Marlis Herberth

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

DNA methylation is an indispensible epigenetic modification required for regulating the expression of mammalian genomes. Immunoprecipitation-based methods for DNA methylome analysis are rapidly shifting the bottleneck in this field from data generation to data analysis, necessitating the development of better analytical tools. In particular, an inability to estimate absolute methylation levels remains a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling. To address this issue, we developed a cross-platform algorithm—Bayesian tool for methylation analysis (Batman)—for analyzing methylated DNA immunoprecipitation (MeDIP) profiles generated using oligonucleotide arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). We developed the latter approach to provide a high-resolution whole-genome DNA methylation profile (DNA methylome) of a mammalian genome. Strong correlation of our data, obtained using mature human spermatozoa, with those obtained using bisulfite sequencing suggest that combining MeDIP-seq or MeDIP-chip with Batman provides a robust, quantitative and cost-effective functional genomic strategy for elucidating the function of DNA methylation.

An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs)

We report a novel resource (methylation profiles of DNA, or mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNA methylation reference profiles of 13 normal somatic tissues, placenta, sperm, and an immortalized cell line, a visualization tool that has been integrated with the Ensembl genome browser and a new algorithm for the analysis of immunoprecipitation-based DNA methylation profiles. We demonstrate the utility of our resource by identifying the first comprehensive genome-wide set of tissue-specific differentially methylated regions (tDMRs) that may play a role in cellular identity and the regulation of tissue-specific genome function. We also discuss the implications of our findings with respect to the regulatory potential of regions with varied CpG density, gene expression, transcription factor motifs, gene ontology, and correlation with other epigenetic marks such as histone modifications.

Increased levels of circulating insulin-related peptides in first-onset, antipsychotic naïve schizophrenia patients

Increased levels of circulating insulin-related peptides in first-onset, antipsychotic naive schizophrenia patients

Peripheral profiling analysis for bipolar disorder reveals markers associated with reduced cell survival

Little is known about the molecular factors that are altered in remitting bipolar disorder (BD) patients. We carried out proteome profiling of peripheral blood mononuclear cells (PBMCs) and serum from BD patients who were not experiencing mania or major depression (euthymia) compared to matched healthy controls using liquid chromatography–mass spectrometry (LC‐MSE) and Multi‐Analyte Profiling (Human Map®) platforms. This resulted in the identification of approximately 60 differentially expressed molecules involved predominantly in cell death/survival pathways. In PBMCs, this was manifested in cytoskeletal and stress response‐associated proteins, whereas most serum analytes were associated with the inflammatory response. The predicted effect of serum analytes on physiological systems was tested by treating PBMCs with serum obtained from the same patients, resulting in reduced cellular survival. These preliminary results suggest that BD patients carry a peripheral fingerprint that has detrimental effects on cell function and that could be used to distinguish BD patients from healthy controls despite being in a remission phase. It is hoped that additional studies of BD patients in the manic and depressed stages could lead to the identification of a molecular fingerprint that could be used for predicting episodic switching and for guiding treatment strategies.

Impaired glycolytic response in peripheral blood mononuclear cells of first onset antipsychotic-naive schizophrenia patients

Little is known about the biological mechanisms underpinning the pathology of schizophrenia. We have analysed the proteome of stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) from schizophrenia patients and controls as a potential model of altered cellular signaling using liquid-chromatography mass spectrometry proteomic profiling. PBMCs from patients and controls were stimulated for 72 h in vitro using staphylococcal enterotoxin B. In total, 18 differentially expressed proteins between first-onset, antipsychotic-naive patients and controls in the unstimulated and stimulated conditions were identified. Remarkably, eight of these proteins were associated with the glycolytic pathway and patient–control differences were more prominent in stimulated compared with unstimulated PBMCs. None of these proteins were altered in chronically ill antipsychotic-treated patients. Non-linear multivariate statistical analysis showed that small subsets of these proteins could be used as a signal for distinguishing first-onset patients from controls with high precision. Functional analysis of PBMCs did not reveal any difference in the glycolytic rate between patients and controls despite increased levels of lactate and the glucose transporter-1, and decreased levels of the insulin receptor in patients. In addition, subjects showed increased serum levels of insulin, consistent with the idea that some schizophrenia patients are insulin resistant. These results show that schizophrenia patients respond differently to PBMC activation and this is manifested at disease onset and may be modulated by antipsychotic treatment. The glycolytic protein signature associated with this effect could therefore be of diagnostic and prognostic value. Moreover, these results highlight the importance of using cells for functional discovery and show that it may not be sufficient to measure protein expression levels in static states.

Elevated S100B levels in schizophrenia are associated with insulin resistance.

Several studies have shown that circulating S100B protein levels are elevated in schizophrenia. This finding has been specifically attributed to glial pathology, as S100B is produced by astroand oligodendroglial cells and is thought to act as a neurotrophic factor with effects on synaptogenesis, dopaminergic and glutamatergic neutrotransmission. However, this study suggests that overweight and insulin resistance may be alternatively responsible. Adipose tissue has already been highlighted by others as an important extracerebral source of S100B, as it contains considerable amounts of S100B. S100B secretion from adipocytes is reduced by insulin and is activated by physiological factors such as stress and fasting. Therefore, weight gain or adipocyte dysfunction, including insulin insensitivity, could offer important alternative explanations for elevated S100B levels, given the increased prevalence of metabolic syndrome in schizophrenic patients and their firstdegree relatives. The assumption of such extracerebral origins of altered S100B levels in schizophrenia is challenged by findings of elevated S100B in cerebrospinal fluid and postmortem brain tissue. However, glial abnormalities in S100B release may also be caused by disrupted energy supply, as suggested by earlier cell culture studies. Accordingly, impaired insulin signaling has been observed in the dorsolateral prefrontal cortex of schizophrenic patients. Consequently, altered peripheral and cerebral glucose usage is an important factor to be studied to clarify its relation to S100B in schizophrenia. Our study thus assessed S100B in both medicated and drug-free schizophrenic patients, along with body mass index (BMI), measures of glucose usage and adipokine levels. After obtaining written informed consent, serum samples were taken from 26 patients with acute paranoid schizophrenia (according to DSM-IV, 17 men/9 women, age 34.7±11.3 years, BMI 27.7±5.7, 16 smokers/10 nonsmokers) and from 32 matched controls (20 men/12 women, age 34.4±10.8, BMI 24.3±3.8, 14 smokers/18 nonsmokers). Eleven patients were unmedicated for X6 weeks; 15 were treated with atypical antipsychotics for 26±21 days. Psychopathology was monitored using the positive and negative syndrome scale. Exclusion criteria consisted of an earlier history of immune diseases, immunomodulating treatment, cancer, chronic terminal disease, cardiovascular disorders, diabetes, substance abuse and severe trauma. Only control cases without a past or family history of neuropsychiatric disorders were included. Blood was collected between 9:00 and 11:00 AM. S100B was measured immunoluminometrically (LIAISON S100, DiaSorin, Dietzenbach, Germany). LINCOplex human adipokine panels A and B (Millipore Corporation, Billerica, MA, USA) were used for the determination of leptin, monocyte chemotactic protein 1, hepatocyte growth factor (HGF), resistin and active plasminogen activator inhibitor 1. Glucose and triglycerides were measured by commercial enzymatic methods. Levels of cotinine, an estimate of tobacco exposure, were quantified by enzymelinked immunosorbent assay (Calbiotech, Spring Valley, CA, USA). Parametric two-tailed tests were applied because of normal distribution of data (Pearson’s correlation, ANCOVA with covariates ‘BMI’ and ‘cotinine level’, unpaired t-test). Schizophrenic patients showed elevated S100B levels (F(1,55) = 6.675, P = 0.012) and impaired insulin sensitivity, as indicated by increased glucose (F(1,55) = 13.785, P < 0.001), C-peptide (F(1,55) = 10.578, P = 0.002) and C-peptide/glucose ratios (F(1,55) = 8.238, P = 0.006). In medicated schizophrenic patients, S100B and BMI were elevated (P = 0.041/P < 0.001), but controls with a BMIX25 were also found to show increased S100B levels (T(1,30) = 2.366, P = 0.025). Moreover, in healthy controls, BMI (r = 0.540, P = 0.001), adipokines (leptin: r = 0.545, P = 0.001, HGF: r = 0.441, P = 0.012, resistin: r = 0.377, P = 0.033) and C-peptide/glucose ratios (r = 0.432, P = 0.014) predicted S100B levels. However, in schizophrenic patients, S100B was elevated in both unmedicated (P = 0.049, closely BMI-matched subgroup: P = 0.028) and medicated patients (P = 0.041). Similarly, the finding of insulin resistance, as assessed by C-peptide/glucose ratios, persisted in unmedicated (P = 0.016, closely BMImatched subgroup: P = 0.015) and medicated patients (P = 0.038). Figure 1 illustrates the results. Our results are suggestive of primary insulin resistance in schizophrenia, resulting in an increased release of S100B from brain and adipose tissue, independent of BMI. The commonly observed weight gain on neuroleptic treatment, however, occurs on the basis of an increased metabolic vulnerability in patients because of medication-independent primary insulin resistance. Future studies in larger samples may focus on drug-naive schizophrenic subjects and the comparison of different standardized treatment regimens to further elucidate the suggested link between S100B and abnormal energy metabolism. Molecular Psychiatry (2010) 15, 3–11 & 2010 Nature Publishing Group All rights reserved 1359-4184/10

Increased alpha-defensins as a blood marker for schizophrenia susceptibility.

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Identification of a molecular profile associated with immune status in first-onset schizophrenia patients.

Schizophrenia is a severe psychotic illness affecting 1% of the general population. There are no consistent pathological features, and the disorder is defined by a complex symptomatology, which overlaps with other psychiatric illnesses. Diagnosis is based on a clinical interview, relying on the patient meeting criteria according to diagnosis manuals, including Diagnostic and Statistical Manual of Mental Disorders, 4th Ed. and International Statistical Classification of Diseases, 10th Revision. Because of the ambiguous symptoms, the diagnostic process can take many months and often years. Rapid and effective treatment has been shown to impact positively on disease progression and outcome, and it is therefore important to identify disease-associated biomarkers allowing early diagnosis. Reliable biomarkers can be used for the development of diagnostic tests and may also help us understand the underlying pathology of this disorder. In the present study, proteins from anti-CD3 stimulated and unstimulated peripheral blood T cell lysates from 15 minimally medicated and unmedicated patients and 15 age-, sex-, race-, and smoking-matched controls were profiled on cation exchange (CM10) chips using SELDI-TOF. Partial least squares discriminate analysis was used to separate patient and control groups according to the expression of 108 detected peaks, and two peaks of 3,374 and 3,450 Da, corresponding to α-defensins based on masses and cationic properties, were found to contribute significantly to the separation of patient and control groups. Reduction of T cell lysates with DTT resulted in a 6-Da shift in the mass of these peaks consistent with the presence of three cysteine bonds in the structure, confirming them as α-defensins. Quantification of α-defensins in T cell lysates from six patients and 18 healthy controls was carried out by ELISA, which also showed that α-defensin levels were significantly increased in patient lysates when compared with matched controls (p = 0.0197). Plasma from 21 monozygotic twins discordant for schizophrenia and eight healthy unaffected twin pairs was also analyzed for the expression of α-defensins by ELISA. Notably both affected and unaffected twins were found to have significantly elevated α-defensin levels compared with healthy control twin pairs (p = 0.0014 and p = 0.0115, respectively). Increased expression of α-defensins in unaffected as well as affected discordant monozygotic twins is of particular interest as monozygotic twins share genes and usually environmental upbringing. The unaffected twin therefore represents the biological and environmental risk of developing schizophrenia in the absence of overt symptomatology and therapeutic medication. These findings suggest that α-defensins could be an important early indicator of the risk of schizophrenia.

Gene body methylation of the dimethylarginine dimethylamino-hydrolase 2 (Ddah2) gene is an epigenetic biomarker for neural stem cell differentiation

OBJECTIVES Alterations in immunological parameters have been reported for schizophrenia although little is known about the effects of inflammatory status on immune-related functional changes at disease onset. Here, we have investigated such T cell-dependent molecular changes in first-onset, antipsychotic-naive schizophrenia patients using a novel ex vivo blood culture system. METHODS Blood samples from patients (n=17) and controls (n=17) were collected into stimulant-containing or null control TruCulture™ tubes, incubated 24 hours and the concentrations of 107 immune and metabolic molecules measured in the conditioned media using the HumanMAP™ immunoassay system. RESULTS Nine molecules showed altered release from schizophrenia blood cells compared to those from controls and this was replicated in an independent cohort. In silico pathway analysis showed that these molecules had roles in endothelial cell function, inflammation, acute phase response and fibrinolysis pathways. Importantly, five of these molecules showed altered release only after stimulation. CONCLUSIONS This study has identified a reproducible peripheral molecular signature associated with altered immune function in first-onset schizophrenia subjects. This suggests that immune status can affect the biomarker profile which could be important for personalized medicine strategies. Furthermore, whole blood culture analysis may be useful in the identification of diagnostic tools or novel treatment strategies due to ease-of-use and clinical accessibility.

Differential effects on T-cell function following exposure to serum from schizophrenia smokers.

DNA methylation is an important epigenetic mark that is involved in the regulation of many cellular processes such as gene expression, genomic imprinting and silencing of repetitive elements. Because of their ability to cause and capture phenotypic plasticity, epigenetic marks such as DNA methylation represent potential biomarkers to distinguish between different types of tissues and stages of differentiation. Here, we have identified differential DNA methylation in the gene body of the nitric oxide inhibitor Ddah2 that discriminates embryonic stem cells from neural stem cells and is positively correlated with differential gene expression.