A randomised clinical pilot trial to test the effectiveness of parent training with video modelling to improve functioning and symptoms in children with autism spectrum disorders and intellectual disability.

BACKGROUND: Poor eye contact and joint attention are early signs of autism spectrum disorder (ASD) and important prerequisites for developing other socio-communicative skills. Teaching parents evidence-based techniques to improve these skills can impact the overall functioning of children with ASD. We aimed to analyse the impact of conducting a group parent-training intervention with video modelling to improve the intelligent quotient (IQ), social and communication functioning and to minimise symptoms in children with ASD and intellectual disability (ID). METHODS: Study design: A multicentre, single-blinded, randomised clinical pilot trial of parent training using video modelling was conducted. SAMPLE: Sixty-seven parents of children with ASD, aged between 3 and 6 years and with IQs between 50 and 70, were randomised: 34 to the intervention group and 33 to the control group. Intervention program: The intervention group received parent training over 22 sessions, and the control group received the standard community treatment. INSTRUMENTS: Pre-evaluation and post-evaluation (week 28), the following were used: Autism Diagnostic Interview, Vineland Adaptive Behaviour Scale I, Snijders-Oomen Nonverbal Intelligence Test, Autism Behaviour Checklist and Hamilton Depression Rating Scale. DATA ANALYSIS: Intention to treat and complier-average causal effect (CACE) were used to estimate the effects of the intervention. RESULTS: There was a statistically significant improvement in the Vineland standardized communication scores in CACE (Cohen's d = 0.260). There was a non-statistically significant decrease in autism symptomatology (Autism Behaviour Checklist total scores) and a significant increase in the non-verbal IQ in the intervention group. After the false discovery rate correction was applied, IQ remained statistically significant under both paradigms. The effect size for this adjusted outcome under the intention-to-treat paradigm was close to 0.4, and when considering adherence (CACE), the effect sizes were more robust (IQ's Cohen's d = 0.433). CONCLUSIONS: Parent training delivered by video modelling can be a useful technique for improving the care given to children with ASD and ID, particularly in countries that lack specialists.

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

UNLABELLED: 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.

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.

Odorant receptor genes are expressed in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a malignant tumor found in the human nasal cavity. These tumors are rare and poorly characterized at the molecular level. In this study, we asked whether olfactory-specific genes are expressed in ONBs by using reverse-transcriptase-polymerase chain reaction. We found that the olfactory marker protein and the RIC-8B genes, which are specifically expressed in mature olfactory neurons, are expressed in ONBs. Importantly, we also found that ONBs express a large variety of odorant receptor genes, representative of different odorant receptor gene subfamilies. Our results show that the ONBs express genes that are normally expressed in mature olfactory neurons and indicate that they are derived from progenitor or immature cells in the olfactory epithelium and not from a clonal expansion of a single or few mature olfactory neurons.

African ancestry protects against Alzheimer's disease-related neuropathology.

Previous studies in dementia epidemiology have reported higher Alzheimer's disease rates in African-Americans when compared with White Americans. To determine whether genetically determined African ancestry is associated with neuropathological changes commonly associated with dementia, we analyzed a population-based brain bank in the highly admixed city of São Paulo, Brazil. African ancestry was estimated through the use of previously described ancestry-informative markers. Risk of presence of neuritic plaques, neurofibrillary tangles, small vessel disease, brain infarcts and Lewy bodies in subjects with significant African ancestry versus those without was determined. Results were adjusted for multiple environmental risk factors, demographic variables and apolipoprotein E genotype. African ancestry was inversely correlated with neuritic plaques (P=0.03). Subjects with significant African ancestry (n=112, 55.4%) showed lower prevalence of neuritic plaques in the univariate analysis (odds ratio (OR) 0.72, 95% confidence interval (CI) 0.55-0.95, P=0.01) and when adjusted for age, sex, APOE genotype and environmental risk factors (OR 0.43, 95% CI 0.21-0.89, P=0.02). There were no significant differences for the presence of other neuropathological alterations. We show for the first time, using genetically determined ancestry, that African ancestry may be highly protective of Alzheimer's disease neuropathology, functioning through either genetic variants or unknown environmental factors. Epidemiological studies correlating African-American race/ethnicity with increased Alzheimer's disease rates should not be interpreted as surrogates of genetic ancestry or considered to represent African-derived populations from the developing nations such as Brazil.

Brazilian psychiatric brain bank: a new contribution tool to network studies.

There is an urgent need for expanding the number of brain banks serving psychiatric research. We describe here the Psychiatric Disorders arm of the Brain Bank of the Brazilian Aging Brain Study Group (Psy-BBBABSG), which is focused in bipolar disorder (BD) and obsessive compulsive disorder (OCD). Our protocol was designed to minimize limitations faced by previous initiatives, and to enable design-based neurostereological analyses. The Psy-BBBABSG first milestone is the collection of 10 brains each of BD and OCD patients, and matched controls. The brains are sourced from a population-based autopsy service. The clinical and psychiatric assessments were done by an expert team including psychiatrists, through an informant. One hemisphere was perfused-fixed to render an optimal fixation for conducting neurostereological studies. The other hemisphere was comprehensively dissected and frozen for molecular studies. In 20 months, we collected 36 brains. A final report was completed for 14 cases: 3 BDs, 4 major depressive disorders, 1 substance use disorder, 1 mood disorder NOS, 3 obsessive compulsive spectrum symptoms, 1 OCD and 1 schizophrenia. The majority were male (64%), and the average age at death was 67.2 ± 9.0 years. The average postmortem interval was 16 h. Three matched controls were collected. The pilot stage confirmed that the protocols are well fitted to reach our goals. Our unique autopsy source makes possible to collect a fairly number of high quality cases in a short time. Such a collection offers an additional to the international research community to advance the understanding on neuropsychiatric diseases.

Temporal blastemal cell gene expression analysis in the kidney reveals new Wnt and related signaling pathway genes to be essential for Wilms' tumor onset.

Wilms' tumors (WTs) originate from metanephric blastema cells that are unable to complete differentiation, resulting in triphasic tumors composed of epithelial, stromal and blastemal cells, with the latter harboring molecular characteristics similar to those of the earliest kidney development stages. Precise regulation of Wnt and related signaling pathways has been shown to be crucial for correct kidney differentiation. In this study, the gene expression profile of Wnt and related pathways was assessed in laser-microdissected blastemal cells in WTs and differentiated kidneys, in human and in four temporal kidney differentiation stages (i.e. E15.5, E17.5, P1.5 and P7.5) in mice, using an orthologous cDNA microarray platform. A signaling pathway-based gene signature was shared between cells of WT and of earliest kidney differentiation stages, revealing genes involved in the interruption of blastemal cell differentiation in WT. Reverse transcription-quantitative PCR showed high robustness of the microarray data demonstrating 75 and 56% agreement in the initial and independent sample sets, respectively. The protein expression of CRABP2, IGF2, GRK7, TESK1, HDGF, WNT5B, FZD2 and TIMP3 was characterized in WTs and in a panel of human fetal kidneys displaying remarkable aspects of differentiation, which was recapitulated in the tumor. Taken together, this study reveals new genes candidate for triggering WT onset and for therapeutic treatment targets.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86 percent of tumors (87 percent sensitivity and 80 percent specificity for predicting response), according to their response to chemotherapy (82 percent in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71 percent samples from the biological validation set were also correctly classified by both trios (72 percent sensitivity; 66 percent specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93 percent of samples from the technical validation group (95 percent sensitivity and 80 percent specificity; 86 percent accuracy by the cross-validation method) and 79 percent from the biological validation group (72 percent sensitivity and 100 percent specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancer patients.

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1, MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1 based trios, with NOTCH1 or NUP210. Both trios correctly separated 86% of tumors (87% sensitivity and 80% specificity for predicting response), according to their response to chemotherapy (82% in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71% samples from the biological validation set were also correctly classified by both trios (72% sensitivity; 66% specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93% of samples from the technical validation group (95% sensitivity and 80% specificity; 86% accuracy by the cross-validation method) and 79% from the biological validation group (72% sensitivity and 100% specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Molecular profiling of isolated histological components of Wilms tumor implicates a commom role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common deregulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Mining ORESTES no-match database: can we still contribute to cancer transcriptome?

The Human Cancer Genome Project generated about 1 million expressed sequence tags by the ORESTES method, principally with the aim of obtaining data from cancer. Of this total, 341,680 showed no similarity with sequences in the public transcript databases, referred to as [quot ]no-match[quot ]. Some of them represent low abundance or difficult to detect human transcripts, but part of these sequences represent genomic contamination or immature mRNA. We performed a bioinformatics pipeline to determine the novelty of ORESTES [quot ]no-match[quot ] datasets from prostate or breast tissues. We started with 14,908 clusters mapped on the human genome. A total of 2226 clusters originating from more than two libraries or singletons with gaps upon genome alignment were selected. Ninety-four clusters with canonical splice sites representing the most stringent criteria to be considered a gene were subjected to manual inspection regarding genomic hits. Of the manually inspected clusters, 49.6% contained new sequences where 42.2% were probable low-expression alternative forms of the characterized genes and 7.4% unpredicted genes. RT-PCR followed by sequencing was performed to validate the largest spliced sequence from 8 clusters, resulting in the confirmation of five sequences as true human transcript fragments. Some of them were differentially expressed between tumor and normal tissue by an in silico analysis. We can conclude that after clean up of the no-match dataset, we still have about 939 new exons and 165 unpredicted genes that could complete the prostate or breast transcriptome.