A Structural Analysis of Gender-Based Violence and Depression in the Lives of Sexual Minority Women and Trans People.

This article explores structural mechanisms that are the context for violence and depression in the lives of sexual minority women and trans people in Ontario, Canada. The article draws on interviews with 14 people who reported experiences of depression in the previous year, foregrounding three representative narratives. Narrative and case study analysis reveal that violence is a repeated and cumulative experience over lifetimes, occurring across different interpersonal contexts and institutional encounters. A common theme across the narratives is that experiences of violence are connected to a broader context in which structural arrangements, cultural norms, and institutional processes create conditions where marginalized people are put in harm's way, perpetrators are empowered, and justice and access to help are elusive. As the violence experienced by these sexual minority women and trans people is rooted in structural and cultural oppression represented in poverty, racism, misogyny, homophobia, and transphobia, the prevention of violence and its consequences for these and other marginalized populations requires systemic transformation of the structures and systems that currently allow and perpetuate harm.

Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

IRF7 and UNC93B1 variants in an infant with recurrent herpes simplex virus infection.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.

Insurance denials and diagnostic rates in a pediatric genomic research cohort.

PURPOSE: This study aimed to assess the amount and types of clinical genetic testing denied by insurance and the rate of diagnostic and candidate genetic findings identified through research in patients who faced insurance denials. METHODS: Analysis consisted of review of insurance denials in 801 patients enrolled in a pediatric genomic research repository with either no previous genetic testing or previous negative genetic testing result identified through cross-referencing with insurance prior-authorizations in patient medical records. Patients and denials were also categorized by type of insurance coverage. Diagnostic findings and candidate genetic findings in these groups were determined through review of our internal variant database and patient charts. RESULTS: Of the 801 patients analyzed, 147 had insurance prior-authorization denials on record (18.3%). Exome sequencing and microarray were the most frequently denied genetic tests. Private insurance was significantly more likely to deny testing than public insurance (odds ratio = 2.03 [95% CI = 1.38-2.99] P = .0003). Of the 147 patients with insurance denials, 53.7% had at least 1 diagnostic or candidate finding and 10.9% specifically had a clinically diagnostic finding. Fifty percent of patients with clinically diagnostic results had immediate medical management changes (5.4% of all patients experiencing denials). CONCLUSION: Many patients face a major barrier to genetic testing in the form of lack of insurance coverage. A number of these patients have clinically diagnostic findings with medical management implications that would not have been identified without access to research testing. These findings support re-evaluation of insurance carriers' coverage policies.

Neonatal hyperoxia induces activated pulmonary cellular states and sex-dependent transcriptomic changes in a model of experimental bronchopulmonary dysplasia.

Hyperoxia disrupts lung development in mice and causes bronchopulmonary dysplasia (BPD) in neonates. To investigate sex-dependent molecular and cellular programming involved in hyperoxia, we surveyed the mouse lung using single cell RNA sequencing (scRNA-seq), and validated our findings in human neonatal lung cells in vitro. Hyperoxia-induced inflammation in alveolar type (AT) 2 cells gave rise to damage-associated transient progenitors (DATPs). It also induced a new subpopulation of AT1 cells with reduced expression of growth factors normally secreted by AT1 cells, but increased mitochondrial gene expression. Female alveolar epithelial cells had less EMT and pulmonary fibrosis signaling in hyperoxia. In the endothelium, expansion of Car4+ EC (Cap2) was seen in hyperoxia along with an emergent subpopulation of Cap2 with repressed VEGF signaling. This regenerative response was increased in females exposed to hyperoxia. Mesenchymal cells had inflammatory signatures in hyperoxia, with a new distal interstitial fibroblast subcluster characterized by repressed lipid biosynthesis and a transcriptomic signature resembling myofibroblasts. Hyperoxia-induced gene expression signatures in human neonatal fibroblasts and alveolar epithelial cells in vitro resembled mouse scRNA-seq data. These findings suggest that neonatal exposure to hyperoxia programs distinct sex-specific stem cell progenitor and cellular reparative responses that underpin lung remodeling in BPD.

The Game of Queer Family Life: Exploring 2SLGBTQI+ Parents' Experiences of Cisheteronormativity, Racism, and Colonialism Through Digital Storytelling in Ontario, Canada.

In this article we describe and analyze five videos created through an arts-informed research project, Precarious Inclusion: Studying Ontarian 2SLGBTQI+ parents' experiences childrearing in a post-legal parity framework. Precarious Inclusion used interviews and digital storytelling to investigate Ontario 2SLGBTQI+ parents' current experiences of inclusion and exclusion when navigating institutional and social interactions in everyday life in a post-legal parity context. The study centrally explored how intersecting identities with regards to sexuality, gender, geography, disability, class, race, Indigeneity, and ethnicity intersect with structural forces to influence 2SLGBTQI+ parents' inclusion and exclusion experiences. We examine research creation activities that supported 2SLGBTQI+ parents in making short videos about their experiences of parenting. Our analysis of the five videos created by Indigenous, racialized, trans, nonbinary, Two-Spirit, and disabled parents show how consistent experiences of exclusion mark 2SLGBTQI+ parents' everyday lives. We deepen theorizations of the material and psychological impacts of exclusion for 2SLGBTQI+ families through foregrounding three themes: 1) the operations of racism, white supremacy, and colonialism in makers' lives; 2) misrecognition and its psychic effects of bifurcation and disjuncture; and 3) love, joy, and multi-species kinship as powerful sites of healing and belonging. We further demonstrate how parents used their videos as self-advocacy for resisting precarious inclusion.

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Navigating return to sex: A qualitative Reddit analysis of parents' perceptions about the timing of resuming sex after a birth.

OBJECTIVE: Given the many transitions that occur in the postpartum period as men and women navigate the return to sex after a birth, this study aimed to explore parents' experiences of resuming sexual activity. METHODS: This was a qualitative study that used posts from both men and women from the online public forum Reddit. Data were analysed using constructivist grounded theory. RESULTS: This study found that the discourse surrounding sexual activity in the postpartum period was dominated by an understanding that medical guidelines prohibited sex prior to six weeks after giving birth. Although some parents disagreed, many Reddit users perceived a high degree of risk in resuming sex prior to six weeks and medical expertise was highly valued as parents negotiated the return to sex. While Reddit users were largely in agreement that penetrative vaginal sex prior to six weeks was a risky activity, there was less consensus as to the risk involved in other sexual activities. CONCLUSION: This study recommends that medical practitioners initiate conversations with men and women about returning to sexual activity after a birth, and that these conversations should consider parents' emotional well-being as they resume sex. Further research is needed to establish evidence-based and comprehensive guidelines to facilitate these conversations.

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes.

PURPOSE: This study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program. METHODS: Extensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes. RESULTS: Diagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases). CONCLUSION: Computational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.

American Medical Society for Sports Medicine Position Statement on the Care of the Athlete and Athletic Person With Diabetes.

ABSTRACT: The American Medical Society for Sports Medicine (AMSSM) developed this position statement to assist physicians and other health professionals in managing athletes and active people with diabetes. The AMSSM selected the author panel through an application process to identify members with clinical and academic expertise in the care of active patients with diabetes. This article reviews the current knowledge and gaps regarding the benefits and risks of various types of exercise and management issues for athletes and physically active people with diabetes, including nutrition and rehabilitation issues. Resistance exercises seem to be beneficial for patients with type 1 diabetes, and the new medications for patients with type 2 diabetes generally do not need adjustment with exercise. In preparing this statement, the authors conducted an evidence review and received open comment from the AMSSM Board of Directors before finalizing the recommendations.

Gait Disorders.

As a child matures so does the child's gait pattern. Gait changes in pediatric patients will be expected and sequential as developmental milestones. Gait changes may also represent normal variations along an appropriate spectrum. There are times when changes in gait are due to urgent orthopedic or medical conditions, and those should not be overlooked. A good understanding of pediatric gait development and a basic understanding of gait assessment are critical for the primary care physician who cares for children.

ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development.

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

Rare Genetic Variants in Immune Genes and Neonatal Herpes Simplex Viral Infections.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with high mortality, particularly when disseminated. Studies in adults and children suggest that susceptibility to herpes simplex encephalitis (HSE) may represent phenotypes for inborn errors in toll-like receptor 3 (TLR3) signaling. However, the genetic basis of susceptibility to neonatal HSV including disseminated disease remains unknown. To test the hypothesis that variants in known HSE-susceptible genes as well as genes mediating HSV immunity will be identified in neonatal HSV, we performed an unbiased exome sequencing study in 10 newborns with disseminated, HSE, and skin, eyes, and mouth disease. Determination of potential impact on function was determined by following American College of Medical Genetics and Genomics guidelines. We identified deleterious and potentially deleterious, rare variants in known HSE-related genes including a stop IRF3 variant (disseminated), nonsynonymous variants in TLR3 and TRAF3 (HSE), STAT1 (skin, eyes, and mouth), and DBR1 (disseminated) in our cohort. Novel and rare variants in other immunodeficiency genes or HSV-related immune genes GRB2, RAG2, PRF1, C6, C7, and MSR1 were found in 4 infants. The variant in GRB2, essential for T-lymphocyte cell responses to HSV, is a novel stop variant not found in public databases. In this pilot study, we identified deleterious or potentially deleterious variants in TLR3 pathway and genes that regulate anti-HSV immunity in neonates with HSV including disseminated disease. Larger, definitive studies incorporating functional analysis of genetic variants are required to validate these data and determine the role of immune genetic variants in neonatal HSV susceptibility.

Single-cell analysis of human adipose tissue identifies depot and disease specific cell types.

The complex relationship between metabolic disease risk and body fat distribution in humans involves cellular characteristics which are specific to body fat compartments. Here we show depot-specific differences in the stromal vascual fraction of visceral and subcutaneous adipose tissue by performing single-cell RNA sequencing of tissue specimen from obese individuals. We characterize multiple immune cells, endothelial cells, fibroblasts, adipose and hematopoietic stem cell progenitors. Subpopulations of adipose-resident immune cells are metabolically active and associated with metabolic disease status and those include a population of potential dysfunctional CD8+ T cells expressing metallothioneins. We identify multiple types of adipocyte progenitors that are common across depots, including a subtype enriched in individuals with type 2 diabetes. Depot-specific analysis reveals a class of adipocyte progenitors unique to visceral adipose tissue, which shares common features with beige preadipocytes. Our human single-cell transcriptome atlas across fat depots provides a resource to dissect functional genomics of metabolic disease.

Prospective Surveillance of Pediatric Invasive Group A Streptococcus Infection.

BACKGROUND: Invasive group A Streptococcus (GAS) disease has an incidence in high-income countries of 3 to 5 per 100000 per annum and a case-fatality ratio of 10% to 15%. Although these rates are comparable to those of invasive meningococcal disease in Australia before vaccine introduction, invasive GAS disease currently requires reporting in only 2 jurisdictions. METHODS: Data were collected prospectively through active surveillance at the Royal Children's Hospital, Melbourne (October 2014 to September 2016). Isolation of GAS from a sterile site was required for inclusion. Comprehensive demographic and clinical data were collected, and emm typing was performed on all isolates. Disease was considered severe if the patient required inotropic support or mechanical ventilation. RESULTS: We recruited 28 patients. The median age of the patients was 3.5 years (range, 4 days to 11 years). Ten (36%) patients had severe disease. Fifteen (54%) children had presented to a medical practitioner for review in the 48 hours before their eventual admission, including 7 of the 10 patients with severe GAS infection. Complications 6 months after discharge persisted in 21% of the patients. emm1 was the most common emm type (29%). CONCLUSION: We found considerable short- and longer-term morbidity associated with pediatric invasive GAS disease in our study. Disease manifestations were frequently severe, and more than one-third of the patients required cardiorespiratory support. More than one-half of the patients attended a medical practitioner for assessment but were discharged in the 48-hour period before admission, which suggests that there might have been a window for earlier diagnosis. Our methodology was easy to implement as a surveillance system.

Clinical genome sequencing in an unbiased pediatric cohort.

PURPOSE: We report for the first time, the use of clinical genome sequencing (GS) in an unbiased pediatric cohort. We describe the clinical validation, patient metrics, ordering patterns, results, reimbursement, and physician retrieval of results for the first consecutive 80 cases. METHODS: Clinical GS was performed for both inpatients and outpatients undergoing etiologic evaluations. Results were reported in the electronic medical record. Evidence of report retrieval by clinicians and whether interpretation was concordant with laboratory report was obtained through retrospective chart review. RESULTS: Twenty definitive diagnoses were made in 19 patients (24%; n = 80). Except for two partial gene deletions, all diagnostic variants would have been detectable by our exome methods. Surprisingly, there was no documentation of communication of results to the family in the medical record for 17.5% of patients, and in 7.5%, physician and laboratory interpretations were discordant. Average insurance reimbursement was 30.2%, with yield for commercial payers significantly higher, at 54.1%. CONCLUSIONS: The detection rate of GS is equivalent and potentially superior to exome sequencing (ES). Reimbursement rates were variable but overall satisfactory for commercial insurers, and poor for government entities. In addition, we identify opportunities for improvement in the communication of results to families, likely translatable to other tests and other institutions.

In spite of the system: A qualitatively-driven mixed methods analysis of the mental health services experiences of LGBTQ people living in poverty in Ontario, Canada.

Lesbian, gay, bisexual, trans, and/or queer (LGBTQ) people face barriers to accessing mental health care; however, we know little about service experiences of low income LGBTQ people. In this qualitatively-driven mixed methods study, over 700 women and/or trans people completed an internet survey, of whom 12 LGBTQ individuals living in poverty participated in interviews. Low income LGBTQ respondents saw more mental health professionals and had more unmet need for care than all other LGBTQ/income groups. Narrative analysis illustrated the work required to take care of oneself in the context of extreme financial constraints. These findings highlight the mechanisms through which inadequate public sector mental health services can serve to reproduce and sustain both poverty and health inequities.