Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.

SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor ß (Tgf-ß) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.

NRG-GY012: Randomized phase 2 study comparing olaparib, cediranib, and the combination of cediranib/olaparib in women with recurrent, persistent, or metastatic endometrial cancer.

PURPOSE: This paper reports the efficacy of the poly (ADP-ribose) polymerase inhibitor olaparib alone and in combination with the antiangiogenesis agent cediranib compared with cediranib alone in patients with advanced endometrial cancer. METHODS: This was open-label, randomized, phase 2 trial (NCT03660826). Eligible patients had recurrent endometrial cancer, received at least one (<3) prior lines of chemotherapy, and were Eastern Cooperative Oncology Group performance status 0 to 2. Patients were randomly assigned (1:1:1), stratified by histology (serous vs. other) to receive cediranib alone (reference arm), olaparib, or olaparib and cediranib for 28-day cycles until progression or unacceptable toxicity. The primary end point was progression-free survival in the intention-to-treat population. Homologous repair deficiency was explored using the BROCA-GO sequencing panel. RESULTS: A total of 120 patients were enrolled and all were included in the intention-to-treat analysis. Median age was 66 (range, 41-86) years and 47 (39.2%) had serous histology. Median progression-free survival for cediranib was 3.8 months compared with 2.0 months for olaparib (hazard ratio, 1.45 [95% CI, 0.91-2.3] p = .935) and 5.5 months for olaparib/cediranib (hazard ratio, 0.7 [95% CI, 0.43-1.14] p = .064). Four patients receiving the combination had a durable response lasting more than 20 months. The most common grade 3/4 toxicities were hypertension in the cediranib (36%) and olaparib/cediranib (33%) arms, fatigue (20.5% olaparib/cediranib), and diarrhea (17.9% cediranib). The BROCA-GO panel results were not associated with response. CONCLUSION: The combination of cediranib and olaparib demonstrated modest clinical efficacy; however, the primary end point of the study was not met. The combination was safe without unexpected toxicity.

Transforming growth factor ß-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-ß (TGF-ß), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-ß-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-ß signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation.

Extended Venous Thromboembolism Prophylaxis after Robotic Staging for Endometrial Cancer.

OBJECTIVES: Our objectives were to estimate the incidence of venous thromboembolism (VTE) after robotic staging for endometrial cancer and to compare the incidence of VTE in patients who received a single dose of preoperative prophylaxis of enoxaparin with those who received extended postoperative prophylaxis. METHODS: This study is a retrospective chart review of patients who underwent robot-assisted surgical staging for endometrial cancer. Patients were categorized into two groups: preoperative prophylaxis (PP), patients who received a single dose of enoxaparin preoperatively, and extended prophylaxis (EP), patients who received 28 days of enoxaparin postoperatively. RESULTS: In total, 148 patients were included, with 117 patients in the PP group and 31 patients in the EP group. The overall incidence of VTE within 30 days postoperatively was 0.67%. No significant difference was found between the PP and the EP groups (0.9% and 0%, respectively; P = 1.00). Most patients in the cohort had endometrioid adenocarcinoma (78%) with low-grade disease (70%), although there were a greater number of patients in the PP group with uterine serous carcinoma compared with the EP group (17% vs 10%; P = 0.034). The PP group had higher estimated blood loss (106 vs 81 mL; P = 0.009) and longer operative times (178 vs 151 min; P = 0.028) compared with the EP group. Significantly more patients in the PP group underwent lymph node dissection compared with the EP group (32% vs 7%; P = 0.008). CONCLUSIONS: The incidence of VTE following robot-assisted surgical staging for endometrial cancer in this study was 0.67%. No significant difference was found in VTE incidence between the PP group compared with the EP group. Mechanical prophylaxis plus a single dose of preoperative pharmacologic prophylaxis may suffice for low-risk patients following robotic surgical staging for endometrial cancer.

Sentinel Lymph Node Sampling in Robot-Assisted Staging of Endometrial Cancer.

OBJECTIVE: Sentinel lymph node (SLN) sampling in endometrial cancer staging has become an acceptable standard. Indocyanine green dye injected into the cervix and detected by near-infrared light is technically simple and sensitive. We aimed to evaluate SLN sampling in robot-assisted surgical staging of endometrial cancer at a university-affiliated teaching hospital. METHODS: A retrospective chart review, from January 2016 to December 2017, of patients who underwent robot-assisted surgical staging with cervical injection of indocyanine green dye detected by near-infrared light. The map rate, sensitivity, false negatives, and negative predictive value were calculated. RESULTS: A total of 105 charts were reviewed; 79 patients met inclusion criteria. The mean age was 65 (range 38-93) and the mean body mass index was 33.3 (range 16-49). Most patients (72.2%) had stage I disease and grade 1 or 2 histology (77.1%). Eight (10.1%) patients had lymph node metastasis. Seventy-two (91.1%) patients had positive mapping to at least 1 SLN. Sixty-two (78.5%) patients had bilateral mapping. Forty-four patients had concurrent pelvic ± para-aortic lymph node dissection and were included in the sensitivity analysis. Five of 44 cases had LN metastasis. The sensitivity was 80%, and the negative predictive value of SLN sampling was 97.5%. CONCLUSIONS: SLN mapping and sampling at a university-affiliated teaching hospital have comparable map rate, sensitivity, and negative predictive value as demonstrated in multiple trials. The technique has the potential to standardize endometrial cancer staging across different practice settings.

Identification of small RNA pathway genes using patterns of phylogenetic conservation and divergence.

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute and Dicer as essential cofactors that process and present small RNAs to their targets. Well-validated small RNA pathway cofactors such as these show distinctive patterns of conservation or divergence in particular animal, plant, fungal and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or short interfering RNA (siRNA)-mediated repression in Caenorhabditis elegans and Drosophila melanogaster, and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction data sets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about one-half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small-RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in those species.

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination.

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell type-dependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases.

Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge.

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.

Genome-wide identification of polycomb-associated RNAs by RIP-seq.

Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.

CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development.

The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.

The spring-loaded genome: nucleosome redistributions are widespread, transient, and DNA-directed.

Nucleosome occupancy plays a key role in regulating access to eukaryotic genomes. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of DNA sequence in this regulation remains unclear, particularly in mammals. To address this problem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV). We show that nucleosome redistribution peaks at 24 h post-KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro-assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 h post-KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans.

Morcellation and the Incidence of Occult Uterine Malignancy: A Dual-Institution Review.

OBJECTIVES: To determine the incidence of unsuspected uterine sarcoma (UtSarc), other uterine malignancies, and potential malignancies at the time of hysterectomy or myomectomy using power morcellation. METHODS: We performed a retrospective cohort study of all women undergoing myomectomy or hysterectomy using power morcellation at 2 institutions between January 1, 2004, and May 31, 2015. The primary outcome was the incidence of uterine malignancy (UM). The predefined secondary outcome was the occurrence of other conditions associated with malignant behavior. For analysis, any UtSarc or endometrial cancer was categorized as a "uterine malignancy," whereas other pathologies with cytologic atypia were categorized as "uterine premalignant disease" (UPM). All other pathological results were classified as "nonmalignant." RESULTS: A total of 1004 women underwent hysterectomy or myomectomy using power morcellation during the studied period. Two women (1/502; 95% confidence interval [CI], 1/4144-1/139) were found to have UM pathology, 2 endometrial carcinomas and none with UtSarc (97.5% CI, 0-1/273). Six (1/167; 95% CI, 1/455-1/77) women were found to have UPM on final pathology: 2 atypical leiomyomas, 1 STUMP (smooth muscle tumors of uncertain malignant potential), and 3 endometrial atypical hyperplasias. Women with UM had uteri that weighed more than those with NM pathology (840 g vs 217.7 g, P = 0.028), and this trend was also seen with UM and UPM (435.0 g vs 217.2 g, P = 0.081). Women with UM and UPM were more likely to have a preoperative surgical indication of "uterine leiomyoma" compared with other benign etiologies (P < 0.001). CONCLUSIONS: Among this cohort, all cases of unsuspected UM at the time of myomectomy or hysterectomy using power morcellation were found to be endometrial carcinoma. Unsuspected UM pathology had an incidence of 1 of 502. Factors associated with increased likelihood of UM or UPM were greater uterine weight and leiomyoma as the surgical indication.

Society of Gynecologic Oncology recommendations for the prevention of ovarian cancer.

Mortality from ovarian cancer may be dramatically reduced with the implementation of attainable prevention strategies. The new understanding of the cells of origin and the molecular etiology of ovarian cancer warrants a strong recommendation to the public and health care providers. This document discusses potential prevention strategies, which include 1) oral contraceptive use, 2) tubal sterilization, 3) risk-reducing salpingo-oophorectomy in women at high hereditary risk of breast and ovarian cancer, 4) genetic counseling and testing for women with ovarian cancer and other high-risk families, and 5) salpingectomy after childbearing is complete (at the time of elective pelvic surgeries, at the time of hysterectomy, and as an alternative to tubal ligation). The Society of Gynecologic Oncology has determined that recent scientific breakthroughs warrant a new summary of the progress toward the prevention of ovarian cancer. This review is intended to emphasize the importance of the fallopian tubes as a potential source of high-grade serous cancer in women with and without known genetic mutations in addition to the use of oral contraceptive pills to reduce the risk of ovarian cancer.

Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations.

X chromosome inactivation (XCI) achieves dosage balance in mammals by repressing one of two X chromosomes in females. During XCI, the long noncoding Xist RNA and Polycomb proteins spread along the inactive X (Xi) to initiate chromosome-wide silencing. Although inactivation is known to commence at the X-inactivation center (Xic), how it propagates remains unknown. Here, we examine allele-specific binding of Polycomb repressive complex 2 (PRC2) and chromatin composition during XCI and generate a chromosome-wide profile of Xi and Xa (active X) at nucleosome-resolution. Initially, Polycomb proteins are localized to ∼150 strong sites along the X and concentrated predominantly within bivalent domains coinciding with CpG islands ("canonical sites"). As XCI proceeds, ∼4000 noncanonical sites are recruited, most of which are intergenic, nonbivalent, and lack CpG islands. Polycomb sites are depleted of LINE repeats but enriched for SINEs and simple repeats. Noncanonical sites cluster around the ∼150 strong sites, and their H3K27me3 levels reflect a graded concentration originating from strong sites. This suggests that PRC2 and H3K27 methylation spread along a gradient unique to XCI. We propose that XCI is governed by a hierarchy of defined Polycomb stations that spread H3K27 methylation in cis.

RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities.

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.

Next-generation sequencing strategies enable routine detection of balanced chromosome rearrangements for clinical diagnostics and genetic research.

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.

The genomic binding sites of a noncoding RNA.

Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to genome-wide mapping of roX2, a well-studied ncRNA involved in dosage compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the male-specific lethal complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to chromatin immunoprecipitation for proteins.

Multiplexed Illumina sequencing libraries from picogram quantities of DNA.

BACKGROUND: High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. RESULTS: We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. CONCLUSIONS: Application of this method allows whole genome studies from samples where material or yields are limiting.