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Background: The Canadian National Vaccine Safety Network conducted active safety surveillance for COVID-19 vaccines. This study aimed to characterize the short-to-medium term safety of mRNA COVID-19 vaccines across the pediatric age spectrum., Methods: In this cohort study, vaccinated and unvaccinated children and adolescents aged 6 months to 19 years from eight Canadian provinces and territories were invited to participate. The outcome was a health event preventing daily activities, resulting in school absenteeism, or requiring medical consultation. Age-stratified multivariable regression models were used to examine health events associated with first and second doses of mRNA COVID-19 vaccines across different age groups: children under 5, children aged 5-11 years and adolescents aged 12-19 years., Findings: From January 2021 through February 2023, a total of 259,361 individuals from the dose one survey, 131,032 from the dose 2 survey, and 1179 from the control survey were included. In the week following dose two, vaccinated adolescents showed a higher proportion of health events [794 (4.6%) of 17,218 BNT162b2 recipients, 98 (8.5%) of 1153 mRNA-1273 recipients, 49 of (10.6%) of 464 heterologous schedule recipients] than unvaccinated adolescents [9 (3.7%) of 242 controls], but most events were self-limited and resolved within 7 days. No significant differences in proportion of health events following mRNA COVID-19 vaccines were observed between vaccinated and unvaccinated groups among adolescents after dose 1, or among children under 5 or those aged 5-11 years after any dose. Reported myocarditis/pericarditis cases within 0-28 days peaked among male adolescents following dose 2, in three of (0.037%) 8088 homologous BNT162b2 recipients, and two of (0.529%) 378 homologous mRNA-1273 recipients., Interpretation: Our findings suggest that reported health events, including myocarditis/pericarditis, vary by pediatric age group. Vaccinated adolescents reported health events more frequently following the second mRNA COVID-19 vaccine dose, while younger age groups did not report events more frequently than their unvaccinated counterparts., Funding: Canadian Immunization Research Network, Canadian Institutes of Health Research; Public Health Agency of Canada; COVID-19 Immunity Task Force., Competing Interests: GDS, HS, HW, LV, MN, KM and OGV have no competing interests. PS received personal payments for consulting from the Joint United Nations Programme on HIV/AIDS outside the submitted work. MS has been an investigator on projects funded by GlaxoSmithKline, Merck, Moderna, Pfizer and Sanofi-Pasteur outside the submitted work. All funds were paid to his institute, and he has not received any personal payments. MPM received payment testifying as an expert with respect to mandatory influenza and COVID-19 vaccination in healthcare settings. JDK has been an investigator on projects funded by Moderna Canada and Alberta Children’s Hospital Research Institute and Government of Alberta, all outside the submitted work. All funds have been paid to his institute, and he has not received any personal payments. KAT reports grants from Canadian Institute of Health Research and the Coalition of Epidemic Preparedness Innovations for COVID-19 vaccine studies. KAT received personal payments for consulting from World Health Organization. JEI has been an investigator on projects funded by GlaxoSmithKline and Sanofi-Pasteur outside the submitted work. All funds were paid to her institute, and she has not received any personal payment. AM reports grants to her institution from Pfizer, Merck, Sanofi, and Seqirus, as well as personal payments for consulting or honoraria from Sienna Senior Living, AstraZeneca, Merck, Biogen, Sanofi, GlaxoSmithKline, Moderna, Medicago, Janssen, Novavax, Pfizer, and Seqirus. JAB has served on the Research Leadership Committee at BC Children’s Hospital Research Institute and the National Advisory Committee on Immunization., (© 2024 The Author(s).)

To evaluate immune responses to COVID-19 vaccines in adults aged 50 years and older, spike protein (S)-specific antibody concentration, avidity, and function (via angiotensin-converting enzyme 2 (ACE2) inhibition surrogate neutralization and antibody dependent cellular phagocytosis (ADCP)), as well as S-specific T cells were quantified via activation induced marker (AIM) assay in response to two-dose series. Eighty-four adults were vaccinated with either: mRNA/mRNA (mRNA-1273 and/or BNT162b2); ChAdOx1-S/mRNA; or ChAdOx1-S/ChAdOx1-S. Anti-S IgG concentrations, ADCP scores and ACE2 inhibiting antibody concentrations were highest at one-month post-second dose and declined by four-months post-second dose for all groups. mRNA/mRNA and ChAdOx1-S/mRNA schedules had significantly higher antibody responses than ChAdOx1-S/ChAdOx1-S. CD8 + T-cell responses one-month post-second dose were associated with increased ACE2 surrogate neutralization. Antibody avidity (total relative avidity index) did not change between one-month and four-months post-second dose and did not significantly differ between groups by four-months post-second dose. In determining COVID-19 correlates of protection, a measure that considers both antibody concentration and avidity should be considered., (© 2024. The Author(s).)

Background and Objectives: Pediatric COVID-19 cases are often mild or asymptomatic, which has complicated estimations of disease burden using existing testing practices. We aimed to determine the age-specific population seropositivity and risk factors of SARS-CoV-2 seropositivity among children and young adults during the pandemic in British Columbia (BC)., Methods: We conducted two cross-sectional serosurveys: phase 1 enrolled children and adults < 25 years between November 2020-May 2021 and phase 2 enrolled children < 10 years between June 2021-May 2022 in BC. Participants completed electronic surveys and self-collected finger-prick dried blood spot (DBS) samples. Samples were tested for immunoglobulin G antibodies against ancestral spike protein (S). Descriptive statistics from survey data were reported and two multivariable analyses were conducted to evaluate factors associated with seropositivity., Results: A total of 2864 participants were enrolled, of which 95/2167 (4.4%) participants were S-seropositive in phase 1 across all ages, and 61/697 (8.8%) unvaccinated children aged under ten years were S-seropositive in phase 2. Overall, South Asian participants had a higher seropositivity than other ethnicities (13.5% vs. 5.2%). Of 156 seropositive participants in both phases, 120 had no prior positive SARS-CoV-2 test. Young infants and young adults had the highest reported seropositivity rates (7.0% and 7.2% respectively vs. 3.0-5.6% across other age groups)., Conclusions: SARS-CoV-2 seropositivity among unvaccinated children and young adults was low in May 2022, and South Asians were disproportionately infected. This work demonstrates the need for improved diagnostics and reporting strategies that account for age-specific differences in pandemic dynamics and acceptability of testing mechanisms., (© 2024. The Author(s).)

Background: Canadian immunization programs for rotavirus started in 2011. We sought to determine their effect on the burden of community-acquired admissions and hospital-acquired rotavirus at pediatric hospitals., Methods: The Canadian Immunization Monitoring Program Active (IMPACT) network conducted active surveillance for rotavirus-positive hospital admissions between 2005 and 2020 at 12 pediatric hospitals. We used yearly rates of community-acquired rotavirus per 10 000 admissions and hospital-acquired rotavirus infections per 1000 patient-days to determine changes in the pre- and post-vaccine program periods., Results: During the 15-year study period, 5691 rotavirus hospital admissions and hospital-acquired infections were detected, including 4323 (76%) community-acquired infections and 1368 (24%) hospital-acquired infections. The average community-acquired rate in the pre-vaccine period was 60.3 (95% confidence interval [CI] 53.7-68.3) per 10 000 admissions, with a decline to 11.0 (95% CI 7.5-15.1) per 10 000 admissions in the post-vaccine period, resulting in an average reduction of 81.7% (95% CI 74.4%-87.8%). The rate of hospital-acquired rotavirus declined from 0.35 (95% CI 0.29-0.41) per 1000 patient-days in the pre-vaccine period to 0.05 (95% CI 0.03-0.07) per 1000 patient-days in the post-vaccine period, resulting in an 85.3% (95% CI 77.7%-91.9%) average decline. Herd protection was present among children aged 2-16 years., Interpretation: Although start dates of rotavirus vaccine programs across provinces varied, there was around an 80% average decrease in both community-acquired and hospital-acquired rotavirus infections at pediatric hospitals in Canada in the 1- to 9-year interval after implementation of rotavirus vaccine programs. Herd protection is an important aspect of rotavirus vaccines for other children who are not vaccine eligible, and rotavirus vaccines continue to provide important benefits both for children and health care systems., Competing Interests: Competing interests: Julie Bettinger reports research funding from the Canadian Institutes of Health Research (CIHR), the British Columbia Ministry of Health, the Public Health Agency of Canada (PHAC) and the COVID-19 Immunity Task Force; travel support from the Public Health Agency of Canada and the Canadian Public Health Association; and volunteer roles with the National Advisory Committee on Immunization and the BC Immunization Committee. Manish Sadarangani reports research funding from GSK, Merck, Moderna, Pfizer, Sanofi-Pasteur and Symvivo. He is chair or deputy chair of 2 data safety monitoring boards for SARS-CoV-2 vaccine trials, involving different vaccines. Scott Halperin reports research funding from GSK, Merck, Sanofi, Moderna, Entos, the Vaccine and Infectious Disease Organization, CIHR, the National Institutes of Health, the United States Centers for Disease Control and Prevention, Pfizer, PHAC, CanSino and Inventprise; consulting fees for serving on ad hoc advisory boards for Merck, Sanofi, Pfizer, AstraZeneca, NovaVax and GSK; payment for expert testimony from the Canadian Medical Protective Association and the Province of Ontario; and participation on data safety monitoring boards for the International AIDS Vaccine Initiative and Medicago. No other competing interests were declared., (© 2023 CMA Impact Inc. or its licensors.)

Background: Understanding of the role of children in COVID-19 transmission has significant implications for school and childcare policies, as well as appropriate targeting of vaccine campaigns. The objective of this systematic review was to identify the role of children in SARS-CoV-2 transmission to other children and adults., Methods: MEDLINE, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials, and Web of Science were electronically searched for articles published before March 31, 2021. Studies of child-to-child and child-to-adult transmission and quantified the incidence of index and resulting secondary attack rates of children and adults in schools, households, and other congregate pediatric settings were identified. All articles describing confirmed transmission of SARS-CoV-2 from a child were included. PRISMA guidelines for data abstraction were followed, with each step conducted by two reviewers., Results: 40 of 6110 articles identified met inclusion criteria. Overall, there were 0.8 secondary cases per primary index case, with a secondary attack rate of 8.4% among known contacts. The secondary attack rate was 26.4% among adult contacts versus 5.7% amongst child contacts. The pooled estimate of a contact of a pediatric index case being infected as secondary case was 0.10 (95% CI 0.03-0.25)., Conclusions: Children transmit COVID-19 at a lower rate to children than to adults. Household adults are at highest risk of transmission from an infected child, more so than adults or children in other settings., (© 2022. The Author(s).)

Objectives: To describe the association between types of cancer and active tuberculosis (TB) risk in migrants. Additionally, in order to better inform latent TB infection (LTBI) screening protocols, we assessed proportion of active TB cases potentially preventable through LTBI screening and treatment in migrants with cancer., Design: Population-based, retrospective cohort study., Setting: British Columbia (BC), Canada., Participants: 1 000 764 individuals who immigrated to Canada from 1985 to 2012 and established residency in BC at any point up to 2015., Primary and Secondary Outcome Measures: Using linked health administrative databases and disease registries, data on demographics, comorbidities, cancer type, TB exposure and active TB diagnosis were extracted. Primary outcomes included: time to first active TB diagnoses, and risks of active TB following cancer diagnoses which were estimated using Cox extended hazard regression models. Potentially preventable TB was defined as active TB diagnosed >6 months postcancer diagnoses., Results: Active TB risk was increased in migrants with cancer ((HR (95% CI)) 2.5 (2.0 to 3.1)), after adjustment for age, sex, TB incidence in country of origin, immigration classification, contact status and comorbidities. Highest risk was observed with lung cancer (HR 11.2 (7.4 to 16.9)) and sarcoma (HR 8.1 (3.3 to 19.5)), followed by leukaemia (HR 5.6 (3.1 to 10.2)), lymphoma (HR 4.9 (2.7 to 8.7)) and gastrointestinal cancers (HR 2.7 (1.7 to 4.4)). The majority (65.9%) of active TB cases were diagnosed >6 months postcancer diagnosis., Conclusion: Specific cancers increase active TB risk to varying degrees in the migrant population of BC, with approximately two-thirds of active TB cases identified as potentially preventable., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct subtype of diffuse large B-cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somatic IL4R mutations in 15 of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8, leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain of function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell-specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantage in vivo. The pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establish IL4R mutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL., (© 2018 by The American Society of Hematology.)

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA sequencing (RNA-seq) provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-seq alone to reveal the full repertoire of spliced species. Here, we present "spliceosome profiling," a strategy based on deep sequencing of RNAs co-purifying with late-stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single-nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus, much as ribosome profiling has transformed our understanding mRNA translation in the cytoplasm., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC / BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy., (© 2017 by The American Society of Hematology.)

Paternal diet can impact metabolic phenotypes in offspring, but mechanisms underlying such intergenerational information transfer remain obscure. Here, we interrogate cytosine methylation patterns in sperm obtained from mice consuming one of three diets, generating whole genome methylation maps for four pools of sperm samples and for 12 individual sperm samples, as well as 61 genome-scale methylation maps. We find that "epivariation," either stochastic or due to unknown demographic or environmental factors, was a far stronger contributor to the sperm methylome than was the diet consumed. Variation in cytosine methylation was particularly dramatic over tandem repeat families, including ribosomal DNA (rDNA) repeats, but rDNA methylation was strongly correlated with genetic variation in rDNA copy number and was not influenced by paternal diet. These results identify loci of genetic and epigenetic lability in the mammalian genome but argue against a direct role for sperm cytosine methylation in dietary reprogramming of offspring metabolism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Follicular lymphoma (FL) is an indolent disease but transforms in 2% to 3% of patients per year into aggressive, large cell lymphoma, a critical event in the course of the disease associated with increased lymphoma-related mortality. Early transformation cannot be accurately predicted at the time of FL diagnosis and the biology of transformed FL (TFL) is poorly understood. Here, we assembled a cohort of 126 diagnostic FL specimens including 40 patients experiencing transformation (<5 years) and 86 patients not experiencing transformation for at least 5 years. In addition, we assembled an overlapping cohort of 155 TFL patients, including 114 cases for which paired samples were available, and assessed temporal changes of routinely available biomarkers, outcome after transformation, as well as molecular subtypes of TFL. We report that the expression of IRF4 is an independent predictor of early transformation (Hazard ratio, 13.3; P < .001). We also show that composite histology at the time of transformation predicts favorable prognosis. Moreover, applying the Lymph2Cx digital gene expression assay for diffuse large B-cell lymphoma (DLBCL) cell-of-origin determination to 110 patients with DLBCL-like TFL, we demonstrate that TFL is of the germinal-center B-cell-like subtype in the majority of cases (80%) but that a significant proportion of cases is of the activated B-cell-like (ABC) subtype (16%). These latter cases are commonly negative for BCL2 translocation and arise preferentially from BCL2 translocation-negative and/or IRF4-expressing FLs. Our study demonstrates the existence of molecular heterogeneity in TFL as well as its relationship to the antecedent FL., (© 2015 by The American Society of Hematology.)

To investigate epigenetic contributions to Huntington's disease (HD) pathogenesis, we carried out genome-wide mapping of the transcriptional mark, trimethyl-histone H3-lysine 4 (H3K4me3) in neuronal nuclei extracted from prefrontal cortex of HD cases and controls using chromatin immunoprecipitation followed by deep-sequencing. Neuron-specific mapping of the genome-wide distribution of H3K4me3 revealed 136 differentially enriched loci associated with genes implicated in neuronal development and neurodegeneration, including GPR3, TMEM106B, PDIA6 and the Notch signaling genes hairy and enhancer of split 4 (HES4) and JAGGED2, supporting the view that the neuronal epigenome is affected in HD. Importantly, loss of H3K4me3 at CpG-rich sequences on the HES4 promoter was associated with excessive DNA methylation, reduced binding of nuclear proteins to the methylated region and altered expression of HES4 and HES4 targeted genes MASH1 and P21 involved in striatal development. Moreover, hypermethylation of HES4 promoter sequences was strikingly correlated with measures of striatal degeneration and age-of-onset in a cohort of 25 HD brains (r = 0.56, P = 0.006). Lastly, shRNA knockdown of HES4 in human neuroblastoma cells altered MASH1 and P21 mRNA expression and markedly increased mutated HTT-induced aggregates and cell death. These findings, taken together, suggest that epigenetic dysregulation of HES4 could play a critical role in modifying HD disease pathogenesis and severity., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high-fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here, we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). Therefore, JNK causes decreased expression of PPARα target genes that increase fatty acid oxidation and ketogenesis and promote the development of insulin resistance. We show that the PPARα target gene fibroblast growth factor 21 (Fgf21) plays a key role in this response because disruption of the hepatic PPARα-FGF21 hormone axis suppresses the metabolic effects of JNK deficiency. This analysis identifies the hepatokine FGF21 as a critical mediator of JNK signaling in the liver., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Excision of introns from pre-mRNAs is mediated by the spliceosome, a multi-megadalton complex consisting of U1, U2, U4/U6, and U5 snRNPs plus scores of associated proteins. Spliceosome assembly and disassembly are highly dynamic processes involving multiple stable intermediates. In this study, we utilized a split TAP-tag approach for large-scale purification of an abundant endogenous U2·U5·U6 complex from Schizosaccharomyces pombe. RNAseq revealed this complex to largely contain excised introns, indicating that it is primarily ILS (intron lariat spliceosome) complexes. These endogenous ILS complexes are remarkably resistant to both high-salt and nuclease digestion. Mass spectrometry analysis identified 68, 45, and 43 proteins in low-salt-, high-salt-, and micrococcal nuclease-treated preps, respectively. The protein content of a S. pombe ILS complex strongly resembles that previously reported for human spliced product (P) and Saccharomyces cerevisiae ILS complexes assembled on single pre-mRNAs in vitro. However, the ATP-dependent RNA helicase Brr2 was either substoichiometric in low-salt preps or completely absent from high-salt and MNase preps. Because Brr2 facilitates spliceosome disassembly, its relative absence may explain why the ILS complex accumulates logarithmically growing cultures and the inability of S. pombe extracts to support in vitro splicing.

Development of prefrontal and other higher-order association cortices is associated with widespread changes in the cortical transcriptome, particularly during the transitions from prenatal to postnatal development, and from early infancy to later stages of childhood and early adulthood. However, the timing and longitudinal trajectories of neuronal gene expression programs during these periods remain unclear in part because of confounding effects of concomitantly occurring shifts in neuron-to-glia ratios. Here, we used cell type-specific chromatin sorting techniques for genome-wide profiling of a histone mark associated with transcriptional regulation--H3 with trimethylated lysine 4 (H3K4me3)--in neuronal chromatin from 31 subjects from the late gestational period to 80 years of age. H3K4me3 landscapes of prefrontal neurons were developmentally regulated at 1,157 loci, including 768 loci that were proximal to transcription start sites. Multiple algorithms consistently revealed that the overwhelming majority and perhaps all of developmentally regulated H3K4me3 peaks were on a unidirectional trajectory defined by either rapid gain or loss of histone methylation during the late prenatal period and the first year after birth, followed by similar changes but with progressively slower kinetics during early and later childhood and only minimal changes later in life. Developmentally downregulated H3K4me3 peaks in prefrontal neurons were enriched for Paired box (Pax) and multiple Signal Transducer and Activator of Transcription (STAT) motifs, which are known to promote glial differentiation. In contrast, H3K4me3 peaks subject to a progressive increase in maturing prefrontal neurons were enriched for activating protein-1 (AP-1) recognition elements that are commonly associated with activity-dependent regulation of neuronal gene expression. We uncovered a developmental program governing the remodeling of neuronal histone methylation landscapes in the prefrontal cortex from the late prenatal period to early adolescence, which is linked to cis-regulatory sequences around transcription start sites., Competing Interests: The authors have declared that no competing interests exist.

Type 1 diabetes (T1D) is a T cell-mediated disease. It is strongly associated with susceptibility haplotypes within the major histocompatibility complex, but this association accounts for an estimated 50% of susceptibility. Other studies have identified as many as 50 additional susceptibility loci, but the effect of most is very modest (odds ratio (OR) <1.5). What accounts for the "missing heritability" is unknown and is often attributed to environmental factors. Here we review new data on the cognate ligand of MHC molecules, the T cell receptor (TCR). In rats, we found that one allele of a TCR variable gene, V β 13A, is strongly associated with T1D (OR >5) and that deletion of V β 13+ T cells prevents diabetes. A role for the TCR is also suspected in NOD mice, but TCR regions have not been associated with human T1D. To investigate this disparity, we tested the hypothesis in silico that previous studies of human T1D genetics were underpowered to detect MHC-contingent TCR susceptibility. We show that stratifying by MHC markedly increases statistical power to detect potential TCR susceptibility alleles. We suggest that the TCR regions are viable candidates for T1D susceptibility genes, could account for "missing heritability," and could be targets for prevention.

Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans., Competing Interests: The authors have declared that no competing interests exist.

Little is known about the regulation of neuronal and other cell-type specific epigenomes from the brain. Here, we map the genome-wide distribution of trimethylated histone H3K4 (H3K4me3), a mark associated with transcriptional regulation, in neuronal and nonneuronal nuclei collected from prefrontal cortex (PFC) of 11 individuals ranging in age from 0.5 to 69 years. Massively parallel sequencing identified 12,732-19,704 H3K4me3 enriched regions (peaks), the majority located proximal to (within 2 kb of) the transcription start site (TSS) of annotated genes. These included peaks shared by neurons in comparison with three control (lymphocyte) cell types, as well as peaks specific to individual subjects. We identified 6,213 genes that show highly enriched H3K4me3 in neurons versus control. At least 1,370 loci, including annotated genes and novel transcripts, were selectively tagged with H3K4me3 in neuronal but not in nonneuronal PFC chromatin. Our results reveal age-correlated neuronal epigenome reorganization, including decreased H3K4me3 at approximately 600 genes (many function in developmental processes) during the first year after birth. In comparison, the epigenome of aging (>60 years) PFC neurons showed less extensive changes, including increased H3K4me3 at 100 genes. These findings demonstrate that H3K4me3 in human PFC is highly regulated in a cell type- and subject-specific manner and highlight the importance of early childhood for developmentally regulated chromatin remodeling in prefrontal neurons.

The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols, and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated.

Mapping DNase I hypersensitive (HS) sites is an accurate method of identifying the location of genetic regulatory elements, including promoters, enhancers, silencers, insulators, and locus control regions. We employed high-throughput sequencing and whole-genome tiled array strategies to identify DNase I HS sites within human primary CD4+ T cells. Combining these two technologies, we have created a comprehensive and accurate genome-wide open chromatin map. Surprisingly, only 16%-21% of the identified 94,925 DNase I HS sites are found in promoters or first exons of known genes, but nearly half of the most open sites are in these regions. In conjunction with expression, motif, and chromatin immunoprecipitation data, we find evidence of cell-type-specific characteristics, including the ability to identify transcription start sites and locations of different chromatin marks utilized in these cells. In addition, and unexpectedly, our analyses have uncovered detailed features of nucleosome structure.

The identification of regulatory elements from different cell types is necessary for understanding the mechanisms controlling cell type-specific and housekeeping gene expression. Mapping DNaseI hypersensitive (HS) sites is an accurate method for identifying the location of functional regulatory elements. We used a high throughput method called DNase-chip to identify 3,904 DNaseI HS sites from six cell types across 1% of the human genome. A significant number (22%) of DNaseI HS sites from each cell type are ubiquitously present among all cell types studied. Surprisingly, nearly all of these ubiquitous DNaseI HS sites correspond to either promoters or insulator elements: 86% of them are located near annotated transcription start sites and 10% are bound by CTCF, a protein with known enhancer-blocking insulator activity. We also identified a large number of DNaseI HS sites that are cell type specific (only present in one cell type); these regions are enriched for enhancer elements and correlate with cell type-specific gene expression as well as cell type-specific histone modifications. Finally, we found that approximately 8% of the genome overlaps a DNaseI HS site in at least one the six cell lines studied, indicating that a significant percentage of the genome is potentially functional., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.