assessed. These results indicate that gene expression profiles obtained from PAXgene™ and Tempus™ differ drastically and should not be analyzed together. These data suggest that researchers must exert caution while interpreting expression profiles obtained through different RNA collection tubes.

Polymorphisms in GRIK4, HTR2A, and FKBP5 show interactive effects in predicting remission to antidepressant treatment.

Single-nucleotide polymorphisms (SNPs) in the FKBP5, GRIK4, and HTR2A genes have been shown to be associated with response to citalopram treatment in the STAR*D sample, but only associations with FKBP5 have so far been tested in the Munich Antidepressant Response Signature (MARS) project. Response and remission of depressive symptoms after 5 weeks of antidepressant treatment were tested against 82 GRIK4 and 37 HTR2A SNPs. Association analysis was conducted in about 300 depressed patients from the MARS project, 10% of whom had bipolar disorder. The most predictive SNPs from these two genes and rs1360780 in FKBP5 were then genotyped in a total of 387 German depressed in-patients to analyze potential additive and interactive effects of these variants. We could not replicate previous findings of the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study in our sample. Although not statistically significant, the effect for the best GRIK4 SNP of STAR*D (rs1954787, p=0.076, pcorrected=0.98) seemed to be in the same direction. On the other hand, the nominally significant association with the top HTR2A SNPs of STAR*D (rs7997012, allelic, p=0.043, pcorrected=0.62) was with the opposite risk allele. The GRIK4 SNP (rs12800734, genotypic, p=0.0019, pcorrected=0.12) and the HTR2A SNP (rs17288723, genotypic, p=0.0011, pcorrected=0.02), which showed the strongest association with remission in our sample, had not been reported previously. Associations across all genetic markers within the GRIK4 (genotypic, p=0.022) or HTR2A (genotypic, p=0.012) locus using the Fishers product method (FPM) were also significant. In all 374 patients, the best predictive model included a main effect for GRIK4 rs12800734 and two significant interactions between GRIK4 rs12800734 and FKBP5 rs1360780, and GRIK4 rs12800734 and HTR2A rs17288723. This three SNP model explained 13.1% of the variance for remission after 5 weeks (p=0.00051 for the model). Analyzing a sub-sample of 194 patients, plasma ACTH (p=0.002) and cortisol (p=0.021) responses of rs12800734 GG (GRIK4) carriers, who also showed favorable treatment response, were significantly lower in the second combined dexamethasone (dex)/corticotrophin-releasing hormone (CRH) test before discharge compared with the other two genotype groups. Despite large differences in ethnicity and design compared with the STAR*D study, our results from the MARS study further support both independent and interactive involvement of GRIK4, HTR2A and FKBP5 in antidepressant treatment response.

Clinical characteristics and treatment outcome in a representative sample of depressed inpatients – Findings from the Munich Antidepressant Response Signature (MARS) project

Depression is a common and often difficult-to-treat clinical condition with a high rate of patients showing insufficient treatment response and persistence of symptoms. We report the characteristics of a representative sample of depressed inpatients participating in the Munich Antidepressant Response Signature (MARS) project. Eight hundred and forty-two inpatients admitted to a psychiatric hospital for treatment of a major depressive episode, recurrent or bipolar depression were thoroughly characterized with respect to demographic factors, clinical history, and the degree of HPA-axis dysregulation evaluated by means of combined dex/CRH tests, and the predictive value of these factors for treatment outcome is investigated. 80.8% of patients responded to treatment (i.e., improvement in symptom severity of at least 50%) and 57.9% reached remission (i.e., near absence of residual depressive symptoms) at discharge after a mean treatment period of 11.8 weeks. Regression analysis identified early partial response (within 2 weeks) as the most important positive predictor for achieving remission. Previous ineffective treatment trials in the current episode and presence of a migration background are potent negative predictors for treatment outcome. In addition, remitters were characterized by a more pronounced normalization of an initially dysregulated HPA-axis. We could show that a large majority of inpatients suffering from depression benefits from antidepressant treatment during hospitalization. However, a considerable number of patients failed to achieve remission. We demonstrated that this subgroup can be characterized by a set of demographic, clinical and neuroendocrine variables allowing to predict unfavorable outcome at an early stage of treatment.

Genetic variation in FKBP5 associated with the extent of stress hormone dysregulation in major depression

The FK506 binding protein 51 or FKBP5 has been implicated in the regulation of glucocorticoid receptor (GR) sensitivity, and genetic variants in this gene have been associated with mood and anxiety disorders. GR resistance and associated stress hormone dysregulation are among the most robust biological findings in major depression, the extent of which may be moderated by FKBP5 polymorphisms. FKBP5 mRNA expression in peripheral blood cells (baseline and following in vivo GR stimulation with 1.5 mg dexamethasone p.o.) was analyzed together with plasma cortisol, ACTH, dexamethasone levels and the FKBP5 polymorphism rs1360780 in 68 depressed patients and 87 healthy controls. We observed a significant (P = 0.02) interaction between disease status and FKBP5 risk allele carrier status (minor allele T) on GR-stimulated FKBP5 mRNA expression. Patients carrying the risk T allele, but not the CC genotype, showed a reduced induction of FKBP5 mRNA. This FKBP5 polymorphism by disease status interaction was paralleled by the extent of plasma cortisol and ACTH suppression following dexamethasone administration, with a reduced suppression only observed in depressed patients carrying the T allele. Only depressed patients carrying the FKBP5 rs1360780 risk allele showed significant GR resistance compared with healthy controls, as measured by dexamethasone-induced FKBP5 mRNA induction in peripheral blood cells and suppression of plasma cortisol and ACTH concentrations. This finding suggests that endocrine alterations in depressed patients are determined by genetic variants and may allow identification of specific subgroups.

Dexamethasone Stimulated Gene Expression in Peripheral Blood is a Sensitive Marker for Glucocorticoid Receptor Resistance in Depressed Patients

Although gene expression profiles in peripheral blood in major depression are not likely to identify genes directly involved in the pathomechanism of affective disorders, they may serve as biomarkers for this disorder. As previous studies using baseline gene expression profiles have provided mixed results, our approach was to use an in vivo dexamethasone challenge test and to compare glucocorticoid receptor (GR)-mediated changes in gene expression between depressed patients and healthy controls. Whole genome gene expression data (baseline and following GR-stimulation with 1.5 mg dexamethasone p.o.) from two independent cohorts were analyzed to identify gene expression pattern that would predict case and control status using a training (N=18 cases/18 controls) and a test cohort (N=11/13). Dexamethasone led to reproducible regulation of 2670 genes in controls and 1151 transcripts in cases. Several genes, including FKBP5 and DUSP1, previously associated with the pathophysiology of major depression, were found to be reliable markers of GR-activation. Using random forest analyses for classification, GR-stimulated gene expression outperformed baseline gene expression as a classifier for case and control status with a correct classification of 79.1 vs 41.6% in the test cohort. GR-stimulated gene expression performed best in dexamethasone non-suppressor patients (88.7% correctly classified with 100% sensitivity), but also correctly classified 77.3% of the suppressor patients (76.7% sensitivity), when using a refined set of 19 genes. Our study suggests that in vivo stimulated gene expression in peripheral blood cells could be a promising molecular marker of altered GR-functioning, an important component of the underlying pathology, in patients suffering from depressive episodes.

Association of genetic variants in the neurotrophic receptor-encoding gene NTRK2 and a lifetime history of suicide attempts in depressed patients.

CONTEXT A consistent body of evidence supports a role of reduced neurotrophic signaling in the pathophysiology of major depressive disorder (MDD) and suicidal behavior. Especially in suicide victims, lower postmortem brain messenger RNA and protein levels of neurotrophins and their receptors have been reported. OBJECTIVE To determine whether the brain-derived neurotrophic factor (BDNF) gene or its high-affinity receptor gene, receptor tyrosine kinase 2 (NTRK2), confer risk for suicide attempt (SA) and MDD by investigating common genetic variants in these loci. DESIGN Eighty-three tagging single-nucleotide polymorphisms (SNPs) covering the genetic variability of these loci in European populations were assessed in a case-control association design. SETTING Inpatients and screened control subjects. PARTICIPANTS The discovery sample consisted of 394 depressed patients, of whom 113 had SA, and 366 matched healthy control subjects. The replication studies comprised 744 German patients with MDD and 921 African American nonpsychiatric clinic patients, of whom 152 and 119 were positive for SA, respectively. INTERVENTIONS Blood or saliva samples were collected from each participant for DNA extraction and genotyping. MAIN OUTCOME MEASURES Associations of SNPs in BDNF and NTRK2 with SA and MDD. RESULTS Independent SNPs within NTRK2 were associated with SA among depressed patients of the discovery sample that could be confirmed in both the German and African American replication samples. Multilocus interaction analysis revealed that single SNP associations within this locus contribute to the risk of SA in a multiplicative and interactive fashion (P = 4.7 x 10(-7) for a 3-SNP model in the combined German sample). The effect size was 4.5 (95% confidence interval, 2.1-9.8) when patients carrying risk genotypes in all 3 markers were compared with those without any of the 3 risk genotypes. CONCLUSIONS Our results suggest that a combination of several independent risk alleles within the NTRK2 locus is associated with SA in depressed patients, further supporting a role of neurotrophins in the pathophysiology of suicide.

Gene Expression Studies in Major Depression

The dramatic technical advances in methods to measure gene expression on a genome-wide level thus far have not been paralleled by breakthrough discoveries in psychiatric disorders—including major depression (MD)—using these hypothesis-free approaches. In this review, we first describe the methodologic advances made in gene expression analysis, from quantitative polymerase chain reaction to next-generation sequencing. We then discuss issues in gene expression experiments specific to MD, ranging from the choice of target tissues to the characterization of the case group. We provide a synopsis of the gene expression studies published thus far for MD, with a focus on studies using mRNA microarray methods. Finally, we discuss possible new strategies for the gene expression studies in MD that circumvent some of the addressed issues.

Genome-Wide Association Study of Antidepressant Treatment-Emergent Suicidal Ideation

Emergence of suicidal ideation (TESI) during treatment with antidepressants in major depression led to a black box warning. We performed a genome-wide association study to identify genetic markers, which increase the risk for this serious side effect. TESI was evaluated in depressed in-patients (N=397) and defined by an emergence of suicidal thoughts during hospitalization without suicidal thoughts at admission using the suicide item (3) of the Hamilton Depression Rating Scale. Genotype distribution of 405.383 single-nucleotide polymorphisms (SNPs) in patients with TESI (N=32/8.1%) was compared to patients without increase in suicidal ideation (N=329/82.9%) and to a subgroup never reported suicidal ideation (N=79/19.9%). Top results were analyzed in an independent sample (N=501). None variant reached genome-wide significance, the best associated SNP was rs1630535 (p-value=1.3 × 10−7). The top 79 SNPs could be analyzed in an independent sample, and 14 variants showed nominal significant association with the same risk allele in the replication sample. A discriminant analysis classifying patients using these 79 SNPs revealed a 91% probability to classify TESI vs non-TESI cases correctly in the replication sample. Although our data need to be interpreted carefully owing to the small numbers in both cohorts, they suggest that a combination of genetic markers might indeed be used to identify patients at risk for TESI.

Genetic Differences in the Immediate Transcriptome Response to Stress Predict Risk-Related Brain Function and Psychiatric Disorders

Summary Depression risk is exacerbated by genetic factors and stress exposure; however, the biological mechanisms through which these factors interact to confer depression risk are poorly understood. One putative biological mechanism implicates variability in the ability of cortisol, released in response to stress, to trigger a cascade of adaptive genomic and non-genomic processes through glucocorticoid receptor (GR) activation. Here, we demonstrate that common genetic variants in long-range enhancer elements modulate the immediate transcriptional response to GR activation in human blood cells. These functional genetic variants increase risk for depression and co-heritable psychiatric disorders. Moreover, these risk variants are associated with inappropriate amygdala reactivity, a transdiagnostic psychiatric endophenotype and an important stress hormone response trigger. Network modeling and animal experiments suggest that these genetic differences in GR-induced transcriptional activation may mediate the risk for depression and other psychiatric disorders by altering a network of functionally related stress-sensitive genes in blood and brain. Video Abstract

Genetic markers within glutamate receptors associated with antidepressant treatment-emergent suicidal ideation.

ajp.psychiatryonline.org cause of space limitations, we were unable to include several key aspects of clinical care. As educators as well as clinicians, we consistently remind those who we teach about the importance of close communication with school personnel regarding the functioning of our patients, wherever they may be on the spectrum of risk to others. In hindsight, including content that stressed the importance of close communication between the clinician and school personnel could have strengthened our article. We agree that the use of a systematic assessment of violence risk is essential. However, it is our opinion that a reductionistic tendency of systems to rely on only one approach of risk assessment—as we have frequently seen in our own clinical practices—often does not lead to optimal placements for young, school-age patients.