Novel intragenic deletion within the FXN gene in a patient with typical phenotype of Friedreich ataxia: may be more prevalent than we think?

Background Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele.Case presentation We report here a patient that presented the typical clinical features of FRDA and genetic analysis of FXN intron 1 led to the assumption that the patient carried the common biallelic expansion. Subsequently, parental sample testing led to the identification of a novel intragenic deletion involving the 5'UTR upstream region and exons 1 and 2 of the FXN gene by MLPA.Conclusions With this case, we want to raise awareness about the potentially higher prevalence of intragenic deletions and underline the essential role of parental sample testing in providing accurate genetic counselling

A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions

Cost-effectiveness analysis of newborn screening for sickle-cell disease in Spain

<p><b>Objectives:</b> This study assesses the cost-effectiveness of adding sickle-cell disease (SCD) to the Spanish newborn screening (NBS) program, and explores the sensitivity of the results to key model parameters.</p> <p><b>Methods:</b> A discrete event simulation model was developed that compared NBS for SCD versus clinical detection. The model followed a simulated cohort of newborns for 10 years, and estimated the impact in costs and life expectancy of prophylactic treatments established after early detection. Probabilistic, one-way and two-way sensitivity analysis were performed.</p> <p><b>Results:</b> NBS was found to be more costly and more effective than clinical detection of SCD. The estimated incremental cost per life year (LY) gained was 34,169.46 €/LY. This result was very sensitive to the cost of the screening test, the birth prevalence and the proportion of severe cases among the affected children.</p> <p><b>Conclusions:</b> There is uncertainty regarding the cost-effectiveness of NBS for SCD in the Spanish context. Our base case estimate of the cost per LY gained lies near the 30,000€/LY commonly cited in Spain.</p

PKCzeta Is Essential for Pancreatic beta-Cell Replication During Insulin Resistance by Regulating mTOR and Cyclin-D2

Adaptive beta-cell replication occurs in response to increased metabolic demand during insulin resistance. The intracellular mediators of this compensatory response are poorly defined and their identification could provide significant targets for beta-cell regeneration therapies. Here we show that glucose and insulin in vitro and insulin resistance in vivo activate protein kinase C zeta (PKCzeta) in pancreatic islets and beta-cells. PKCzeta is required for glucose- and glucokinase activator-induced proliferation of rodent and human beta-cells in vitro. Furthermore, either kinase-dead PKCzeta expression (KD-PKCzeta) or disruption of PKCzeta in mouse beta-cells blocks compensatory beta-cell replication when acute hyperglycemia/hyperinsulinemia is induced. Importantly, KD-PKCzeta inhibits insulin resistance-mediated mammalian target of rapamycin (mTOR) activation and cyclin-D2 upregulation independent of Akt activation. In summary, PKCzeta activation is key for early compensatory beta-cell replication in insulin resistance by regulating the downstream signals mTOR and cyclin-D2. This suggests that alterations in PKCzeta expression or activity might contribute to inadequate beta-cell mass expansion and beta-cell failure leading to type 2 diabetes

Does Plant Breeding for Antioxidant-Rich Foods Have an Impact on Human Health?

Given the general beneficial effects of antioxidants-rich foods on human health and disease prevention, there is a continuous interest in plant secondary metabolites conferring attractive colors to fruits and grains and responsible, together with others, for nutraceutical properties. Cereals and Solanaceae are important components of the human diet, thus, they are the main targets for functional food development by exploitation of genetic resources and metabolic engineering. In this review, we focus on the impact of antioxidants-rich cereal and Solanaceae derived foods on human health by analyzing natural biodiversity and biotechnological strategies aiming at increasing the antioxidant level of grains and fruits, the impact of agronomic practices and food processing on antioxidant properties combined with a focus on the current state of pre-clinical and clinical studies. Despite the strong evidence in in vitro and animal studies supporting the beneficial effects of antioxidants-rich diets in preventing diseases, clinical studies are still not sufficient to prove the impact of antioxidant rich cereal and Solanaceae derived foods on huma

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors

International audienceSeveral obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human Tlymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy)