HIF1A and MIF as potential predictive mRNA biomarkers of pre-eclampsia: a longitudinal prospective study in high risk population.

BACKGROUND: Pre-eclampsia (PE) is a hypertensive multisystem disorder, causing significant fetal-maternal mortality and morbidity worldwide. This study aims to define possible longitudinal predictive mRNA markers involved in the main pathogenic pathways of PE: inflammation [macrophage migration inhibitory factor (MIF)], hypoxia and oxidative stress [hypoxia inducible factor 1-α subunit (HIF1A) and ß-site APP-cleaving enzyme-2 (BACE2)] and endothelial dysfunction [endoglin (ENG), fms-related tyrosine kinase-1 (FLT1) and vascular endothelial growth factor (VEGF)]. METHODS: Peripheral blood was collected from 33 singleton pregnancies characterized by a high cardiovascular profile risk sampled consecutively at 6-16; 17-23; 24-30; 31-34; ≥35 weeks followed by the Obstetrics and Gynecology Unit of the San Raffaele Hospital in Milan. A real-time quantitative PCR reaction was performed on plasma RNA. RESULTS: Of the 33 women enrolled, nine developed PE. Until 23 weeks HIF1A was significantly higher in women who later developed PE compared to women who did not (p=0.049 and p=0.012 in the first and second blood collection). In the third time interval MIF (p=0.0005), FLT1 (p=0.024), ENG (p=0.0034) and BACE2 (p=0.044) appeared to be significantly increased while HIF1A was elevated even from 24 week onwards but not reaching the statistical significance. In the fourth time interval ENG mRNA still remained increased (p=0.037). CONCLUSIONS: HIF1A, marker of hypoxia and oxidative stress, and MIF, marker of inflammation, seemed to be the most promising RNA markers, suggesting that hypoxia, principally, and inflammation may play an important role in PE pathogenesis.

Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation.

BACKGROUND: Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. DESIGN AND METHODS: We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. RESULTS: Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. CONCLUSIONS: Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation.

Evaluation of a panel of circulating DNA, RNA and protein potential markers for pathologies of pregnancy.

BACKGROUND: Among markers of pregnancy complications, corticotropin-releasing hormone (CRH) mRNA, long pentraxin 3 (PTX3) protein and fetal and total DNA had been reported to be increased in the plasma of women with overt preeclampsia (PE). We developed an optimized protocol to evaluate whether concentrations of CRH mRNA, PTX3 mRNA and protein, fetal and/or total DNA are increased in fetal growth restriction (FGR), and whether they predict complications of pregnancy. METHODS: The protocol included a preamplification step to enrich rare mRNA species. CRH and PTX3 mRNA, DNA and PTX3 protein were measured in the plasma of women with PE or FGR, in women at risk of developing these pathologies and in healthy women matched for gestational age. RESULTS: CRH mRNA, fetal and/or total DNA and PTX3 protein were significantly increased in women with overt PE when compared to controls. Pregnant women who later developed PE or FGR during pregnancy showed total DNA levels that were significantly increased before the onset of both pathologies, while RNA markers were increased only in women who later developed PE. CONCLUSIONS: Our protocol for plasma RNA quantification may allow for the extension of a panel of predictive markers to be investigated in larger patient cohorts.

High-sensitive microarray substrates specifically designed to improve sensitivity for the identification of fetal paternally inherited sequences in maternal plasma.

BACKGROUND: The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application. METHODS: A combined approach based on innovative microarray slides coated with a special copolymer was developed for the identification of three polymorphisms located in the causative gene for cystic fibrosis (CF). This technique was applied to the analysis of fetal DNA in maternal plasma from four couples that carried different allelic variants. RESULTS: The use of highly sensitive slides correctly identified fetal paternally inherited alleles without any enrichment strategy. CONCLUSIONS: Our results confirm that the high fluorescence signal provided by the optimized substrate may be applied to the identification of any fetal paternally inherited sequence. This helps extend the application of non-invasive prenatal diagnosis to genetic diseases caused by predominant mutations or minor rare molecular defects.

Italian Rett database and biobank.

Rett syndrome is the second most common cause of severe mental retardation in females, with an incidence of approximately 1 out of 10,000 live female births. In addition to the classic form, a number of Rett variants have been described. MECP2 gene mutations are responsible for about 90% of classic cases and for a lower percentage of variant cases. Recently, CDKL5 mutations have been identified in the early onset seizures variant and other atypical Rett patients. While the high percentage of MECP2 mutations in classic patients supports the hypothesis of a single disease gene, the low frequency of mutated variant cases suggests genetic heterogeneity. Since 1998, we have performed clinical evaluation and molecular analysis of a large number of Italian Rett patients. The Italian Rett Syndrome (RTT) database has been developed to share data and samples of our RTT collection with the scientific community (http://www.biobank.unisi.it). This is the first RTT database that has been connected with a biobank. It allows the user to immediately visualize the list of available RTT samples and, using the "Search by" tool, to rapidly select those with specific clinical and molecular features. By contacting bank curators, users can request the samples of interest for their studies. This database encourages collaboration projects with clinicians and researchers from around the world and provides important resources that will help to better define the pathogenic mechanisms underlying Rett syndrome.