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Adults previously infected with SARS-CoV-2 develop short-term immunity and may have increased reactogenicity to COVID-19 vaccines. This prospective, multi-center active surveillance cohort study examined the short-term safety of COVID-19 vaccines in adults with a prior history of SARS-CoV-2.Canadian adults vaccinated between December 22, 2020 and November 27, 2021 were sent an electronic questionnaire 7 days post dose 1, dose 2 and dose 3 vaccination. The main outcome was health events occurring in the first 7 days after each vaccination that prevented daily activities, resulted in work absenteeism or required a medical consultation, including hospitalization.Among 684,998 vaccinated individuals, 2.6% (18,127/684,998) reported a prior history of SARS-CoV-2 infection a median of 4 months (interquartile range 2-6 months) previously. After dose 1, individuals with moderate (bedridden) to severe (hospitalized) COVID-19 who received BNT162b2, mRNA1273 or ChAdox1-S vaccines had higher odds of a health event preventing daily activities, resulting in work absenteeism or requiring medical consultation; adjusted odds ratio (AOR) 3.96 (95% CI 3.67-4.28) for BNT162b2, 5.01 (4.57-5.50) for mRNA1273 and 1.84 (1.54-2.20) for ChAdox1-S compared to no infection. Following dose 2 and 3, the greater risk associated with previous infection was also present but attenuated compared to dose 1. For all doses, the association was lower or absent after mild or asymptomatic infection.Adults with moderate or severe previous SARS-CoV-2 infection were more likely to have a health event sufficient to impact routine activities or require medical assessment in the week following each vaccine doses.

Background The online Tuberculin Skin Test/Interferon Gamma Release Assay (TST/IGRA) Interpreter V3.0 (TSTin3D), a tool for estimating the risk of active tuberculosis (TB) in individuals with latent TB infection (LTBI), has been in use for more than a decade, but its predictive performance has never been evaluated. Methods People with a positive TST or IGRA result from 1985 to 2015 were identified using a health data linkage that involved migrants to British Columbia, Canada. Comorbid conditions at the time of LTBI testing were identified from physician claims, hospitalizations, vital statistics, outpatient prescriptions, and kidney and HIV databases. The risk of developing active TB within 2 and 5 years was estimated using TSTin3D. The discrimination and calibration of these estimates were evaluated. Results A total of 37 163 individuals met study inclusion criteria; 10.4% were tested by IGRA. Generally, the TSTin3D algorithm assigned higher risks to demographic and clinical groups known to have higher active TB risks. Concordance estimates ranged from 0.66 to 0.68 in 2- and 5-year time frames. Comparing predicted to observed counts suggests that TSTin3D overestimates active TB risks and that overestimation increases over time (with relative bias of 3% and 12% in 2- and 5-year periods, respectively). Calibration plots also suggest that overestimation increases toward the upper end of the risk spectrum. Conclusions TSTin3D can discriminate adequately between people who developed and did not develop active TB in this linked database of migrants with predominately positive skin tests. Further work is needed to improve TSTin3D’s calibration.

Pregnant individuals have been receiving COVID-19 vaccines following pre-authorisation clinical trials in non-pregnant people. This study aimed to determine the frequency and nature of significant health events among pregnant females after COVID-19 vaccination, compared with unvaccinated pregnant controls and vaccinated non-pregnant individuals.We did an observational cohort study, set in seven Canadian provinces and territories including Ontario, Quebec, British Columbia, Alberta, Nova Scotia, Yukon, and Prince Edward Island. Eligibility criteria for vaccinated individuals were a first dose of a COVID-19 vaccine within the previous 7 days; an active email address and telephone number; ability to communicate in English or French; and residence in the aforementioned provinces or territories. Study participants were pregnant and non-pregnant females aged 15-49 years. Individuals were able to participate as controls if they were unvaccinated and fulfilled the other criteria. Data were collected primarily by self-reported survey after both vaccine doses, with telephone follow-up for those reporting any medically attended event. Participants reported significant health events (new or worsening of a health event sufficient to cause work or school absenteeism, medical consultation, or prevent daily activities) occurring within 7 days of vaccination or within the past 7 days for unvaccinated individuals. We employed multivariable logistic regression to examine significant health events associated with mRNA vaccines, adjusting for age group, previous SARS-CoV-2 infection, and trimester, as appropriate.As of Nov 4, 2021, 191 360 women aged 15-49 years with known pregnancy status had completed the first vaccine dose survey and 94 937 had completed the second dose survey. 180 388 received one dose and 94 262 received a second dose of an mRNA vaccine, with 5597 pregnant participants receiving dose one and 3108 receiving dose two, and 174 765 non-pregnant participants receiving dose one and 91 131 receiving dose two. Of 6179 included unvaccinated control participants, 339 were pregnant and 5840 were not pregnant. Overall, 226 (4·0%) of 5597 vaccinated pregnant females reported a significant health event after dose one of an mRNA vaccine, and 227 (7·3%) of 3108 after dose two, compared with 11 (3·2%) of 339 pregnant unvaccinated females. Pregnant vaccinated females had an increased odds of a significant health event within 7 days of the vaccine after dose two of mRNA-1273 (adjusted odds ratio [aOR] 4·4 [95% CI 2·4-8·3]) compared with pregnant unvaccinated controls within the past 7 days, but not after dose one of mRNA-1273 or any dose of BNT162b2. Pregnant vaccinated females had decreased odds of a significant health event compared with non-pregnant vaccinated females after both dose one (aOR 0·63 [95% CI 0·55-0·72]) and dose two (aOR 0·62 [0·54-0·71]) of any mRNA vaccination. There were no significant differences in any analyses when restricted to events which led to medical attention.COVID-19 mRNA vaccines have a good safety profile in pregnancy. These data can be used to appropriately inform pregnant people regarding reactogenicity of COVID-19 vaccines during pregnancy, and should be considered alongside effectiveness and immunogenicity data to make appropriate recommendations about best use of COVID-19 vaccines in pregnancy.Canadian Institutes of Health Research, Public Health Agency of Canada.

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.

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.

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. Integrative analysis in patients with diffuse large B-cell lymphoma uncovers that biallelic mutations on TMEM30A are associated with a favorable outcome and enhanced sensitivity to CD47 blockade.

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 (

SUMMARYFull understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although 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 inS. pombeand 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 like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

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 somaticIL4Rmutations in 15 out 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 advantagein vivoThe pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establishIL4Rmutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

SummaryThe 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.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype worldwide, accounting for 40% of all non-Hodgkin lymphomas. DLBCL presents as an aggressive disease requiring immediate treatment. Although significant improvement in outcome has been achieved, ~40% of patients still experience treatment failure. Here, we characterized the recurrent genetic alterations and transcriptomic signatures in diagnostic biopsies from a population registry-based cohort of 347 patients with de novo DLBCL uniformly treated with R-CHOP. This analysis revealed bi-allelic loss of function mutations of TMEM30A that were associated with favorable treatment outcome. TMEM30A is a chaperone protein, involved in maintaining the asymmetric distribution of phosphatidylethanolamine and phosphatidylserine, an integral component of the plasma membrane and “eat-me” signal recognized by macrophages. Using TMEM30A knockout systems by CRISPR genome editing techniques, we have functionally characterized this loss-of-function mutation in representative human and mouse DLBCL cell line models. We have discovered that TMEM30A loss is associated with increased B-cell signaling following antigen stimulation, including a two-fold increase in the diffusion rate of B-cell receptor (BCR) clustering, using high resolution Single Particle Tracking (SPT) technology. In addition, we have measured three-fold increase in chemotherapeutic drug accumulation in both knockout cell lines and randomly selected patient biopsies with TMEM30A biallelic loss. This observation was validated in a xenograft mouse model, which presented improved survival and limited tumor growth following vincristine treatment in mice injected with TMEM30A null DLBCL cell lines compared with native cell lines. This phenotype explains the improved prognosis observed in DLBCL patients following R-CHOP treatment. Furthermore, we have observed over two fold higher numbers of tumor-associated macrophages in B-cell lymphoma syngeneic mouse models with Tmem30a loss-of-function, prior to any form of treatment, suggesting the existence of “hot” and primed tumors. Our data highlight a multi-faceted role for TMEM30A and plasma membrane physiology in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited. Characterization of these mechanisms will address a missing link in the cancer field as related insights in lymphoma will outline therapeutic approaches that can be extended to cancer therapy in general. Citation Format: Shannon Healy, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Christoffer Hother, Anja Mottok, Fong Chun Chan, Lauren Chong, Robert Kridel, Merrill Boyle, Barbara Meissner, Tomohiro Aoki, Katsuyoshi Takata, Bruce W. Woolcock, Elena Vigano, Libin Abraham, Michael Gold, Adele Telenius, Pedro Farinha, Graham Slack, Susana Ben-Neriah, Daniel Lai, Allen W. Zhang, Sohrab Salehi, Hennady P. Shulha, Derek S. Chiu, Sara Mostafavi, Alina S. Gerrie, Diego Villa, Laurie H. Sehn, Kerry J. J. Savage, Andrew J. J. Mungall, Andrew P. Weng, Marcel Bally, Ryan D. Morin, Gabriela V. Cohen Freue, Joseph M. Connors, Marco A. Marra, Sohrab P. Shah, Randy D. Gascoyne1, David W. Scott, Christian Steidl, Ulrich Steidl. TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3480.

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.

The clinical significance of

Promoters are important regulatory elements that contain the necessary sequence features for cells to initiate transcription. To functionally characterize a large set of human promoters, we measured the transcriptional activities of 4575 putative promoters across eight cell lines using transient transfection reporter assays. In parallel, we measured gene expression in the same cell lines and observed a significant correlation between promoter activity and endogenous gene expression (r = 0.43). As transient transfection assays directly measure the promoting effect of a defined fragment of DNA sequence, decoupled from epigenetic, chromatin, or long-range regulatory effects, we sought to predict whether a promoter was active using sequence features alone. CG dinucleotide content was highly predictive of ubiquitous promoter activity, necessitating the separation of promoters into two groups: high CG promoters, mostly ubiquitously active, and low CG promoters, mostly cell line–specific. Computational models trained on the binding potential of transcriptional factor (TF) binding motifs could predict promoter activities in both high and low CG groups: average area under the receiver operating characteristic curve (AUC) of the models was 91% and exceeded the AUC of CG content by an average of 23%. Known relationships, for example, between HNF4A and hepatocytes, were recapitulated in the corresponding cell lines, in this case the liver-derived cell line HepG2. Half of the associations between tissue-specific TFs and cell line–specific promoters were new. Our study underscores the importance of collecting functional information from complementary assays and conditions to understand biology in a systematic framework.

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.

Follicular lymphoma is a clinically and genetically heterogeneous disease, but the prognostic value of somatic mutations has not been systematically assessed. We aimed to improve risk stratification of patients receiving first-line immunochemotherapy by integrating gene mutations into a prognostic model.We did DNA deep sequencing to retrospectively analyse the mutation status of 74 genes in 151 follicular lymphoma biopsy specimens that were obtained from patients within 1 year before beginning immunochemotherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). These patients were recruited between May 4, 2000, and Oct 20, 2010, as part of a phase 3 trial (GLSG2000). Eligible patients had symptomatic, advanced stage follicular lymphoma and were previously untreated. The primary endpoints were failure-free survival (defined as less than a partial remission at the end of induction, relapse, progression, or death) and overall survival calculated from date of treatment initiation. Median follow-up was 7·7 years (IQR 5·5-9·3). Mutations and clinical factors were incorporated into a risk model for failure-free survival using multivariable L1-penalised Cox regression. We validated the risk model in an independent population-based cohort of 107 patients with symptomatic follicular lymphoma considered ineligible for curative irradiation. Pretreatment biopsies were taken between Feb 24, 2004, and Nov 24, 2009, within 1 year before beginning first-line immunochemotherapy consisting of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP). Median follow-up was 6·7 years (IQR 5·7-7·6).We established a clinicogenetic risk model (termed m7-FLIPI) that included the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the Follicular Lymphoma International Prognostic Index (FLIPI), and Eastern Cooperative Oncology Group (ECOG) performance status. In the training cohort, m7-FLIPI defined a high-risk group (28%, 43/151) with 5-year failure-free survival of 38·29% (95% CI 25·31-57·95) versus 77·21% (95% CI 69·21-86·14) for the low-risk group (hazard ratio [HR] 4·14, 95% CI 2·47-6·93; p0·0001; bootstrap-corrected HR 2·02), and outperformed a prognostic model of only gene mutations (HR 3·76, 95% CI 2·10-6·74; p0·0001; bootstrap-corrected HR 1·57). The positive predictive value and negative predictive value for 5-year failure-free survival were 64% and 78%, respectively, with a C-index of 0·80 (95% CI 0·71-0·89). In the validation cohort, m7-FLIPI again defined a high-risk group (22%, 24/107) with 5-year failure-free survival of 25·00% (95% CI 12·50-49·99) versus 68·24% (58·84-79·15) in the low-risk group (HR 3·58, 95% CI 2·00-6·42; p0.0001). The positive predictive value for 5-year failure-free survival was 72% and 68% for negative predictive value, with a C-index of 0·79 (95% CI 0·69-0·89). In the validation cohort, risk stratification by m7-FLIPI outperformed FLIPI alone (HR 2·18, 95% CI 1·21-3·92), and FLIPI combined with ECOG performance status (HR 2·03, 95% CI 1·12-3·67).Integration of the mutational status of seven genes with clinical risk factors improves prognostication for patients with follicular lymphoma receiving first-line immunochemotherapy and is a promising approach to identify the subset at highest risk of treatment failure.Deutsche Krebshilfe, Terry Fox Research Institute.

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 growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes ( ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D ) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.

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) β13A, is strongly associated with T1D (OR >5) and that deletion of Vβ13+ 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 hypothesisin silicothat 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.

Mapping histone methylation landscapes in neurons from human, chimpanzee, and macaque brains reveals coordinated, human-specific epigenetic regulation at hundreds of regulatory sequences., 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., Author Summary Primate and human genomes comprise billions of base pairs, but we are unlikely to gain a deeper understanding of brain functions unique to human (including cognitive abilities and psychiatric diseases) merely by comparing linear DNA sequences. Such determinants of species-specific function might instead be found in the so-called “epigenetic” characteristics of genomic regions; differences in the protein-packaged chromatin state in which genomic DNA exists in the cell. Here, we examine neurons from the prefrontal cortex, a brain region closely associated with the evolution of the primate brain, and identify hundreds of short DNA sequences defined by human-specific changes in chromatin structure and function when compared to non-human primates. These changes included species-specific regulation of methylation marks on the histone proteins around which genomic DNA is wrapped. Sequences subject to human-specific epigenetic regulation showed significant spatial clustering, and despite being separated by hundreds of thousands of base pairs on the linear genome, were in direct physical contact with each other through chromosomal looping and other higher order chromatin features. This observation raises the intriguing possibility that coordinated epigenetic regulation via newly derived chromatin features at gene transcription start sites could play an important role in the emergence of human-specific gene expression networks in the brain. Finally, we identified a strong genetic footprint of hominid evolution in a small subset of transcription start sites defined by human-specific gains in histone methylation, with particularly strong enrichment in prefrontal cortex neurons. For example, the base pair sequence of DPP10 (a gene critically important for normal human brain development) not only showed distinct human-specific changes, but also evidence for more recent selective pressures within the human population.

Context Neuronal dysfunction in cerebral cortex and other brain regions could contribute to the cognitive and behavioral defects in autism. Objective To characterize epigenetic signatures of autism in prefrontal cortex neurons. Design We performed fluorescence-activated sorting and separation of neuronal and nonneuronal nuclei from postmortem prefrontal cortex, digested the chromatin with micrococcal nuclease, and deeply sequenced the DNA from the mononucleosomes with trimethylated H3K4 (H3K4me3), a histone mark associated with transcriptional regulation. Approximately 15 billion base pairs of H3K4me3-enriched sequences were collected from 32 brains. Setting Academic medical center. Participants A total of 16 subjects diagnosed as having autism and 16 control subjects ranging in age from 0.5 to 70 years. Main Outcome Measures Identification of genomic loci showing autism-associated H3K4me3 changes in prefrontal cortex neurons. Results Subjects with autism showed no evidence for generalized disruption of the developmentally regulated remodeling of the H3K4me3 landscape that defines normal prefrontal cortex neurons in early infancy. However, excess spreading of H3K4me3 from the transcription start sites into downstream gene bodies and upstream promoters was observed specifically in neuronal chromatin from 4 of 16 autism cases but not in controls. Variable subsets of autism cases exhibit altered H3K4me3 peaks at numerous genes regulating neuronal connectivity, social behaviors, and cognition, often in conjunction with altered expression of the corresponding transcripts. Autism-associated H3K4me3 peaks were significantly enriched in genes and loci implicated in neurodevelopmental diseases. Conclusions Prefrontal cortex neurons from subjects with autism show changes in chromatin structures at hundreds of loci genome-wide, revealing considerable overlap between genetic and epigenetic risk maps of developmental brain disorders.

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.

We developed a strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated ‘knock-in’. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation–microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.

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 about chromatin structure has emerged, including its interrelationship 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 novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

Background: MYC and BCL2 are critical driver genes for non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). However, the clinical impact of MYC and BCL2 genetic alterations, apart from translocations, has not been comprehensively investigated using high-resolution genetic assays, such as next generation sequencing and high-resolution SNP arrays. Moreover, correlations with cell of origin (COO) subtype, determined by gene expression profiling, have not been widely studied in a large homogeneously treated cohort. We determined the frequency and clinical impact of MYC and BCL2 genetic aberrations in DLBCL in a large population-based cohort uniformly treated with R-CHOP. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were treated with R-CHOP in BC. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of MYC and BCL2 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Dual positivity for MYC (cut off; 40%) and BCL2 (cut off; 50%) proteins identified a dual protein expresser (DPE) phenotype. COO classification was achieved using the Lymph2Cx assay based on NanoString technology in 299 patients and the Hans algorithm in 32 cases with low tumor content ( Results: COO determination revealed 193 cases to be GCB subtype, 107 cases ABC/non-GCB and 30 were unclassifiable. Using next generation sequencing, 310 SNVs were detected in MYC (29/347; 8% of cases) and BCL2 (88/347; 25% of cases), with mean redundant coverage depths of 633-fold. All MYC and 98% of BCL2 mutations were misssense mutations. Analysis of copy number alterations using GISTIC 2.0 revealed significant focal gain/amplification (gain/amp) peaks affecting 8q24, including the MYC locus (68/341; 20% of cases) and 18q21, including the BCL2 locus (82/341; 24% of cases). MYC and BCL2 translocations were detected in 38/283 (13%) and 90/300 (30%) of tumors, respectively. MYC gain/amp, BCL2 mutation, BCL2 translocations and double MYC/BCL2 translocation (DHIT) were seen significantly more often in GCB-DLBCL (26% vs 10%, p=0.001; 35% vs 11%, p With a median follow up of 6.5 years for living patients, the presence of MYC translocation, BCL2 gain/amp and BCL2 mutation were associated with an inferior 5y-time to progression (TTP, 53% vs 30%, p=0.019; 60% vs 32%, p=0.009 and 52% vs 17%, p=0.026, respectively) in ABC subtype. In GCB subtype, BCL2 translocation, MYC gain/amp and MYC translocation were associated with an inferior 5y-TTP (85% vs 62%, p=0.001; 80% vs 63%, p=0.011; and 79% vs 61%, p=0.013, respectively). In a multivariate Cox model of TTP including IPI and DPE, BCL2 gain/amp remained prognostic (HR=2.4 [1.3-4.5], p=0.008) independent of IPI and DPE in ABC-DLBCL. In GCB-DLBCL, BCL2 translocation and/or MYC gain/amp showed strong prognostic value (HR=3.0 [1.4-6.4], p=0.006) independent of IPI and DPE. In the IPI high risk group (IPI=3-5), the presence of a BCL2 translocation and/or MYC gain/amp defined a remarkably poor outcome group in GCB-DLBCL (5y-TTP; 29%). Similar poor outcome was observed in ABC-DLBCL cases that harbored BCL2 gain/amp (5y-TTP; 22%). Conclusions: The DHIT genotype was only seen in GCB-DLBCL. High-resolution genomic assays identified extremely poor prognostic groups within each COO subtype on the basis of MYC and BCL2 genetic status in a large uniformly R-CHOP-treated population-based cohort of DLBCL. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Figure 1. TTP according to MYC/BCL2 genetic alterations with IPI in ABC-DLBC (n=101) and GCB-DLBCL patients (n=166) treated with R-CHOP. Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Background: Diffuse large B cell lymphoma (DLBCL) is divided into two distinct molecular subtypes, germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. Genetic landscape studies of DLBCL have revealed several GCB-DLBCL specific mutations, including CREBBP, GNA13, EZH2, TNFRSF14, BCL2 and MEF2B. Functional studies have recently shown that the inactivation of Gα13 signaling pathway genes, including GNA13, together with BCL2 over-expression, allows GC B-cells to escape the germinal center niche and widely disseminate. Although these findings revealed a critical role of genetic alterations of Gα13 signaling pathway in GC-driven mouse models of lymphomagenesis, clinical correlation is lacking. Here we analyzed the clinical impact of genetic alterations of Gα13 signaling pathway in a large population-based DLBCL cohort. Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of GNA13, P2RY8, ARHGEF1, S1PR2 and RHOA was performed using a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Cell-of-origin classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014; 123) in 299 patients and the Hans algorithm (Hans, Blood 2004; 103) in 32 cases with low tumor content ( Results: Using next generation sequencing, 225 SNVs and 5 Indels were detected in GNA13 (16%), P2RY8 (18%), ARHGEF1 (6%), S1PR2 (3%) and RHOA (6%). SNP 6.0 microarrays revealed heterozygous deletions in GNA13 (2%), ARHGEF1 (1%), S1PR2 (4%) and RHOA (8%), but homozygous deletion was not found in any of these five loci. GNA13, P2RY8 and ARHGEF1 mutations were significantly more frequent in the GCB subtype than ABC subtype (26% vs. 6%; p In the cases with mutations of any of the five Gα13 signaling pathway genes, BCL2 over-expression (cut off; 50%) and translocation was associated with increasing stage (p=.018 and p=.005, respectively), but not in wt cases (p=.53 and p=.63, respectively). Specifically, in the cases with GNA13 and P2RY8 mutations individually, BCL2 over-expression was associated with advanced stage (stage III/IV, p=.018 and p=.037, respectively), but not in wild type (wt) cases. Importantly, BCL2 over-expression in the cases harboring Gα13 pathway mutations was not significantly associated with other poor risk features, including any other IPI factors or bone marrow involvement, indicating that genetic alterations in Gα13 signaling pathway accompanied by BCL2 over-expression might promote lymphoma dissemination into lymph nodes but not extranodal sites. With a median follow up of 6.5 years for living patients, there was no prognostic impact of harboring any isolated Gα13 pathway mutation in GCB-DLBCL patients. However, in cases with any Gα13 pathway mutations, BCL2 over-expression was significantly associated with an inferior 5y-time to progression (TTP; 90% vs 62%, p=.003) and disease-specific survival (DSS; 90% vs 71%, p=.042), but not in wt cases (Fig 1). In a Cox model of TTP including the IPI, BCL2 over-expression remained prognostic in the cases harboring any Gα13 pathway mutations (HR=4.13 [1.42-12.01], p=.009), but not in wt cases (HR=1.70 [0.62-4.68], p=.31). In cases with any Gα13 pathway alterations including copy number loss, BCL2 over-expression was also significantly associated with an inferior TTP (HR=3.64 [1.39-9.57], p=.009) independent of IPI, but not in the cases without genetic alterations (HR=1.75 [0.57-5.34], p=.33). Conclusions: Genetic alterations in Gα13 signaling pathway genes cooperate with BCL2 over-expression to promotes lymphoma dissemination to nodal sites and is associated with the poor outcome in GCB-DLBCL Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Figure 1. TTP and DSS according to BCL2 over-expression with/without Gα13 signaling pathway mutations in GCB-DLBCL patients (n=185) treated with R-CHOP Disclosures Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Background: Although R-CHOP has significantly improved outcome in diffuse large B-cell lymphoma (DLBCL), 40% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Recent studies using next-generation sequencing strategies have described the landscape of recurrent mutations in DLBCL. However, with the exception of TP53 and FOXO1, little is known about the clinical relevance of recurrent mutations and importantly, the interactions of these genetic alterations in DLBCL. Moreover, an integrated analysis of copy number alterations and recurrent mutations annotated across cell-of-origin (COO) distinctions for a large cohort of DLBCL cases who have received uniform therapy is lacking. The present study examined the frequency and clinical impact of recurrent genetic aberrations of DLBCL using high-resolution technologies in a large population-based DLBCL cohort. Methods: We analyzed 348 newly diagnosed DLBCL cases that were uniformly treated with R-CHOP at the BC Cancer Agency (Vancouver) with available DNA extracted from fresh frozen biopsy material (tumor content >30%). Matched germ line DNA was available for 67 patients. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Targeted re-sequencing of the coding exons of 56 genes was performed using a Truseq Custom Amplicon assay. Gene selection was based on mutational frequencies that have been previously described in DLBCL mutational landscape publications. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Tissue microarrays were constructed using duplicate 0.6mm cores from 332 cases, and breakapart FISH assays for MYC, BCL2 and BCL6 and IHC staining for MYC, BCL2 and cell of origin proteins were performed. COO classification was available in 331 cases, according to gene expression profiling by the Lymph2Cx assay using the NanoString platform (Scott, Blood 2014;123) in 299 patients as well as Hans algorithm (Hans, Blood 2004;103) in 32 cases with low tumor content. 194 cases were assigned to GCB subtype, 107 cases, ABC/non-GCB and 30 were unclassifiable. Results: In the mutation analysis, we identified 2,757 SNVs and 245 small indels. The mean depth of coverage was 634. Recurrent mutation frequencies varied between 0 and 58, with a mean of 8.25 per case. 98% of cases harbored at least one mutation and 95% of cases multiple mutations. 10 mutated genes were detected significantly more frequently in the GCB subtype including CREBBP, GNA13, EZH2, TNFRSF14, IRF8,STAT3, BCL2, SGK1, MEF2B and CD83, and 4 mutated genes, MYD88, CD79B, PRDM1 and PIM1, in the ABC subtype. In the copy number analysis, 45 significant amplification peaks and 57 deletion peaks were revealed by the GISTIC algorithm. As previously reported, 9p21.3, including CDKN2A,were more frequently detected in the ABC subtypes. With a median follow up of 6.5 years for living patients, the 5 y disease specific survival (DSS) and time to progression (TTP) of all patients were 72% and 64%, respectively. The clinical cohort was representative of registry data from BC based on a comparison of patient characteristics and survival outcomes with 1,194 control DLBCL R-CHOP patients. The ABC subtype was significantly associated with an inferior DSS and TTP (both p Conclusions: Our approach using next generation sequencing and high resolution SNP array provides an accurate estimation of frequency and clinical significance of recurrent genetic alterations of DLBCL in a uniformly R-CHOP-treated large population-based cohort of patients. Disclosures No relevant conflicts of interest to declare.

SummaryPaternal 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.

SummaryMapping 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.