Gisele Rodrigues Gouveia

Comparison of Two Methods of RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue Specimens

The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables—such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested—on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens.

Pathophysiology and molecular aspects of diffuse large B-cell lymphoma.

Diffuse large B-Cell lymphoma is the most common subtype of non-Hodgkin lymphoma in the West. In Brazil, it is the fifth cause of cancer, with more than 55,000 cases and 26,000 deaths per year. At Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - HCFMUSP, diffuse large B-Cell lymphoma represents 49.7% of all non-Hodgkin lymphoma cases. Initially, the classification of non-Hodgkin lymphoma was based on morphology, but advances in immunology and molecular medicine allowed the introduction of a biological classification for these diseases. As for other cancers, non-Hodgkin lymphoma involves patterns of multifactorial pathogenesis with environmental factors, as well as genetic, occupational and dietary factors, contributing to its development. Multiple lesions involving molecular pathways of B-cell proliferation and differentiation may result in the activation of oncogenes such as the BCL2, BCL6, and MYC genes and the inactivation of tumor suppressor genes such as p53 and INK4, as well as other important transcription factors such as OCT-1 and OCT-2. A dramatic improvement in survival was seen after the recent introduction of the anti-CD20 monoclonal antibody. The association of this antibody to the cyclophosphamide, hydroxydaunorubicin, oncovin and prednisolone (CHOP) regimen has increased overall survival of diffuse large B-Cell lymphoma and follicular lymphoma patients by 20%. However, 50% of all diffuse large B-Cell lymphoma patients remain incurable, creating a demand for more research with new advances in treatment. Thus, it is important to know and understand the key factors and molecular pathways involved in the pathogenesis of diffuse large B-Cell lymphoma.

Prevalence of non-Hodgkin lymphomas in São Paulo, Brazil

The incidence of NHL worldwide is increasing by 4% per year and is thus a concern for public health. This increase may be attributed to several factors including the early diagnosis of more cases of the indolent lymphoma, increases in the use of immunosuppressive drugs and the aging of the

Sex differences in DNA methylation of the cord blood are related to sex-bias psychiatric diseases

Sex differences in the prevalence of psychiatric disorders are well documented, with exposure to stress during gestation differentially impacting females and males. We explored sex-specific DNA methylation in the cord blood of 39 females and 32 males born at term and with appropriate weight at birth regarding their potential connection to psychiatric outcomes. Mothers were interviewed to gather information about environmental factors (gestational exposure) that could interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. Excluding XYS probes, there were 2,332 differentially methylated CpG sites (DMSs) between sexes, which were enriched within brain modules of co-methylated CpGs during brain development and also differentially methylated in the brains of boys and girls. Genes associated with the DMSs were enriched for neurodevelopmental disorders, particularly for CpG sites found differentially methylated in brain tissue between patients with schizophrenia and controls. Moreover, the DMS had an overlap of 890 (38%) CpG sites with a cohort submitted to toxic exposition during gestation. This study supports the evidences that sex differences in DNA methylation of autosomes act as a primary driver of sex differences that are found in psychiatric outcomes.

Epigenetics insights into chronic pain: DNA hypomethylation in fibromyalgia - A controlled pilot-study

Abstract To evaluate changes in DNA methylation profiles in patients with fibromyalgia (FM) compared to matched healthy controls (HCs). All individuals underwent full clinical and neurophysiological assessment by cortical excitability (CE) parameters measured by transcranial magnetic stimulation. DNA from the peripheral blood of patients with FM (n = 24) and HC (n = 24) were assessed using the Illumina-HumanMethylation450 BeadChips. We identified 1610 differentially methylated positions (DMPs) in patients with FM displaying a nonrandom distribution in regions of the genome. Sixty-nine percent of DMP in FM were hypomethylated compared to HC. Differentially methylated positions were enriched in 5 genomic regions (1p34; 6p21; 10q26; 17q25; 19q13). The functional characterization of 960 genes related to DMPs revealed an enrichment for MAPK signaling pathway (n = 18 genes), regulation of actin cytoskeleton (n = 15 genes), and focal adhesion (n = 13 genes). A gene–gene interaction network enrichment analysis revealed the participation of DNA repair pathways, mitochondria-related processes, and synaptic signaling. Even though DNA was extracted from peripheral blood, this set of genes was enriched for disorders such as schizophrenia, mood disorders, bulimia, hyperphagia, and obesity. Remarkably, the hierarchical clusterization based on the methylation levels of the 1610 DMPs showed an association with neurophysiological measurements of CE in FM and HC. Fibromyalgia has a hypomethylation DNA pattern, which is enriched in genes implicated in stress response and DNA repair/free radical clearance. These changes occurred parallel to changes in CE parameters. New epigenetic insights into the pathophysiology of FM may provide the basis for the development of biomarkers of this disorder.

Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina

INTRODUCTION: Formalin fixed paraffin embedded (FFPE) tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders Molecular Biology tests once it fragments and chemically modifies nucleic acids, particularly RNA. OBJECTIVE: To compare the efficiency of three different RNA extraction protocols for gene expression analysis of FFEP tissues. MATERIAL AND METHODS: RNA was extracted from FFPE samples of human lymph by means of Ambion and Arcturus Bioscience kits and the classical Trizol method. After extraction, RNA was quantified and tested for amplification through real time polymerase chain reaction (RT-PCR) using glyceraldehydes-3 phosphate dehydrogenase (GAPDH) endogenous gene primers. DISCUSSION/CONCLUSION: All the protocols produced sufficient and adequate amounts of total RNA. However, only protocols using Arcturus and Ambion kits generated suitable RNA for PCR amplification.

An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder

Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two “in silico” protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.

Nucleic Acids Extraction from Formalin-Fixed and Paraffin-Embedded Tissues

Formalin-fixed paraffin-embedded (FFPE) tissues are an important sample source for ret‐ rospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders molecular biology tests since it fragments and chemically modifies nucleic acids, especially RNA. Although several studies describe techniques that allow extracting nu‐ cleic acids from FFPE tissues, so far there is no consensus in the literature about the best protocol to be used in this type of material. Thus, the current chapter aims to describe the factors affecting the FFPE tissue nucleic acid extracting process, compare the available protocols and to describe the modifications developed by our group in some protocols. Such modifications enable nucleic acids obtainment in satisfactory quantity and quality for molecular biology studies.

Erratum: An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder (Scientific Reports (2016) 6 (22851) DOI: 10.1038/srep22851)

Leandro de Araújo Lima, Ana Cecília Feio-dos-Santos, Sintia Iole Belangero, Ary Gadelha, Rodrigo Affonseca Bressan, Giovanni Abrahão Salum, Pedro Mario Pan, Tais Silveira Moriyama, Ana Soledade Graeff-Martins, Ana Carina Tamanaha, Pedro Alvarenga, Fernanda Valle Krieger, Bacy Fleitlich-Bilyk, Andrea Parolin Jackowski, Elisa Brietzke, João Ricardo Sato, Guilherme Vanoni Polanczyk, Jair de Jesus Mari, Gisele Gus Manfro, Maria Conceição do Rosário, Eurípedes Constantino Miguel, Renato David Puga, Ana Carolina Tahira, Viviane Neri Souza, Thais Chile, Gisele Rodrigues Gouveia, Sérgio Nery Simões, Xiao Chang, Renata Pellegrino, Lifeng Tian, Joseph T. Glessner, Ronaldo Fumio Hashimoto, Luis Augusto Rohde, Patrick M. A. Sleiman, Hakon Hakonarson & Helena Brentani