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.
Genetic disorders can be detected by prenatal diagnosis using Chorionic Villus Sampling, but the 1:100 chance to result in miscarriage restricts the use to fetuses that are suspected to have an ...aberration. Detection of trisomy 21 cases noninvasively is now possible owing to the upswing of next-generation sequencing (NGS) because a small percentage of fetal DNA is present in maternal plasma. However, detecting other trisomies and smaller aberrations can only be realized using high-coverage NGS, making it too expensive for routine practice. We present a method, WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR), which detects small aberrations using low-coverage NGS. The increased detection resolution was achieved by comparing read counts within the tested sample of each genomic region with regions on other chromosomes that behave similarly in control samples. This within-sample comparison avoids the need to re-sequence control samples. WISECONDOR correctly identified all T13, T18 and T21 cases while coverages were as low as 0.15-1.66. No false positives were identified. Moreover, WISECONDOR also identified smaller aberrations, down to 20 Mb, such as del(13)(q12.3q14.3), +i(12)(p10) and i(18)(q10). This shows that prevalent fetal copy number aberrations can be detected accurately and affordably by shallow sequencing maternal plasma. WISECONDOR is available at bioinformatics.tudelft.nl/wisecondor.
Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA ...sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using pairedend massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.
The adult heart has almost completely lost the proliferative potential of the fetal heart. Instead, loss of cardiomyocytes due to myocardial infarction (MI) leads to a limited, and often ...insufficient, hypertrophic response of cardiomyocytes in the spared myocardium. This response is still characterized by a partial reexpression of the fetal gene program. Because of the suggested involvement of microRNAs (miRNAs) in cardiac remodeling, we examined the miRNA expression profile of the spared left ventricular myocardium using a MI mouse model. C57Bl/6J mice of either sex were randomly assigned to the sham-operated group or MI group. MI was induced by ligation of the left coronary artery. One week after surgery RNA was isolated from the left ventricle. MiRNA analysis was performed using the Taqman Megaplex rodent array. Unexpectedly, we found a set of 29 up-regulated miRNAs originating from the Dlk1-Dio3 genomic imprinted region, which has been identified as a hallmark of pluripotency and proliferation. This miRNA signature was associated with a 6-fold increase in expression of the deiodinase type 3 gene (Dio3) located in this region. Dio3 is a fetally expressed thyroid hormone-inactivating enzyme associated with cell proliferation, which was shown to be up-regulated in cardiomyocytes creating a local hypothyroid condition in the spared myocardium in this model. These data suggest that a regenerative process is initiated, but not completed, in adult cardiomyocytes after MI. The identified miRNA signature could provide new ways to manipulate the in vivo response of adult cardiomyocytes to stress and to increase the regenerative capacity of the injured myocardium.
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 discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be ...universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.
•Circulating tumour DNA (ctDNA) can be detected in patients with HNSCC.•Comprehensive analyses of molecular alterations improves ctDNA detection.•Levels of ctDNA correlate with disease stage.
...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.
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.
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.
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.
Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing ...approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.
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