Germline DNA copy number variation in familial and early-onset breast cancer

Introduction: Genetic factors predisposing individuals to cancer remain elusive in the majority of patients with a familial or clinical history suggestive of hereditary breast cancer. Germline DNA copy number variation (CNV) has recently been implicated in predisposition to cancers such as neuroblastomas as well as prostate and colorectal cancer. We evaluated the role of germline CNVs in breast cancer susceptibility, in particular those with low population frequencies (rare CNVs), which are more likely to cause disease." Methods: Using whole-genome comparative genomic hybridization on microarrays, we screened a cohort of women fulfilling criteria for hereditary breast cancer who did not carry BRCA1/BRCA2 mutations. Results: The median numbers of total and rare CNVs per genome were not different between controls and patients. A total of 26 rare germline CNVs were identified in 68 cancer patients, however, a proportion that was significantly different (P = 0.0311) from the control group (23 rare CNVs in 100 individuals). Several of the genes affected by CNV in patients and controls had already been implicated in cancer. Conclusions: This study is the first to explore the contribution of germline CNVs to BRCA1/2-negative familial and early-onset breast cancer. The data suggest that rare CNVs may contribute to cancer predisposition in this small cohort of patients, and this trend needs to be confirmed in larger population samples.Brazilian National Institute of Science and Technology in Oncogenomics [FAPESP 2008/57887-9, CNPq 573589/08-9, FAPESP (2009/00898-1)]Brazilian National Institute of Science and Technology in Oncogenomic

Studies of gene co-expression networks of the frontal cortex and striatum (post mortem study) of individuals with OCD and controls

O transtorno obsessivo compulsivo (TOC) é um transtorno psiquiátrico, caracterizado pela presença de obsessões e / ou compulsões. Estudos de neuroimagem funcional indicam que o TOC é um distúrbio heterogêneo relacionado ao circuito talâmico cortico-estriatal (CSTC) e as áreas que compõem este circuito incluem o nucleus accumbens (NAC), putâmen (PT), núcleo caudado (CN), córtex orbitofrontal (OFC) e o córtex cingulado anterior (ACC). As principais características do CSTC são a inervação do córtex frontal em direção ao estriado e cada pequeno circuito possui características específicas: afetiva/límbica, cognitivo e associativo dorsal e cognitivo ventral e motor. Neste trabalho comparamos o transcriptoma de casos e controles das três áreas estriatais (CN, NAC e PT) separadamente de tecido cerebral post mortem e as redes de co-expressão do striatum e de dois circuitos envolvidos no transtorno. Os resultados mostraram que diferentes processos biológicos, bem como a desregulação da conectividade de rede, são específicos para cada região do estriado e estão de acordo com o modelo tripartido do estriado e contribuem de diferentes formas para a fisiopatologia do TOC. Especificamente, a regulação dos níveis de neurotransmissores, processo pré-sináptico envolvido na transmissão sináptica química foram compartilhados entre NAC e PT. A resposta celular ao estímulo químico, resposta ao estímulo externo, resposta à substância orgânica, regulação da plasticidade sináptica e modulação da transmissão sináptica foram compartilhadas entre CN e PT. A maioria dos genes que possuem variantes comuns e / ou raras previamente associadas ao TOC que são diferencialmente expressas ou que fazem parte de módulos de co-expressão menos preservados em nosso estudo também sugerem especificidade de cada região estriatal. Os módulos de co-expressão preservados e menos preservados nos circuitos afetivo e cognitivo ventral corroboram com as assinaturas transcricionais de cada área e de cada circuito no TOC e nos controles. Este é o primeiro trabalho com a proposta de avaliar a expressão gênica em áreas estriatais, analisadas individualmente, envolvidas com o TOC, bem como as redes de co-expressão do estriado e dos circuitos individualmenteObsessive compulsive disorder (OCD) is a psychiatric disorder, characterized by the presence of obsessions and/or compulsions. Functional neuroimaging studies indicate that OCD is a heterogeneous disorder related the cortical-striatal thalamic circuitry (CSTC) and the areas that compose this circuitry include the nucleus accumbens (NAC), putamen (PT), caudate nucleus (CN), orbitofrontal cortex (OFC) and subgenual cingulate gyri (ACC). The main characteristics of CSTC is the innervation of the frontal cortex in direction of the striatum and each small circuitries have specific characteristics in the affective, dorsal cognitive and ventral cognitive motor. In this work we compared the cases and controls transcriptome of the three striatal areas (CN, NAC and PT) separately from post mortem brain tissue and the co-expression networks of the striatum and of two circuits involved in the disorder. Results showed that different biological process as well as networks connectivity deregulation were specific for each striatum region according to the striatum tripartite model and contribute in different ways to OCD pathophysiology. Specifically, regulation of neurotransmitter levels, presynaptic process involved in chemical synaptic transmission were shared between NAC and PT. Cellular response to chemical stimulus, response to external stimulus, response to organic substance, regulation of synaptic plasticity, and modulation of synaptic transmission were shared between CN and PT. Most genes harboring common and/or rare variants previously associated with OCD that are differentially expressed or part of a least preserved co-expression modules in our study also suggest striatum sub regions specificity. The co-expression modules preserved and least preserved in affective and ventral cognitive circuitry corroborate with transcriptional signatures of each area and each circuitry in OCD and controls. This is the first work with the proposal to evaluate the gene expression in striatum areas individually, involved with OCD as well evaluate the coexpression networks in striatum and each circuitr

Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide

Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. in this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) the 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of I amino acid. (ii) the conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.Universidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Interdisciplinary Ctr Gene Therapy, São Paulo, BrazilUniv São Paulo, Fac Med, Dept Radiol, São Paulo, BrazilUniv São Paulo, ICB, Dept Pharmacol, BR-09500900 São Paulo, BrazilButantan Inst, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Interdisciplinary Ctr Gene Therapy, São Paulo, BrazilWeb of Scienc

Epigenetic evidence for involvement of the oxytocin receptor gene in obsessive–compulsive disorder

Abstract Background Obsessive–compulsive disorder (OCD) is a chronic neurodevelopmental disorder that affects up to 3% of the general population. Although epigenetic mechanisms play a role in neurodevelopment disorders, epigenetic pathways associated with OCD have rarely been investigated. Oxytocin is a neuropeptide involved in neurobehavioral functions. Oxytocin has been shown to be associated with the regulation of complex socio-cognitive processes such as attachment, social exploration, and social recognition, as well as anxiety and other stress-related behaviors. Oxytocin has also been linked to the pathophysiology of OCD, albeit inconsistently. The aim of this study was to investigate methylation in two targets sequences located in the exon III of the oxytocin receptor gene (OXTR), in OCD patients and healthy controls. We used bisulfite sequencing to quantify DNA methylation in peripheral blood samples collected from 42 OCD patients and 31 healthy controls. Results We found that the level of methylation of the cytosine-phosphate-guanine sites in two targets sequences analyzed was greater in the OCD patients than in the controls. The higher methylation in the OCD patients correlated with OCD severity. We measured DNA methylation in the peripheral blood, which prevented us from drawing any conclusions about processes in the central nervous system. Conclusion To our knowledge, this is the first study investigating DNA methylation of the OXTR in OCD. Further studies are needed to evaluate the roles that DNA methylation and oxytocin play in OCD

Brain areas involved with obsessive-compulsive disorder present different DNA methylation modulation

BackgroundObsessive-compulsive disorder (OCD) is characterized by intrusive thoughts and repetitive actions, that presents the involvement of the cortico-striatal areas. The contribution of environmental risk factors to OCD development suggests that epigenetic mechanisms may contribute to its pathophysiology. DNA methylation changes and gene expression were evaluated in post-mortem brain tissues of the cortical (anterior cingulate gyrus and orbitofrontal cortex) and ventral striatum (nucleus accumbens, caudate nucleus and putamen) areas from eight OCD patients and eight matched controls.ResultsThere were no differentially methylated CpG (cytosine-phosphate-guanine) sites (DMSs) in any brain area, nevertheless gene modules generated from CpG sites and protein-protein-interaction (PPI) showed enriched gene modules for all brain areas between OCD cases and controls. All brain areas but nucleus accumbens presented a predominantly hypomethylation pattern for the differentially methylated regions (DMRs). Although there were common transcriptional factors that targeted these DMRs, their targeted differentially expressed genes were different among all brain areas. The protein-protein interaction network based on methylation and gene expression data reported that all brain areas were enriched for G-protein signaling pathway, immune response, apoptosis and synapse biological processes but each brain area also presented enrichment of specific signaling pathways. Finally, OCD patients and controls did not present significant DNA methylation age differences.ConclusionsDNA methylation changes in brain areas involved with OCD, especially those involved with genes related to synaptic plasticity and the immune system could mediate the action of genetic and environmental factors associated with OCD

Putative contributions of the sex chromosome proteins SOX3 and SRY to neurodevelopmental disorders

Coordenação de Aperfeiçoamento de Pessoalde Nível Superior, Grant/Award Number: DSProgramDS-1750212PROEX-1669479PROEX-33002010073P7; Fundaçãode Amparo à Pesquisa do Estado de São Paulo,Grant/Award Number: 2011/04956-62011/14658-22014/00041-12014/00591-12014/10488-32015/06281-7; Universidade Federaldo ABC, Grant/Award Number: InstitutionalScholarship; UFABC; CAPES, Grant/AwardNumber: DS-1750212; FAPESP, Grant/AwardNumbers: 2014/10488-3, 2011/04956-6,2014/00591-1, 2014/00041-1,2015/06281-7, 2011/14658-2.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil / University of São Paulo. Inter-institutional Grad Program on Bioinformatics. São Paulo, SP, Brazil.Federal University of ABC. Center of Mathematics, Computing and Cognition. Santo André, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil.Brazilian Center for Research in Energy and Materials. Brazilian Biosciences National Laboratory. Campinas, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Laboratório de Pesquisas Básicas em Malária - Entomologia. Ananindeua, PA, Brasil.Brazilian Center for Research in Energy and Materials. Brazilian Biosciences National Laboratory. Campinas, SP, Brazil.Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria, Hospital das Clinicas HCFMUSP. Laboratório de Psicopatologia e Terapêutica Psiquiátrica . São Paulo, SP, Brazil / University of São Paulo. Inter-institutional Grad Program on Bioinformatics. São Paulo, SP, Brazil / Universidade de São Paulo. Faculdade de Medicina. Instituto de Psiquiatria. Hospital das Clinicas HCFMUSP. São Paulo, SP, Brazil / National Institute of Developmental Psychiatry for Children and Adolescents (INPD). São Paulo, SP, Brazil / Universidade de São Paulo. Faculdade de Medicina FMUSP. São Paulo, SP, Brazil.The male-biased prevalence of certain neurodevelopmental disorders and the sex-biased outcomes associated with stress exposure during gestation have been previously described. Here, we hypothesized that genes distinctively targeted by only one or both homologous proteins highly conserved across therian mammals, SOX3 and SRY, could induce sexual adaptive changes that result in a differential risk for neurodevelopmental disorders. ChIP-seq/chip data showed that SOX3/SRY gene targets were expressed in different brain cell types in mice. We used orthologous human genes in rodent genomes to extend the number of SOX3/SRY set (1,721). These genes were later found to be enriched in five modules of coexpressed genes during the early and mid-gestation periods (FDR < 0.05), independent of sexual hormones. Genes with differential expression (24, p < 0.0001) and methylation (40, p < 0.047) between sexes were overrepresented in this set. Exclusive SOX3 or SRY target genes were more associated with the late gestational and postnatal periods. Using autism as a model sex-biased disorder, the SOX3/SRY set was enriched in autism gene databases (FDR ≤ 0.05), and there were more de novo variations from the male autism spectrum disorder (ASD) samples under the SRY peaks compared to the random peaks (p < 0.024). The comparison of coexpressed networks of SOX3/SRY target genes between male autism and control samples revealed low preservation in gene modules related to stress response (99 genes) and neurogenesis (78 genes). This study provides evidence that while SOX3 is a regulatory mechanism for both sexes, the male-exclusive SRY also plays a role in gene regulation, suggesting a potential mechanism for sex bias in ASD

Characterization of germline mutations of MLH1 and MSH2 in unrelated south American suspected Lynch syndrome individuals

Lynch syndrome (LS) is an autosomal dominant syndrome that predisposes individuals to development of cancers early in life. These cancers are mainly the following: colorectal, endometrial, ovarian, small intestine, stomach and urinary tract cancers. LS is caused by germline mutations in DNA mismatch repair genes (MMR), mostly MLH1 and MSH2, which are responsible for more than 85% of known germline mutations. To search for germline mutations in MLH1 and MSH2 genes in 123 unrelated South American suspected LS patients (Bethesda or Amsterdam Criteria) DNA was obtained from peripheral blood, and PCR was performed followed by direct sequencing in both directions of all exons and intron-exon junctions regions of the MLH1 and MSH2 genes. MLH1 or MSH2 pathogenic mutations were found in 28.45% (34/123) of the individuals, where 25/57 (43.85%) fulfilled Amsterdam I, II and 9/66 (13.63%) the Bethesda criteria. The mutations found in both genes were as follows: nonsense (35.3%), frameshift (26.47%), splicing (23.52%), and missense (9%). Thirteen alterations (35.14%) were described for the first time. The data reported in this study add new information about MLH1 and MSH2 gene mutations and contribute to better characterize LS in Brazil, Uruguay and Argentina. The high rate of novel mutations demonstrates the importance of defining MLH1 and MSH2 mutations in distinct LS populations

KRAS insertions in colorectal cancer: What do we know about unusual KRAS mutations?

Introduction: KRAS mutations are negative predictors of the response to anti-EGFR therapy in colorectal carcinomas (CRCs). Point mutations in codons 12,13, and 61 are the most common KRAS mutations in CRC There are few reports on insertions in KRAS, and little is known about its ability to activate the RAS pathway. The scarcity of data regarding insertion frequencies and nucleotide additions in KRAS impedes the management of patients with such mutations. We present data on KRAS insertions in CRC and discuss a case.Materials and methods: Pyrosequencing and Sanger sequencing were performed to identify KRAS and BRAF mutations in paraffin-embedded samples of CRC Expression of mismatch repair proteins was examined by immunohistochemistry.Results: We detected a GGT insertion between codons 12 and 13 (c.36_37insGGT;p.G12_G13insG) in a CRC patient. We found that insertions in KRAS is very rare in CRC and that the most frequent type of insertion is c36_37insGGT.Conclusions: KRAS gene insertions represent a diagnostic and clinical challenge due to the difficult and unusual pyrosequencing findings and the lack of information regarding its clinical impact. (C) 2014 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Integrative Variation Analysis Reveals that a Complex Genotype May Specify Phenotype in Siblings with Syndromic Autism Spectrum Disorder

<div><p>It has been proposed that copy number variations (CNVs) are associated with increased risk of autism spectrum disorder (ASD) and, in conjunction with other genetic changes, contribute to the heterogeneity of ASD phenotypes. Array comparative genomic hybridization (aCGH) and exome sequencing, together with systems genetics and network analyses, are being used as tools for the study of complex disorders of unknown etiology, especially those characterized by significant genetic and phenotypic heterogeneity. Therefore, to characterize the complex genotype-phenotype relationship, we performed aCGH and sequenced the exomes of two affected siblings with ASD symptoms, dysmorphic features, and intellectual disability, searching for <i>de novo</i> CNVs, as well as for <i>de novo</i> and rare inherited point variations—single nucleotide variants (SNVs) or small insertions and deletions (indels)—with probable functional impacts. With aCGH, we identified, in both siblings, a duplication in the 4p16.3 region and a deletion at 8p23.3, inherited by a paternal balanced translocation, t(4, 8) (p16; p23). Exome variant analysis found a total of 316 variants, of which 102 were shared by both siblings, 128 were in the male sibling exome data, and 86 were in the female exome data. Our integrative network analysis showed that the siblings’ shared translocation could explain their similar syndromic phenotype, including overgrowth, macrocephaly, and intellectual disability. However, exome data aggregate genes to those already connected from their translocation, which are important to the robustness of the network and contribute to the understanding of the broader spectrum of psychiatric symptoms. This study shows the importance of using an integrative approach to explore genotype-phenotype variability.</p></div