The IL-2/JAK3/STAT-5 signaling pathway is involved on the initiation and maintenance of the transcription factor Foxp3 in regulatory T cells (Treg) and has been associated with demethylation of the ...intronic Conserved Non Coding Sequence-2 (CNS2). However, the role of the JAK/STAT pathway in controlling Foxp3 in the short term has been poorly investigated. Using two different JAK/STAT pharmacological inhibitors, we observed a detectable loss of Foxp3 after 10 min. of treatment that affected 70% of the cells after one hour. Using cycloheximide, a general inhibitor of mRNA translation, we determined that Foxp3, but not CD25, has a high turnover in IL-2 stimulated Treg. This reduction was correlated with a rapid reduction of Foxp3 mRNA. This loss of Foxp3 was associated with a loss in STAT-5 binding to the CNS2, which however remains demethylated. Consequently, Foxp3 expression returns to normal level upon restoration of basal JAK/STAT signaling in vivo. Reduced expression of several genes defining Treg identity was also observed upon treatment. Thus, our results demonstrate that Foxp3 has a rapid turn over in Treg partly controlled at the transcriptional level by the JAK/STAT pathway.
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A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. We illustrate that expression profiles derived from whole tissue RNA ...highly reflect the degenerating tissues' altered cellular composition, not necessarily transcriptional regulation. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Using peripheral endotoxemia to establish the method, we reveal highly specific transcriptional responses and altered RNA processing in each cell type, with Tnfr1 required for the astrocytic response. Extending the method to an Alzheimer's disease model, we confirm that transcriptomic changes observed in whole tissue are driven primarily by cell type composition, not transcriptional regulation, and identify hundreds of cell type-specific changes undetected in whole tissue RNA. Applying similar methods to additional models and patient tissues will transform our understanding of aberrant gene expression in neurological disease.
The BCR::ABL1 negative myeloproliferative neoplasms are sequentially tested for JAK2 p.V617F, followed by CALR exon 9 pathogenic variants. Historically, these variants were thought to be mutually ...exclusive. However, recent reports indicate coexisting JAK2 p.V617F and CALR exon 9 somatic variants.
Analysis of JAK2 p.V617F and CALR exon 9 variant was performed by polymerase chain reaction (PCR)-based assays. Subsequent testing was performed on the Genexus integrated sequencer (ThermoFisher) using the Oncomine myeloid assay GX v2.
CALR exon 9 variants were positive in 3 cases, while 2 were positive for JAK2 p.V617F on PCR-based assays. Next-generation sequencing confirmed the JAK2 P.V617F status in all cases. CALR variants resulting in in-frame deletions were identified in 2 cases at a variant allele frequency of 52.16% and 50.91%, while the third case had an intronic CALR variant c.-48G>A at a variant allele frequency of 51.1%. Thus, CALR variants in all 3 cases were interpreted as potentially germline. Of the 228 cases that underwent JAK2 p.V617F and CALR cotesting in the past 2 years, only these 2 cases were positive for both JAK2 p.V617F and CALR exon 9 variants.
These cases highlight the importance of understanding the pitfalls of molecular techniques in current practice.
Objective
To evaluate the utility of prenatal exome sequencing (ES) for isolated increased nuchal translucency (NT) and to investigate factors that increase diagnostic yield.
Design
Retrospective ...analysis of data from two prospective cohort studies.
Setting
Fetal medicine centres in the UK and USA.
Population
Fetuses with increased NT ≥3.5 mm at 11–14 weeks of gestation recruited to the Prenatal Assessment of Genomes and Exomes (PAGE) and Columbia fetal whole exome sequencing studies (n = 213).
Methods
We grouped cases based on (1) the presence of additional structural abnormalities at presentation in the first trimester or later in pregnancy, and (2) NT measurement at presentation. We compared diagnostic rates between groups using Fisher exact test.
Main outcome measures
Detection of diagnostic genetic variants considered to have caused the observed fetal structural anomaly.
Results
Diagnostic variants were detected in 12 (22.2%) of 54 fetuses presenting with non‐isolated increased NT, 12 (32.4%) of 37 fetuses with isolated increased NT in the first trimester and additional abnormalities later in pregnancy, and 2 (1.8%) of 111 fetuses with isolated increased NT in the first trimester and no other abnormalities on subsequent scans. Diagnostic rate also increased with increasing size of NT.
Conclusions
The diagnostic yield of prenatal ES is low for fetuses with isolated increased NT but significantly higher where there are additional structural anomalies. Prenatal ES may not be appropriate for truly isolated increased NT but timely, careful ultrasound scanning to identify other anomalies emerging later can direct testing to focus where there is a higher likelihood of diagnosis.
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Prenatal ES has a low diagnostic rate (<2%) for isolated increased NT but is significantly more likely to yield a diagnosis where there are additional fetal structural anomalies.
Linked article This article is commented on by AN Talati and NL Vora, p. 61–62 in this issue. To view this mini commentary visit https://doi.org/10.1111/1471-0528.16942.
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Myeloid neoplasms require comprehensive characterization of genetic abnormalities, including single-nucleotide variants, small insertions and deletions, and fusions and translocations for management. ...The Oncomine Myeloid Assay GX v2 (Thermo Fisher Scientific) analyzes 17 full genes, 28 hotspot genes, 30 fusion driver genes, and 5 expression genes.
The validation set included 192 DNA samples, 28 RNA samples, and 9 cell lines and contrived controls. The DNA and RNA were extracted from both peripheral blood and bone marrow. Library preparation, templating, and sequencing was performed on the fully automated Genexus Integrated Sequencer (Thermo Fisher Scientific). The sequencing data were analyzed by manual curation, default Oncomine filters and the Oncomine Reporter (Thermo Fisher Scientific).
Of the 600 reference pathogenic DNA variants targeted by the assay, concordance was seen in 98.3% of unfiltered variant call format files. Precision and reproducibility were 100%, and the lower limit of detection was 2% variant allele frequency for DNA. Inability to detect variants in long homopolymer regions intrinsic to the Ion Torrent chemistry led to 7 missed variants; 100% concordance was seen with reference RNA samples.
This extensive clinical validation of the Oncomine Myeloid Assay GX v2 on the Genexus Integrated Sequencer with its built-in bioinformatics pipeline and Ion Torrent Oncomine Reporter shows robust performance in terms of variant calling accuracy, precision, and reproducibility, with the advantage of a rapid turnaround time of 2 days. The greatest limitation is the inability to detect variants in long homopolymer regions.
Biliary tract cancer is an uncommon cancer with a poor outcome. We assembled data from the National Cancer Research Institute (UK) ABC-02 study and 10 international studies to determine prognostic ...outcome characteristics for patients with advanced disease.
Multivariable analyses of the final dataset from the ABC-02 study were carried out. All variables were simultaneously included in a Cox proportional hazards model, and backward elimination was used to produce the final model (using a significance level of 10%), in which the selected variables were associated independently with outcome. This score was validated externally by receiver operating curve (ROC) analysis using the independent international dataset.
A total of 410 patients were included from the ABC-02 study and 753 from the international dataset. An overall survival (OS) and progression-free survival (PFS) Cox model was derived from the ABC-02 study. White blood cells, haemoglobin, disease status, bilirubin, neutrophils, gender, and performance status were considered prognostic for survival (all with P < 0.10). Patients with metastatic disease {hazard ratio (HR) 1.56 95% confidence interval (CI) 1.20–2.02} and Eastern Cooperative Oncology Group performance status (ECOG PS) 2 had worse survival HR 2.24 (95% CI 1.53–3.28). In a dataset restricted to patients who received cisplatin and gemcitabine with ECOG PS 0 and 1, only haemoglobin, disease status, bilirubin, and neutrophils were associated with PFS and OS. ROC analysis suggested the models generated from the ABC-02 study had a limited prognostic value 6-month PFS: area under the curve (AUC) 62% (95% CI 57–68); 1-year OS: AUC 64% (95% CI 58–69).
These data propose a set of prognostic criteria for outcome in advanced biliary tract cancer derived from the ABC-02 study that are validated in an international dataset. Although these findings establish the benchmark for the prognostic evaluation of patients with ABC and confirm the value of longheld clinical observations, the ability of the model to correctly predict prognosis is limited and needs to be improved through identification of additional clinical and molecular markers.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Unconventional gas drilling (UGD) has enabled extraordinarily rapid growth in the extraction of natural gas. Despite frequently expressed public concern, human health studies have not kept pace. We ...investigated the association of proximity to UGD in the Marcellus Shale formation and perinatal outcomes in a retrospective cohort study of 15,451 live births in Southwest Pennsylvania from 2007-2010. Mothers were categorized into exposure quartiles based on inverse distance weighted (IDW) well count; least exposed mothers (first quartile) had an IDW well count less than 0.87 wells per mile, while the most exposed (fourth quartile) had 6.00 wells or greater per mile. Multivariate linear (birth weight) or logistical (small for gestational age (SGA) and prematurity) regression analyses, accounting for differences in maternal and child risk factors, were performed. There was no significant association of proximity and density of UGD with prematurity. Comparison of the most to least exposed, however, revealed lower birth weight (3323 ± 558 vs 3344 ± 544 g) and a higher incidence of SGA (6.5 vs 4.8%, respectively; odds ratio: 1.34; 95% confidence interval: 1.10-1.63). While the clinical significance of the differences in birth weight among the exposure groups is unclear, the present findings further emphasize the need for larger studies, in regio-specific fashion, with more precise characterization of exposure over an extended period of time to evaluate the potential public health significance of UGD.
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Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive ...database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases.
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•A searchable database of eQTL from macula and peripheral retina and RPE/choroid•Single-cell expression from human retina and RPE with cell subtype resolution•Integrative analysis of bulk and single-cell transcriptomics in the human eye•Co-localization of GWAS and eQTL identified putative causal genes for AMD
To find genes underlying risk for age-related macular degeneration, Orozco et al. analyze human ocular transcriptomes in conjunction with genotypes and build a single-nucleus atlas. They identify 15 putative causal genes, of which TSPAN10 and TRPM1 were enriched in retinal pigment epithelium, the site of early disease pathology.
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Despite widespread use, the accuracy of the diagnostic test for SARS-CoV-2 infection is poorly understood. The aim of our work was to better quantify misclassification errors in identification of ...true cases of COVID-19 and to study the impact of these errors in epidemic curves using publicly available surveillance data from Alberta, Canada and Philadelphia, USA.
We examined time-series data of laboratory tests for SARS-CoV-2 viral infection, the causal agent for COVID-19, to try to explore, using a Bayesian approach, the sensitivity and specificity of the diagnostic test.
Our analysis revealed that the data were compatible with near-perfect specificity, but it was challenging to gain information about sensitivity. We applied these insights to uncertainty/bias analysis of epidemic curves under the assumptions of both improving and degrading sensitivity. If the sensitivity improved from 60 to 95%, the adjusted epidemic curves likely falls within the 95% confidence intervals of the observed counts. However, bias in the shape and peak of the epidemic curves can be pronounced, if sensitivity either degrades or remains poor in the 60-70% range. In the extreme scenario, hundreds of undiagnosed cases, even among the tested, are possible, potentially leading to further unchecked contagion should these cases not self-isolate.
The best way to better understand bias in the epidemic curves of COVID-19 due to errors in testing is to empirically evaluate misclassification of diagnosis in clinical settings and apply this knowledge to adjustment of epidemic curves.
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