Noninvasive Prenatal Test for ß-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

BACKGROUND: Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the ß-hemoglobinopathies to reduce the risk to the fetus. METHOD: Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with ß-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. RESULTS: Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The ß-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. CONCLUSION: This probe capture NGS assay demonstrates the potential of NIPT for ß-hemoglobinopathies.

The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Integration of published information into a resistance-associated mutation database for Mycobacterium tuberculosis.

Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings.

Induction of a unique isoform of the NCOA7 oxidation resistance gene by interferon ß-1b.

We demonstrate that interferon (IFN)-ß-1b induces an alternative-start transcript containing the C-terminal TLDc domain of nuclear receptor coactivator protein 7 (NCOA7), a member of the OXR family of oxidation resistance proteins. IFN-ß-1b induces NCOA7-AS (alternative start) expression in peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals and multiple sclerosis patients and human fetal brain cells, astrocytoma, neuroblastoma, and fibrosarcoma cells. NCOA7-AS is a previously undocumented IFN-ß-inducible gene that contains only the last 5 exons of full-length NCOA7 plus a unique first exon (exon 10a) that is not found in longer forms of NCOA7. This exon encodes a domain closely related to an important class of bacterial aldo-keto oxido-reductase proteins that play a critical role in regulating redox activity. We demonstrate that NCOA7-AS is induced by IFN and LPS, but not by oxidative stress and exhibits, independently, oxidation resistance activity. We further demonstrate that induction of NCOA7-AS by IFN is dependent on IFN-receptor activation, the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway, and a canonical IFN-stimulated response element regulatory sequence upstream of exon 10a. We describe a new role for IFN-ßs involving a mechanism of action that leads to an increase in resistance to inflammation-mediated oxidative stress.

Cutting edge: Vitamin D regulates lipid metabolism in Mycobacterium tuberculosis infection.

Vitamin D has long been linked to resistance to tuberculosis, an infectious respiratory disease that is increasingly hard to treat because of multidrug resistance. Previous work established that vitamin D induces macrophage antimicrobial functions against Mycobacterium tuberculosis. In this article, we report a novel, metabolic role for vitamin D in tuberculosis identified through integrated transcriptome and mechanistic studies. Transcriptome analysis revealed an association between vitamin D receptor (VDR) and lipid metabolism in human tuberculosis and infected macrophages. Vitamin D treatment of infected macrophages abrogated infection-induced accumulation of lipid droplets, which are required for intracellular M. tuberculosis growth. Additional transcriptomics results showed that vitamin D downregulates the proadipogenic peroxisome proliferator-activated receptor γ (PPARγ) in infected macrophages. PPARγ agonists reversed the antiadipogenic and the antimicrobial effects of VDR, indicating a link between VDR and PPARγ signaling in regulating both vitamin D functions. These findings suggest the potential for host-based, adjunct antituberculosis therapy targeting lipid metabolism.

Differential gene expression in thrombomodulin (TM; CD141)(+) and TM(-) dendritic cell subsets.

Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin(+) and thrombomodulin(-) dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin(+) dendritic cells compared to control dendritic cells and thrombomodulin(-) dendritic cells. Thrombomodulin-treated and thrombomodulin(+) dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin(+) dendritic cells produced more lipoxins than thrombomodulin(-) dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin(+) dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin(+) dendritic cells contained more cells actively dividing than those of thrombomodulin(-) dendritic cells. Production of IL-10 is increased in thrombomodulin(+) dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin(+) dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin(+) dendritic cells, which have significantly altered gene expression compared to thrombomodulin(-) dendritic cells in key immune function pathways.

Evidence for postinitiation regulation of mRNA biogenesis in tuberculosis.

Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to IFN-γ, a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN-γ. The results indicate that negative postinitiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN-γ. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals downregulation of postinitiation mRNA biogenesis processes and pathways by infection, with and without IFN-γ stimulation. Clinical relevance for regulation of postinitiation mRNA biogenesis is demonstrated by studies of donor samples showing that postinitiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared with cured disease and in active tuberculosis compared with ongoing treatment or with latent tuberculosis. For active disease and latent infection donors from two populations (London, U.K., and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward downregulation of postinitiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of postinitiation mRNA biogenesis can constrain the macrophage response to infection and overall host defense against tuberculosis.

Early postmyocardial infarction survival in Murphy Roths Large mice is mediated by attenuated apoptosis and inflammation but depends on genetic background.

The Murphy Roths Large (MRL) mouse, a strain capable of regenerating right ventricular myocardium, has a high postmyocardial infarction (post-MI) survival rate compared with C57BL/6J (C57) mice. The biological processes responsible for this survival advantage are unknown. To assess the effect of genetic background, the LG/J strain, which harbours 75% of the MRL composite genome, was included in the study. The MRL survival advantage versus C57 mice (92 versus 68%, P < 0.05) occurred primarily in the first 5 days; LG/J survival was intermediate (P = n.s.). Microarray data analysis revealed an attenuation of apoptotic (P < 0.05) and stress response transcripts in MRL hearts compared with C57 hearts post-MI. Supporting the microarray results, there were fewer TUNEL-positive cells 1 day post-MI in MRL infarcts compared with C57 infarcts (P = 0.001) and fewer CD45-positive cells in the MRL infarct border zone 2 days post-MI (P < 0.01); the LG/J results were intermediate (P = n.s.). The MRL hearts had smaller infarct scars and attenuated ventricular dilatation 30 days post-MI compared with C57 hearts (P < 0.05). We conclude that the early post-MI survival advantage of MRL mice over the C57 strain is mediated at least in part by reductions in apoptosis and inflammatory infiltration, and that these reductions may influence chronic remodelling. The intermediate survival, apoptosis and inflammation profile of LG/J mice suggests that this high tolerance for MI in the MRL mouse could be derived from its shared genetic background with the LG/J mouse.

Interferonß-1b Induces the Expression of RGS1 a Negative Regulator of G-Protein Signaling.

We present evidence of a link between interferonß-1b (IFN-ß) and G-protein signaling by demonstrating that IFN-ß can induce the expression of the negative regulator of G-protein signaling 1 (RGS1). RGS1 reduces G-protein activation and immune cell migration by interacting with heterotrimeric G-proteins and enhancing their intrinsic GTPase activity. In this study, IFN-ß treatment resulted in the induction of RGS1 in peripheral blood mononuclear cells (PBMCs), monocytes, T cells, and B cells. Induction of RGS1 by IFN-ß was concentration dependent and observed at both the RNA and protein level. Other members of the RGS family were not induced by IFN-ß, and induction of RGS1 required the activation of the IFN receptor. In addition, RGS1 induction was observed in PBMCs obtained from IFN-ß-treated multiple sclerosis patients suggesting a possible, as yet unexplored, involvement of G-protein regulation in disease treatment. The upregulation of RGS1 by IFN-ß has not been previously reported.

IFN-beta1b induces transient and variable gene expression in relapsing-remitting multiple sclerosis patients independent of neutralizing antibodies or changes in IFN receptor RNA expression.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Interferon-beta (IFN-beta) therapy for MS is hypothesized to cause short-term and long-term changes in gene expression that shift the inflammation from Th1 to Th2. In vivo gene induction to define kinetics of response to IFN-beta therapy in a large cohort of MS patients is described. Differential gene expression in peripheral blood mononuclear cells (PBMCs) obtained from relapsing-remitting MS patients (RRMS) was assessed using high content microarrays. Rapid onset of gene expression appeared within 4 h of subcutaneous IFN-beta administration, returning to baseline levels at 42 h in clinically stable RRMS. IFN-beta therapy in vivo rapidly but transiently induced strong upregulation of genes mediating immune modulation, IFN signaling, and antiviral responses. RT-PCR showed significant patient-to-patient variation in the magnitude of expression of multiple genes, especially for IFN-beta-inducible genes, such as MxA, IRF7, and CCL8, a Th1 product. Variation among patients in IFN-beta-induced RNA transcription was not explained by neutralizing antibodies or IFN receptor expression. Surprisingly, genes regulated in vivo by IFN-beta therapy do not support a simple Th1 to Th2 shift. A complex interplay between both proinflammatory and anti-inflammatory immune regulatory genes is likely to act in concert in the treatment of RRMS.

IFN-beta-regulated genes show abnormal expression in therapy-naïve relapsing-remitting MS mononuclear cells: gene expression analysis employing all reported protein-protein interactions.

The molecular mechanism by which interferon beta (IFN-beta) is effective in treating multiple sclerosis is not well understood. Mononuclear cells from therapy-naïve MS patients, IFN-beta-1b-treated MS patients, and healthy controls were analyzed to examine mRNA changes that characterize both the disease and its treatment. The scientific literature was comprehensively searched for all protein-protein interactions. In MS patients who had never been treated with IFN-beta, statistical analysis revealed coordinate changes in mRNA expression for proteins reported in the literature as "regulated by IFN-beta." As a positive control for this approach, samples from a separate MS patient cohort showed significant change of these same genes during in vivo treatment with IFN-beta-1b.The strength of effect observed for regulation by IFN-beta was greater than for IFN-alpha, IFN-gamma (Th1), or IL-4 (Th2). Of the sets we investigated, the most strongly affected by disease was the subset defined by regulation by both IFN-beta and IFN-alpha. Changes in cells from therapy-naïve MS patients thus anticipated the importance of IFN-beta in therapy. These findings are a significant step towards marrying MS disease etiology and IFN-beta mechanism of action at a molecular level.

The ubiquitin-conjugating enzyme E2-EPF is overexpressed in primary breast cancer and modulates sensitivity to topoisomerase II inhibition.

We identified the ubiquitin-conjugating enzyme E2-EPF mRNA as differentially expressed in breast tumors relative to normal tissues and performed studies to elucidate its putative role in cancer. We demonstrated that overexpression of E2-EPF protein correlated with estrogen receptor (ER) negativity in breast cancer specimens and that its expression is cell cycle-regulated, suggesting a potential function for E2-EPF in cell cycle progression. However, reduction of E2-EPF protein levels by > 80% using RNAi had no significant effects on the proliferation of HeLa cervical cancer cells or ER(-) MDA-MB-231 or MDA-MB-453 breast cancer cells. Because E2-EPF protein levels were elevated during the G(2)/M phase of the cell cycle and because E2-EPF mRNA in tumor specimens was frequently coexpressed with genes involved in cell cycle control, spindle assembly, and mitotic surveillance, the possibility that E2-EPF might have a function in the cellular response to agents that induce a G(2) checkpoint or an M checkpoint was investigated. E2-EPF knockdown sensitized HeLa cells to the topoisomerase (topo) II inhibitors etoposide and doxorubicin and also increased topo IIalpha protein levels. These data suggest that combined administration of topo II-directed drugs and E2-EPF inhibitors may enhance their clinical effectiveness.