Renato David Puga

Transcriptome signature of the adult mouse choroid plexus.

BackgroundAlthough the gene expression profile of several tissues in humans and in rodent animal models has been explored, analysis of the complete choroid plexus (CP) transcriptome is still lacking. A better characterization of the CP transcriptome can provide key insights into its functions as one of the barriers that separate the brain from the periphery and in the production of cerebrospinal fluid.MethodsThis work extends further what is known about the mouse CP transcriptome through a microarray analysis of CP tissue from normal mice under physiological conditions.ResultsWe found that the genes most highly expressed are those implicated in energy metabolism (oxidative phosphorylation, glycolysis/gluconeogenesis) and in ribosomal function, which is in agreement with the secretory nature of the CP. On the other hand, genes encoding for immune mediators are among those with lower expression in basal conditions. In addition, we found genes known to be relevant during brain development, and not previously identified to be expressed in the CP, including those encoding for various axonal guidance and angiogenesis molecules and for growth factors. Some of these are known to influence the neural stem cell niche in the subventricular zone, highlighting the involvement of the CP as a likely modulator of neurogenesis. Interestingly, our observations confirm that the CP transcriptome is unique, displaying low homology with that of other tissues. Of note, we describe here that the closest similarity is with the transcriptome of the endothelial cells of the blood-brain barrier.ConclusionsBased on the data presented here, it will now be possible to further explore the function of particular proteins of the CP secretome in health and in disease.

Comprehensive Analysis of BRCA1, BRCA2 and TP53 Germline Mutation and Tumor Characterization: A Portrait of Early-Onset Breast Cancer in Brazil

Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most important disease-causing issues in young breast cancer patients worldwide. The specific defective biological processes that trigger germline mutation-associated and -negative tumors remain unclear. To delineate an initial portrait of Brazilian early-onset breast cancer, we performed an investigation combining both germline and tumor analysis. Germline screening of the BRCA1, BRCA2, CHEK2 (c.1100delC) and TP53 genes was performed in 54 unrelated patients <35 y; their tumors were investigated with respect to transcriptional and genomic profiles as well as hormonal receptors and HER2 expression/amplification. Germline mutations were detected in 12 out of 54 patients (22%) [7 in BRCA1 (13%), 4 in BRCA2 (7%) and one in TP53 (2%) gene]. A cancer familial history was present in 31.4% of the unrelated patients, from them 43.7% were carriers for germline mutation (37.5% in BRCA1 and in 6.2% in the BRCA2 genes). Fifty percent of the unrelated patients with hormone receptor-negative tumors carried BRCA1 mutations, percentage increasing to 83% in cases with familial history of cancer. Over-representation of DNA damage-, cellular and cell cycle-related processes was detected in the up-regulated genes of BRCA1/2-associated tumors, whereas cell and embryo development-related processes were over-represented in the up-regulated genes of BRCA1/2-negative tumors, suggesting distinct mechanisms driving the tumorigenesis. An initial portrait of the early-onset breast cancer patients in Brazil was generated pointing out that hormone receptor-negative tumors and positive familial history are two major risk factors for detection of a BRCA1 germline mutation. Additionally, the data revealed molecular factors that potentially trigger the tumor development in young patients.

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

BackgroundHuman homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated.MethodsReal time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively.ResultsOverexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle.ConclusionThe present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.

Poly (A)+ Transcriptome Assessment of ERBB2-Induced Alterations in Breast Cell Lines

We report the first quantitative and qualitative analysis of the poly (A)+ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

Whole-exome sequencing in obsessive-compulsive disorder identifies rare mutations in immunological and neurodevelopmental pathways

Studies of rare genetic variation have identified molecular pathways conferring risk for developmental neuropsychiatric disorders. To date, no published whole-exome sequencing studies have been reported in obsessive-compulsive disorder (OCD). We sequenced all the genome coding regions in 20 sporadic OCD cases and their unaffected parents to identify rare de novo (DN) single-nucleotide variants (SNVs). The primary aim of this pilot study was to determine whether DN variation contributes to OCD risk. To this aim, we evaluated whether there is an elevated rate of DN mutations in OCD, which would justify this approach toward gene discovery in larger studies of the disorder. Furthermore, to explore functional molecular correlations among genes with nonsynonymous DN SNVs in OCD probands, a protein–protein interaction (PPI) network was generated based on databases of direct molecular interactions. We applied Degree-Aware Disease Gene Prioritization (DADA) to rank the PPI network genes based on their relatedness to a set of OCD candidate genes from two OCD genome-wide association studies (Stewart et al., 2013; Mattheisen et al., 2014). In addition, we performed a pathway analysis with genes from the PPI network. The rate of DN SNVs in OCD was 2.51 × 10−8 per base per generation, significantly higher than a previous estimated rate in unaffected subjects using the same sequencing platform and analytic pipeline. Several genes harboring DN SNVs in OCD were highly interconnected in the PPI network and ranked high in the DADA analysis. Nearly all the DN SNVs in this study are in genes expressed in the human brain, and a pathway analysis revealed enrichment in immunological and central nervous system functioning and development. The results of this pilot study indicate that further investigation of DN variation in larger OCD cohorts is warranted to identify specific risk genes and to confirm our preliminary finding with regard to PPI network enrichment for particular biological pathways and functions.

MicroRNAs discriminate familial from sporadic non-BRCA1/2 breast carcinoma arising in patients ≤35 years

The influence of genetic factors may contribute to the poor prognosis of breast cancer (BC) at a very young age. However BRCA1/2 mutations could not explain the majority of cases arising in these patients. MicroRNAs (miRs) have been implicated in biological processes associated with BC. Therefore, we investigated differences in miRs expression between tumors from young patients (≤35 years) with sporadic or familial history and non-carriers of BRCA1/2 mutations. Thirty-six young Brazilian patients were divided into 2 groups: sporadic (NF-BC) or familial breast cancer (F-BC). Most of the samples were classified as luminal A and B and the frequency of subtypes did not differ between familial or sporadic cases. Using real time qPCR and discriminant function analysis, we identified 9 miRs whose expression levels rather than miR identity can discriminate between both patient groups. Candidate predicted targets were determined by combining results from miRWalk algorithms with mRNA expression profiles (n = 91 differently expressed genes). MiR/mRNA integrated analysis identified 91 candidate genes showing positive or negative correlation to at least 1 of the 9 miRs. Co-expression analysis of these genes with 9 miRs indicated that 49 differentially co-expressed miR-gene interactions changes in F-BC tumors as compared to those of NF-BC tumors. Out of 49, 17 (34.6%) of predicted miR-gene interactions showed an inverse correlation suggesting that miRs act as post-transcriptional regulators, whereas 14 (28.6%) miR-gene pairs tended to be co-expressed in the same direction indicating that the effects exerted by these miRs pointed to a complex level of target regulation. The remaining 18 pairs were not predicted by our criteria suggesting involvement of other regulators. MiR–mRNA co-expression analysis allowed us to identify changes in the miR-mRNA regulation that were able to distinguish tumors from familial and sporadic young BC patients non-carriers of BRCA mutations.

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.

Profile of apoptotic proteins in oral squamous cell carcinoma: A cluster analysis of 171 cases

BackgroundOral squamous cell carcinoma (OSCC) is the eighth most prevalent cancer worldwide. In recent large-scale studies, by immunohistochemistry and cluster analysis, several markers were associated with patient survival in various tumors. The aim of this study was to analyze the expression profiles of 23 proteins that have been linked to the inhibition (Bcl-2, Bcl-x, Bcl-xL, Bcl-2-related protein A1, BAG-1, and survivin) and promotion (Bak, Bax, Bim/Bod, Bim-Long, Bad, Bid, PUMA, Apaf-1, caspase-2, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, Smac/DIABLO, and cytochrome c) of apoptosis in OSCC.MethodsTwo-hundred and twenty nine cases of OSCC, arranged in a tissue microarray, were immunohistochemically analyzed, and the results were quantified on an automated imaging system. The data were analyzed using a random forest clustering method.ResultsOverall protein expression patterns defined two chief clusters: an anti-apoptotic cluster (142 cases) and a pro-apoptotic cluster (29 cases). These groups could not be explained by any clinical or pathological characteristic, and overall and disease-free survival did not differ between them.ConclusionsAlthough there was no association with survival, the cluster analysis demonstrated specific protein profiles that could be of interest for using targeted therapies: in one of the clusters, the expression of pro-apoptotic proteins was more prominent, demonstrating a pro-apoptotic profile and highlighting the importance of apoptosis during OSCC development.

A Set of miRNAs, Their Gene and Protein Targets and Stromal Genes Distinguish Early from Late Onset ER Positive Breast Cancer

Breast cancer (BC) in young adult patients (YA) has a more aggressive biological behavior and is associated with a worse prognosis than BC arising in middle aged patients (MA). We proposed that differentially expressed miRNAs could regulate genes and proteins underlying aggressive phenotypes of breast tumors in YA patients when compared to those arising in MA patients. Objective: Using integrated expression analyses of miRs, their mRNA and protein targets and stromal gene expression, we aimed to identify differentially expressed profiles between tumors from YA-BC and MA-BC. Methodology and Results: Samples of ER+ invasive ductal breast carcinomas, divided into two groups: YA-BC (35 years or less) or MA-BC (50–65 years) were evaluated. Screening for BRCA1/2 status according to the BOADICEA program indicated low risk of patients being carriers of these mutations. Aggressive characteristics were more evident in YA-BC versus MA-BC. Performing qPCR, we identified eight miRs differentially expressed (miR-9, 18b, 33b, 106a, 106b, 210, 518a-3p and miR-372) between YA-BC and MA-BC tumors with high confidence statement, which were associated with aggressive clinicopathological characteristics. The expression profiles by microarray identified 602 predicted target genes associated to proliferation, cell cycle and development biological functions. Performing RPPA, 24 target proteins differed between both groups and 21 were interconnected within a network protein-protein interactions associated with proliferation, development and metabolism pathways over represented in YA-BC. Combination of eight mRNA targets or the combination of eight target proteins defined indicators able to classify individual samples into YA-BC or MA-BC groups. Fibroblast-enriched stroma expression profile analysis resulted in 308 stromal genes differentially expressed between YA-BC and MA-BC. Conclusion: We defined a set of differentially expressed miRNAs, their mRNAs and protein targets and stromal genes that distinguish early onset from late onset ER positive breast cancers which may be involved with tumor aggressiveness of YA-BC.

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