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Experimental evidence indicates differences between women and men in several medical areas, including susceptibility to metabolic diseases. Sexual dimorphism could be influenced by microRNA (miRNA)-mediated regulation of gene expression, and miRNAs have also been implicated in fetal programming of obesity. Our previous finding of altered proteome in human amniotic stem cells (hA-MSCs) during obesity (Ob-) prompted us to look for gender-related differences in the miRNA regulation of gene expression in Ob-hA-MSCs that might be involved in metabolic changes. Using small RNA-sequencing we studied the miRNomes of 7 Control (Co-) hA-MSCs and 13 Ob-hA-MSCs from mothers of boys (3 M-Co, 6 M-Ob-) or girls (4 F-Co-, 7 F-Ob-). Most miRNAs were similarly expressed in the Co-hA-MSCs regardless of offspring gender, whereas 13 miRNAs were down-expressed and miR-146a was up-expressed in F-Ob-hA-MSCs vs M-Ob-hA-MSCs (p

Next-generation sequencing (NGS) technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology’s flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics.

By analyzing multiple gene panels, next-generation sequencing is more effective than conventional procedures in identifying disease-related mutations that are useful for clinical decision-making. Here, we aimed to test the efficacy of an 84 genes customized-panel in BRCA1 and BRCA2 mutation-negative patients. Twenty-four patients were enrolled in this study. DNA libraries were prepared using a picodroplet PCR-based approach and sequenced with the MiSeq System. Highly putative pathogenic mutations were identified in genes other than the commonly tested BRCA1/2: 2 pathogenic mutations one in TP53 and one in MUTYH; 2 missense variants in MSH6 and ATM, respectively; 2 frameshift variants in KLLN, and ATAD2, respectively; an intronic variant in ANPEP, and 3 not functionally known variants (a frameshift variant in ATM a nonsense variant in ATM and a missense variant in NFE2L2). Our results show that this molecular screening will increase diagnostic sensitivity leading to a better risk assessment in breast cancer patients and their families. This strategy could also reveal genes that have a higher penetrance for breast and ovarian cancers by matching gene mutation with familial and clinical data, thereby increasing information about hereditary breast and ovarian cancer genetics and improving cancer prevention measures or therapeutic approaches.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3. Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3. 10.1093/brain/awy039-video1 awy039media1 5742053534001

BACKGROUND: DNA methylation of the Th1 and Th2 cytokine genes is altered during cow's milk allergy (CMA). Forkhead box transcription factor 3 (FoxP3) is essential for the development and function of regulatory T cells (Tregs) and is involved in oral tolerance acquisition. We assessed whether tolerance acquisition in children with IgE-mediated CMA is associated with DNA demethylation of the Treg-specific demethylated region (TSDR) of FoxP3. RESULTS: Forty children (aged 3-18 months) were enrolled: 10 children with active IgE-mediated CMA (group 1), 10 children who outgrew CMA after dietary treatment with an extensively hydrolyzed casein formula containing the probiotic Lactobacillus rhamnosus GG (group 2), 10 children who outgrew CMA after treatment with other formulas (group 3), and 10 healthy controls (group 4). FoxP3 TSDR demethylation and expression were measured in mononuclear cells purified from peripheral blood of the four groups of children. FoxP3 TSDR demethylation was significantly lower in children with active IgE-mediated CMA than in either children who outgrew CMA or in healthy children. Formula selection influenced the FoxP3 TSDR demethylation profile. The FoxP3 TSDR demethylation rate and expression level were correlated. CONCLUSIONS: Tolerance acquisition in children with IgE-mediated CMA involves epigenetic regulation of the FoxP3 gene. This feature could be a new target for preventive and therapeutic strategies against CMA.

Background Epigenetic changes in DNA methylation could regulate the expression of several allergy-related genes. We investigated whether tolerance acquisition in children with immunoglobulin E (IgE)-mediated cow’s milk allergy (CMA) is characterized by a specific DNA methylation profile of Th2 (IL-4, IL-5) and Th1 (IL-10, IFN-γ)-associated cytokine genes. Results DNA methylation of CpGs in the promoting regions of genes from peripheral blood mononuclear cells and serum level of IL-4, IL-5, IL-10 and INF-γ were assessed in children with active IgE-mediated CMA (group 1), in children who acquired tolerance to cow’s milk proteins (group 2) and in healthy children (group 3). Forty children (24 boys, aged 3 to 18 months) were enrolled: 10 in group 1, 20 in group 2, and 10 in the control group. The DNA methylation profiles clearly separated active CMA patients from healthy controls. We observed an opposite pattern comparing subjects with active IgE-mediated CMA with healthy controls and group 2 children who outgrew CMA. The IL-4 and IL-5 DNA methylation was significantly lower, and IL-10 and INF-γ DNA methylation was higher in active IgE-mediated CMA patients. Gene promoter DNA methylation rates of all cytokines and respective serum levels were strongly correlated. Formula selection significantly influenced cytokine DNA methylation profiles in group 2. Conclusions Tolerance acquisition in children with IgE-mediated CMA is characterized by a distinct Th1 and Th2 cytokine gene DNA methylation pattern. These results suggest that DNA methylation may be a target for CMA prevention and treatment.

Next-generation sequencing (NGS) technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology’s flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics.