Hypoxia-specific imaging in patients with lymphoma undergoing CAR-T therapy.

PURPOSE: Intratumoral hypoxia in non-Hodgkin's Lymphoma (NHL) may interfere with chimeric antigen receptor T-cell (CAR-T) function. We conducted a single-center pilot study (clinicaltrials.gov ID NCT04409314) of [18F]fluoroazomycin arabinoside, a hypoxia-specific radiotracer abbreviated as [18F]FAZA, to assess the feasibility of this positron emission tomography (PET) imaging modality in this population. METHODS: Patients with relapsed NHL being evaluated for CAR-T therapy received a one-time [18F]FAZA PET scan before pre-CAR-T lymphodepletion. A tumor to mediastinum (T/M) ratio of 1.2 or higher with regard to [18F]FAZA uptake was defined as positive for intratumoral hypoxia. We planned to enroll 30 patients with an interim futility analysis after 16 scans. RESULTS: Of 16 scanned patients, 3 had no evidence of disease by standard [18F]fluorodeoxyglucose PET imaging before CAR-T therapy. Six patients (38%) had any [18F]FAZA uptake above background. Using a T/M cutoff of 1.20, only one patient (a 68-year-old male with relapsed diffuse large B-cell lymphoma) demonstrated intratumoral hypoxia in an extranodal chest wall lesion (T/M 1.35). Interestingly, of all 16 scanned patients, he was the only patient with progressive disease within 1 month of CAR-T therapy. However, because of our low overall proportion of positive scans, our study was stopped for futility. CONCLUSIONS: Our pilot study identified low-level [18F]FAZA uptake in a small number of patients with NHL receiving CAR-T therapy. The only patient who met our pre-specified threshold for intratumoral hypoxia was also the only patient with early CAR-T failure. Future plans include exploration of [18F]FAZA in a more selected patient population.

Genetic and Epigenetic Fine Mapping of Complex Trait Associated Loci in the Human Liver.

Deciphering the impact of genetic variation on gene regulation is fundamental to understanding common, complex human diseases. Although histone modifications are important markers of gene regulatory elements of the genome, any specific histone modification has not been assayed in more than a few individuals in the human liver. As a result, the effects of genetic variation on histone modification states in the liver are poorly understood. Here, we generate the most comprehensive genome-wide dataset of two epigenetic marks, H3K4me3 and H3K27ac, and annotate thousands of putative regulatory elements in the human liver. We integrate these findings with genome-wide gene expression data collected from the same human liver tissues and high-resolution promoter-focused chromatin interaction maps collected from human liver-derived HepG2 cells. We demonstrate widespread functional consequences of natural genetic variation on putative regulatory element activity and gene expression levels. Leveraging these extensive datasets, we fine-map a total of 74 GWAS loci that have been associated with at least one complex phenotype. Our results reveal a repertoire of genes and regulatory mechanisms governing complex disease development and further the basic understanding of genetic and epigenetic regulation of gene expression in the human liver tissue.

IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins.

The IL1A and IL1B genes lie in close proximity on chromosome 2 near the gene for their natural inhibitor, IL1RN Despite diverse functions, they are all three inducible through TLR4 signaling but with distinct kinetics. This study analyzed transcriptional induction kinetics, chromosome looping, and enhancer RNA production to understand the distinct regulation of these three genes in human cells. IL1A, IL1B, and IL1RN were rapidly induced after stimulation with LPS; however, IL1B mRNA production was less inhibitable by iBET151, suggesting it does not use pause-release regulation. Surprisingly, chromatin looping contacts between IL1A and IL1B were highly intermingled, although those of IL1RN were distinct, and we focused on comparing IL1A and IL1B transcriptional pathways. Our studies demonstrated that enhancer RNAs were produced from a subset of the regulatory regions, that they were critical for production of the mRNAs, and that they bound a diverse array of RNA binding proteins, including p300 but not CBP. We, furthermore, demonstrated that recruitment of p300 was dependent on MAPKs. Integrator is another RNA binding protein recruited to the promoters and enhancers, and its recruitment was more dependent on NF-κB than MAPKs. We found that integrator and NELF, an RNA polymerase II pausing protein, were associated with RNA in a manner that facilitated interaction. We conclude that IL1A and IL1B share many regulatory contacts, signaling pathways, and interactions with enhancer RNAs. A complex of protein interactions with enhancer RNAs emphasize the role of enhancer RNAs and the overall structural aspects of transcriptional regulation.

A Brief Survey of the COVID-19 Pandemic's Impact on the Chronic Pain Experience.

BACKGROUND: The COVID-19 pandemic has forced sweeping social and behavioral changes that have adversely affected the general population. Many changes, such as business closures, working from home, increased psychological distress, and delayed access to health care, could have unique adverse effects on patients diagnosed with chronic pain (CP). The present study sought to examine perceived changes in the CP experience brought about by the COVID-19 pandemic. DESIGN: Participants included 487 self-reported patients with musculoskeletal, neuropathic, or postsurgical pain recruited using CloudResearch. A 53-item survey was created to assess changes in perceived pain, mood, control over pain, physical activity, employment, and medical access since the onset of the pandemic. RESULTS: Results suggested a worsening of the pain experience, particularly for women, with greater pain, negative affect, sedentary functioning, perceived decline in treatment quality, and increased treatment delays. Of note, pandemic-related declines in control over pain, which represents an important clinical target, are associated with other pandemic-related declines and also mediates relevant associations. CONCLUSIONS: For frontline treatment providers, particularly primary care nurses and physicians, these findings may be relevant in order to reduce the likelihood of a worsening of symptoms, loss of self-efficacy regarding management of pain and/or potential maladaptive increase in the use of pain medications.

Coupling with COVID: The Role of Dyadic Coping in Relationship Satisfaction and Psychological Distress during the COVID-19 Pandemic.

Models of dyadic coping suggest that facing a stressful situation, such as the COVID-19 pandemic, with one's partner to meet their needs is associated with positive outcomes. This study explored dyadic coping and its association with relationship satisfaction and distress in the time of the COVID-19 pandemic. Data were collected online from 564 participants. Participants completed measures of dyadic coping, relationship satisfaction, COVID anxiety, and OCD, and asked to describe their experience in an open-ended question. Results showed that experiences were quite polarized. Significant gender differences and differences for couples with/without children were noted for distress and relationship satisfaction. There was a significant interaction between dyadic coping and relationship satisfaction for women when predicting COVID OCD; however, post-hoc analysis showed that this interaction was only significant for women with children. The potential exponential burden that female couple members may face during COVID-19 as well as implications for intervention, are discussed.

Couples' Relationship Satisfaction and Its Association with Depression and Spouse Responses Within the Context of Chronic Pain Adjustment.

BACKGROUND: Chronic pain is one specific health condition where couple relationships have been directly linked to physical and psychological outcomes. Understanding how relationship satisfaction, couple dynamics, and pain adjustment interrelate is crucial for nurses who provide patient-centered care for patients with pain. AIMS: The current study was aimed at examining the associations of depressive symptoms and spouse response styles with relationship satisfaction in the context of West Haven-Yale Multidimensional Pain Inventory classifications. METHODS: Seventy-eight middle-aged outpatients with chronic pain (average pain duration of 8.98 years (SD = 9.51)) were recruited from a pain clinic in southeastern Michigan. Participants completed the Multidimensional Pain Inventory, from which pain adjustment classifications (adaptive, dysfunctional, interpersonally distressed) and spouse response styles were derived, the Dyadic Adjustment Scale to assess relationship satisfaction, and the Mood and Anxiety Symptom Questionnaire to assess depressive symptoms. RESULTS: Interpersonally distressed patients scored lower on relationship satisfaction than those classified as either adaptive or dysfunctional, F(2,66) = 6.38, p < .01. Significant inverse associations were found between punishing spouse response styles and relationship satisfaction for both interpersonally distressed and dysfunctional adjustment classifications: r = -.53, p < .05; r = -.57, p < .01, respectively. CONCLUSIONS: Overall, findings indicate that relationship satisfaction may be an area of concern among interpersonally distressed pain patients and that spouse response style may be a particularly important area of clinical attention.

The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5.

AIMS/HYPOTHESIS: One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. RESULTS: We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. CONCLUSIONS/INTERPRETATION: This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.

Perceptions of Plagiarism by STEM Graduate Students: A Case Study.

Issues of academic integrity, specifically knowledge of, perceptions and attitudes toward plagiarism, are well documented in post-secondary settings using case studies for specific courses, recording discourse with focus groups, analyzing cross-cultural education philosophies, and reviewing the current literature. In this paper, the authors examine the perceptions of graduate students in science, technology, engineering, and mathematics (STEM) disciplines at the University of Florida regarding misconduct and integrity issues. Results revealed students' perceptions of the definition and seriousness of potential academic misconduct, knowledge of institutional procedures, and views on faculty actions, all with a focus on divergences between U.S. and internationally-educated students. The open-ended questions provide anecdotal evidence to highlight personal experiences, positive and negative, aimed at the faculty, international students and undergraduates. Combined, these findings outline an important part of the campus academic integrity culture at a major American university. Recommendations for local actions also are discussed.

Distinct roles for N-Cadherin linked c-Src and fyn kinases in lens development.

BACKGROUND: Src family tyrosine kinases (SFKs) are often coincidently expressed but few studies have dissected their individual functions in the same cell during development. Using the classical embryonic lens as our model, we investigated SFK signaling in the regulation of both differentiation initiation and morphogenesis, and the distinct functions of c-Src and Fyn in these processes. RESULTS: Blocking SFK activity with the highly specific inhibitor PP1 induced initiation of the lens differentiation program but blocked lens fiber cell elongation and organization into mini lens-like structures called lentoids. These dichotomous roles for SFK signaling were discovered to reflect distinct functions of c-Src and Fyn and their differentiation-state-specific recruitment to and action at N-cadherin junctions. c-Src was highly associated with the nascent N-cadherin junctions of undifferentiated lens epithelial cells. Its siRNA knockdown promoted N-cadherin junctional maturation, blocked proliferation, and induced lens cell differentiation. In contrast, Fyn was recruited to mature N-cadherin junctions of differentiating lens cells and siRNA knockdown suppressed differentiation-specific gene expression and blocked morphogenesis. CONCLUSIONS: Through inhibition of N-cadherin junction maturation, c-Src promotes lens epithelial cell proliferation and the maintenance of the lens epithelial cell undifferentiated state, while Fyn, signaling downstream of mature N-cadherin junctions, promotes lens fiber cell morphogenesis.

Association between pain catastrophizing, spouse responses to pain, and blood pressure in chronic pain patients: a pathway to potential comorbidity.

BACKGROUND: Chronic pain has been shown to be highly comorbid with other medical conditions. Theoretical and empirical associations between pain and cardiovascular health can be made based on the current literature. Psychosocial variables associated with the pain experience may, however, have an impact on cardiovascular health. PURPOSE: The purpose of this study was to examine how cognitive and interpersonal aspects of chronic pain, including pain catastrophizing (PC) and negative spouse responses (NSR), relate to systolic and diastolic blood pressure (SBP, DBP) as cardiovascular risk factors. METHODS: Data were collected from 57 treatment-seeking patients with chronic musculoskeletal pain. Participants completed the West Haven-Yale Multidimensional Pain Inventory, Pain Catastrophizing Scale, and pain severity ratings based on an analog pain scale. In addition, participants consented to a medical chart review to collect blood pressure and prescribed medication data. Hierarchical linear regressions were used to test associations between PC and NSR, and blood pressure while controlling cardiac medication status. RESULTS: A positive association between PC and both SBP and DBP was found. A positive association was also found for NSR and SBP. These findings suggest that psychosocial aspects of chronic pain may represent a direct risk for elevated blood pressure and, thus, increased risk for cardiovascular health problems. CONCLUSIONS: Psychosocial aspects of pain may constitute a form of chronic stress as described in the cardiovascular reactivity literature. The findings highlight the need for comprehensive multidimensional treatments of pain.

Relationship between Posttraumatic Stress Disorder and Sleep Disturbances in Syrian Refugees in the United States.

Background Posttraumatic stress disorder (PTSD) is associated with disturbed sleep. However, the impact of sleep disturbances and PTSD symptomology in refugee populations is not well known. This study examined how PTSD-related sleep symptoms and overall sleep quality were impacted by previous and current traumatic and stressful experiences. Methods Adult Syrian refugees living in Southeast Michigan were assessed via scheduled in-home interviews. Overall sleep quality was measured using the Pittsburgh Sleep Quality Index. PTSD-related sleep disturbances were measured using the Pittsburgh Sleep Quality Index Addendum. The presence of PTSD symptomatology was assessed via self-report using the Posttraumatic Stress Disorder Checklist. The Life Events Checklist for the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition-5 screened for prior traumatic events experienced and the Postmigration Living Difficulties Questionnaire was assessed for postmigration stressors. Correlational analysis was conducted between overall sleep quality, PTSD symptom severity, and previous trauma experienced. A stepwise linear regression analysis was conducted to examine the role of overall sleep quality, PTSD-specific sleep disturbances, current living difficulties, and the number of preimmigration traumatic events directly experienced or witnessed due to the presence of overall PTSD symptomology. Results A total of 53 adults completed the study. PTSD-disturbed sleep was found to be positively associated with overall poor sleep quality ( r = 0.42, p < 0.01), PTSD symptomology ( r = 0.65, p < 0.01), and current living difficulties ( r = 0.37, p < 0.05). The PTSD-related sleep disturbances (B = 0.66, p < 0.01) and postmigration living difficulties (B = 0.44, p < 0.01) were found to be the strongest predictors of PTSD symptoms. Conclusion Disturbed sleep is strongly associated with current stressful experiences and PTSD symptomology among Syrian refugees.

Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens.

Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked ß-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo.

Implicating effector genes at COVID-19 GWAS loci using promoter-focused Capture-C in disease-relevant immune cell types.

BACKGROUND: SARS-CoV-2 infection results in a broad spectrum of COVID-19 disease, from mild or no symptoms to hospitalization and death. COVID-19 disease severity has been associated with some pre-existing conditions and the magnitude of the adaptive immune response to SARS-CoV-2, and a recent genome-wide association study (GWAS) of the risk of critical illness revealed a significant genetic component. To gain insight into how human genetic variation attenuates or exacerbates disease following SARS-CoV-2 infection, we implicated putatively functional COVID risk variants in the cis-regulatory landscapes of human immune cell types with established roles in disease severity and used high-resolution chromatin conformation capture to map these disease-associated elements to their effector genes. RESULTS: This functional genomic approach implicates 16 genes involved in viral replication, the interferon response, and inflammation. Several of these genes (PAXBP1, IFNAR2, OAS1, OAS3, TNFAIP8L1, GART) were differentially expressed in immune cells from patients with severe versus moderate COVID-19 disease, and we demonstrate a previously unappreciated role for GART in T cell-dependent antibody-producing B cell differentiation in a human tonsillar organoid model. CONCLUSIONS: This study offers immunogenetic insight into the basis of COVID-19 disease severity and implicates new targets for therapeutics that limit SARS-CoV-2 infection and its resultant life-threatening inflammation.

An Implementation Science Approach to Promote Optimal Implementation, Adoption, Use, and Spread of Continuous Clinical Monitoring System Technology.

OBJECTIVES: This study aimed to apply implementation science tenets to guide the deployment and use of in-hospital Clinical Monitoring System Technology (CMST) and to develop a toolkit to promote optimal implementation, adoption, use, and spread of CMST. METHODS: Six steps were carried out to (1) establish leadership support; (2) identify, educate, and sustain champions; (3) enlist clinical staff users to learn barriers and facilitators; (4) examine initial qualitative data from 11 clinician group interviews; (5) validate barriers/facilitators to CMST use and toolkit content; and (6) propose a toolkit to promote utilization. Clinical Monitoring System Technology output before and after implementation were compared. RESULTS: The top 3 barriers to effective CMST use were as follows: (1) inadequate education/training/support, (2) clinical workflow challenges, and (3) lack of communication. Facilitators to CMST implementation and adoption included the following: (1) providing comprehensive and consistent CMST education, (2) presenting evidence early and often, (3) tailoring device and usage expectations to individual environments, and (4) providing regular feedback about progress. Empirical data drove the development of a CMST implementation toolkit covering 6 areas: (1) why, (2) readiness, (3) readiness and implementation, (4) patient/family introduction, (5) champions, (6) care team saves, and (7) troubleshooting. Clinical Monitoring System Technology positively impacted failure to rescue events. Monthly median cardiac alert responses decreased from 30 to 3.64 minutes (87.9%), and respiratory alert responses decreased from 26 to 4.85 minutes (81.4%). CONCLUSIONS: Using implementation science tenets to concurrently guide deployment and study performance of 2 CMST devices and impact on workload was effective for both learning CMST efficacy at 2 hospital systems and developing a toolkit to promote optimal implementation, adoption, use, and spread.

3D promoter architecture re-organization during iPSC-derived neuronal cell differentiation implicates target genes for neurodevelopmental disorders.

Neurodevelopmental disorders are thought to arise from interrupted development of the brain at an early age. Genome-wide association studies (GWAS) have identified hundreds of loci associated with susceptibility to neurodevelopmental disorders; however, which noncoding variants regulate which genes at these loci is often unclear. To implicate neuronal GWAS effector genes, we performed an integrated analysis of transcriptomics, epigenomics and chromatin conformation changes during the development from Induced pluripotent stem cell-derived neuronal progenitor cells (NPCs) into neurons using a combination of high-resolution promoter-focused Capture-C, ATAC-seq and RNA-seq. We observed that gene expression changes during the NPC-to-neuron transition were highly dependent on both promoter accessibility changes and long-range interactions which connect distal cis-regulatory elements (enhancer or silencers) to developmental-stage-specific genes. These genome-scale promoter-cis-regulatory-element atlases implicated 454 neurodevelopmental disorder-associated, putative causal variants mapping to 600 distal targets. These putative effector genes were significantly enriched for pathways involved in the regulation of neuronal development and chromatin organization, with 27 % expressed in a stage-specific manner. The intersection of open chromatin and chromatin conformation revealed development-stage-specific gene regulatory architectures during neuronal differentiation, providing a rich resource to aid characterization of the genetic and developmental basis of neurodevelopmental disorders.

Biological constraints on GWAS SNPs at suggestive significance thresholds reveal additional BMI loci.

To uncover novel significant association signals (p<5×10-8), genome-wide association studies (GWAS) requires increasingly larger sample sizes to overcome statistical correction for multiple testing. As an alternative, we aimed to identify associations among suggestive signals (5 × 10-8≤p<5×10-4) in increasingly powered GWAS efforts using chromatin accessibility and direct contact with gene promoters as biological constraints. We conducted retrospective analyses of three GIANT BMI GWAS efforts using ATAC-seq and promoter-focused Capture C data from human adipocytes and embryonic stem cell (ESC)-derived hypothalamic-like neurons. This approach, with its extremely low false-positive rate, identified 15 loci at p<5×10-5 in the 2010 GWAS, of which 13 achieved genome-wide significance by 2018, including at NAV1, MTIF3, and ADCY3. Eighty percent of constrained 2015 loci achieved genome-wide significance in 2018. We observed similar results in waist-to-hip ratio analyses. In conclusion, biological constraints on sub-significant GWAS signals can reveal potentially true-positive loci for further investigation in existing data sets without increasing sample size.

Variant-to-Gene-Mapping Analyses Reveal a Role for the Hypothalamus in Genetic Susceptibility to Inflammatory Bowel Disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a polygenic disorder characterized principally by dysregulated inflammation impacting the gastrointestinal tract. However, there also is increasing evidence for a clinical association with stress and depression. Given the role of the hypothalamus in stress responses and in the pathogenesis of depression, useful insights could be gleaned from understanding its genetic role in IBD. METHODS: We conducted genetic correlation analyses on publicly available genome-wide association study summary statistics for depression and IBD traits to identify genetic commonalities. We used partitioned linkage disequilibrium score regression, leveraging our ATAC sequencing and promoter-focused Capture C data, to measure enrichment of IBD single-nucleotide polymorphisms within promoter-interacting open chromatin regions of human embryonic stem cell-derived hypothalamic-like neurons (HNs). Using the same data sets, we performed variant-to-gene mapping to implicate putative IBD effector genes in HNs. To contrast these results, we similarly analyzed 3-dimensional genomic data generated in epithelium-derived colonoids from rectal biopsy specimens from donors without pathologic disease noted at the time of colonoscopy. Finally, we conducted enrichment pathway analyses on the implicated genes to identify putative IBD dysfunctional pathways. RESULTS: We found significant genetic correlations (rg) of 0.122 with an adjusted P (Padj) = 1.4 × 10-4 for IBD: rg = 0.122; Padj = 2.5 × 10-3 for ulcerative colitis and genetic correlation (rg) = 0.094; Padj = 2.5 × 10-3 for Crohn's disease, and significant approximately 4-fold (P = .005) and approximately 7-fold (P = .03) enrichment of IBD single-nucleotide polymorphisms in HNs and colonoids, respectively. We implicated 25 associated genes in HNs, among which CREM, CNTF, and RHOA encode key regulators of stress. Seven genes also additionally were implicated in the colonoids. We observed an overall enrichment for immune and hormonal signaling pathways, and a colonoid-specific enrichment for microbiota-relevant terms. CONCLUSIONS: Our results suggest that the hypothalamus warrants further study in the context of IBD pathogenesis.

Genome-wide association study implicates novel loci and reveals candidate effector genes for longitudinal pediatric bone accrual.

BACKGROUND: Bone accrual impacts lifelong skeletal health, but genetic discovery has been primarily limited to cross-sectional study designs and hampered by uncertainty about target effector genes. Here, we capture this dynamic phenotype by modeling longitudinal bone accrual across 11,000 bone scans in a cohort of healthy children and adolescents, followed by genome-wide association studies (GWAS) and variant-to-gene mapping with functional follow-up. RESULTS: We identify 40 loci, 35 not previously reported, with various degrees of supportive evidence, half residing in topological associated domains harboring known bone genes. Of several loci potentially associated with later-life fracture risk, a candidate SNP lookup provides the most compelling evidence for rs11195210 (SMC3). Variant-to-gene mapping combining ATAC-seq to assay open chromatin with high-resolution promoter-focused Capture C identifies contacts between GWAS loci and nearby gene promoters. siRNA knockdown of gene expression supports the putative effector gene at three specific loci in two osteoblast cell models. Finally, using CRISPR-Cas9 genome editing, we confirm that the immediate genomic region harboring the putative causal SNP influences PRPF38A expression, a location which is predicted to coincide with a set of binding sites for relevant transcription factors. CONCLUSIONS: Using a new longitudinal approach, we expand the number of genetic loci putatively associated with pediatric bone gain. Functional follow-up in appropriate cell models finds novel candidate genes impacting bone accrual. Our data also raise the possibility that the cell fate decision between osteogenic and adipogenic lineages is important in normal bone accrual.

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits.

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.