Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia

Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia

Epikarstic crustaceans from some Italian caves: endemisms and spatial scales.

The epikarst crustacean fauna from four Sicilian caves (Conza, Entella, Molara, and Zubbia del Cavallo caves) and four caves in the Lessinian Massif in the Venetian Prealps (Covolo della Croce, Ponte di Veja, Roverè Mille, Buso della Rana caves) was recently investigated. The two groups of caves differ in their environmental conditions: the Sicilian caves are fossil except one which has an active branch; they are all fed by strongly intermittent and scarce rainfall peaking in the fall. The Lessinian caves are fed by more abundant rainfall, with two yearly peaks (May-June and October-November); two of them are active, one has a temporary stream, one is fossil. The crustacean fauna found in the epikarst drip of each of the studied caves is characterized by interesting endemic harpacticoid and cyclopoid copepods, and one bathynellacean syncarid, often collected in only one cave. Higher diversity of stygobiotic taxa was recorded for the Lessinian caves (9 species of copepods in the Lessinian, and 6 species of copepods and one bathynellacean in Sicily); most of the taxa collected in Sicily are endemic to one cave. Spatial analysis showed very different distributions over short spatial scales (tens of kilometers) and, within each cave, the distribution also varied over distances of a few meters. Our data correspond with other studies where many epikarst crustaceans showed a distribution with a linear extent of only a few hundred meters: the epikarst fauna is not uniformly distributed but rather divided in “blocks” probably characterized by different environmental conditions and, as a consequence, by different taxocoenoses. The data highlight the epikarst as a source of “hidden” biodiversity, and the importance of management protection plans which include not only the caves, but also the epikarst overlying layer and the water sources that feed it

Genetic Variants Identified by Whole Exome Sequencing in a Large Italian Family with High Plasma Levels of Factor VIII and Von Willebrand Factor

High plasma levels of factor VIII (FVIII) and von Willebrand factor (VWF) have been indicated as independent risk factors for venous thromboembolism. However, the genetic factors responsible for their increase remain poorly known. In a large Italian family with high FVIII/VWF levels and thrombotic episodes, whole exome sequencing (WES) was performed on 12 family members to identify variants/genes involved in FVIII/VWF increase. Twenty variants spread over a 8300 Kb region on chromosome 5 were identified in 12 genes, including the low frequency rs13158382, located upstream of the MIR143/145 genes, which might affect miR-143/145 transcription or processing. The expression of miR-143/145 and VWF mRNA were evaluated in the peripheral blood mononuclear cells of six family members. Members with the variant (n = 3) showed lower levels of both miRNAs and higher levels of VWF mRNA compared to members without the variant (n = 3). An analysis of genetic and expression data from a larger cohort of individuals from the 1000 Genomes and GEUVADIS project confirmed a statistically significant reduction (p-value = 0.023) in miR-143 in heterozygous (n = 35) compared to homozygous wild-type individuals (n = 386). This family-based study identified a new genetic variant potentially involved in VWF increase by affecting miR-143/145 expression

Histone acetylation deficits in lymphoblastoid cell lines from patients with rubinstein-Taybi syndrome

Background: Rubinstein-Taybi syndrome (RSTS) is a congenital neurodevelopmental disorder defined by postnatal growth deficiency, characteristic skeletal abnormalities and mental retardation and caused by mutations in the genes encoding for the transcriptional co-activators with intrinsic lysine acetyltransferase (KAT) activity CBP and p300. Previous studies have shown that neuronal histone acetylation is reduced in mouse models of RSTS. Methods: The authors identified different mutations at the CREBBP locus and generated lymphoblastoid cell lines derived from nine patients with RSTS carrying distinct CREBBP mutations that illustrate different grades of the clinical severity in the spectrum of the syndrome. They next assessed whether histone acetylation levels were altered in these cell lines. Results: The comparison of CREBBP-mutated RSTS cell lines with cell lines derived from patients with an unrelated mental retardation syndrome or healthy controls revealed significant deficits in histone acetylation, affecting primarily histone H2B and histone H2A. The most severe defects were observed in the lines carrying the whole deletion of the CREBBP gene and the truncating mutation, both leading to a haploinsufficiency state. Interestingly, this deficit was rescued by treatment with an inhibitor of histone deacetylases (HDACi). Conclusions: The authors' results extend to humans the seminal observations in RSTS mouse models and point to histone acetylation defects, mainly involving H2B and H2A, as relevant molecular markers of the disease.This study was supported by the ItalyeSpain bilateral MIUR project (referred to as IT08143C4F in Italy and HI2007-0203 in Spain). Research at Larizza’s lab is supported by a grant from ASM (Associazione Studio Malformazioni). Research at Barco’s lab is supported by the grants from the Spanish Ministry of Science and Innovation BFU2008-00611, CSD2007-00023 and SAF2008-03194-E (part of the coordinated ERA-Net NEURON project Epitherapy) and a grant from Fundación Ramón Areces. JLA has a Juan de la Cierva contract given by the Spanish Ministry of Science and Innovation.Peer Reviewe

Dual Role of G-runs and hnRNP F in the Regulation of a Mutation-Activated Pseudoexon in the Fibrinogen Gamma-Chain Transcript

<div><p>Most pathological pseudoexon inclusion events originate from single activating mutations, suggesting that many intronic sequences are on the verge of becoming exons. However, the precise mechanisms controlling pseudoexon definition are still largely unexplored. Here, we investigated the <i>cis</i>-acting elements and <i>trans</i>-acting regulatory factors contributing to the regulation of a previously described fibrinogen gamma-chain (<i>FGG</i>) pseudoexon, which is activated by a deep-intronic mutation (IVS6-320A>T). This pseudoexon contains several G-run elements, which may be bound by heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H. To explore the effect of these proteins on <i>FGG</i> pseudoexon inclusion, both silencing and overexpression experiments were performed in eukaryotic cells. While hnRNP H did not significantly affect pseudoexon splicing, hnRNP F promoted pseudoexon inclusion, indicating that these two proteins have only partially redundant functions. To verify the binding of hnRNP F and the possible involvement of other <i>trans</i>-acting splicing modulators, pulldown experiments were performed on the region of the pseudoexon characterized by both a G-run and enrichment for exonic splicing enhancers. This 25-bp-long region strongly binds hnRNP F/H and weakly interacts with Serine/Arginine-rich protein 40, which however was demonstrated to be dispensable for <i>FGG</i> pseudoexon inclusion in overexpression experiments. Deletion analysis, besides confirming the splicing-promoting role of the G-run within this 25-bp region, demonstrated that two additional hnRNP F binding sites might instead function as silencer elements. Taken together, our results indicate a major role of hnRNP F in regulating <i>FGG</i> pseudoexon inclusion, and strengthen the notion that G-runs may function either as splicing enhancers or silencers of the same exon.</p> </div

Functional dissection of G-run elements within the pseudoexon sequence.

<p>(top) The complete 75-bp-long pseudoexon sequence and flanking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the star indicates the IVS6-320A>T mutation; the deleted sequences (shaded in gray) are indicated. (bottom) Histograms representing the relative amount of transcripts including or skipping the pseudoexon, calculated for each deletion mutant as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059333#pone-0059333-g002" target="_blank">Figure 2B</a>. Bars represent mean ± SD of 3 independent experiments, each performed in triplicate. The results were analyzed by unpaired t-test. Statistical significance was calculated referring to the M construct (*P<0.05; **P<0.01; ***P<0.001).</p

Schematic representation of the 75-bp <i>FGG</i> pseudoexon activated by the IVS6-320A>T mutation.

<p>(top) The fibrinogen cluster; boxes and lines represent exons and intronic/intergenic regions, respectively (only exons are drawn to scale); the two parallel slanted lines indicate breaks in the scale. (middle) The <i>FGG</i> minigene (M) cloned in pTargeT vector; the star marks the IVS6-320A>T mutation. (bottom) The complete 75-bp-long pseudoexon sequence and flaking splice sites; nucleotides belonging to the pseudoexon are in capital letters; the strength of pseudoexon splice sites, calculated by using the NNSPLICE 0.9 (<a href="http://www.fruitfly.org/seq_tools/splice.html" target="_blank">http://www.fruitfly.org/seq_tools/splice.html</a>) and the Netgene2 (<a href="http://www.cbs.dtu.dk/services/NetGene2/" target="_blank">http://www.cbs.dtu.dk/services/NetGene2/</a>) software is reported below the corresponding sequence; G-stretches are shaded in gray.</p