A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection.

Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.

Neurite outgrowth deficits caused by rare PLXNB1 mutation in pediatric bipolar disorder.

Pediatric bipolar disorder (PBD) is a severe mood dysregulation condition that affects 0.5-1% of children and teens in the United States. It is associated with recurrent episodes of mania and depression and an increased risk of suicidality. However, the genetics and neuropathology of PBD are largely unknown. Here, we used a combinatorial family-based approach to characterize cellular, molecular, genetic, and network-level deficits associated with PBD. We recruited a PBD patient and three unaffected family members from a family with a history of psychiatric illnesses. Using resting-state functional magnetic resonance imaging (rs-fMRI), we detected altered resting-state functional connectivity in the patient as compared to an unaffected sibling. Using transcriptomic profiling of patient and control induced pluripotent stem cell (iPSC)-derived telencephalic organoids, we found aberrant signaling in the molecular pathways related to neurite outgrowth. We corroborated the presence of neurite outgrowth deficits in patient iPSC-derived cortical neurons and identified a rare homozygous loss-of-function PLXNB1 variant (c.1360C>C; p.Ser454Arg) responsible for the deficits in the patient. Expression of wild-type PLXNB1, but not the variant, rescued neurite outgrowth in patient neurons, and expression of the variant caused the neurite outgrowth deficits in cortical neurons from PlxnB1 knockout mice. These results indicate that dysregulated PLXNB1 signaling may contribute to an increased risk of PBD and other mood dysregulation-related disorders by disrupting neurite outgrowth and functional brain connectivity. Overall, this study established and validated a novel family-based combinatorial approach for studying cellular and molecular deficits in psychiatric disorders and identified dysfunctional PLXNB1 signaling and neurite outgrowth as potential risk factors for PBD.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3ß) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3ß in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3ß-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3ß levels, allowing us to study GSK3ß function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3ß. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3ß-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3ß in regulating NK cell metabolism.

Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

A common allele of HLA mediates asymptomatic SARS-CoV-2 infection.

Despite some inconsistent reporting of symptoms, studies have demonstrated that at least 20% of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will remain asymptomatic. Although most global efforts have focused on understanding factors underlying severe illness in COVID-19 (coronavirus disease of 2019), the examination of asymptomatic infection provides a unique opportunity to consider early disease and immunologic features promoting rapid viral clearance. Owing to its critical role in the immune response, we postulated that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection. We enrolled 29,947 individuals registered in the National Marrow Donor Program for whom high-resolution HLA genotyping data were available in the UCSF Citizen Science smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n=1428) was comprised of unvaccinated, self-identified subjects who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci (HLA-A, -B, -C, -DRB1, -DQB1) with disease course and identified a strong association of HLA-B*15:01 with asymptomatic infection, and reproduced this association in two independent cohorts. Suggesting that this genetic association is due to pre-existing T-cell immunity, we show that T cells from pre-pandemic individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF, and 100% of the reactive cells displayed memory phenotype. Finally, we characterize the protein structure of HLA-B*15:01-peptide complexes, demonstrating that the NQKLIANQF peptide from SARS-CoV-2, and the highly homologous NQKLIANAF from seasonal coronaviruses OC43-CoV and HKU1-CoV, share similar ability to be stabilized and presented by HLA-B*15:01, providing the molecular basis for T-cell cross-reactivity and HLA-B*15:01-mediated pre-existing immunity.

The history and geographic distribution of a KCNQ1 atrial fibrillation risk allele.

The genetic architecture of atrial fibrillation (AF) encompasses low impact, common genetic variants and high impact, rare variants. Here, we characterize a high impact AF-susceptibility allele, KCNQ1 R231H, and describe its transcontinental geographic distribution and history. Induced pluripotent stem cell-derived cardiomyocytes procured from risk allele carriers exhibit abbreviated action potential duration, consistent with a gain-of-function effect. Using identity-by-descent (IBD) networks, we estimate the broad- and fine-scale population ancestry of risk allele carriers and their relatives. Analysis of ancestral migration routes reveals ancestors who inhabited Denmark in the 1700s, migrated to the Northeastern United States in the early 1800s, and traveled across the Midwest to arrive in Utah in the late 1800s. IBD/coalescent-based allele dating analysis reveals a relatively recent origin of the AF risk allele (~5000 years). Thus, our approach broadens the scope of study for disease susceptibility alleles to the context of human migration and ancestral origins.

Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.

Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient's healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient's iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.

Genetic Mapping of a new Hippo allele, HpoN.1.2, in Drosophila melanogaster.

Genetic screens provide a mechanism to identify genes involved with different cellular and organismal processes. Using a Flp/FRT screen in the Drosophila eye we identified mutations that result in alterations and de-regulation of cell growth and division. From this screen a group of undergraduate researchers part of the Fly-CURE consortium mapped and characterized a new allele of the gene Hippo, HpoN.1.2.

Postseptic Cognitive Impairment and Expression of APOE in Peripheral Blood: The Cognition After SepsiS (CASS) Observational Pilot Study.

BACKGROUND: Cognitive impairment after sepsis is an important clinical problem. Determinants of postseptic cognitive impairment are not well understood. We thus undertook a systems biology approach to exploring a possible role for apolipoprotein E (APOE) in postseptic cognitive impairment. DESIGN: Prospective, observational cohort. SETTING: Intermountain Medical Center, a tertiary referral center in Utah. PATIENTS/PARTICIPANTS: Patients with sepsis admitted to study intensive care units. INTERVENTIONS: None. METHODS: We obtained peripheral blood for deep sequencing of RNA and followed up survivors at 6 months with a battery of cognitive instruments. We defined cognitive impairment based on the 6-month Hayling test of executive function. In our primary analysis, we employed weighted network analysis. Secondarily, we compared variation in gene expression between patients with normal versus impaired cognition. MEASUREMENTS AND MAIN RESULTS: We enrolled 40 patients, of whom 34 were follow-up eligible and 31 (91%) completed follow-up; 1 patient's RNA sample was degraded-the final analytic cohort was 30 patients. Mean Hayling test score was 5.8 (standard deviation 1.1), which represented 20% with impaired executive function. The network module containing APOE was dominated by low-expression genes, with no association on primary analysis (P = .8). Secondary analyses suggested several potential lines of future investigation, including oxidative stress. CONCLUSIONS: In this prospective pilot cohort, executive dysfunction affected 1 in 5 survivors of sepsis. The APOE gene was sparsely transcribed in peripheral leukocytes and not associated with cognitive impairment. Future lines of research are suggested.

Genome-wide analysis reveals the unique stem cell identity of human amniocytes.

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage.

14-3-3ε plays a role in cardiac ventricular compaction by regulating the cardiomyocyte cell cycle.

Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G(2)/M and the accumulation of cardiomyocytes in the G(0)/G(1) phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27(Kip1), possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27(Kip1). These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.

Focal adhesion kinase regulates smooth muscle cell recruitment to the developing vasculature.

OBJECTIVE: The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. METHODS AND RESULTS: We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. CONCLUSIONS: FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.

Loss of microRNAs in neural crest leads to cardiovascular syndromes resembling human congenital heart defects.

OBJECTIVE: Congenital heart defects represent the most common human birth defects. Even though the genetic cause of these syndromes has been linked to candidate genes, the underlying molecular mechanisms are still largely unknown. Disturbance of neural crest cell (NCC) migration into the derivatives of the pharyngeal arches and pouches can account for many of the developmental defects. The goal of this study was to investigate the function of microRNA (miRNA) in NCCs and the cardiovascular system. METHODS AND RESULTS: We deleted Dicer from the NCC lineage and showed that Dicer conditional mutants exhibit severe defects in multiple craniofacial and cardiovascular structures, many of which are observed in human neuro-craniofacial-cardiac syndrome patients. We found that cranial NCCs require Dicer for their survival and that deletion of Dicer led to massive cell death and complete loss of NCC-derived craniofacial structures. In contrast, Dicer and miRNAs were not essential for the survival of cardiac NCCs. However, the migration and patterning of these cells were impaired in Dicer knockout mice, resulting in a spectrum of cardiovascular abnormalities, including type B interrupted aortic arch, double-outlet right ventricle, and ventricular septal defect. We showed that Dicer loss of function was, at least in part, mediated by miRNA-21 (miR-21) and miRNA-181a (miR-181a), which in turn repressed the protein level of Sprouty 2, an inhibitor of Erk1/2 signaling. CONCLUSIONS: Our results uncovered a central role for Dicer and miRNAs in NCC survival, migration, and patterning in craniofacial and cardiovascular development which, when mutated, lead to congenital neuro-craniofacial-cardiac defects.

Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis.

Plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA(2) is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA(2) and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA(2) protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA(2) mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA(2) were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA(2) were negatively correlated (r = - 0.578). The ratio of Lp-PLA(2) to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA(2) existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA(2) epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA(2) and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.

Chapter 9. Development of coronary vessels.

This chapter summarizes experimental techniques used to study coronary vessel development from its origins in the proepicardium (PE) to the final assembled network of arteries, veins, and capillaries present in the mature heart. Methods are described for microdissection and culture of the PE and embryonic epicardial cells, isolation of total RNA from single PE primordia and analysis by RT-PCR, imaging of the epicardium and coronary vessels by whole-mount confocal microscopy and by scanning electron microscopy, and the preparation of coronary vascular corrosion casts to visualize the entire coronary artery network structure. These techniques form the basic tools to study the cellular and molecular pathways that guide development and remodeling of coronary vessels.

A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells.

We characterize a sonic hedgehog (Shh) signaling domain restricted to the adventitial layer of artery wall that supports resident Sca1-positive vascular progenitor cells (AdvSca1). Using patched-1 (Ptc1(lacZ)) and patched-2 (Ptc2(lacZ)) reporter mice, adventitial Shh signaling activity was first detected at embryonic day (E) 15.5, reached the highest levels between postnatal day 1 (P1) and P10, was diminished in adult vessels, and colocalized with a circumferential ring of Shh protein deposited between the media and adventitia. In Shh(-/-) mice, AdvSca1 cells normally found at the aortic root were either absent or greatly diminished in number. Using a Wnt1-cre lineage marker that identifies cells of neural crest origin, we found that neither the adventitia nor AdvSca1 cells were labeled in arteries composed of neural crest-derived smooth muscle cells (SMCs). Although AdvSca1 cells do not express SMC marker proteins in vivo, they do express transcription factors thought to be required for SMC differentiation, including serum response factor (SRF) and myocardin family members, and readily differentiate to SMC-like cells in vitro. However, AdvSca1 cells also express potent repressors of SRF-dependent transcription, including Klf4, Msx1, and FoxO4, which may be critical for maintenance of the SMC progenitor phenotype of AdvSca1 cells in vivo. We conclude that a restricted domain of Shh signaling is localized to the arterial adventitia and may play important roles in maintenance of resident vascular SMC progenitor cells in the artery wall.

Absence of Msx2 does not affect cardiac conduction or rescue conduction defects associated with Nkx2-5 mutation.

INTRODUCTION: The transcription factors governing embryonic development of the AV conduction system are largely unknown. Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause AV conduction defects, which are associated with anatomic hypoplasia of the conduction system. In situ expression patterns of Msx2 in the mouse and chick embryonic heart have suggested a developmental function for this transcription factor. Homozygous Nkx2-5 knockout mouse embryos express Msx2 ectopically throughout the myocardium, suggesting Msx2 affects conduction system development through a transcriptional cascade starting with Nkx2-5. Several observations support a model in which Msx2 negatively regulates formation of the conduction system and inappropriate Msx2 up-regulation causes the conduction defects associated with Nkx2-5 mutation. METHODS AND RESULTS: We obtained surface ECGs and performed intracardiac electrophysiologic studies in Msx2 knockout mice and in Nkx2-5 wild-type and heterozygous null mutant mice in an Msx2 null mutant background. Msx2 null mutant mice had normal cardiac conduction and no increased vulnerability to inducible arrhythmia. Absence of Msx2 did not alter the conduction defects observed in heterozygous Nkx2-5 knockout mice. CONCLUSION: Msx2 likely does not contribute to development of the conduction system. Abnormal Msx2 expression likely does not cause the AV conduction defects present in Nkx2-5 knockout mice.

Function follows form: cardiac conduction system defects in Nkx2-5 mutation.

Mutations of Nkx2-5 cause congenital heart disease and atrioventricular block in man. The altered expression of an electrophysiologic protein regulated by Nkx2-5 was originally presumed to cause the conduction defect, but when no such protein was found, an alternative hypothesis was considered. In pediatric patients, the association of certain cardiac malformations with congenital atrioventricular block suggests that errors in specific developmental pathways could cause both an anatomic and a physiologic defect. We therefore hypothesized that Nkx2-5 insufficiency perturbs the conduction system during development, which in turn manifests as a postnatal conduction defect. Experimental results from Nkx2-5 knockout mouse models support the developmental hypothesis. Hypoplasia of the atrioventricular node, His bundle, and Purkinje system can explain in whole or in part specific conduction and electrophysiologic defects present in Nkx2-5 haploinsufficiency.