Determination of KCNQ1OT1 and H19 methylation levels in BWS and SRS patients using methylation-sensitive high-resolution melting analysis

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are caused by imprinting defects on chromosome 11p15.5. Standard diagnostic tests for these syndromes include methylation analysis of the differential methylated regions of the H19 and KCNQ1OT1 genes. Traditionally this has been conducted by Southern blot analysis. PCR-based methods greatly improve the turn around time of the test and require less DNA. One of the newly emerging techniques for SNP genotyping and mutation scanning, high-resolution melting (HRM) analysis, has been shown to be also applicable for methylation analysis. We tested methylation-sensitive HRM analysis as a method for the detection of methylation defects in a group of 16 BWS and SRS patients with known methylation status (determined previously by Southern blotting), as well as 45 normal controls. HRM analysis was able to detect all methylation aberrations in the patients and appeared to be more sensitive than Southern blotting. Variation in normal controls is minimal and the presence of SNPs in the amplified fragment does not influence the outcome of the test. We conclude that methylation-sensitive HRM analysis is a robust, fast, sensitive and cost effective method for methylation analysis in BWS and SRS.     

Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells

The Beckwith–Wiedemann syndrome (BWS) is a growth disorder for which an increased frequency of monozygotic (MZ) twinning has been reported. With few exceptions, these twins are discordant for BWS and for females. Here, we describe the molecular and phenotypic analysis of 12 BWS twins and a triplet; seven twins are MZ, monochorionic and diamniotic, three twins are MZ, dichorionic and diamniotic and three twins are dizygotic. Twelve twins are female. In the majority of the twin pairs (11 of 13), the defect on chromosome 11p15 was hypomethylation of the paternal allele of DMR2. In 5 of 10 twins, there was additional hypomethylation of imprinted loci; in most cases, the loci affected were maternally methylated, but in two cases, hypomethylation of the paternally methylated DLK1 and H19 DMRs was detected, a novel finding in BWS. In buccal swabs of the MZ twins who share a placenta, the defect was present only in the affected twin; comparable hypomethylation in lymphocytes was detected in both the twins. The level of hypomethylation reached levels below 25%. The exchange of blood cells through vascular connections cannot fully explain the degree of hypomethylation found in the blood cell of the non-affected twin. We propose an additional mechanism through which sharing of aberrant methylation patterns in discordant twins, limited to blood cells, might occur. In a BWS-discordant MZ triplet, an intermediate level of demethylation was found in one of the non-affected sibs; this child showed mild signs of BWS. This finding supports the theory that a methylation error proceeds and possibly triggers the twinning process.     

Apoptosis in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy

To test the hypothesis that an apoptotic process plays a role in the pathogenesis of cerebral lesions in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), we examined samples from frontal, temporal, insular, and occipital regions, basal ganglia, and cerebellum from 4 patients with CADASIL, 2 with Binswanger disease, and 3 controls. Apoptotic cells were identified using in situ end labeling and activated caspase 3 immunostaining. Immunolabeling for Notch3, the beta-amyloid protein precursor, and phosphorylated neurofilament protein was performed on successive sections. Apoptosis of vascular cells was markedly increased in status cribrosus in CADASIL, both in basal ganglia and subcortical white matter, suggesting that concomitantly with Notch3 deposition it may play a causative role in the dilatation of Virchow-Robin spaces. Neuronal apoptosis was found in CADASIL, mostly in cortical layers 3 and 5. Its severity correlated semiquantitatively with the extent of ischemic lesions and axonal damage in the underlying white matter. It was more severe in demented patients. Only occasional apoptotic neurons were found in the Binswanger cases and none in the controls. This supports the view that neuronal apoptosis may contribute to cortical atrophy and cognitive impairment in patients with CADASIL and that it may, at least partly, result from axonal damage in the underlying white matter.     

Antiphosphatidylethanolamine antibodies are associated with an increased odds ratio for thrombosis. A multicenter study with the participation of the European Forum on antiphospholipid antibodies

A multicenter study was set up to evaluate the prevalence, clinical and biological significance of antiphosphatidylethanolamine antibodies (aPE) in thrombotic patients with or without the main known clinical and biological risk factors for thrombosis. APE and antibodies, defined as the laboratory criteria of antiphospholipid syndrome (APS) -lupus anticoagulant, anticardiolipin and anti-beta(2)-GPI antibodies were measured in 270 patients with thrombosis (234 venous and 37 arterial) and 236 matched controls. APE were found in 15% of thrombotic patients compared to 3% of controls (p < 0.001) with no predominant isotype, no association with the main known clinical or biological risk factors for thrombosis neither with a type of thrombosis, arterial or venous. In a multivariate logistic regression analysis of antibodies, aPE showed the highest association with thrombosis (odds ratio [OR]: 4.2, p < 0.001). Moreover, using a multivariate analysis in a case-control subgroup study on 158 patients, IgGaPE were found to be significantly associated with venous thrombosis (OR:6;p = 0.005). Interestingly, 25 of the 40 aPE-positive patients (63%) were negative for the APS laboratory criteria. Most of them (21/25) had venous thrombosis, recurrent in ten of them. Four patients also suffered from early or late miscarriages. Our results underline the strength of the association between the presence of aPE and thrombosis and suggest their measurement in thrombotic patients, especially when lupus anticoagulant, anticardiolipin or anti-beta(2)-GPI antibodies are absent.     

Valproic acid induces apoptosis in chronic lymphocytic leukemia cells through activation of the death receptor pathway and potentiates TRAIL response

OBJECTIVE: Chronic lymphocytic leukemia (CLL) cells develop chemoresistance over time associated with defects in apoptosis pathway. Novel treatment strategies are required to overcome resistance of cells to commonly used agents. The effects of valproic acid (VPA), an antiepileptic drug with histone deacetylase inhibitory activity, on mononuclear cells isolated from 40 CLL patients were evaluated. METHODS: CLL cells were treated with increasing doses of VPA (0.5, 1, 2, and 5 mM). The mode of cytotoxic drug action was determined by annexin binding, DNA fragmentation, and caspase activation. RESULTS: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in the 40 CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IgV(H) mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA significantly increased sensitivity of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and led to downregulation of c-FLIP (L) expression. VPA caused no potentialization of TRAIL-induced apoptosis on normal B cells. In addition, VPA overcame the prosurvival effects of bone marrow stromal cells. CONCLUSION: These findings point out that the combination of TRAIL and VPA, at clinically relevant concentration, may be valuable in the treatment of CLL.     

Macrophage NADPH oxidase activation, impaired cholesterol fluxes, and increased cholesterol biosynthesis in diabetic mice: a stimulatory role for D-glucose

Diabetes is clearly associated with accelerated atherosclerosis development, but molecular mechanisms involved in diabetes-induced atherosclerosis remain to be clarified. The aim of this study was to identify cellular mechanisms involved in diabetes-induced macrophage foam cell formation, the hallmark of early atherogenesis. Mouse peritoneal macrophages (MPMs) isolated from Balb-C streptozotocin-induced diabetic mice, exhibited significantly higher total peroxides, lipid peroxides and paraoxonase 2 (PON2) activity by 290%, 61% and 55%, respectively, compared to non-diabetic mice. In vitro studies revealed that glucose-induced oxidative stress was obtained by D-glucose, but not by L-glucose and it involved activation of the NADPH oxidase complex, and up-regulation of the macrophage PON2. Next, MPMs isolated from Balb-C diabetic mice, compared to control Balb-C mice, demonstrated increased cholesterol content by 4.2-fold associated with increased cholesterol biosynthesis and increased uptake of oxidized LDL (Ox-LDL) by 5.9-fold and 31%, respectively. These effects on cellular cholesterol metabolism were associated with up-regulation of the scavenger receptors for Ox-LDL (CD-36 and SR-A), and of HMG-CoA reductase (cholesterol biosynthesis rate limiting enzyme). Finally, using pravastatin (inhibitor of HMG-CoA reductase) and the antioxidant Vitamin E, we have shown that D-glucose-induced macrophage oxidative stress is secondary to its stimulatory effect on macrophage cholesterol biosynthesis. In conclusion, macrophages from diabetic mice demonstrate increased oxidative stress associated with activation of NADPH oxidase and up-regulation of cellular PON2, as well as increased macrophages cholesterol uptake and biosynthesis (increased expression of CD-36 and HMG-CoA reductase). The above mechanisms in diabetic mice could be the result of the effect of high D-glucose on macrophages.     

Effect of weight loss after weight loss surgery on plasma N-terminal pro-B-type natriuretic peptide levels

Natriuretic peptides have multiple beneficial cardiovascular effects. Previous cross-sectional studies have indicated that obese subjects have lower natriuretic peptide concentrations than those of normal weight. It is not known whether this relative natriuretic peptide deficiency is reversible with weight loss. We studied 132 obese subjects undergoing weight loss surgery with serial measurement of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations at preoperative, early (1 to 2 months), and late postoperative (6 months) points. In addition, 20 subjects also underwent echocardiography at baseline and 6 months after surgery. Significant weight loss was observed after surgery (median body mass index 45.1, 41.0, and 32.9 kg/m(2) for the 3 corresponding points, analysis of variance p <0.001). The median NT-proBNP levels increased substantially (31.6, 66.9, and 84.9 pg/ml; p <0.001). The average intrasubject increase in NT-proBNP at the 2 postoperative points was 3.4 and 5.0 times the preoperative level (p <0.001 for both points vs preoperatively). In the multivariate regression models adjusted for clinical characteristics and insulin resistance, the strongest predictor of the change in NT-proBNP level 6 months after weight loss surgery was the change in weight (p = 0.03). Echocardiography showed a mean intrasubject reduction in left ventricular mass index of 18% (p <0.001) and mild improvements in diastolic function, with no change in ejection fraction. In conclusion, we have demonstrated that weight loss is associated with early and sustained increases in NT-proBNP concentrations, despite evidence of preserved systolic and improved diastolic function. These findings suggest a direct, reversible relation between obesity and reduced natriuretic peptide levels.     

Burden of Malaria during Pregnancy at the Time of IPTp/SP Implementation in Gabon

The new recommendations to prevent malaria in pregnant women have recently been implemented in Gabon. There is little information on the pregnancy indicators that are useful for their evaluation. A cross-sectional study for the assessment of the prevalence of peripheral, placental, and cord malaria and anemia among delivering women was performed at the largest public hospital of Gabon. Malaria prevalence was 34.4%, 53.6%, and 18.2% for maternal peripheral, placental, and cord blood respectively, with no difference between primigravidae and multigravidae. Submicroscopic infections were frequent and concerned all the positive cord samples. Maternal peripheral, late placental, and cord infections were all associated with a reduced mean birth weight in primigravidae (P = 0.02). Anemia prevalence was 53%, low birth rate was 13%, and prematurity was 25%. The use of intermittent preventive treatment with sulfadoxine-pyrimethamine (greater than or equal to one dose) combined with bed net was associated with a reduction in infection only in multigravidae and with a reduced risk of maternal anemia.     

Transgenic rhesus monkeys produced by gene transfer into early-cleavage–stage embryos using a simian immunodeficiency virus-based vector

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage–stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.