Prediction of labour outcomes using prelabour computerised cardiotocogram and maternal and fetal Doppler indices: A prospective cohort study.

OBJECTIVES: To investigate the association and the potential value of prelabour fetal heart rate short-term variability (STV) determined by computerised cardiotocography (cCTG) and maternal and fetal Doppler in predicting labour outcomes. DESIGN: Prospective cohort study. SETTING: The Prince of Wales Hospital, a tertiary maternity unit, in Hong Kong SAR. POPULATION: Women with a term singleton pregnancy in latent phase of labour or before labour induction were recruited during May 2019-November 2021. METHODS: Prelabour ultrasonographic assessment of fetal growth, Doppler velocimetry and prelabour cCTG monitoring including Dawes-Redman CTG analysis were registered shortly before induction of labour or during the latent phase of spontaneous labour. MAIN OUTCOME MEASURES: Umbilical cord arterial pH, emergency delivery due to pathological CTG during labour and neonatal intensive care unit (NICU)/special care baby unit (SCBU) admission. RESULTS: Of the 470 pregnant women invited to participate in the study, 440 women provided informed consent and a total of 400 participants were included for further analysis. Thirty-four (8.5%) participants underwent emergency delivery for pathological CTG during labour. A total of 6 (1.50%) and 148 (37.00%) newborns required NICU and SCBU admission, respectively. Middle cerebral artery pulsatility index (MCA-PI) and MCA-PI z-score were significantly lower in pregnancies that required emergency delivery for pathological CTG during labour compared with those that did not (1.23 [1.07-1.40] versus 1.40 [1.22-1.64], p = 0.002; and 0.55 ± 1.07 vs. 0.12 ± 1.06), p = 0.049]. This study demonstrated a weakly positive correlation between umbilical cord arterial pH and prelabour log10 STV (r = 0.107, p = 0.035) and the regression analyses revealed that the contributing factors for umbilical cord arterial pH were smoking (p = 0.006) and prelabour log10 STV (p = 0.025). CONCLUSIONS: In pregnant women admitted in latent phase of labour or for induction of labour at term, prelabour cCTG STV had a weakly positive association with umbilical cord arterial pH but was not predictive of emergency delivery due to pathological CTG during labour.

Healthcare resource utilization, costs and treatment associated with myasthenia gravis exacerbations among patients with myasthenia gravis in the USA: a retrospective analysis of claims data.

Aim: There are limited data on the clinical and economic burden of exacerbations in patients with myasthenia gravis (MG). We assessed patient clinical characteristics, treatments and healthcare resource utilization (HCRU) associated with MG exacerbation. Patients & methods: This was a retrospective analysis of adult patients with MG identified by commercial, Medicare or Medicaid insurance claims from the IBM® MarketScan® database. Eligible patients had two or more MG diagnosis codes, without evidence of exacerbation or crisis in the baseline period (12 months prior to index [first eligible MG diagnosis]). Clinical characteristics were evaluated at baseline and 12 weeks before each exacerbation. Number of exacerbations, MG treatments and HCRU costs associated with exacerbation were described during a 2-year follow-up period. Results: Among 9352 prevalent MG patients, 34.4% (n = 3218) experienced ≥1 exacerbation after index: commercial, 53.0% (n = 1706); Medicare, 39.4% (n = 1269); and Medicaid, 7.6% (n = 243). During follow-up, the mean (standard deviation) number of exacerbations per commercial and Medicare patient was 3.7 (7.0) and 2.7 (4.1), respectively. At least two exacerbations were experienced by approximately half of commercial and Medicare patients with ≥1 exacerbation. Mean total MG-related healthcare costs per exacerbation ranged from $26,078 to $51,120, and from $19,903 to $49,967 for commercial and Medicare patients, respectively. AChEI use decreased in patients with multiple exacerbations, while intravenous immunoglobulin use increased with multiple exacerbations. Conclusion: Despite utilization of current treatments for MG, MG exacerbations are associated with a high clinical and economic burden in both commercial and Medicare patients. Additional treatment options and improved disease management may help to reduce exacerbations and disease burden.

Heat shock induces alternative polyadenylation through dynamic DNA methylation-regulated chromatin looping.

Alternative cleavage and polyadenylation (APA) is a gene regulatory mechanism used by cells under stress to upregulate proteostasis-promoting transcripts, but how cells achieve this remains poorly understood. Previously, we elucidated a DNA methylation-regulated APA mechanism, in which gene body DNA methylation enhances distal poly(A) isoform expression by blocking CTCF binding and chromatin loop formation at APA control regions. We hypothesized that DNA methylation-regulated APA is one mechanism cells employ to induce proteostasis-promoting poly(A) isoforms. At the DNAJB6 co-chaperone gene locus, acute heat shock resulted in binding of stress response transcription factors HSF1, ATF6, and YY1 at the APA control region and an increase in the expression of the proximal poly(A) isoform known to prevent protein aggregation. Furthermore, TET1 was recruited to rapidly demethylate DNA, facilitating CTCF binding and chromatin loop formation, thereby reinforcing preferential proximal poly(A) isoform expression. As cells recovered, the transcription factors vacated the APA control region, and DNMT1 was recruited to remethylate the region. This process resolved chromatin looping and reset the poly(A) isoform expression pattern. Our findings unveil an epigenetic mechanism enabling cells to dynamically modulate poly(A) isoforms in response to stress while shedding light on the interplay between DNA methylation, transcription factors, and chromatin looping.

Protocols for single-cell RNA-seq and spatial gene expression integration and interactive visualization.

There is a wealth of software that utilizes single-cell RNA-seq (scRNA-seq) data to deconvolve spatial transcriptomic spots, which currently are not yet at single-cell resolution. Here we provide protocols for implementing Seurat and Giotto packages to elucidate cell-type distribution in our example human ureter scRNA-seq dataset. We also describe how to create a stand-alone interactive web application using Seurat libraries to visualize and share our results. For complete details on the use and execution of this protocol, please refer to Fink et al. (2022).1.

KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability. SIGNIFICANCE: A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

A real-world analysis of factors associated with high healthcare resource utilization and costs in patients with myasthenia gravis receiving second-line treatment.

Despite current treatments, patients with myasthenia gravis (MG) experience unpredictable and inadequately controlled symptoms, lending to variability in the clinical and economic burden of disease. However, limited data are available on MG healthcare costs, and specifically, no data on patients initiating second-line therapy. Using claims data from the IBM® MarketScan® database, we assessed patient characteristics, healthcare resource utilization, and costs among MG patients initiating second-line therapy, and identified potential factors associated with high healthcare costs over a two-year follow-up period. We identified 1498 patients, of whom 49% and 31% received chronic steroids and non-steroidal immunosuppressants (NSISTs) as their second-line therapy, respectively. During follow-up, 49% experienced ≥1 MG exacerbation. Among all patients, mean all-cause total healthcare cost was $106,821 per patient during follow-up, with $88,040 and $18,780 attributed to medical and pharmacy costs, respectively. In a multivariable analysis, variables significantly associated with high cost included use of high-dose steroids, chronic intravenous immunoglobulin (IVIg, ≥6 cycles), and 1 and ≥ 4 (but not 2-3) MG exacerbations in the first year after second-line therapy initiation. Any number of exacerbations were associated with high cost in a univariable analysis. A stratified cost analysis showed that patients with >1 exacerbation, ≥1 treatment switch, and high-dose steroid use in this first year experienced $198,487, $114,037, and $79,752 mean MG-related total healthcare spend during follow-up, respectively. These data suggest that patients receiving chronic IVIg or NSISTs for MG experience significant economic burden. Disease characteristics including exacerbation and treatment history may be an indicator of future high costs.

Single-cell and spatial mapping Identify cell types and signaling Networks in the human ureter.

Tissue engineering offers a promising treatment strategy for ureteral strictures, but its success requires an in-depth understanding of the architecture, cellular heterogeneity, and signaling pathways underlying tissue regeneration. Here, we define and spatially map cell populations within the human ureter using single-cell RNA sequencing, spatial gene expression, and immunofluorescence approaches. We focus on the stromal and urothelial cell populations to enumerate the distinct cell types composing the human ureter and infer potential cell-cell communication networks underpinning the bi-directional crosstalk between these compartments. Furthermore, we analyze and experimentally validate the importance of the sonic hedgehog (SHH) signaling pathway in adult progenitor cell maintenance. The SHH-expressing basal cells support organoid generation in vitro and accurately predict the differentiation trajectory from basal progenitor cells to terminally differentiated umbrella cells. Our results highlight the essential processes involved in adult ureter tissue homeostasis and provide a blueprint for guiding ureter tissue engineering.

Physician-Reported Perspectives on Myasthenia Gravis in the United States: A Real-World Survey.

INTRODUCTION: Myasthenia gravis (MG) is a rare, debilitating, chronic disorder caused by the production of pathogenic immunoglobulin G autoantibodies against the neuromuscular junction. A lack of real-world studies in rare diseases reflects a relatively limited understanding of the significant unmet needs and burden of disease for patients. We aimed to provide comprehensive real-world insights into the management and burden of MG from treating physicians in the United States (US). METHODS: Data were collected using the Adelphi Real World MG Disease Specific Programme™, a point-in-time survey of physicians and their patients with MG, in the US between March and July 2020. Physician-reported clinical data, including demographics, comorbidities, symptoms, disease history, treatments, and healthcare resource utilization, were collected. RESULTS: In total, 456 patient record forms were completed by 78 physicians based in the US. At time of survey completion, patient mean age was 54.5 years. Mean time from symptom onset to diagnosis was 9.0 months (n = 357). Ocular symptoms were reported in 71.7% of patients. General fatigue affected 47.1% of patients and over half of those reported the severity as moderate or severe (59.5%, n = 128). Acetylcholinesterase inhibitors and/or steroids were the most frequently prescribed first-line treatment type among patients receiving treatment at time of survey completion and with moderate-to-severe symptoms (77.9%, n = 159/204). High-dose steroids (n = 14) and intravenous immunoglobulin (n = 13) were the most prescribed acute treatments among those receiving an acute treatment at time of survey completion (n = 36), with symptom exacerbations or myasthenic crises being the most common reasons for acute treatment. On average, 2.5 healthcare professionals were involved in patient management and 5.0 consultations were made per patient over the last 12 months. CONCLUSIONS: Our findings indicated that, despite treatment, there is a proportion of patients with MG in the US who had a significant need for improved disease management.

Processing and cryopreservation of human ureter tissues for single-cell and spatial transcriptomics assays.

Characterizing the cellular heterogeneity of human ureter tissues using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics provides a detailed atlas of cell types, signaling networks, and potential cell-cell cross talk underlying developmental and regenerative pathways. We describe an optimized protocol for generating, cryopreserving, and thawing single-cell suspensions from ureter tissues isolated post-cystectomy for scRNA-seq. In addition, we describe an optimized protocol for cryopreserving human ureter tissues for 10x Genomics Visium spatial gene expression platform. For complete details on the use and execution of this protocol, please refer to Fink et al. (2022).1.

DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex.

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Epigenetically regulated miR-1247 functions as a novel tumour suppressor via MYCBP2 in methylator colon cancers.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with distinct clinical subsets based on underlying genetic and epigenetic changes. DNA hypermethylation yields a unique CRC subset with a distinct phenotype and clinical behaviour, but this oncogenic pathway is not fully characterised. This study identifies and characterises miR-1247 as a novel tumour suppressor microRNA in methylated human colon cancers. METHOD: Tumour samples from patients with hypermethylated and non-methylated colon cancer and cell lines were evaluated for miR-1247 expression and function. A murine subcutaneous xenograft model was used for in vivo functional studies. RESULTS: miR-1247 was methylated and underexpressed in methylator colon cancers. Overexpression of miR-1247 significantly inhibited cell proliferation, decreased tumour cell motility, induced apoptosis, and mitigated tumour formation capacity both in vivo and in vitro. Pharmacologic demethylation increased miR-1247 expression and produced similar anti-tumour activities. Mechanistic investigations revealed that MYCBP2, a member of the c-myc oncogene family, is a direct functional target of miR-1247. Furthermore, in CRC patients, MYCBP2 protein levels are associated with miR-1247 levels and survival. CONCLUSIONS: miR-1247 acts as a tumour suppressor by inhibiting MYCBP2 in methylator colon cancer. The MYCBP2/c-myc axis may underlie the anti-tumour activities of miR-1247 and is a potential therapeutic target via demethylation agents.

Cancer-predicting transcriptomic and epigenetic signatures revealed for ulcerative colitis in patient-derived epithelial organoids.

Ulcerative colitis (UC) is a prevalent form of inflammatory bowel disease (IBD) whose pathogenic mechanisms remain unclear. Elucidating these mechanisms is important to reduce UC symptoms and to prevent UC progression into colitis-associated colon cancer (CAC). Our goal was to develop and validate faithful, human-derived, UC models and analyze them at histologic, transcriptomic and epigenetic levels to allow mechanistic studies of UC and CAC pathogenesis. We generated patient-derived primary-organoid cultures from UC and non-IBD colonic epithelium. We phenotyped them histologically and used next-generation-sequencing approaches to profile whole transcriptomes and epigenomes of organoids and primary tissues. Tissue organization and expression of mucin 2 (MUC2) and lysozyme (LYZ) demonstrated histologic faithfulness of organoids to healthy and diseased colonic epithelium. Transcriptomic analyses showed increased expression of inflammatory pathways in UC patient-derived organoids and tissues. Profiling for active enhancers using the H3K27ac histone modification revealed UC-derived organoid enrichment for pathways indicative of gastrointestinal cancer, including S100 calcium-binding protein P (S100P), and revealed novel markers for GI cancer, including both LYZ and neuropeptide S receptor 1 (NPSR1). Immunolocalization showed increased levels of LYZ, S100P, and NPSR1 proteins in UC and CAC. In conclusion, primary colonic organoid cultures from UC and non-IBD patients can be established that faithfully represent diseased or normal colonic states. These models reveal precancerous molecular pathways that are already activated in UC. The findings demonstrate the suitability of primary organoids for dissecting UC and CAC pathogenic mechanisms and suggest new targets for therapeutic intervention.

Treg-specific IL-27Rα deletion uncovers a key role for IL-27 in Treg function to control autoimmunity.

Dysregulated Foxp3+ Treg functions result in uncontrolled immune activation and autoimmunity. Therefore, identifying cellular factors modulating Treg functions is an area of great importance. Here, using Treg-specific Il27ra-/- mice, we report that IL-27 signaling in Foxp3+ Tregs is essential for Tregs to control autoimmune inflammation in the central nervous system (CNS). Following experimental autoimmune encephalomyelitis (EAE) induction, Treg-specific Il27ra-/- mice develop more severe EAE. Consistent with the severe disease, the numbers of IFNγ- and IL-17-producing CD4 T cells infiltrating the CNS tissues are greater in these mice. Treg accumulation in the inflamed CNS tissues is not affected by the lack of IL-27 signaling in Tregs, suggesting a functional defect of Il27ra-/- Tregs. IL-10 production by conventional CD4 T cells and their CNS accumulation are rather elevated in Treg-specific Il27ra-/- mice. Analysis with Treg fate-mapping reporter mice further demonstrates that IL-27 signaling in Tregs may control stability of Foxp3 expression. Finally, systemic administration of recombinant IL-27 in Treg-specific Il27ra-/- mice fails to ameliorate the disease even in the presence of IL-27-responsive conventional CD4 T cells. These findings uncover a previously unknown role of IL-27 in regulating Treg function to control autoimmune inflammation.

WOMEN IN CANCER THEMATIC REVIEW: Diverse functions of DNA methylation: implications for prostate cancer and beyond.

Prostate cancer is one of the most common malignancies in men worldwide. Current clinical screening ensures that most prostate cancers are diagnosed while still organ confined, but disease outcome is highly variable. Thus, a better understanding of the molecular features contributing to prostate cancer aggressiveness is being sought. For many cancers, aberrant genome-wide patterns of cytosine DNA methylation in CpG dinucleotides distinguish tumor from normal tissue and contribute to disease progression by altering the transcriptome. In prostate cancer, recent genomic studies identified cancer and high grade-specific differential DNA methylation in gene promoters, gene bodies, gene 3' ends and at distal regulatory elements. Using examples from developmental and disease systems, we will discuss how DNA methylation in each of these genomic contexts can contribute to transcriptome diversity by modulating transcription initiation, alternative transcription start site selection, alternative pre-mRNA splicing and alternative polyadenylation. Alternative transcripts from the same gene often exhibit altered protein-coding potential, translatability, stability and/or localization. All of these can have functional consequences in cells. In future work, it will be important to determine if DNA methylation abnormalities in prostate cancer modify the transcriptome through some or all of these mechanisms and if these DNA methylation-mediated transcriptome alterations impact prostate tumorigenesis and aggressiveness.

Goldmine integrates information placing genomic ranges into meaningful biological contexts.

Bioinformatic analysis often produces large sets of genomic ranges that can be difficult to interpret in the absence of genomic context. Goldmine annotates genomic ranges from any source with gene model and feature contexts to facilitate global descriptions and candidate loci discovery. We demonstrate the value of genomic context by using Goldmine to elucidate context dynamics in transcription factor binding and to reveal differentially methylated regions (DMRs) with context-specific functional correlations. The open source R package and documentation for Goldmine are available at http://jeffbhasin.github.io/goldmine.

Genome-wide analysis of DNA methylation and gene expression defines molecular characteristics of Crohn's disease-associated fibrosis.

BACKGROUND: Fibrosis of the intestine is a common and poorly understood complication of Crohn's disease (CD) characterized by excessive deposition of extracellular matrix and accompanied by narrowing and obstruction of the gut lumen. Defining the molecular characteristics of this fibrotic disorder is a vital step in the development of specific prediction, prevention, and treatment strategies. Previous epigenetic studies indicate that alterations in DNA methylation could explain the mechanism by which mesenchymal cells adopt the requisite pro-fibrotic phenotype that promotes fibrosis progression. However, to date, genome-wide analysis of the DNA methylome of any type of human fibrosis is lacking. We employed an unbiased approach using deep sequencing to define the DNA methylome and transcriptome of purified fibrotic human intestinal fibroblasts (HIF) from the colons of patients with fibrostenotic CD. RESULTS: When compared with normal fibroblasts, we found that the majority of differential DNA methylation was within introns and intergenic regions and not associated with CpG islands. Only a low percentage occurred in the promoters and exons of genes. Integration of the DNA methylome and transcriptome identified regions in three genes that inversely correlated with gene expression: wingless-type mouse mammary tumor virus integration site family, member 2B (WNT2B) and two eicosanoid synthesis pathway enzymes (prostacyclin synthase and prostaglandin D2 synthase). These findings were independently validated by RT-PCR and bisulfite sequencing. Network analysis of the data also identified candidate molecular interactions relevant to fibrosis pathology. CONCLUSIONS: Our definition of a genome-wide fibrosis-specific DNA methylome provides new gene networks and epigenetic states by which to understand mechanisms of pathological gene expression that lead to fibrosis. Our data also provide a basis for development of new fibrosis-specific therapies, as genes dysregulated in fibrotic Crohn's disease, following functional validation, can serve as new therapeutic targets.

MethylAction: detecting differentially methylated regions that distinguish biological subtypes.

DNA methylation differences capture substantial information about the molecular and gene-regulatory states among biological subtypes. Enrichment-based next generation sequencing methods such as MBD-isolated genome sequencing (MiGS) and MeDIP-seq are appealing for studying DNA methylation genome-wide in order to distinguish between biological subtypes. However, current analytic tools do not provide optimal features for analyzing three-group or larger study designs. MethylAction addresses this need by detecting all possible patterns of statistically significant hyper- and hypo- methylation in comparisons involving any number of groups. Crucially, significance is established at the level of differentially methylated regions (DMRs), and bootstrapping determines false discovery rates (FDRs) associated with each pattern. We demonstrate this functionality in a four-group comparison among benign prostate and three clinical subtypes of prostate cancer and show that the bootstrap FDRs are highly useful in selecting the most robust patterns of DMRs. Compared to existing tools that are limited to two-group comparisons, MethylAction detects more DMRs with strong differential methylation measurements confirmed by whole genome bisulfite sequencing and offers a better balance between precision and recall in cross-cohort comparisons. MethylAction is available as an R package at http://jeffbhasin.github.io/methylaction.

Methylome-wide Sequencing Detects DNA Hypermethylation Distinguishing Indolent from Aggressive Prostate Cancer.

A critical need in understanding the biology of prostate cancer is characterizing the molecular differences between indolent and aggressive cases. Because DNA methylation can capture the regulatory state of tumors, we analyzed differential methylation patterns genome-wide among benign prostatic tissue and low-grade and high-grade prostate cancer and found extensive, focal hypermethylation regions unique to high-grade disease. These hypermethylation regions occurred not only in the promoters of genes but also in gene bodies and at intergenic regions that are enriched for DNA-protein binding sites. Integration with existing RNA-sequencing (RNA-seq) and survival data revealed regions where DNA methylation correlates with reduced gene expression associated with poor outcome. Regions specific to aggressive disease are proximal to genes with distinct functions from regions shared by indolent and aggressive disease. Our compendium of methylation changes reveals crucial molecular distinctions between indolent and aggressive prostate cancer.