DYT-TUBB4A (DYT4 Dystonia): New Clinical and Genetic Observations.

OBJECTIVE: To report 4 novel TUBB4A mutations leading to laryngeal and cervical dystonia with frequent generalization. METHODS: We screened 4 families including a total of 11 definitely affected members with a clinical picture resembling the original description. RESULTS: Four novel variants in the TUBB4A gene have been identified: D295N, R46M, Q424H, and R121W. In silico modeling showed that all variants have characteristics similar to R2G. The variants segregate with the disease in 3 of the families with evidence of incomplete penetrance in 2 of them. All 4 variants would be classified as likely pathogenic. The clinical picture particularly included laryngeal dystonia (often the site of onset), associated with cervical and upper limb dystonia and frequent generalization. Laryngeal dystonia was extremely prevalent (>90%) both in the original cases and in this case series. The hobby horse gait was evident in only 1 patient in this case series. CONCLUSIONS: Our interpretation is that laryngeal involvement is a hallmark feature of DYT-TUBB4A. Nevertheless, TUBB4A mutations remain an exceedingly rare cause of laryngeal or other isolated dystonia.

Noninvasive prenatal paternity determination using microhaplotypes: a pilot study.

BACKGROUND: The use of noninvasive techniques to determine paternity prenatally is increasing because it reduces the risks associated with invasive procedures. Current methods, based on SNPs, use the analysis of at least 148 markers, on average. METHODS: To reduce the number of regions, we used microhaplotypes, which are chromosomal segments smaller than 200 bp containing two or more SNPs. Our method employs massively parallel sequencing and analysis of microhaplotypes as genetic markers. We tested 20 microhaplotypes and ascertained that 19 obey Hardy-Weinberg equilibrium and are independent, and data from the 1000 Genomes Project were used for population frequency and simulations. RESULTS: We performed simulations of true and false paternity, using the 1000 Genomes Project data, to confirm if the microhaplotypes could be used as genetic markers. We observed that at least 13 microhaplotypes should be used to decrease the chances of false positives. Then, we applied the method in 31 trios, and it was able to correctly assign the fatherhood in cases where the alleged father was the real father, excluding the inconclusive results. We also cross evaluated the mother-plasma duos with the alleged fathers for false inclusions within our data, and we observed that the use of at least 15 microhaplotypes in real data also decreases the false inclusions. CONCLUSIONS: In this work, we demonstrated that microhaplotypes can be used to determine prenatal paternity by using only 15 regions and with admixtures of DNA.

Initial findings of striatum tripartite model in OCD brain samples based on transcriptome analysis.

Obsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by obsessions and/or compulsions. Different striatal subregions belonging to the cortico-striato-thalamic circuitry (CSTC) play an important role in the pathophysiology of OCD. The transcriptomes of 3 separate striatal areas (putamen (PT), caudate nucleus (CN) and accumbens nucleus (NAC)) from postmortem brain tissue were compared between 6 OCD and 8 control cases. In addition to network connectivity deregulation, different biological processes are specific to each striatum region according to the tripartite model of the striatum and contribute in various ways to OCD pathophysiology. Specifically, regulation of neurotransmitter levels and presynaptic processes involved in chemical synaptic transmission were shared between NAC and PT. The Gene Ontology terms 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 were differentially expressed or part of a least preserved coexpression module in our study also suggest striatum subregion specificity. At the transcriptional level, our study supports differences in the 3 circuit CSTC model associated with OCD.

Short-Term Effects of Sepsis and the Impact of Aging on the Transcriptional Profile of Different Brain Regions.

Among the clinical manifestations observed in septic patients, sepsis-associated encephalopathy (SAE) is probably the most obscure and poorly explored. It is well established, however, that SAE is more prevalent in aged individuals and related to a worse outcome. In this context, we decided to investigate the acute effects of sepsis, induced by cecal ligation and puncture (CLP), on the cerebral transcriptional profile of young and old rats. The idea was to highlight important signaling pathways possibly implicated in the early stages of SAE. Global gene expression analysis of three different brain regions (hippocampus, cerebellum, and cortex) indicated a relatively small interference of sepsis at the transcriptional level. Cerebellum tissue was the least affected by sepsis in aged rats. The increased expression of S100a8, Upp1, and Mt2a in all three brain regions of young septic rats indicate that these genes may be involved in the first line of response to sepsis in the younger brain. On the other hand, altered expression of a network of genes involved in sensory perception of smell in the cortex of aged rats, but not in young ones, indicates an earlier disruption of cortex function, possibly more sensitive to the systemic inflammation. The expression of S100a8 at the protein level was confirmed in all brain regions, with clear-up regulation in septic aged cortex. Taken together, our results indicate that the transcriptional response of the central nervous system to early sepsis varies between distinct brain regions and that the cortex is affected earlier in aged animals, in line with early neurological manifestations observed in older patients.

The prevalence of PRKRA mutations in idiopathic dystonia.

INTRODUCTION: DYT-PRKRA (DYT16) is considered a rare cause of dystonia-parkinsonism. The significance of this gene as a cause of dystonia and its phenotypical characterization must be determined in larger cohorts. We aimed to investigate the role of PRKRA in patients with dystonia. METHODS: We sequenced PRKRA in 153 unrelated Brazilian patients with idiopathic dystonia. The frequency of novel missense variants was investigated in healthy Brazilian controls and in public databases. Homozygosity in the PRKRA region was assessed through polymorphic markers. RESULTS: PRKRA variants were identified in seven probands with isolated dystonia, including a novel c.C795A variant in compound heterozygosity with the previously described c.C665T variant. Heterozygosity in the gene region was observed in two probands who were homozygous for c.C665T, indicating that this mutation originated from independent events, suggesting a hotspot. CONCLUSION: PRKRA is not an unusual cause of idiopathic dystonia. In this cohort, it was responsible for 4.5% of the total of cases (4.9% of the isolated dystonia cases). The most common phenotype was early-onset isolated focal dystonia followed by generalization, parkinsonism was not observed. This is first report of PRKRA causing adulthood-onset dystonia. Screenings of large cohorts are recommended to investigate the role of this gene in isolated dystonia, as well as in dystonia-parkinsonism cases worldwide.

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

Oral 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. METHODS: Two-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. RESULTS: Overall 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. CONCLUSIONS: Although 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.

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.

Linear mRNA amplification approach for RNAseq from limited amount of RNA.

Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)(+) mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3' end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

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.

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

BACKGROUND: Human 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. METHODS: Real 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. RESULTS: Overexpression 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. CONCLUSION: The 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.

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.

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.

Plant-antivenom: database of anti-venom medicinal plants

Plant-antivenom is a computational Websystem about medicinal plants with anti-venom properties. The system consists of a database of these plants, including scientific publications on this subject and amino acid sequences of active principles from venomous animals. The system relates these data allowing their integration through different search applications. For the development of the system, the first surveys were conducted in scientific literature, allowing the creation of a publication database in a library for reading and user interaction. Then, classes of categories were created, allowing the use of tags and the organization of content. This database on medicinal plants has information such as family, species, isolated compounds, activity, inhibited animal venoms, among others. Provision is made for submission of new information by registered users, by the use of wiki tools. Content submitted is released in accordance to permission rules defined by the system. The database on biological venom protein amino acid sequences was structured from the essential information from National Center for Biotechnology Information (NCBI). Plant-antivenom's interface is simple, contributing to a fast and functional access to the system and the integration of different data registered on it. Plant-antivenom system is available on the Internet at http://gbi.fmrp.usp.br/plantantivenom.