TULIPs decorate the three-dimensional genome of PFA ependymoma.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

SMARCA4 vulnerability in H3K27M midline glioma: A silver bullet for a lethal disease.

To investigate epigenetic dependencies and identify therapeutic vulnerabilities, Mo et al.1 and Panditharatna et al.2 performed CRISPR screens and show that deadly H3K27M gliomas are dependent on mammalian BAF (SWI/SNF) chromatin remodeling complex.

Histone H3.3 K27M and K36M mutations de-repress transposable elements through perturbation of antagonistic chromatin marks.

Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. We show that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon de-repression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. Our results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks.

Understanding a community's needs for an emergency department-based childhood injury prevention programme: a mixed-methods study.

OBJECTIVE: Unintentional injuries are the most common cause of childhood death in the USA and are preventable. We developed a framework for an injury prevention programme using local injury data and understanding stakeholder perspectives. METHODS: We used a mixed-methods approach. We performed a retrospective cross-sectional analysis of children presenting to an academic hospital system between January 2019 and December 2020 with an injury-related diagnosis. The primary outcome was encounters with an injury-related ICD-10 code. We conducted a thematic analysis by interviewing caregivers and emergency department (ED) providers. RESULTS: There were 10 193 unique injury-related encounters. Most common injuries were natural/environmental (22.9%), falls (20.0%) and striking an object (5.1%). Highest rates of injury were seen in children who identified as Native Hawaiian or Pacific Islander (154 injuries per 10 000 children per year), followed by Hispanic or Latino (148). Three out of 20 zip code areas represented 43.4% of all injuries and correlated with lower household income. Twenty-five caregivers and eight ED providers participated in interviews that resulted in four major themes: perceptions of injury risk, caregiver receipt of injury prevention information, barriers and provider counselling. CONCLUSION: Clear differences exist within the injury burden in San Francisco by demographics, geography and type of injury. The findings from the study will guide the first steps in designing a strategic paediatric injury prevention centre. The methods may guide future investigations into the dynamic needs of clinicians and caregivers regarding injury. A strategic programme focused on the community's unique needs and barriers may effectively reduce injury rates.

Toxic Exposures Among Young Children One Year into the COVID-19 Pandemic: A Retrospective Review of Three San Francisco Bay Area Emergency Departments.

BACKGROUND: Daycare and school closures prompted by shelter-in-place orders may have increased opportunities for unintentional ingestions among young children. OBJECTIVES: We examined emergency department (ED) presentations for toxic exposures among young children during the COVID-19 pandemic in the San Francisco Bay Area, which had some of the strictest and most prolonged shelter-in-place policies in the United States. METHODS: We performed a retrospective cross-sectional study of children 0 to 5 years of age who presented with an ED International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnosis code of toxic exposure within a tertiary care hospital system between March 16, 2016 and March 15, 2021. We considered the period after March 16, 2020 to represent the pandemic. RESULTS: During the pandemic, the absolute number of poisonings among young children remained stable. Overall, ED encounters within this cohort decreased by 55%, which doubled the relative toxic exposure rate per 1000 ED encounters from 4.99 (95% confidence interval [CI] 3.19-5.90) to 9.79 (95% CI 8.09-11.49). Rates of admission, severe medical complications, operating room case requests, and length of stay were not significantly different. Shelter-in-place was associated with significantly higher odds of cannabis ingestion (odds ratio = 2.70, 95% CI 1.60-4.49). CONCLUSION: Despite dramatic decreases in overall ED patient volumes, the absolute number and severity of toxic exposures were similar during the pandemic compared with previous years. © 2022 Elsevier Inc.

H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2.

Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants. Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1-/- and Ehmt2-/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells.

Development of a simulation-based curriculum for Pediatric prehospital skills: a mixed-methods needs assessment.

BACKGROUND: The assessment and treatment of pediatric patients in the out-of-hospital environment often presents unique difficulties and stress for EMS practitioners. OBJECTIVE: Use a mixed-methods approach to assess the current experience of EMS practitioners caring for critically ill and injured children, and the potential role of a simulation-based curriculum to improve pediatric prehospital skills. METHODS: Data were obtained from three sources in a single, urban EMS system: a retrospective review of local pediatric EMS encounters over one year; survey data of EMS practitioners' comfort with pediatric skills using a 7-point Likert scale; and qualitative data from focus groups with EMS practitioners assessing their experiences with pediatric patients and their preferred training modalities. RESULTS: 2.1% of pediatric prehospital encounters were considered "critical," the highest acuity level. A total of 136 of approximately 858 prehospital providers responded to the quantitative survey; 34.4% of all respondents either somewhat disagree (16.4%), disagree (10.2%), or strongly disagree (7.8%) with the statement: "I feel comfortable taking care of a critically ill pediatric patient." Forty-seven providers participated in focus groups that resulted in twelve major themes under three domains. Specific themes included challenges in medication dosing, communication, and airway management. Participants expressed a desire for more repetition and reinforcement of these skills, and they were receptive to the use of high-fidelity simulation as a training modality. CONCLUSIONS: Critically ill pediatric prehospital encounters are rare. Over one third of EMS practitioners expressed a low comfort level in managing critically ill children. High-fidelity simulation may be an effective means to improve the comfort and skills of prehospital providers.

Comparison of the effectiveness of hands-on versus online education in child passenger safety.

BACKGROUND: Community paediatricians' knowledge of appropriate child safety seat (CSS) use in vehicles may be inadequate. We compared the effectiveness of hands-on and online education in improving and retaining child passenger safety (CPS) knowledge and skills among paediatric trainees. METHODS: Paediatric trainees were randomised to receive hands-on skills training versus a 1-hour online module in CPS. CSS knowledge and installation skills were assessed using a validated 10-item/point questionnaire and an assessment tool respectively at baseline and after 6 months. Preintervention and postintervention knowledge improvement and CSS installation skills between groups were assessed using paired t-tests and effect size (d). RESULTS: Forty-eight students agreed to participate and were randomised. Thirty-nine completed training (hands-on: 23 and online: 15). At entry, no significant differences in learners' demographics and prior CPS education existed. Baseline CPS knowledge scores did not differ significantly between groups (p=0.26). Postintervention, both groups demonstrated a significant increase in knowledge scores (hands-on=3.1 (95% CI 2.4 to 3.7), p<0.0001; online=2.6 (95% CI 1.9 to 3.3), p<0.0001), though the pre-post gain in knowledge scores were not significantly different between groups (p=0.35). At follow-up, both groups demonstrated a significant increase in knowledge scores (hands-on=1.8 (95% CI 1.2 to 2.4), p<0.0001; online=1.1 (95% CI 0.7 to 1.6), p<0.0001) with the hands-on group scores significantly better than the online group (p<0.02). The long-term gain in knowledge scores was not significantly different between groups (p=0.12).Baseline CSS installation skill scores did not significantly differ between groups for forward-facing seats (p=0.16) and rear-facing seats (p=0.51). At follow-up, mean CSS installation skill scores significantly increased for the hands-on group (forward-facing seat: 0.8 (95% CI 0.16 to 1.44), p<0.02; rear-facing seat: 1.2 (95% CI 0.6 to 1.7), p<0.001) but not for the online group (forward-facing seat: 0.9 (95% CI -0.08 to 1.9), p=0.07); rear-facing seat: -0.2 (95% CI -1.1 to 0.7), p=0.6). CONCLUSIONS: Among paediatric trainees, hands-on and online CPS education are both effective in improving long-term CPS knowledge. Long-term installation skills for forward-facing and rear-facing CSS persist for hands-on education but are inconclusive for online education.

Idiopathic Pulmonary Hemosiderosis Presenting as Anemia, Failure to Thrive, and Jaundice in a Toddler.

Idiopathic pulmonary hemosiderosis (IPH) is a rare disease characterized by the triad of hemoptysis, pulmonary infiltrates on chest radiograph, and anemia. Its diagnosis should be considered in any child presenting with moderate to severe anemia and failure to thrive of unclear etiology. Consideration of the differential diagnosis in such a child should include the review of both extravascular and intravascular causes of hemolysis. Systemic treatment of IPH with glucocorticoids has been shown to decrease morbidity, mortality, and disease progression to pulmonary fibrosis. Thus, diagnostic delays can impact prognosis. Here, we present a case of a 15-month-old boy with IPH who presented with anemia, jaundice, and failure to thrive, as well as a history of hemoptysis that was not initially elicited.

Oncohistones in brain tumors: the soil and seed.

Recurrent somatic mutations in histone 3 (H3) variants (termed 'oncohistones') have been identified in high-grade gliomas (HGGs) in children and young adults and induce tumorigenesis through disruption of chromatin states. Oncohistones occur with exquisite neuroanatomical specificity and are associated with specific age distribution and epigenome landscapes. Here, we review the known intrinsic ('seed') and the extrinsic ('soil') factors needed for their optimal oncogenic effect and highlight the many unresolved questions regarding their effects on development and crosstalk with the tumor microenvironment. The 'seed and soil' analogy, used to explain tumor metastatic niches, also applies to oncohistones, which mainly thrive and flourish in specific chromatin states during very narrow windows of development, creating exquisite vulnerabilities, which could provide effective therapies for these deadly cancers.

Using Geocoding to Identify COVID-19 Outbreaks in Congregate Residential Settings: San Francisco's Outbreak Response in Single-Room Occupancy Hotels.

More than 500 single-room occupancy hotels (SROs), a type of low-cost congregate housing with shared bathrooms and kitchens, are available in San Francisco. SRO residents include essential workers, people with disabilities, and multigenerational immigrant families. In March 2020, with increasing concerns about the potential for rapid transmission of COVID-19 among a population with disproportionate rates of comorbidity, poor access to care, and inability to self-isolate, the San Francisco Department of Public Health formed an SRO outbreak response team to identify and contain COVID-19 clusters in this congregate residential setting. Using address-matching geocoding, the team conducted active surveillance to identify new cases and outbreaks of COVID-19 at SROs. An outbreak was defined as 3 separate households in the SRO with a positive test result for COVID-19. From March 2020 through February 2021, the SRO outbreak response team conducted on-site mass testing of all residents at 52 SROs with outbreaks identified through geocoding. The rate of positive COVID-19 tests was significantly higher at SROs with outbreaks than at SROs without outbreaks (12.7% vs 6.4%; P < .001). From March through May 2020, the rate of COVID-19 cases among SRO residents was higher than among residents of other settings (ie, non-SRO residents), before decreasing and remaining at an equal level to non-SRO residents during later periods of 2020. The annual case fatality rate for SRO residents and non-SRO residents was similar (1.8% vs 1.5%). This approach identified outbreaks in a setting at high risk of COVID-19 and facilitated rapid deployment of resources. The geocoding surveillance approach could be used for other diseases and in any setting for which a list of addresses is available.

Emerging and Re-emerging Infections in Children: COVID/ MIS-C, Zika, Ebola, Measles, Varicella, Pertussis ... Immunizations.

The role of the emergency provider lies at the forefront of recognition and treatment of novel and re-emerging infectious diseases in children. Familiarity with disease presentations that might be considered rare, such as vaccine-preventable and non-endemic illnesses, is essential in identifying and controlling outbreaks. As we have seen thus far in the novel coronavirus pandemic, susceptibility, severity, transmission, and disease presentation can all have unique patterns in children. Emergency providers also have the potential to play a public health role by using lessons learned from the phenomena of vaccine hesitancy and refusal.

Invasive Bacterial Infections in Afebrile Infants Diagnosed With Acute Otitis Media.

OBJECTIVES: To determine the prevalence of invasive bacterial infections (IBIs) and adverse events in afebrile infants with acute otitis media (AOM). METHODS: We conducted a 33-site cross-sectional study of afebrile infants ≤90 days of age with AOM seen in emergency departments from 2007 to 2017. Eligible infants were identified using emergency department diagnosis codes and confirmed by chart review. IBIs (bacteremia and meningitis) were determined by the growth of pathogenic bacteria in blood or cerebrospinal fluid (CSF) culture. Adverse events were defined as substantial complications resulting from or potentially associated with AOM. We used generalized linear mixed-effects models to identify factors associated with IBI diagnostic testing, controlling for site-level clustering effect. RESULTS: Of 5270 infants screened, 1637 met study criteria. None of the 278 (0%; 95% confidence interval [CI]: 0%-1.4%) infants with blood cultures had bacteremia; 0 of 102 (0%; 95% CI: 0%-3.6%) with CSF cultures had bacterial meningitis; 2 of 645 (0.3%; 95% CI: 0.1%-1.1%) infants with 30-day follow-up had adverse events, including lymphadenitis (1) and culture-negative sepsis (1). Diagnostic testing for IBI varied across sites and by age; overall, 278 (17.0%) had blood cultures, and 102 (6.2%) had CSF cultures obtained. Compared with infants 0 to 28 days old, older infants were less likely to have blood cultures (P < .001) or CSF cultures (P < .001) obtained. CONCLUSION: Afebrile infants with clinician-diagnosed AOM have a low prevalence of IBIs and adverse events; therefore, outpatient management without diagnostic testing may be reasonable.

H3.3 G34W Promotes Growth and Impedes Differentiation of Osteoblast-Like Mesenchymal Progenitors in Giant Cell Tumor of Bone.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR-Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. SIGNIFICANCE: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775.

Effectiveness of a Pediatric Emergency Medicine Curriculum in a Public Tanzanian Referral Hospital.

INTRODUCTION: The World Health Organization recently recognized the importance of emergency and trauma care in reducing morbidity and mortality. Training programs are essential to improving emergency care in low-resource settings; however, a paucity of comprehensive curricula focusing specifically on pediatric emergency medicine (PEM) currently exists. The African Federation for Emergency Medicine (AFEM) developed a PEM curriculum that was pilot-tested in a non-randomized, controlled study to evaluate its effectiveness in nurses working in a public Tanzanian referral hospital. METHODS: Fifteen nurses were recruited to participate in a two-and-a-half-day curriculum of lectures, skill sessions, and simulation scenarios covering nine topics; they were matched with controls. Both groups completed pre- and post-training assessments of their knowledge (multiple-choice test), self-efficacy (Likert surveys), and behavior. Changes in behavior were assessed using a binary checklist of critical actions during observations of live pediatric resuscitations. RESULTS: Participant-rated pre-training self-efficacy and knowledge test scores were similar in both control and intervention groups. However, post-training, self-efficacy ratings in the intervention group increased by a median of 11.5 points (interquartile range [IQR]: 6-16) while unchanged in the control group. Knowledge test scores also increased by a median of three points (IQR: 0-4) in the nurses who received the training while the control group's results did not differ in the two periods. A total of 1192 pediatric resuscitation cases were observed post-training, with the intervention group demonstrating higher rates of performance of three of 27 critical actions. CONCLUSION: This pilot study of the AFEM PEM curriculum for nurses has shown it to be an effective tool in knowledge acquisition and improved self-efficacy of pediatric emergencies. Further evaluation will be needed to assess whether it is currently effective in changing nurse behavior and patient outcomes or whether curricular modifications are needed.

H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.

Pervasive H3K27 Acetylation Leads to ERV Expression and a Therapeutic Vulnerability in H3K27M Gliomas.

High-grade gliomas defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.