Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

The association between blood MxA mRNA and long-term disease activity in early multiple sclerosis.

Background: Myxovirus resistance protein A (MxA) is a protein that is upregulated by interferon-beta. Homeostatic MxA mRNA levels are potentially correlated with inflammatory disease activity in multiple sclerosis (MS) and could have an important role in MS pathology. Aim: To investigate the association between myxovirus resistance protein A (MxA) mRNA levels in blood and disease activity and progression in MS over a long-term follow-up period. Methods: Baseline blood MxA mRNA levels were determined in a prospective cohort of 116 untreated patients with a clinically isolated syndrome (CIS) or early relapsing remitting MS (RRMS), and related to long-term relapses, radiological disease activity, clinical scores [Expanded Disability Status Scale (EDSS), timed-25-foot walk (T25FW), 9-hole-peg test (9HPT)], MS type, and disease modifying therapy (DMT) use. Results: Low MxA mRNA levels were associated with the occurrence of ≥9 T2-lesions on MRI imaging and the occurrence of relapses during long-term follow-up (median 11 years, IQR 5.91-13.69 years). MxA mRNA levels were not associated with EDSS, T25FW, 9HPT, and MS subtype. Conclusion: Baseline MxA mRNA levels are associated with long-term development of T2-lesions on MRI-scans in our cohort. This confirms the relevance of the endogenous interferon-beta system in the occurrence of MS disease activity.

The Analysis of Platelet-Derived circRNA Repertoire as Potential Diagnostic Biomarker for Non-Small Cell Lung Cancer.

Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription-quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies.

Circular RNA Sequencing of Maternal Platelets: A Novel Tool for the Identification of Pregnancy-Specific Biomarkers.

BACKGROUND: In the first trimester of pregnancy, the maternal platelet is directly involved in a positive feedback mechanism that facilitates invasion of the extravillous trophoblast into the maternal spiral arteries. Dysfunctional trophoblast invasion with defective deep placentation is primordial in the etiology of the "great obstetrical syndromes." METHODS: In this proof-of-concept study, using transcriptome analysis of circular RNA (circRNA) following RNA sequencing of maternal platelets, we tested whether pregnancy-specific circRNA markers could be identified in the first trimester of normal pregnancies. Differential transcript expression analysis of circRNAs, as predicted by Accurate CircRNA Finder Suite, CircRNA Identifier (version 2), and Known and Novel Isoform Explorer, was done using thromboSeq.R with variation of multiple settings. Test performance was checked for (a) de novo circRNA identification using the novel platelet-specific Plt-circR4 as a positive control, (b) complete segregation of groups (pregnant vs nonpregnant) after heat map-dendrogram clustering, (c) identification of pregnancy-specific circRNA markers at a false discovery rate (FDR) <0.05, and (d) confirmation of differentially expressed circRNA markers with an FDR <0.05 by an independent method, reverse transcription-quantitative PCR. RESULTS: Of the differentially expressed circRNAs with P values <0.05, 41 circRNAs were upregulated (logFC >2), and 52 circRNAs were downregulated (logFC less than -2) in first-trimester platelet RNA. Of these, nuclear receptor-interacting protein 1 circRNA covering exons 2 and 3 of the 5'-untranslated region was pregnancy specific with upregulation in first-trimester maternal platelets compared to nonpregnant controls. CONCLUSION: CircRNA sequencing of first-trimester maternal platelets permits the identification of novel pregnancy-specific RNA biomarkers. Future use could include the assessment of maternal and fetal well-being.

The bivariate NRIP1/ZEB2 RNA marker permits non-invasive presymptomatic screening of pre-eclampsia.

Using genome-wide transcriptome analysis by RNA sequencing of first trimester plasma RNA, we tested whether the identification of pregnancies at risk of developing pre-eclampsia with or without preterm birth or growth restriction is possible between weeks 9-14, prior to the appearance of clinical symptoms. We implemented a metaheuristic approach in the self-learning SVM algorithm for differential gene expression analysis of normal pregnancies (n = 108), affected pregnancies (n = 34) and non-pregnant controls (n = 19). Presymptomatic candidate markers for affected pregnancies were validated by RT-qPCR in first trimester samples (n = 34) from an independent cohort. PRKG1 was significantly downregulated in a subset of pregnancies with birth weights below the 10thpercentile as shared symptom. The NRIP1/ZEB2 ratio was found to be upregulated in pregnancies with pre-eclampsia or trisomy 21. Complementary quantitative analysis of both the linear and circular forms of NRIP1 permitted discrimination between pre-eclampsia and trisomy 21. Pre-eclamptic pregnancies showed an increase in linear NRIP1 compared to circular NRIP1, while trisomy 21 pregnancies did not.

Comprehensive multiparameter genetic analysis improves circulating tumor DNA detection in head and neck cancer patients.

INTRODUCTION: Tumor-specific genetic aberrations in cell-free DNA (cfDNA) from plasma are promising biomarkers for diagnosis of recurrent head and neck squamous cell carcinoma (HNSCC). However, the sensitivity when using somatic mutations only in cfDNA is suboptimal. Here, we combined detection of copy number aberrations (CNAs), human papillomavirus (HPV) DNA and somatic mutations in a single sequencing workflow. METHODS: Pretreatment plasmas of 40 patients and 20 non-cancer controls were used for analysis. Plasma DNA underwent low-coverage whole genome sequencing (lcWGS) to detect both CNAs and HPV-DNA, and deep sequencing to detect mutations in 12 frequently altered cancer driver genes in HNSCC using the same sequencing library. A specific analysis pipeline line was developed for data mining. The corresponding tumors were analyzed using slightly adapted protocols. RESULTS: Using the developed method, somatic mutations and CNAs were detected in plasma DNA of HNSCC patients in 67% and 52%, respectively. HPV-DNA in plasma was detected in 100% of patients with HPV-positive tumors, and not in plasma of patients with HPV-negative tumors or non-cancer controls. Combined analysis increased the detection rate of tumor DNA in plasma to 78%. The detection rate was significantly associated with the stage of disease of the tumor. Neither HPV status nor location of the primary tumor influenced detection of CNAs or somatic mutations in plasma. CONCLUSIONS: This study demonstrates that the combined analysis of CNAs, HPV and somatic mutations in plasma of HNSCC patients is feasible and contributes to a higher sensitivity of the assay compared to single modality analyses.

The effect of maternal NODAL on STOX1 expression in extravillous trophoblasts is mediated by IGF1.

The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.

Comparing methods for fetal fraction determination and quality control of NIPT samples.

OBJECTIVE: To compare available analysis methods for determining fetal fraction on single read next generation sequencing data. This is important as the performance of non-invasive prenatal testing (NIPT) procedures depends on the fraction of fetal DNA. METHODS: We tested six different methods for the detection of fetal fraction in NIPT samples. The same clinically obtained data were used for all methods, allowing us to assess the effect of fetal fraction on the test result, and to investigate the use of fetal fraction for quality control. RESULTS: We show that non-NIPT methods based on body mass index (BMI) and gestational age are unreliable predictors of fetal fraction, male pregnancy specific methods based on read counts on the Y chromosome perform consistently and the fetal sex-independent new methods SeqFF and SANEFALCON are less reliable but can be used to obtain a basic indication of fetal fraction in case of a female fetus. CONCLUSION: We recommend the use of a combination of methods to prevent the issue of reports on samples with insufficient fetal DNA; SANEFALCON to check for presence of fetal DNA, SeqFF for estimating the fetal fraction for a female pregnancy and any Y-based method for estimating the fetal fraction for a male pregnancy. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Identification of HIF-2α-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro.

During prolonged hypoxic conditions, endothelial cells change their gene expression to adjust to the low oxygen environment. This process is mainly regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Although endothelial cells do not form sprouts during prolonged hypoxic culturing, silencing of HIF-2α partially restores sprout formation. The present study identifies novel HIF-2α-target genes that may regulate endothelial sprouting during prolonged hypoxia. The gene expression profile of primary human microvascular endothelial cells (hMVECs) that were cultured at 20 % oxygen was compared to hMVECs that were cultured at 1 % oxygen for 14 days by using genome-wide RNA-sequencing. The differentially regulated genes in hypoxia were compared to the genes that were differentially regulated upon silencing of HIF-2α in hypoxia. Surprisingly, KEGG pathway analysis showed that metabolic pathways were enriched within genes upregulated in response to hypoxia and enriched within genes downregulated upon HIF-2α silencing. Moreover, 51 HIF-2α-regulated genes were screened for their role in endothelial sprouting in hypoxia, of which four genes ARRDC3, MME, PPARG and RALGPS2 directly influenced endothelial sprouting during prolonged hypoxic culturing. The manipulation of specific downstream targets of HIF-2α provides a new, but to be further evaluated, perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization of implanted tissue-engineered scaffolds.

Noncoding RNA-regulated gain-of-function of STOX2 in Finnish pre-eclamptic families.

The familial forms of early onset pre-eclampsia and related syndromes (HELLP) present with hypertension and proteinuria in the mother and growth restriction of the fetus. Genetically, these clinically similar entities are caused by different founder-dependent, placentally-expressed paralogous genes. All susceptibility genes (STOX1, lincHELLP, INO80B) identified so far are master control genes that regulate an essential trophoblast differentiation pathway, but act at different entry points. Many genes remain to be identified. Here we demonstrate that a long non-coding RNA (lncRNA) within intron 3 of the STOX2 gene on 4q35.1 acts as a permissive cis-acting regulator of alternative splicing of STOX2. When this lncRNA is mutated or absent, an alternative exon (3B) of STOX2 is included. This introduces a stop codon resulting in the deletion of a highly conserved domain of 64 amino acids in the C-terminal of the STOX2 protein. A mutation present within a regulatory region within intron 1 of STOX2 has the same effect after blocking with CRISPR technology: transcripts with exon 3B are upregulated. This proces appears related to transcriptional control by a chromatin-splicing adaptor complex as described for FGFR2. For STOX2, CHD5, coding for a chromodomain helicase DNA binding protein, qualifies as the chromatin modifier in this process.

Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

MicroRNA 214 Is a Potential Regulator of Thyroid Hormone Levels in the Mouse Heart Following Myocardial Infarction, by Targeting the Thyroid-Hormone-Inactivating Enzyme Deiodinase Type III.

Cardiac thyroid-hormone signaling is a critical determinant of cellular metabolism and function in health and disease. A local hypothyroid condition within the failing heart in rodents has been associated with the re-expression of the fetally expressed thyroid-hormone-inactivating enzyme deiodinase type III (Dio3). While this enzyme emerges as a common denominator in the development of heart failure, the mechanism underlying its regulation remains largely unclear. In the present study, we investigated the involvement of microRNAs (miRNAs) in the regulation of Dio3 mRNA expression in the remodeling left ventricle (LV) of the mouse heart following myocardial infarction (MI). In silico analysis indicated that of the miRNAs that are differentially expressed in the post-MI heart, miR-214 has the highest potential to target Dio3 mRNA. In accordance, a luciferase reporter assay, including the full-length 3'UTR of mouse Dio3 mRNA, showed a 30% suppression of luciferase activity by miR-214. In the post-MI mouse heart, miR-214 and Dio3 protein were shown to be co-expressed in cardiomyocytes, while time-course analysis revealed that Dio3 mRNA expression precedes miR-214 expression in the post-MI LV. This suggests that a Dio3-induced decrease of T3 levels is involved in the induction of miR-214, which was supported by the finding that cardiac miR-214 expression is down regulated by T3 in mice. In vitro analysis of human DIO3 mRNA furthermore showed that miR-214 is able to suppress both mRNA and protein expression. Dio3 mRNA is a target of miR-214 and the Dio3-dependent stimulation of miR-214 expression in post-MI cardiomyocytes supports the involvement of a negative feedback mechanism regulating Dio3 expression.

Calculating the fetal fraction for noninvasive prenatal testing based on genome-wide nucleosome profiles.

OBJECTIVE: While large fetal copy number aberrations can generally be detected through sequencing of DNA in maternal blood, the reliability of tests depends on the fraction of DNA that originates from the fetus. Existing methods to determine this fetal fraction require additional work or are limited to male fetuses. We aimed to create a sex-independent approach without additional work. METHODS: DNA fragments used for noninvasive prenatal testing are cut only by natural processes; thus, influences on cutting by the packaging of DNA in nucleosomes will be preserved in sequencing. As cuts are expected to be made preferentially in linker regions, the shorter fetal fragments should be enriched for reads starting in nucleosome covered positions. RESULTS: We generated genome-wide nucleosome profiles based on single end sequencing of cell-free DNA. We found a difference between DNA digestion of fetal cell-free DNA and maternal cell-free DNA and used this to calculate the fraction of fetal DNA in maternal plasma for both male and female fetuses. CONCLUSION: Our method facilitates cost-effective noninvasive prenatal testing, as the fetal DNA fraction can be estimated without the need for expensive paired-end sequencing or additional tests. The methodology is implemented as a tool, which we called SANEFALCON (Single reAds Nucleosome-basEd FetAL fraCtiON). It is available for academic and non-profit purposes under Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License. github.com/rstraver/sanefalcon. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Genome-Wide Identification of Epigenetic Hotspots Potentially Related to Cardiovascular Risk in Adult Women after a Complicated Pregnancy.

BACKGROUND: The physiological demands of pregnancy on the maternal cardiovascular system can catapult women into a metabolic syndrome that predisposes to atherosclerosis in later life. We sought to identify the nature of the epigenomic changes associated with the increased cardiovascular disease (CVD) risk in adult women following pre-eclampsia. FINDINGS: We assessed the genome wide epigenetic profile by methyl-C sequencing of monozygotic parous twin sister pairs discordant for a severe variant of pre-eclampsia. In the adult twin sisters at risk for CVD as a consequence of a complicated pregnancy, a set of 12 differentially methylated regions with at least 50% difference in methylation percentage and the same directional change was found to be shared between the affected twin sisters and significantly different compared to their unaffected monozygous sisters. CONCLUSION: The current epigenetic marker set will permit targeted analysis of differentially methylated regions potentially related to CVD risk in large cohorts of adult women following complicated pregnancies.

Differential Expression of microRNA in Cerebrospinal Fluid as a Potential Novel Biomarker for Alzheimer's Disease.

BACKGROUND AND OBJECTIVE: The need to find a better reflection of Alzheimer's disease (AD) pathophysiology led us to investigate differential expression of microRNA (miRNA) in cerebrospinal fluid (CSF) of AD patients compared to matched controls, using a genome-wide data-driven approach. METHODS: From the Amsterdam Dementia Cohort, we selected 19 AD patients with CSF indicative of AD pathophysiology and 19 age and gender-matched controls without CSF evidence of AD (67 ± 6 years old, 20 [53%] female). We measured 754 miRNA in CSF using qRT-PCR (Taqman Array MicroRNA cards A and B, v3.0) according to the Megaplex Taqman protocol. Hierarchical cluster analysis was performed and groups were compared using Linear Models for Microarray Data, a modified t-test. We performed validation analysis using qRT-PCR single assays. RESULTS: 144 ± 66 miRNA could be detected using Megaplex array analysis (19% ). Mean Ct (average 32.4 ± 0.5) was correlated to age (r = 0.52, p = 0.001). Five miRNA were differentially expressed in CSF of AD patients. None of these could be replicated. After stratification by age, seven miRNA showed differential expression in late-onset AD, of which lower abundance of let-7a was replicated (log10RQ -1.46, p <  0.05). In early-onset AD, twelve miRNA were differentially expressed of which lower abundance of miRNA-532-3p remained borderline significant (log10RQ -1.27, p = 0.05). CONCLUSION: Although we could not consistently separate AD patients and controls in the whole group, we have found indications miRNA in CSF are able to reflect aging and perhaps also heterogeneity in AD. Further investigation requires optimizing RNA input, while maintaining strict age matching.

Mutations within the LINC-HELLP non-coding RNA differentially bind ribosomal and RNA splicing complexes and negatively affect trophoblast differentiation.

LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo.

Maternal plasma RNA sequencing.

This review discusses the question: which steps are essential or need to be optimized so that maternal plasma RNA sequencing for pre-eclampsia and related syndromes will become as specific and sensitive as maternal plasma DNA sequencing for trisomy?