Renato Puga

Co-expression network of neural-differentiation genes shows specific pattern in schizophrenia

BackgroundSchizophrenia is a neurodevelopmental disorder with genetic and environmental factors contributing to its pathogenesis, although the mechanism is unknown due to the difficulties in accessing diseased tissue during human neurodevelopment. The aim of this study was to find neuronal differentiation genes disrupted in schizophrenia and to evaluate those genes in post-mortem brain tissues from schizophrenia cases and controls.MethodsWe analyzed differentially expressed genes (DEG), copy number variation (CNV) and differential methylation in human induced pluripotent stem cells (hiPSC) derived from fibroblasts from one control and one schizophrenia patient and further differentiated into neuron (NPC). Expression of the DEG were analyzed with microarrays of post-mortem brain tissue (frontal cortex) cohort of 29 schizophrenia cases and 30 controls. A Weighted Gene Co-expression Network Analysis (WGCNA) using the DEG was used to detect clusters of co-expressed genes that werenon-conserved between adult cases and controls brain samples.ResultsWe identified methylation alterations potentially involved with neuronal differentiation in schizophrenia, which displayed an over-representation of genes related to chromatin remodeling complex (adjP = 0.04). We found 228 DEG associated with neuronal differentiation. These genes were involved with metabolic processes, signal transduction, nervous system development, regulation of neurogenesis and neuronal differentiation. Between adult brain samples from cases and controls there were 233 DEG, with only four genes overlapping with the 228 DEG, probably because we compared single cell to tissue bulks and more importantly, the cells were at different stages of development. The comparison of the co-expressed network of the 228 genes in adult brain samples between cases and controls revealed a less conserved module enriched for genes associated with oxidative stress and negative regulation of cell differentiation.ConclusionThis study supports the relevance of using cellular approaches to dissect molecular aspects of neurogenesis with impact in the schizophrenic brain. We showed that, although generated by different approaches, both sets of DEG associated to schizophrenia were involved with neocortical development. The results add to the hypothesis that critical metabolic changes may be occurring during early neurodevelopment influencing faulty development of the brain and potentially contributing to further vulnerability to the illness.

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

Gene expression of peripheral blood lymphocytes may discriminate patients with schizophrenia from controls

To identify a classifier in schizophrenia, blood gene expression profiling was applied to patients with schizophrenia under different treatments and to controls. Expression of six genes discriminated patients with sensitivity of 89.3% and specificity of 90%, supporting the use of peripheral blood as biological material for diagnosis in schizophrenia.

microRNA Portraits in Human Vulvar Carcinoma

Unregulated expression of microRNAs is well known and has already been demonstrated in many tumor types. However, in vulvar carcinoma this field has been unknown territory. Our study characterizes microRNA in vulvar tumors through an expression profile of 754 miRNAs, relating this with clinical and anatomopathologic data, and presence of HPV infection. Twenty HPV-negative and 20 HPV-positive samples, genotyped for high-risk HPVs (HPV16, 18, 31, 33) and a pool of seven normal vulvar skin samples were used for the identification of differentially expressed miRNAs by TLDA Quantitative Real Time PCR (qRT-PCR). Twenty-five differentially expressed microRNAs between HPV-positive and HPV-negative groups and 79 differentially expressed on the tumor compared with normal samples were obtained. A network between microRNA expression profiles and putative target mRNAs predicted by target prediction algorithms and previously demonstrated as relevant in vulvar carcinomas, such as TP53, RB, PTEN, and EGFR was constructed. Downregulation of both miR-223-5p and miR-19-b1-5p were correlated with the presence of lymph node metastasis; downregulation of miR-100-3p and miR-19-b1-5p were correlated with presence of vascular invasion; overexpression of miR-519b and miR-133a were associated with advanced FIGO staging. In conclusion, our study demonstrates that microRNAs may be clinically important in vulvar carcinomas and our findings may help for further studies on functional implications of miRNA deregulation in this type of cancer. Cancer Prev Res; 6(11); 1231–41. ©2013 AACR.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

OBJECTIVE To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. DESIGN Comparison between oocyte groups and between early embryo stages. SETTING Laboratories of embryo manipulation and molecular biology from Departamento de Genética (FMRP) and Departamento de Ciências Básicas (FZEA)--University of São Paulo. SAMPLE(S) Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. MAIN OUTCOME MEASURE(S) Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). RESULT(S) Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good- and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. CONCLUSION(S) Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development.

Granulocyte whole exome sequencing and endothelial JAK2V617F in patients with JAK2V617F positive Budd‐Chiari Syndrome without myeloproliferative neoplasm

Budd-Chiari Syndrome (BCS) is characterized by hepatic venous outflow obstruction. JAK2-positive myeloproliferative neoplasms (JAK2 MPN) are one of the most frequent thrombotic conditions underlying a diagnosis of BCS (Smalberg et al, 2012). A subset of BCS patients harbour the JAK2 mutation in peripheral blood (PB) granulocytes without evidence of overt MPN (JAK2 MPN-neg). While the occurrence of other myeloid-associated mutations in genes are common in patients with JAK2 MPN, it is unknown if such mutations are present in patients with JAK2 MPN-neg. In addition, it remains to be determined if there are differences in JAK2 allele burden between JAK2 MPN and JAK2 MPN-neg. Recently, it has been demonstrated that a subset of patients with JAK2 MPN have endothelial cells (ECs) that are positive for JAK2(Teofili et al, 2011) and that these cells may possibly contribute to the prothrombotic state. However, the presence of JAK2-positive endothelial colony-forming cells (E-CFC) in JAK2 MPN-neg patients with BCS has not been evaluated. The objective of this study was to assess the presence of JAK2 in BM E-CFC from JAK2 MPN-neg patients with BCS and to evaluate the JAK2 allele burden and the presence of additional mutations. We selected patients diagnosed with BCS and the presence of JAK2 mutation in the absence of other thrombophilic conditions at Hospital Israelita Albert Einstein, Sao Paulo, Brazil, between March 2013 and June 2015. Patients were investigated for the presence of an associated MPN and diagnosed according to the 2001 World Health Organization criteria. This study was approved by the Institutional Review Board and conducted according to the Principles of the Declarations of Helsinki. Paired DNA (sorted CD66b-granulocytes/skin biopsy) was subjected to whole exome sequencing (WES) on the HiSeq 2000 platform (Illumina Inc., San Diego, CA) using the SureSelect library preparation kit (Agilent, Santa Clara, CA). Somatic variant calls were generated by combining the output of SomaticSniper (http://gmt.genome.wustl.edu/packages/somatic-sniper/), MuTect (https://www.broadinstitute.org/cancer/cga/mutec) and Pindel (www.sanger.ac.uk/ science/tools/pindel) plus additional in-house criteria (minimum coverage at both tumour/germline ≥8 reads; ratio of allele fraction tumour:germline >2). Bone marrow (BM) mononuclear cells were obtained by density centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Approximately 20 ml of BM was washed twice with phosphate-buffered saline and 2% fetal bovine serum and then suspended in Endocult liquid medium (STEMCELL Technologies Canada Inc., Vancouver, BC, Canada), as previously described (Wu et al, 2012), but extending the culture time by 7 days. ECs grown in culture were then evaluated by flow cytometry (ECs were considered CD146+, CD144+, CD34 , CD117 ) (Ozdogu et al, 2007). ECs were isolated by fluorescence-activated cell sorting (FACS) in a FACSAria (BD Biosciences, San Jose, CA). DNA was extracted from sorted ECs and tested for the presence of the JAK2 mutation by an allele-specific polymerase chain reaction (PCR). Data on JAK2 allele burden was extracted from WES results obtained from a cohort of 77 patients with JAK2-positive MPN (including 4 patients with JAK2 MPN-neg) (Santos et al, 2014) and from 110 patients previously reported by Nangalia et al (2013). We identified 32 cases of BCS, 10 (31 2%) of who were positive for the JAK2 mutation. Three of these patients passed away before inclusion in the study and 7 patients were included; their baseline features are summarized in Table I. Two patients were diagnosed with primary myelofibrosis (PMF) (Patients 1 and 7) during evaluation and the remaining 5 cases were JAK2 MPN-neg. Samples from Patients 1–6) were analysed by WES. In the 5 JAK2MPN-neg patients (Patients 2–6), the only known pathogenic somatic mutation found was JAK2. We found no additional myeloid-associated mutations in these patients. The median JAK2 variant allele frequency Table I. Demographic features of BCS patients and JAK2 alleleburden.

A novel mechanism of NPM1 cytoplasmic localization in acute myeloid leukemia: the recurrent gene fusion NPM1–HAUS1

NPM1 heterozygous mutations are present in roughly a third of patients with acute myeloid leukemia (AML), making it one of the most frequent genomic alterations in these patients.[1][1] The mutations are characterized by frameshift insertions in the region encoding the C-terminus of the protein,

Identification of ANLN as ETV6 partner gene in recurrent t(7;12)(p15;p13): a possible role of deregulated ANLN expression in leukemogenesis

The ETV6 gene encodes an ETS family transcription factor that is involved in a myriad of chromosomal rearrangements found in hematological malignancies and other neoplasms. A recurrent ETV6 translocation, previously described in patients with acute myeloid leukemia (AML) (Genes Chromosomes Cancer 51:328–337,2012, Leuk Res 35:e212-214, 2011), whose partner has not been identified is t(7;12)(p15;p13). We herein report that the t(7;12)(p15;p13) fuses ETV6 to ANLN, a gene not previously implicated in the pathogenesis of hematological malignancies, and we demonstrate that this translocation leads to high expression of the fusion transcript in the myeloid and lymphoid lineages.

Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis

Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years after diagnosis. In the majority of patients with PMF somatic mutations were detected either in Janus Kinase 2 (JAK2; in 60% of patients), Calreticulin (CALR; in 25% of patients) or MPL (in 5% of patients) genes. Neither mutation was detected 5% to 10% of PMF patients. Patients with mutated JAK2 are known to have a more aggressive disease compared to patients with mutated CALR. However patients with mutated JAK2 and high allele burden have a favorable outcome compared to patients with a low mutated JAK2 burden. Aim: To develop a model that uses genetic information to predict survival outcome of patients with PMF. Patients and Methods: Bone marrow samples were collected from 344 patients with PMF that were followed at MD Anderson Cancer Center between 2000 and 2013 (157 months). All samples were screened for JAK2 and for mutations in CALR. Patients who did not have a mutation in either gene were also screened for mutations in MPL. Results: In 226 patients (66%) JAK2 was detected and in 43 (13%) CALR was mutated. Of the 75 patients who did not have JAK2 or CALR mutations, 16 (21%) had mutated MPL. In 59 patients (17%), none of those mutations was detected. In the 226 patients who harbored the JAK2 mutation, a cut-point of 50% dichotomized patients into those with a high JAK2 burden and a favorable overall survival (OS; median OS: 80 months) and those with a low JAK2 burden and an adverse OS (median OS: 50 months). Age (above 65 years) and mutation status (low JAK2 burden or triple-negative) were independent risk factors. Patients with a favorable mutation status and age below 65 had a median survival of 126 months (n 1⁄4 82). Patients with either one risk factor, age above 65 (n 1⁄4 88) or adverse mutation status (n 1⁄4 87) had intermediate survival expectancy. The two risk factors were additive and patients age > 65 years and adverse mutation status (n 1⁄4 87) had a median survival of 35 months. Conclusions: Age and mutation status are independent predictors of survival in patients with PMF and stratify patients into 4 groups of equal size with very different survival outcome. 703 Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis Fabio Santos, Renato Puga, Bianca Lisboa, Welbert Pereira, Mariana Miyagi, Evelyn Mata, Tarcila Datoguia, Isabel Bello, Michelli Diniz, Sandra Nakashima, Guilherme Perini, Ricardo Helman, Nelson Hamerschlak, Paulo Campregher Hematology/Oncology, Hospital Israelita Albert Einstein

Abstract 516: S100P: Cause or effect of vulvar carcinoma prognostic status

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Vulvar squamous cell carcinoma (VSCC) is a rare disease accounting for 3 to 5% of female genital-tract malignancy. Conventional surgical treatment of VSCC can be very aggressive and mutilating, resulting in relevant psychosexual implications for patients. S100 family comprises genes involved in several molecular pathways including proliferation, differentiation and migration. Among them, S100P has a significant role during the development and progression of different cancers being able to promote migration. Aims: To evaluate the role of S100P in VSCC and associate it with clinical outcomes. Methods: 5 samples of frozen VSCC tissue were microdissected after carefully revision of the HE slides. Two areas of the same tumor - invasion front (IF) and center of the tumor (CT)- were selected for different dissections. Each of the samples had their total RNA extracted, amplified and analyzed using Whole Human Genome 8×60K array (Agilent Technologies). Results of RNA expression were compared between both groups (IF and CT) from the same tumor. The differential expressed genes were selected by RankProduct methodology, which pointed out S100P as one of the main ones. Then, 66 VSCC formalin-fixed paraffin-embbebed samples were used for immunohistochemical (IHC) analysis of S100P. The stained slides were digitalized and evaluated using Pannoramic250 Flash II and Pannoramic Viewer, respectively (3DHistechTM). HScore was calculated based on the percentage and the intensity of stained cells. The statistical analysis was performed on SPSS® Statistics software, version 21.0.0.0, with a confidence interval of 95%. Results: The gene wide expression analysis presented a ranked list with 69 genes, one of them, S100P. The IHC results showed that IF had significant (p = 0,002) higher HScore values (mean = 132,79) compared to CT (mean = 112,34). Analysis of IF staining hot spots showed that higher expression of S100P is related with worse prognosis (p = 0,041). In addition, the greater is the difference in S100P expression between IF compared to CT of the same tumor, the worse is the patient prognosis in cancer specific survival analysis (p = 0,035). Conclusion: S100P is a marker of worse prognosis in VSCC. Also, its heterogeneity of expression between IF and CT seems to be related to a more aggressive component of the disease, maybe due to its relation with cell motility and migration. A comprehensive IHC assessment of S100P can bring important contribution for stablishing prognosis of women with vulvar cancer. Note: This abstract was not presented at the meeting. Citation Format: Mayara C. Botelho, Andre M. Lavorato-Rocha, Iara S. Rodrigues, Beatriz M. Maia, Katia C. Carvalho, Renato Puga, Fernando A. Soares, Rafael M. Rocha. S100P: Cause or effect of vulvar carcinoma prognostic status. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 516. doi:10.1158/1538-7445.AM2015-516