Epidemiological, Clinical, and Genomic Profile in Head and Neck Cancer Patients and Their Families.

Inherited cancer predisposition genes are described as risk factors in head and neck cancer (HNC) families. To explore the clinical and epidemiological data and their association with a family history of cancer, we recruited 74 patients and 164 relatives affected by cancer. The germline copy number alterations were evaluated in 18 patients using array comparative genomic hybridization. Two or more first-degree relatives with HNC, tobacco-associated tumor sites (lung, esophagus, and pancreas), or other related tumors (breast, colon, kidney, bladder, cervix, stomach carcinomas, and melanoma) were reported in 74 families. Ten index patients had no exposure to any known risk factors. Family members presented tumors of 19 topographies (30 head and neck, 26 breast, 21 colon). In first-degree relatives, siblings were frequently affected by cancer (n = 58, 13 had HNC). Breast cancer (n = 21), HNC (n = 19), and uterine carcinoma (n = 15) were commonly found in first-degree relatives and HNC in second-degree relatives (n = 11). Nineteen germline genomic imbalances were detected in 13 patients; three presented gains of WRD genes. The number of HNC patients, the degree of kinship, and the tumor types detected in each relative support the role of heredity in these families. Germline alterations may potentially contribute to cancer development.

Hippocampal CA3 transcriptional modules associated with granule cell alterations and cognitive impairment in refractory mesial temporal lobe epilepsy patients.

In about a third of the patients with epilepsy the seizures are not drug-controlled. The current limitation of the antiepileptic drug therapy derives from an insufficient understanding of epilepsy pathophysiology. In order to overcome this situation, it is necessary to consider epilepsy as a disturbed network of interactions, instead of just looking for changes in single molecular components. Here, we studied CA3 transcriptional signatures and dentate gyrus histopathologic alterations in hippocampal explants surgically obtained from 57 RMTLE patients submitted to corticoamygdalohippocampectomy. By adopting a systems biology approach, integrating clinical, histopathological, and transcriptomic data (weighted gene co-expression network analysis), we were able to identify transcriptional modules highly correlated with age of disease onset, cognitive dysfunctions, and granule cell alterations. The enrichment analysis of transcriptional modules and the functional characterization of the highly connected genes in each trait-correlated module allowed us to unveil the modules' main biological functions, paving the way for further investigations on their roles in RMTLE pathophysiology. Moreover, we found 15 genes with high gene significance values which have the potential to become novel biomarkers and/or therapeutic targets in RMTLE.

Prevalence of Inflammatory Pathways Over Immuno-Tolerance in Peripheral Blood Mononuclear Cells of Recent-Onset Type 1 Diabetes.

Background: Changes in innate and adaptive immunity occurring in/around pancreatic islets had been observed in peripheral blood mononuclear cells (PBMC) of Caucasian T1D patients by some, but not all researchers. The aim of our study was to investigate whether gene expression patterns of PBMC of the highly admixed Brazilian population could add knowledge about T1D pathogenic mechanisms. Methods: We assessed global gene expression in PBMC from two groups matched for age, sex and BMI: 20 patients with recent-onset T1D (≤ 6 months from diagnosis, in a time when the autoimmune process is still highly active), testing positive for one or more islet autoantibodies and 20 islet autoantibody-negative healthy controls. Results: We identified 474 differentially expressed genes between groups. The most expressed genes in T1D group favored host defense, inflammatory and anti-bacterial/antiviral effects (LFT, DEFA4, DEFA1, CTSG, KCNMA1) and cell cycle progression. Several of the downregulated genes in T1D target cellular repair, control of inflammation and immune tolerance. They were related to T helper 2 pathway, induction of FOXP3 expression (AREG) and immune tolerance (SMAD6). SMAD6 expression correlated negatively with islet ZnT8 antibody. The expression of PDE12, that offers resistance to viral pathogens was decreased and negatively related to ZnT8A and GADA levels. The increased expression of long non coding RNAs MALAT1 and NEAT1, related to inflammatory mediators, autoimmune diseases and innate immune response against viral infections reinforced these data. Conclusions: Our analysis suggested the activation of cell development, anti-infectious and inflammatory pathways, indicating immune activation, whereas immune-regulatory pathways were downregulated in PBMC from recent-onset T1D patients with a differential genetic profile.

Age-related transcriptional modules and TF-miRNA-mRNA interactions in neonatal and infant human thymus.

The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1st and 2nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0-30 days (A); 31days-6 months (B); 7-12 months (C); 13-18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.

Age-related transcriptional modules and TF-miRNA-mRNA interactions in neonatal and infant human thymus

The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1st and 2nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0-30 days (A); 31days-6 months (B); 7-12 months (C); 13-18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.

Minipuberty and Sexual Dimorphism in the Infant Human Thymus.

AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the effects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the first six months of life - could affect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for differentially expressed genes and miRNA-target analysis revealed sex differences in thymic tissue during minipuberty, but such differences were not detected in the thymic tissue of infants aged 7-18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several differences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and genomic mechanisms interact in shaping thymic sexual dimorphism along the first six months of life.

Minipuberty and Sexual Dimorphism in the Infant Human Thymus

AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the efects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the frst six months of life - could afect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for diferentially expressed genes and miRNA-target analysis revealed sex diferences in thymic tissue during minipuberty, but such diferences were not detected in the thymic tissue of infants aged 7­18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several diferences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and genomic mechanisms interact in shaping thymic sexual dimorphism along the frst six months of life.

Modular transcriptional repertoire and MicroRNA target analyses characterize genomic dysregulation in the thymus of Down syndrome infants.

Trisomy 21-driven transcriptional alterations in human thymus were characterized through gene coexpression network (GCN) and miRNA-target analyses. We used whole thymic tissue--obtained at heart surgery from Down syndrome (DS) and karyotipically normal subjects (CT)--and a network-based approach for GCN analysis that allows the identification of modular transcriptional repertoires (communities) and the interactions between all the system's constituents through community detection. Changes in the degree of connections observed for hierarchically important hubs/genes in CT and DS networks corresponded to community changes. Distinct communities of highly interconnected genes were topologically identified in these networks. The role of miRNAs in modulating the expression of highly connected genes in CT and DS was revealed through miRNA-target analysis. Trisomy 21 gene dysregulation in thymus may be depicted as the breakdown and altered reorganization of transcriptional modules. Leading networks acting in normal or disease states were identified. CT networks would depict the "canonical" way of thymus functioning. Conversely, DS networks represent a "non-canonical" way, i.e., thymic tissue adaptation under trisomy 21 genomic dysregulation. This adaptation is probably driven by epigenetic mechanisms acting at chromatin level and through the miRNA control of transcriptional programs involving the networks' high-hierarchy genes.

Modular transcriptional repertoire and MicroRNA target analyses characterize genomic dysregulation in the thymus of Down syndrome infants

Trisomy 21-driven transcriptional alterations in human thymus were characterized through gene coexpression network (GCN) and miRNA-target analyses. We used whole thymic tissue - obtained at heart surgery from Down syndrome(DS) and karyotipically normal subjects (CT) - and a network-based approach forGCN analysis that allows the identification of modular transcriptional repertoires(communities) and the interactions between all the system’s constituents through community detection. Changes in the degree of connections observed for hierarchically important hubs/genes in CT and DS networks corresponded to community changes. Distinct communities of highly interconnected genes were topologically identified inthese networks. The role of miRNAs in modulating the expression of highly connected genes in CT and DS was revealed through miRNA-target analysis. Trisomy 21 genedys regulation in thymus may be depicted as the breakdown and altered reorganization of transcriptional modules. Leading networks acting in normal or disease states were identified. CT networks would depict the “canonical” way of thymus functioning. Conversely, DS networks represent a “non-canonical” way, i.e., thymic tissue adaptation under trisomy 21 genomic dysregulation. This adaptation is probablydriven by epigenetic mechanisms acting at chromatin level and through the miRNAcontrol of transcriptional programs involving the networks’ high-hierarchy genes...

Community structure analysis of transcriptional networks reveals distinct molecular pathways for early- and late-onset temporal lobe epilepsy with childhood febrile seizures.

Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A network-based approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO) - all valid GO annotated transcripts - GCNs for the E and L groups; ii) the study of interactions between all the system's constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early onset epilepsies, although impacting more severely the hippocampus, are associated to compensatory mechanisms, while in late MTLE development the brain is less able to generate adaptive mechanisms, what has implications for epilepsy management and drug discovery.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.

Caracterização da região 22q13 31 e sua associação com genes supressores de tumor em carcinomas de cabeça e pescoço / Characterization of 22q13 31 region and its association with tumor suppressor genes in head and neck carcinomas

Os fatores de risco mais importantes descritos para os carcinomas de células escamosas de cabeça e pescoço (CCECP) são o consumo de álcool e o uso de tabaco, além da infecção pelo vírus do papiloma humano. Entretanto, os agentes etiológicos conhecidos não explicam uma parcela significativa de casos. O objetivo deste estudo foi caracterizar a região 22q13.31 como contendo um candidato a supressor tumoral associado com os CCECP. Para isso foi inicialmente selecionado o gene PHF21B para avaliação do número de cópias gênicas e do perfil de metilação em amostras pareadas de DNA tumoral e do sangue periférico, assim como em linhagens celulares de CCECP. Além disso, foi analisada a expressão dos miRNAs hsa-let-7a-3 e hsa-let-7b. Foram detectadas perdas do PHF21B em 43/75 amostras tumorais: 36/43 casos apresentavam história de câncer na família, sendo que 22/36 casos tinham história de câncer em parentes de 1º grau (P=0,049). Entre as amostras de sangue periférico, 6/49 casos apresentaram perdas para o PHF21B: 2/6 casos relataram história familial de câncer. O sequenciamento direto das amostras de sangue e tumor de um subgrupo de casos não revelou alterações nos éxons estudados (3, 6, 7-9, 11). Na ausência de mutações, foi questionado o envolvimento de metilação anormal como um mecanismo alternativo de inativação de um supressor tumoral. A ilha CpG presente no promotor do gene também foi avaliada e se mostrou metilada em 13/37 amostras tumorais. Destes 13 casos, cinco apresentavam história de câncer em parentes em 1º grau e oito apresentaram perda do segmento genômico avaliado para o PHF21B. Em conjunto, estes dados sugerem que o gene PHF21B é um potencial supressor tumoral o qual pode ser inativado por um mecanismo genético (deleção) e epigenético (silenciado por metilação). Na avaliação dos miRNAs, a expressão do hsa-let-7a-3 foi menor no sangue (p=0,002) e no tumor (p=0,006) dos pacientes com história familial de câncer em relação aos controles...