Michael D. Spain

Identification of a biological signature for schizophrenia in serum

Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure serum concentrations of 181 proteins and small molecules in 250 first and recent onset SCZ, 35 major depressive disorder (MDD), 32 euthymic bipolar disorder (BPD), 45 Asperger syndrome and 280 control subjects. Preliminary analysis resulted in identification of a signature comprised of 34 analytes in a cohort of closely matched SCZ (n=71) and control (n=59) subjects. Partial least squares discriminant analysis using this signature gave a separation of 60–75% of SCZ subjects from controls across five independent cohorts. The same analysis also gave a separation of ∼50% of MDD patients and 10–20% of BPD and Asperger syndrome subjects from controls. These results demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This study lays the groundwork for development of a diagnostic test that can be used as an aid for distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric illnesses with overlapping symptoms.

Validation of a Blood-Based Laboratory Test to Aid in the Confirmation of a Diagnosis of Schizophrenia

We describe the validation of a serum-based test developed by Rules-Based Medicine which can be used to help confirm the diagnosis of schizophrenia. In preliminary studies using multiplex immunoassay profiling technology, we identified a disease signature comprised of 51 analytes which could distinguish schizophrenia (n = 250) from control (n = 230) subjects. In the next stage, these analytes were developed as a refined 51-plex immunoassay panel for validation using a large independent cohort of schizophrenia (n = 577) and control (n = 229) subjects. The resulting test yielded an overall sensitivity of 83% and specificity of 83% with a receiver operating characteristic area under the curve (ROC-AUC) of 89%. These 51 immunoassays and the associated decision rule delivered a sensitive and specific prediction for the presence of schizophrenia in patients compared to matched healthy controls.

Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia

Recently, we showed that the circulating levels of insulin-related peptides and the secretory granule protein chromogranin A were increased in small cohorts of first onset schizophrenia patients. Assuming that this effect was associated with impaired insulin signalling, we investigated the possibility that secretion of other hormones is also affected in schizophrenia. Multiplex immunoassay analysis of 21 hormones and hormone-related molecules was carried out using sera from 236 first and recent onset schizophrenia patients and 230 matched controls. Serum concentrations of insulin and chromogranin A were increased in schizophrenia subjects, consistent with our previous study. In addition, we found elevated concentrations of pancreatic polypeptide, prolactin, progesterone and cortisol, and decreased levels of growth hormone. We also found that growth hormone levels were decreased in post-mortem pituitaries obtained from chronic schizophrenia patients. It will be important to determine whether any of these molecules are involved in the pathosphysiology of schizophrenia or if they reflect the associated insulin resistance. We conclude that function of multiple components of the hypothalamic-pituitary-adrenal-gonadal axis may be affected in schizophrenia. This could have important implications for future biomarker discovery efforts and personalized medicine strategies based on patient stratification for the treatment of this debilitating disorder.

Sex-specific serum biomarker patterns in adults with Asperger's syndrome

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviors. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Aspergers syndrome (AS) compared with controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83, respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the ‘extreme male brain’ and androgen theories of autism spectrum conditions. We conclude that to understand the etiology and development of autism spectrum conditions, stratification by sex is essential.

Identification of a blood-based biological signature in subjects with psychiatric disorders prior to clinical manifestation

Abstract Objectives. To determine whether a molecular signature is present in blood of patients with psychiatric disorders before manifestation of symptoms. Methods. Multiplex immunoassay analyses were carried out using serum obtained from two case-control studies of schizophrenia (n = 75) and bipolar disorder (n = 110) patients and their matched controls. The samples were drawn within 1 month before estimated onset of illness. Results. This led to identification of 20 molecules which were altered in pre-schizophrenia and 14 molecules in pre-bipolar disorder subjects compared to controls. Only two of these molecular changes were identical in both data sets and predictive testing confirmed that the biomarker signatures for pre-schizophrenia and pre-bipolar disorder were dissimilar. Conclusion. The present results suggest that there are distinct serum alterations that occur before clinical manifestation of schizophrenia and bipolar disorder. These findings could lead to development of diagnostic tests to help clinical psychiatrists identify and classify vulnerable patients early in the disease process, allowing for earlier and more effective therapeutic intervention.

Analysis of serum and plasma identifies differences in molecular coverage, measurement variability, and candidate biomarker selection

To compare the use of serum and plasma in multiplex immunoassay analyses of 190 proteins and small molecules, and associated molecular pathways. We also tested whether differences between these biofluids can influence the identification of potential biomarkers in a preliminary study comparing bipolar disorder patients with controls.

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

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.

Rapid, economical testing in the clinical laboratory: A new flow cytometry-based multiplex system

Summary Luminex Corp. has developed a multiplex assay system (FlowMetrix) that incorporates the most advanced digital signal processing technology into an established assay platform, enhancing its speed, economy, sensitivity, precision, and throughput. Microsphere-based assays are now multiplexed assays, capable of performing up to 64 discrete tests in a single tube from the same sample at the same time. The flow cytometer becomes an absolute measurement instrument, acquiring and analyzing the data generated by thousands of microspheres every second, and reporting the values in real time. With applications in routine immunoassay and advanced molecular diagnostics, this system should provide significant benefit to the clinical laboratory today and in the future.