Differences in white blood cell proportions between schizophrenia cases and controls are influenced by medication and variations in time of day

Cases with schizophrenia (SCZ) and healthy controls show differences in white blood cell (WBC) counts and blood inflammation markers. Here, we investigate whether time of blood draw and treatment with psychiatric medications are related to differences in estimated WBC proportions between SCZ cases and controls. DNA methylation data from whole blood was used to estimate proportions of six subtypes of WBCs in SCZ patients (n = 333) and healthy controls (n = 396). We tested the association of case-control status with estimated cell-type proportions and the neutrophil-to-lymphocyte ratio (NLR) in 4 models: with/without adjusting for time of blood draw, and then compared results from blood samples drawn during a 12-h (07:00–19:00) or 7-h (07:00-14:00) period. We also investigated WBC proportions in a subgroup of medication-free patients (n = 51). Neutrophil proportions were significantly higher in SCZ cases (mean=54.1%) vs. controls (mean=51.1%; p = <0.001), and CD8+T lymphocyte proportions were lower in SCZ cases (mean=12.1%) vs. controls (mean=13.2%; p = 0.001). The effect sizes in the 12-h sample (07:00–19:00) showed a significant difference between SCZ vs. controls for neutrophils, CD4+T, CD8+T, and B-cells, which remained significant after adjusting for time of blood draw. In the samples matched for time of blood draw during 07.00–14.00, we also observed an association with neutrophils, CD4+T, CD8+T, and B-cells that was unaffected by further adjustment for time of blood draw. In the medication-free patients, we observed differences that remained significant in neutrophils (p = 0.01) and CD4+T (p = 0.01) after adjusting for time of day. The association of SCZ with NLR was significant in all models (range: p < 0.001 to p = 0.03) in both medicated and unmedicated patients. In conclusion, controlling for pharmacological treatment and circadian cycling of WBC is necessary for unbiased estimates in case-control studies. Nevertheless, the association of WBC with SCZ remains, even after adjusting for the time of day.publishedVersio

Análise da expressão de genes relacionados à depressão em sangue de crianças e adolescentes deprimidos

Major Depressive Disorder (MDD) is a complex disorder caused by interactions among genetic and environmental factors, affecting several biological systems. Evidences have shown that the majority of the first depressive episode happened before adulthood. However, the difficulties in diagnosing MDD in children and adolescents are higher than those found in older individuals, because the current diagnostic criteria were developed for the adult population, neglecting many of the developmental differences between children/adolescents and adults. Our aims was to evaluate the gene expression of 13 depression related genes in blood of children and adolescents with major depressive disorder (MDD group), children and adolescents with depressive symptoms without MDD diagnosis (depressive group) and children and adolescents controls (control group). We also correlated gene expression with global psychopathology (CBCL DSM-oriented scale for affective problems) and compared gene expression between children with MDD diagnosis and higher global psychopathology and children with MDD diagnosis and lower global psychopathology. Gene expression analysis was performed using Taqman Low Density Array (TLDA) technology. The ?Crt genes values of each group were compared using Multivariate General Linear Model (GLM), followed by a Scheffe post-hoc test and a Bonferroni correction for multiple comparisons. The Type I error was set at 5%. We found that GLUL, NR3C1, TNF, TNFR1 and IL1B mRNA levels were decreased in MDD group compared to control and depressive groups. We have not found gene expression differences between depressive and control groups neither between children with higher global psychopathology and children with lower global psychopathology. Moreover, ?Crt values were not correlated with CBCL DSM-oriented score. Although there are few studies that investigated gene expression in blood of adolescent with MDD diagnosis, to our knowledge, this is the first study investigating the gene expression in children with MDD. Our results suggest five genes as a possible genetic biomarkers for MDD diagnosis in childhood and adolescence. Moreover, we have not found correlation between ?Crt values and CBCL DSM-oriented score, suggesting that gene expression were not associated with depressive symptoms, corroborating our results. Furthermore, there are no differences in gene expression children with MDD diagnosis and higher global psychopathology and children with MDD diagnosis and lower global psychopathology. These results suggest that the reduction of gene expression in children with MDD is not state-dependent but a trait-dependent finding in MDD. Therefore, we concluded that the GLUL, NR3C1, TNF, TNFR1 and IL1B gene expression may be a possible genetic biomarkers for MDD diagnosis in childhood and adolescence. However, more studies are necessary to elucidate the role of these genes in children and adolescents with MDD.O Transtorno Depressivo Maior (TDM) é uma doença complexa cujo desenvolvimento depende da interação de fatores genéticos e ambientais. O seu diagnóstico é definido apenas por critérios clínicos, sem referência a nenhum biomarcador ou aspecto fisiopatológico da doença. Estudos mostram que a grande maioria dos primeiros episódios do TDM começam antes da vida adulta. Entretanto, o diagnóstico do TDM em crianças e adolescentes é mais difícil de ser realizado do que em adultos, pois o atual critério foi desenvolvido para a população adulta, negligenciando várias diferenças de desenvolvimento que existem entre essas faixas etárias. Dessa forma, o objetivo geral do presente estudo foi identificar potenciais marcadores genéticos de diagnóstico do TDM e/ou de sintomas depressivos na infância e na adolescência, por meio de: 1) comparação da expressão de 13 genes relacionados à depressão no sangue de 23 crianças e adolescentes com diagnóstico de TDM (grupo com diagnóstico), 49 crianças e adolescentes com sintomas depressivos mas sem diagnóstico da doença (grupo com sintomas de depressão), e 62 crianças e adolescentes controles (grupo controle); 2) comparação da expressão dos mesmos genes entre crianças com diagnóstico de TDM que apresentavam mais sintomas depressivos e crianças com diagnóstico de TDM que apresentavam menos sintomas depressivos, avaliados a partir da subescala de psicopatologia Child Behavior Checklist (CBCL) DSM-oriented para transtornos afetivos (CBCL DSM/TDM); 3) Correlação dos dados de expressão gênica com os escores da CBCL DSM/TDM, tanto na amostra inteira quanto nos grupos separadamente. A análise da expressão gênica foi realizada pela técnica de RT-qPCR em tempo real utilizando os cartões de TaqMan® Low Density Array (TLDA). Observamos que as expressões dos genes GLUL, NR3C1, TNF, TNFR1 e IL1B estão diminuídas no sangue de crianças e adolescentes com diagnóstico de TDM em comparação com as crianças e adolescentes dos grupos controle e com sintomas de depressão. Nenhum dos genes avaliados apresentou expressão diferencial entre os grupos com sintomas de depressão e controle e nem entre os subgrupos de crianças com diagnóstico de TDM. Além disso, não há correlação entre os dados de expressão dos genes analisados e os escores de sintomas da CBCL DSM/TDM na amostra inteira e nos três grupos analisados separadamente. A comparação entre os grupos com diagnóstico com o controle e a comparação entre crianças com sintomas de depressão com crianças controles nos dá possibilidade de investigar potenciais marcadores genéticos de, respectivamente, diagnóstico do TDM e sintomas depressivos na infância e na adolescência. Dessa forma, nossos resultados sugerem 5 genes como potenciais marcadores genéticos de diagnóstico do TDM em crianças e adolescentes. Ainda, esses cinco genes também estão diminuídos nas crianças do grupo com diagnóstico em comparação com as do grupo com sintomas de depressão, confirmando o potencial desses genes como marcador genético de traço da doença (apresentar diagnóstico ou não), uma vez que ambos os grupos apresentam sintomas depressivos mas diferem em relação ao diagnóstico do TDM. Além disso, a não correlação entre os dados de expressão gênica e os escores de psicopatologia da CBCL DSM/TDM indicam que os cinco genes diferencialmente expressos parecem não estar relacionados com os sintomas do TDM. O fato de não termos encontrado diferenças de expressão gênica entre o subgrupos de crianças com diagnóstico de TDM sugerem também que os genes diferencialmente expressos parecem não estar associados ao estado depressivo (estar remitido ou não). Para o nosso conhecimento, embora haja alguns estudos que tenham investigado a expressão gênica em sangue de adolescentes com TDM, este é o primeiro estudo a utilizar sangue de crianças com TDM para caracterizar a expressão de genes relacionados à depressão. Portanto, concluímos que as expressões dos genes GLUL, NR3C1, TNF, TNFR1 e IL1B podem ser potenciais marcadores genéticos de diagnóstico do TDM na infância e na adolescência e parecem não estar relacionados com a sintomatologia do TDM nessa faixa etária. Contudo, mais trabalhos são necessários para confirmar o possível papel desses genes como marcadores genéticos de diagnóstico do TDM no sangue de crianças e adolescentes.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Análises de metilação do DNA e de expressão gênica associados a sintomas psiquiátricos e a fatores ambientais em adolescentes

Psychiatric disorders are complex phenotypes influenced by genetics and environmental factors and by the interaction between them. Studies suggest that early adverse life events can lead to changes in gene expression through epigenetic mechanisms (such as DNA methylation) that, in turn, alter stress reactivity, brain function, and at last the behavior. One way of measuring altered behavior is to assess the levels of psychopathology, i.e., the presence or frequency of psychiatric symptoms. The main aim of this doctoral thesis was to investigate the relationship among early adverse life events, changes in DNA methylation, changes in gene expression and the emergence of dimensional psychopathology in adolescents over a 3-year follow-up. For this purpose, we performed two studies investigating adolescents (n=24) who presented a significant increase in psychopathology after 3 years of follow-up and who had biological samples collected before and after this follow-up. In the study 1, we explored the relationship among psychopathology, DNA methylation and gene expression by an initial screening analysis to identify differentially methylated positions (DMPs) and differentially methylated regions (DMRs) associated with the emergence of psychopathology. We identified 663 DMPs and 90 DMRs associated with the emergence of psychopathology. We observed that 15 DMPs were mapped to genes that were differentially expressed in the blood. Of the DMRs, three genes were differentially expressed: ASCL2, HLA-E and RPS6KB1. In the study 2, we aimed to find a link between life adversities and the emergence of psychopathology, with the hypothesis that life adversities may dysregulate blood gene expression associated with psychopathology through DNA methylation. Life adversity variables were generated using a latent modelling approach with a bifactor structure, considering general and specifics life experiences. We found that adversity factors related to school or health/loss events were associated with changes in DNA methylation of EST1, FYTTD1 e FAM117B genes. These changes in DNA methylation, in turn, were associated with changes in the expression of these genes. Finally, the expression of these genes was associated with the xxiii emergence of psychopathology. Overall, the results of these two studies showed that the emergence of psychopathology in adolescents along a 3-year follow-up appeared concurrently with changes in the patterns of DNA methylation and gene expression patterns, and that these changes were related. Moreover, we have shown that life adversities that occurred over the 3 years evaluated may have influenced DNA methylation patterns, which in turn may have changed the expression patterns of the genes associated with the emergence of psychopathology. This study highlight the influence of gene expression and DNA methylation in the development of psychopathology in adolescents.Transtornos psiquiátricos são fenótipos complexos causados pela influencia de fatores genéticos, fatores ambientais e pela interação entre eles. Estudos sugerem que eventos adversos na infância podem alterar a expressão gênica por meio de mecanismos epigenéticos (metilação do DNA por exemplo) que, por sua vez, pode alterar a reatividade ao estresse, função cerebral e, por fim, o comportamento, aumentando o risco de transtornos psiquiátricos. Uma forma de medir alterações do comportamento é avaliando os níveis de psicopatologia, ou seja, a presença e a frequência de sintomas psiquiátricos. O objetivo geral desta tese foi investigar ao longo de 3 anos de seguimento a relação entre eventos adversos de vida, alterações dos padrões de metilação do DNA, alterações dos padrões de expressão gênica e o aumento da psicopatologia em adolescentes. Para isso, realizamos dois estudos investigando adolescentes(n=24) que apresentaram um aumento significativo da psicopatologia após 3 anos de acompanhamento e que tiveram amostras biológicas coletas antes e após esse acompanhamento. No estudo 1, exploramos a relação entre psicopatologia, metilação do DNA e expressão gênica a partir de uma análise inicial de rastreio para identificar sítios e regiões diferencialmente metiladas associadas ao aumento de psicopatologia. Encontramos 663 sítios CpG e 90 regiões CpG diferencialmente metiladas associadas ao aumento de psicopatologia. Desses, 15 sítios e 3 regiões estão localizados em genes cuja expressão também foi associada ao aumento de psicopatologia. Essas três regiões estão mapeadas nos seguintes genes: ASCL2, HLA-E e RPS6KB1. No estudo 2, investigamos a relação à nível molecular entre eventos adversos de vida e aumento de psicopatologia, com a hipótese de que eventos adversos de vida influenciam mudanças nos padrões de metilação do DNA que, por sua vez, influenciam os níveis de expressão de genes associados ao aumento de psicopatologia. Encontramos que adversidades de vida relacionadas a eventos de saúde/luto e de contexto escolar foram associadas a mudanças dos padrões de metilação do DNA dos genes EST1, FYTTD1 e FAM117B. Essas mudanças de metilação, por sua vez, foram associadas a mudanças dos níveis de xxi expressões desses genes. Por fim, a expressão desses genes foi associada ao aumento de psicopatologia. De maneira geral, os resultados desses dois estudos mostraram que o aumento de psicopatologia em adolescentes ao longo de três anos ocorreu, concomitantemente, com alterações dos padrões de metilação do DNA e de expressão gênica, e que essas alterações estão relacionadas. Além disso, mostramos que adversidades de vida que aconteceram ao longo dos 3 anos avaliados podem ter influenciado os padrões de metilação que, por sua vez, podem ter mudado os padrões de expressão dos genes associados ao aumento de psicopatologia. Este trabalho reforça a influência de diferentes aspectos moleculares no desenvolvimento de psicopatologia em adolescentes.Dados abertos - Sucupira - Teses e dissertações (2019

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K-i = 3.29 nM) and human cathepsin L (K-i= 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan. (C) 2011 Elsevier Inc. All rights reserved

Expression profile of neurotransmitter receptor and regulatory genes in the prefrontal cortex of spontaneously hypertensive rats: Relevance to neuropsychiatric disorders

The spontaneously hypertensive rat (SHR) strain was shown to be a useful animal model to study several behavioral, pathophysiological and pharmacological aspects of schizophrenia and attention-deficit/hyperactivity disorder. To further understand the genetic underpinnings of this model, our primary goal in this study was to compare the gene expression profile of neurotransmitter receptors and regulators in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) of SHR and Wistar rats (control group). in addition, we investigated DNA methylation pattern of promoter region of the genes differentially expressed. We performed gene expression analysis using a PCRarray technology, which simultaneously measures the expression of 84 genes related to neurotransmission. Four genes were significantly downregulated in the PFC of SHR compared to Wistar rats (Gad2, Chrnb4, Slc5a7, and Qrfpr) and none in nucleus accumbens. Gad2 and Qrfpr have CpG islands in their promoter region. for both, the promoter region was hypomethylated in SHR group, and probably this mechanism is not related with the downregulation of these genes. in summary, we identified genes that are downregulated in the PFC of SHR, and might be related to the behavioral abnormalities exhibited by this strain. (C) 2014 Elsevier Ireland Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fed Univ São Paulo UNIFESP, Div Genet, Dept Morphol & Genet, São Paulo, BrazilInterdisciplinmy Lab Clin Neurosci LiNC, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, São Paulo, BrazilFed Univ São Paulo UNIFESP, Invest Ctr Gene Therapy, São Paulo, BrazilFed Univ São Paulo UNIFESP, Div Genet, Dept Morphol & Genet, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, São Paulo, BrazilFed Univ São Paulo UNIFESP, Invest Ctr Gene Therapy, São Paulo, BrazilWeb of Scienc

Evaluation of neurotransmitter receptor gene expression identifies GABA receptor changes: A follow-up study in antipsychotic-naive patients with first-episode psychosis

A study of the gene expression levels in the blood of individuals with schizophrenia in the beginning of the disease, such as first-episode psychosis (FEP), is useful to detect gene expression changes in this disorder in response to treatment. Although a large number of genetic studies on schizophrenia have been conducted, little is known about the effects of antipsychotic treatment on gene expression. the aim of the present study was to examine differences in the gene expression in the blood of antipsychotic-naive FEP patients before and after risperidone treatment (N = 44) and also to verify the correlation with treatment response. in addition, we determined the correlations between differentially expressed genes and clinical variables. the expression of 40 neurotransmitter and neurodevelopment-associated genes was assessed using the RT2 Profiler (TM) PCR Array. the results indicated that the GABRR2 gene was downregulated after risperidone treatment, but no genes were associated with response to treatment and clinical variables after Bonferroni correction. GABRR2 downregulation after treatment can both suggest an effect of risperidone treatment or processes related to disease progression, either not necessarily associated with the improvement of symptoms. Despite this change was observed in blood, this decrease in GABRR2 mRNA levels might be an effect of changes in GABA concentrations or other systems interplay consequently to D2 blockage induced by risperidone, for example. Thus, it is important to consider that antipsychotics or the progression of psychotic disorders might interfere with gene expression. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo UNIFESP, Dept Morphol & Genet, Div Genet, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, LiNC Interdisciplinary Lab Clin Neurosci, BR-05039032 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilISCMSP, Dept Psychiat, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilUniv Fed ABC, Ctr Math Computat & Cognit, Santo Andre, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Morphol & Genet, Div Genet, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, LiNC Interdisciplinary Lab Clin Neurosci, BR-05039032 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilFAPESP: 2010/08968-6FAPESP: 2010/19176-3FAPESP: 2011/50740-5FAPESP: 2013/10498-6Web of Scienc

Evaluation of neurotransmitter receptor gene expression identifies GABA receptor changes: A follow-up study in antipsychotic-naive patients with first-episode psychosis

A study of the gene expression levels in the blood of individuals with schizophrenia in the beginning of the disease, such as first-episode psychosis (FEP), is useful to detect gene expression changes in this disorder in response to treatment. Although a large number of genetic studies on schizophrenia have been conducted, little is known about the effects of antipsychotic treatment on gene expression. the aim of the present study was to examine differences in the gene expression in the blood of antipsychotic-naive FEP patients before and after risperidone treatment (N = 44) and also to verify the correlation with treatment response. in addition, we determined the correlations between differentially expressed genes and clinical variables. the expression of 40 neurotransmitter and neurodevelopment-associated genes was assessed using the RT2 Profiler (TM) PCR Array. the results indicated that the GABRR2 gene was downregulated after risperidone treatment, but no genes were associated with response to treatment and clinical variables after Bonferroni correction. GABRR2 downregulation after treatment can both suggest an effect of risperidone treatment or processes related to disease progression, either not necessarily associated with the improvement of symptoms. Despite this change was observed in blood, this decrease in GABRR2 mRNA levels might be an effect of changes in GABA concentrations or other systems interplay consequently to D2 blockage induced by risperidone, for example. Thus, it is important to consider that antipsychotics or the progression of psychotic disorders might interfere with gene expression. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo UNIFESP, Dept Morphol & Genet, Div Genet, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, LiNC Interdisciplinary Lab Clin Neurosci, BR-05039032 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilISCMSP, Dept Psychiat, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilUniv Fed ABC, Ctr Math Computat & Cognit, Santo Andre, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Morphol & Genet, Div Genet, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, LiNC Interdisciplinary Lab Clin Neurosci, BR-05039032 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Psychiat, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Pharmacol, São Paulo, BrazilFAPESP: 2010/08968-6FAPESP: 2010/19176-3FAPESP: 2011/50740-5FAPESP: 2013/10498-6Web of Scienc