Rising Mortality in Pediatric Self-Inflicted Firearm Trauma Associated With Distinct Anatomic Injury.

INTRODUCTION: Self-inflicted injuries are the second leading cause of pediatric (10-18 y old) mortality. Self-inflicted firearm trauma (SIFT) was responsible for up to half of these deaths in certain age groups. We hypothesized that SIFT prevalence has increased and is associated with specific demographics, injury patterns, and outcomes. MATERIALS AND METHODS: Data were abstracted from the 2007-2018 American College of Surgeons (ACS) Trauma Quality Programs Participant Use Files (TQP-PUF). Pediatric (1-17 yold) victims of firearm violence were eligible. Age, race, gender, anatomic region, and intent were abstracted. Variables were analyzed using chi-squared tests, t-tests, and single-variate linear regression models. Temporal trends were analyzed using ANCOVA tests. Multivariate logistic regressions were conducted to identify factors influencing mortality. Significance was P < 0.05. RESULTS: There were 41,239 pediatric firearm trauma patients (SIFT: 5.5% [n = 2272]). SIFT incidence increased over the 12-y period (2007 (n = 67) versus 2018 (n = 232), P < 0.05). SIFT was significantly associated with Caucasian race, 67% (n = 1537), teenagers, 90% (n = 2056), male gender, 87% (n = 1978), and a higher median injury severity score (ISS) than other intents of injury (SIFT: 20.0 (IQR: 9.0, 25.0) versus other: 9.0 (IQR: 1.0-13.0), P < 0.001). The SIFT mortality rate was 44% (n = 1005). On multivariate regression head gunshot wounds (OR: 21.1, 95% C.I.: 9.9-45.2, P = 0.001), and ISS (OR:1.1, 95% C.I.: 1.1-1.1, P = 0.001) were significantly associated with mortality. Compared to other intents, SIFT mortality rates increased at a higher annual rate (P < 0.001). CONCLUSIONS: Comprehensive local and federal policy changes to reduce firearms access and increase pediatric mental health support may mitigate these injuries.

Knockdown of GADD34 in neonatal mutant SOD1 mice ameliorates ALS.

Mutations in Cu/Zn superoxide dismutase (SOD1) cause ~20% of familial ALS (FALS), which comprises 10% of total ALS cases. In mutant SOD1- (mtSOD1-) induced ALS, misfolded aggregates of SOD1 lead to activation of the unfolded protein response/integrated stress response (UPR/ISR). Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a kinase that phosphorylates eukaryotic translation initiator factor 2α (p-eIF2α), coordinates the response by causing a global suppression of protein synthesis. Growth arrest and DNA damage 34 (GADD34) dephosphorylates p-eIF2α, allowing protein synthesis to return to normal. If the UPR/ISR is overwhelmed by the amount of misfolded protein, CCAAT/enhancer-binding homologous protein (CHOP) is activated leading to apoptosis. In the current study we investigated the effect of knocking down CHOP and GADD34 on disease of G93A and G85R mtSOD1 mice. Although a CHOP antisense oligonucleotide had no effect on survival, an intravenous injection of GADD34 shRNA encoded in adeno-associated virus 9 (AAV9) into neonatal G93A as well as neonatal G85R mtSOD1 mice led to a significantly increased survival. G85R mtSOD1 mice had a reduction in SOD1 aggregates/load, astrocytosis, and microgliosis. In contrast, there was no change in disease phenotype when GADD34 shRNA was delivered to older G93A mtSOD1 mice. Our current study shows that GADD34 shRNA is effective in ameliorating disease when administered to neonatal mtSOD1 mice. Targeting the UPR/ISR may be beneficial in mtSOD1-induced ALS as well as other neurodegenerative diseases in which misfolded proteins and ER stress have been implicated.

Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Genome-wide association study identifies 30 obsessive-compulsive disorder associated loci.

Obsessive-compulsive disorder (OCD) affects ~1% of the population and exhibits a high SNP-heritability, yet previous genome-wide association studies (GWAS) have provided limited information on the genetic etiology and underlying biological mechanisms of the disorder. We conducted a GWAS meta-analysis combining 53,660 OCD cases and 2,044,417 controls from 28 European-ancestry cohorts revealing 30 independent genome-wide significant SNPs and a SNP-based heritability of 6.7%. Separate GWAS for clinical, biobank, comorbid, and self-report sub-groups found no evidence of sample ascertainment impacting our results. Functional and positional QTL gene-based approaches identified 249 significant candidate risk genes for OCD, of which 25 were identified as putatively causal, highlighting WDR6, DALRD3, CTNND1 and genes in the MHC region. Tissue and single-cell enrichment analyses highlighted hippocampal and cortical excitatory neurons, along with D1- and D2-type dopamine receptor-containing medium spiny neurons, as playing a role in OCD risk. OCD displayed significant genetic correlations with 65 out of 112 examined phenotypes. Notably, it showed positive genetic correlations with all included psychiatric phenotypes, in particular anxiety, depression, anorexia nervosa, and Tourette syndrome, and negative correlations with a subset of the included autoimmune disorders, educational attainment, and body mass index.. This study marks a significant step toward unraveling its genetic landscape and advances understanding of OCD genetics, providing a foundation for future interventions to address this debilitating disorder.

Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp.

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq ("Patch-Seq"). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries - especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.

Gene Expression in Patient-Derived Neural Progenitors Implicates WNT5A Signaling in the Etiology of Schizophrenia.

BACKGROUND: Genome-wide association studies of schizophrenia have demonstrated that variations in noncoding regions are responsible for most of the common variation heritability of the disease. It is hypothesized that these risk variants alter gene expression. Therefore, studying alterations in gene expression in schizophrenia may provide a direct approach to understanding the etiology of the disease. In this study we use cultured neural progenitor cells derived from olfactory neuroepithelium (CNON cells) as a genetically unaltered cellular model to elucidate the neurodevelopmental aspects of schizophrenia. METHODS: We performed a gene expression study using RNA sequencing of CNON cells from 111 control subjects and 144 individuals with schizophrenia. Differentially expressed genes were identified with DESeq2 software, using covariates to correct for sex, age, library batches, and 1 surrogate variable component. RESULTS: A total of 80 genes were differentially expressed (false discovery rate < 10%), showing enrichment in cell migration, cell adhesion, developmental process, synapse assembly, cell proliferation, and related Gene Ontology categories. Cadherin and Wnt signaling pathways were positive in overrepresentation test, and, in addition, many genes were specifically involved in WNT5A signaling. The differentially expressed genes were modestly, but significantly, enriched in the genes overlapping single nucleotide polymorphisms with genome-wide significant association from the Psychiatric Genomics Consortium genome-wide association study of schizophrenia. We also found substantial overlap with genes associated with other psychiatric disorders or brain development, enrichment in the same Gene Ontology categories as genes with mutations de novo in schizophrenia, and studies of induced pluripotent stem cell-derived neural progenitor cells. CONCLUSIONS: CNON cells are a good model of the neurodevelopmental aspects of schizophrenia and can be used to elucidate the etiology of the disorder.

Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation.

As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.