Ultrarare Variants in DNA Damage Repair Genes in Pediatric Acute-Onset Neuropsychiatric Syndrome or Acute Behavioral Regression in Neurodevelopmental Disorders.

Introduction: Acute onset of severe psychiatric symptoms or regression may occur in children with premorbid neurodevelopmental disorders, although typically developing children can also be affected. Infections or other stressors are likely triggers. The underlying causes are unclear, but a current hypothesis suggests the convergence of genes that influence neuronal and immunological function.

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

Background: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies.

ETS1 is a novel transcriptional regulator of adult T-cell leukemia/lymphoma of North American descent.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell lymphoma associated with the human T-cell lymphotropic virus type 1 virus endemic in regions including Japan, the Caribbean islands, and Latin America. Although progress has been made to understand the disease, survival outcomes with current standard therapy remain extremely poor particularly in acute ATLL, underlying the need for better understanding of its biology and identification of novel therapeutic targets. Recently, it was demonstrated that ATLL of North American-descendent patients (NA-ATLL) is both clinically and molecularly distinct from Japanese-descendent (J-ATLL), with inferior prognosis and higher incidence of epigenetic-targeting mutations compared with J-ATLL.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine.

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1 mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells.

A Cellular Stress Response Induced by the CRISPR-dCas9 Activation System Is Not Heritable Through Cell Divisions.

The CRISPR-Cas9 system can be modified to perform "epigenetic editing" by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions-an epigenetic regulatory property.

Misidentification of and other mutations in cancer due to highly homologous genomic regions.

The gene has been shown to be recurrently mutated in many malignancies including in families with acute myeloid leukemia. We demonstrate that many variant calls made by exome sequencing are false positives due to misalignment to homologous regions, including a region on chr21, and can only be validated by long-range PCR. Numerous other recurrently mutated genes reported in COSMIC and TCGA databases have pseudogenes and cannot also be validated by conventional short read-based sequencing approaches.

The nutritional environment determines which and how intestinal stem cells contribute to homeostasis and tumorigenesis.

Sporadic colon cancer accounts for approximately 80% of colorectal cancer (CRC) with high incidence in Western societies strongly linked to long-term dietary patterns. A unique mouse model for sporadic CRC results from feeding a purified rodent Western-style diet (NWD1) recapitulating intake for the mouse of common nutrient risk factors each at its level consumed in higher risk Western populations. This causes sporadic large and small intestinal tumors in wild-type mice at an incidence and frequency similar to that in humans.

Detecting, quantifying, and discriminating the mechanism of mosaic chromosomal aneuploidies using MAD-seq.

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost, or the need to culture cells. We describe the mosaic aneuploidy detection by massively parallel sequencing (MAD-seq) capture assay and the analytical approach that allow low (<10%) levels of mosaicism for chromosomal aneuploidy or regional loss of heterozygosity to be detected, assigned to a meiotic or mitotic origin, and quantified as a proportion of the cells in the sample. We show results from a multi-ethnic MAD-seq (meMAD-seq) capture design that works equally well in populations of diverse racial and ethnic origins and how the analytical approach can be applied to exome or whole-genome sequencing data, revealing previously unrecognized aneuploidy or copy number neutral loss of heterozygosity in samples studied by the 1000 Genomes Project, cell lines from public repositories, and one of the Illumina Platinum Genomes samples.

Detection of circulating tumor DNA in patients with osteosarcoma.

Identification and quantification of somatic alterations in plasma-derived, circulating tumor DNA (ctDNA) is gaining traction as a non-invasive and cost effective method of disease monitoring in cancer patients, particularly to evaluate response to treatment and monitor for disease recurrence. To our knowledge, genetic analysis of ctDNA in osteosarcoma has not yet been studied. To determine whether somatic alterations can be detected in ctDNA and perhaps applied to patient management in this disease, we collected germline, tumor, and serial plasma samples from pediatric, adolescent, and young adult patients with osteosarcoma and used targeted Next Generation Sequencing (NGS) to identify somatic single nucleotide variants (SNV), insertions and deletions (INDELS), and structural variants (SV) in 7 genes commonly mutated in osteosarcoma.

Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways.

Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues.

The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements.

Unlabelled: Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear.

RNASeq in C. elegans Following Manganese Exposure.

Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT-1 and ferroportin, are known, it is presumed that many more are involved and as yet unknown.

Transcriptional and metabolic signatures of Arabidopsis responses to chewing damage by an insect herbivore and bacterial infection and the consequences of their interaction.

Plants use multiple interacting signaling systems to identify and respond to biotic stresses. Although it is often assumed that there is specificity in signaling responses to specific pests, this is rarely examined outside of the gene-for-gene relationships of plant-pathogen interactions. In this study, we first compared early events in gene expression and later events in metabolite profiles of Arabidopsis thaliana following attack by either the caterpillar Spodoptera exigua or avirulent (DC3000 avrRpm1) Pseudomonas syringae pv.

Mosaic epigenetic dysregulation of ectodermal cells in autism spectrum disorder.

DNA mutational events are increasingly being identified in autism spectrum disorder (ASD), but the potential additional role of dysregulation of the epigenome in the pathogenesis of the condition remains unclear. The epigenome is of interest as a possible mediator of environmental effects during development, encoding a cellular memory reflected by altered function of progeny cells. Advanced maternal age (AMA) is associated with an increased risk of having a child with ASD for reasons that are not understood.

Myeloma is characterized by stage-specific alterations in DNA methylation that occur early during myelomagenesis.

Epigenetic changes play important roles in carcinogenesis and influence initial steps in neoplastic transformation by altering genome stability and regulating gene expression. To characterize epigenomic changes during the transformation of normal plasma cells to myeloma, we modified the HpaII tiny fragment enrichment by ligation-mediated PCR assay to work with small numbers of purified primary marrow plasma cells. The nano-HpaII tiny fragment enrichment by ligation-mediated PCR assay was used to analyze the methylome of CD138(+) cells from 56 subjects representing premalignant (monoclonal gammopathy of uncertain significance), early, and advanced stages of myeloma, as well as healthy controls.

High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis.

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation.

Direct, genome-wide assessment of DNA mutations in single cells.

DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors.

RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders.

Genome-wide expression analysis using next generation sequencing (RNA-Seq) provides an opportunity for in-depth molecular profiling of fundamental biological processes, such as cellular differentiation and malignant transformation. Differentiating human neurons derived from induced pluripotent stem cells (iPSCs) provide an ideal system for RNA-Seq since defective neurogenesis caused by abnormalities in transcription factors, DNA methylation, and chromatin modifiers lie at the heart of some neuropsychiatric disorders. As a preliminary step towards applying next generation sequencing using neurons derived from patient-specific iPSCs, we have carried out an RNA-Seq analysis on control human neurons.

Dampened activity of E2F1-DP and Myb-MuvB transcription factors in Drosophila endocycling cells.

The endocycle is a variant cell cycle comprised of alternating gap (G) and DNA synthesis (S) phases (endoreplication) without mitosis (M), which results in DNA polyploidy and large cell size. Endocycles occur widely in nature, but much remains to be learned about the regulation of this modified cell cycle. Here, we compared gene expression profiles of mitotic cycling larval brain and disc cells with the endocycling cells of fat body and salivary gland of the Drosophila larva.

The Einstein Center for Epigenomics: studying the role of epigenomic dysregulation in human disease.

There is increasing interest in the role of epigenetic and transcriptional dysregulation in the pathogenesis of a range of human diseases, not just in the best-studied example of cancer. It is, however, quite difficult for an individual investigator to perform these studies, as they involve genome-wide molecular assays combined with sophisticated computational analytical approaches of very large datasets that may be generated from various resources and technologies. In 2008, the Albert Einstein College of Medicine in New York, USA established a Center for Epigenomics to facilitate the research programs of its investigators, providing shared resources for genome-wide assays and for data analysis.

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress.

Initiation of DNA replication at origins more than once per cell cycle results in rereplication and has been implicated in cancer. Here we use Drosophila to examine the checkpoint responses to rereplication in a developmental context. We find that increased Double-parked (Dup), the Drosophila ortholog of Cdt1, results in rereplication and DNA damage.

Transformation of oats and its application to improving osmotic stress tolerance.

Oat (Avena sativa L.), a worldwide temperate cereal crop, is deficient in tolerance to osmotic stress due to drought and/or salinity. To genetically transform the available commercial oat cultivars, a genotype-independent and efficient regeneration system from shoot apical meristems was developed using four oat cultivars: Prairie, Porter, Ogle, and Pacer.

Arabidopsis GH3-LIKE DEFENSE GENE 1 is required for accumulation of salicylic acid, activation of defense responses and resistance to Pseudomonas syringae.

In Arabidopsis, the GH3-like gene family consists of 19 members, several of which have been shown to adenylate the plant hormones jasmonic acid, indole acetic acid and salicylic acid (SA). In some cases, this adenylation has been shown to catalyze hormone conjugation to amino acids. Here we report molecular characterization of the GH3-LIKE DEFENSE GENE 1 (GDG1), a member of the GH3-like gene family, and show that GDG1 is an important component of SA-mediated defense against the bacterial pathogen Pseudomonas syringae.

Overexpression of CRK13, an Arabidopsis cysteine-rich receptor-like kinase, results in enhanced resistance to Pseudomonas syringae.

Protein kinases play important roles in relaying information from perception of a signal to the effector genes in all organisms. Cysteine-rich receptor-like kinases (CRKs) constitute a sub-family of plant receptor-like kinases (RLKs) with more than 40 members that contain the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. Here we report molecular characterization of one member of this gene family, CRK13.