Genome-wide analysis of spina bifida risk variants in a case-control study from Bangladesh.

BACKGROUND: Human studies of genetic risk factors for neural tube defects, severe birth defects associated with long-term health consequences in surviving children, have predominantly been restricted to a subset of candidate genes in specific biological pathways including folate metabolism. METHODS: In this study, we investigated the association of genetic variants spanning the genome with risk of spina bifida (i.e., myelomeningocele and meningocele) in a subset of families enrolled from December 2016 through December 2022 in a case-control study in Bangladesh, a population often underrepresented in genetic studies. Saliva DNA samples were analyzed using the Illumina Global Screening Array. We performed genetic association analyses to compare allele frequencies between 112 case and 121 control children, 272 mothers, and 128 trios. RESULTS: In the transmission disequilibrium test analyses with trios only, we identified three novel exonic spina bifida risk loci, including rs140199800 (SULT1C2, p = 1.9 × 10-7), rs45580033 (ASB2, p = 4.2 × 10-10), and rs75426652 (LHPP, p = 7.2 × 10-14), after adjusting for multiple hypothesis testing. Association analyses comparing cases and controls, as well as models that included their mothers, did not identify genome-wide significant variants. CONCLUSIONS: This study identified three novel single nucleotide polymorphisms involved in biological pathways not previously associated with neural tube defects. The study warrants replication in larger groups to validate findings and to inform targeted prevention strategies.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

Heterozygous loss-of-function SMC3 variants are associated with variable and incompletely penetrant growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 13 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated a milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, some instead having intriguing symptomatologies with rational biological links to SMC3 including bone marrow failure, acute myeloid leukemia, and Coats retinal vasculopathy. Analyses of transcriptomic and epigenetic data suggest that SMC3 pLoF variants reduce SMC3 expression but do not result in a blood DNA methylation signature clustering with that of CdLS, and that the global transcriptional signature of SMC3 loss is model-dependent. Our finding of substantial population-scale LoF intolerance in concert with variable penetrance in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multi-layered genomic data paired with careful phenotyping.

Mendelian Disorders in an Interstitial Cystitis/Bladder Pain Syndrome Cohort.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic pain disorder causing symptoms of urinary frequency, urgency, and bladder discomfort or pain. Although this condition affects a large population, little is known about its etiology. Genetic analyses of whole exome sequencing are performed on 109 individuals with IC/BPS. One family has a previously reported SIX5 variant (ENST00000317578.6:c.472G>A, p.Ala158Thr), consistent with Branchiootorenal syndrome 2 (BOR2). A likely pathogenic heterozygous variant in ATP2A2 (ENST00000539276.2:c.235G>A, p.Glu79Lys) is identified in two unrelated probands, indicating possible Darier-White disease. Two private heterozygous variants are identified in ATP2C1 (ENST00000393221.4:c.2358A>T, p.Glu786Asp (VUS/Likely Pathogenic) and ENST00000393221.4:c.989C>G, p.Thr330Ser (likely pathogenic)), indicative of Hailey-Hailey Disease. Sequence kernel association test analysis finds an increased burden of rare ATP2C1 variants in the IC/BPS cases versus a control cohort (p = 0.03, OR = 6.76), though does not survive Bonferroni correction. The data suggest that some individuals with IC/BPS may have unrecognized Mendelian syndromes. Comprehensive phenotyping and genotyping aid in understanding the range of diagnoses in the population-based IC/BPS cohort. Conversely, ATP2C1, ATP2A2, and SIX5 may be candidate genes for IC/BPS. Further evaluation with larger numbers is needed. Genetically screening individuals with IC/BPS may help diagnose and treat this painful disorder due to its heterogeneous nature.

The SET oncoprotein promotes estrogen-induced transcription by facilitating establishment of active chromatin.

SET is a multifunctional histone-binding oncoprotein that regulates transcription by an unclear mechanism. Here we show that SET enhances estrogen-dependent transcription. SET knockdown abrogates transcription of estrogen-responsive genes and their enhancer RNAs. In response to 17ß-estradiol (E2), SET binds to the estrogen receptor α (ERα) and is recruited to ERα-bound enhancers and promoters at estrogen response elements (EREs). SET functions as a histone H2 chaperone that dynamically associates with H2A.Z via its acidic C-terminal domain and promotes H2A.Z incorporation, ERα, MLL1, and KDM3A loading and modulates histone methylation at EREs. SET depletion diminishes recruitment of condensin complexes to EREs and impairs E2-dependent enhancer-promoter looping. Thus, SET boosts E2-induced gene expression by establishing an active chromatin structure at ERα-bound enhancers and promoters, which is essential for transcriptional activation.

Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition.

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition.

Clinical Phenotypes and Outcomes in Monogenic Versus Non-monogenic Very Early Onset Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Over 80 monogenic causes of very early onset inflammatory bowel disease [VEOIBD] have been identified. Prior reports of the natural history of VEOIBD have not considered monogenic disease status. The objective of this study is to describe clinical phenotypes and outcomes in a large single-centre cohort of patients with VEOIBD and universal access to whole exome sequencing [WES]. METHODS: Patients receiving IBD care at a single centre were prospectively enrolled in a longitudinal data repository starting in 2012. WES was offered with enrollment. Enrolled patients were filtered by age of diagnosis <6 years to comprise a VEOIBD cohort. Monogenic disease was identified by filtering proband variants for rare, loss-of-function, or missense variants in known VEOIBD genes inherited according to standard Mendelian inheritance patterns. RESULTS: This analysis included 216 VEOIBD patients, followed for a median of 5.8 years. Seventeen patients [7.9%] had monogenic disease. Patients with monogenic IBD were younger at diagnosis and were more likely to have Crohn's disease phenotype with higher rates of stricturing and penetrating disease and extraintestinal manifestations. Patients with monogenic disease were also more likely to experience outcomes of intensive care unit [ICU] hospitalisation, gastrostomy tube, total parenteral nutrition use, stunting at 3-year follow-up, haematopoietic stem cell transplant, and death. A total of 41 patients [19.0%] had infantile-onset disease. After controlling for monogenic disease, patients with infantile-onset IBD did not have increased risk for most severity outcomes. CONCLUSIONS: Monogenic disease is an important driver of disease severity in VEOIBD. WES is a valuable tool in prognostication and management of VEOIBD.

Single-cell transcriptome profile of mouse skin undergoing antigen-driven allergic inflammation recapitulates findings in atopic dermatitis skin lesions.

BACKGROUND: Allergic skin inflammation elicited in mice by epicutaneous (EC) sensitization with antigen shares characteristics with human atopic dermatitis (AD). OBJECTIVE: We characterized gene expression by single cells in mouse skin undergoing antigen-driven allergic inflammation and compared the results with findings in AD skin lesions. METHODS: Mice were EC sensitized by application of ovalbumin (OVA) or saline to tape-stripped skin. Single-cell RNA sequencing was performed on skin cells 12 days later. Flow cytometry analysis was performed to validate results. RESULTS: Sequencing identified 7 nonhematopoietic and 6 hematopoietic cell subsets in EC-sensitized mouse skin. OVA sensitization resulted in the expansion in the skin of T cells, dendritic cells, macrophages, mast cells/basophils, fibroblasts, and myocytes cell clusters, and in upregulation of TH2 cytokine gene expression in CD4+ T cells and mast cells/basophils. Genes differentially expressed in OVA-sensitized skin included genes important for inflammation in dendritic cells and macrophages, collagen deposition, and leukocyte migration in fibroblasts, chemotaxis in endothelial cells and skin barrier integrity, and differentiation in KCs-findings that recapitulate those in AD skin lesions. Unexpectedly, mast cells/basophils, rather than T cells, were the major source of Il4 and ll13 in OVA-sensitized mouse skin. In addition, our results suggest novel pathways in fibroblast and endothelial cells that may contribute to allergic skin inflammation. CONCLUSION: The gene expression profile of single cells in mouse skin undergoing antigen-driven shares many features with that in AD skin lesions and unveils novel pathways that may be involved in allergic skin inflammation.

Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Maternal Iron Deficiency Modulates Placental Transcriptome and Proteome in Mid-Gestation of Mouse Pregnancy.

BACKGROUND: Maternal iron deficiency (ID) is associated with poor pregnancy and fetal outcomes. The effect is thought to be mediated by the placenta but there is no comprehensive assessment of placental responses to maternal ID. Additionally, whether the influence of maternal ID on the placenta differs by fetal sex is unknown. OBJECTIVES: To identify gene and protein signatures of ID mouse placentas at mid-gestation. A secondary objective was to profile the expression of iron genes in mouse placentas across gestation. METHODS: We used a real-time PCR-based array to determine the mRNA expression of all known iron genes in mouse placentas at embryonic day (E) 12.5, E14.5, E16.5, and E19.5 (n = 3 placentas/time point). To determine the effect of maternal ID, we performed RNA sequencing and proteomics in male and female placentas from ID and iron-adequate mice at E12.5 (n = 8 dams/diet). RESULTS: In female placentas, 6 genes, including transferrin receptor (Tfrc) and solute carrier family 11 member 2, were significantly changed by maternal ID. An additional 154 genes were altered in male ID placentas. A proteomic analysis quantified 7662 proteins in the placenta. Proteins translated from iron-responsive element (IRE)-containing mRNA were altered in abundance; ferritin and ferroportin 1 decreased, while TFRC increased in ID placentas. Less than 4% of the significantly altered genes in ID placentas occurred both at the transcriptional and translational levels. CONCLUSIONS: Our data demonstrate that the impact of maternal ID on placental gene expression in mice is limited in scope and magnitude at mid-gestation. We provide strong evidence for IRE-based transcriptional and translational coordination of iron gene expression in the mouse placenta. Finally, we discover sexually dimorphic effects of maternal ID on placental gene expression, with more genes and pathways altered in male compared with female mouse placentas.

Regulation of the glucocorticoid receptor via a BET-dependent enhancer drives antiandrogen resistance in prostate cancer.

In prostate cancer, resistance to the antiandrogen enzalutamide (Enz) can occur through bypass of androgen receptor (AR) blockade by the glucocorticoid receptor (GR). In contrast to fixed genomic alterations, here we show that GR-mediated antiandrogen resistance is adaptive and reversible due to regulation of GR expression by a tissue-specific enhancer. GR expression is silenced in prostate cancer by a combination of AR binding and EZH2-mediated repression at the GR locus, but is restored in advanced prostate cancers upon reversion of both repressive signals. Remarkably, BET bromodomain inhibition resensitizes drug-resistant tumors to Enz by selectively impairing the GR signaling axis via this enhancer. In addition to revealing an underlying molecular mechanism of GR-driven drug resistance, these data suggest that inhibitors of broadly active chromatin-readers could have utility in nuanced clinical contexts of acquired drug resistance with a more favorable therapeutic index.

Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development.

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that the DNMT3A mutational hotspot at Arg882 (DNMT3A(R882H)) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3A(R882H) directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1, and Hoxa gene cluster. DNMT3A(R882H) induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3A(R882H)-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3A(R882H)-induced gene-expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice.

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility. Here we show that super-enhancers underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters ('epicentres') of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia.

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999's on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

Comparison of REST cistromes across human cell types reveals common and context-specific functions.

Recent studies have shown that the transcriptional functions of REST are much broader than repressing neuronal genes in non-neuronal systems. Whether REST occupies similar chromatin regions in different cell types and how it interacts with other transcriptional regulators to execute its functions in a context-dependent manner has not been adequately investigated. We have applied ChIP-seq analysis to identify the REST cistrome in human CD4+ T cells and compared it with published data from 15 other cell types. We found that REST cistromes were distinct among cell types, with REST binding to several tumor suppressors specifically in cancer cells, whereas 7% of the REST peaks in non-neuronal cells were ubiquitously called and <25% were identified for ≥ 5 cell types. Nevertheless, using a quantitative metric directly comparing raw ChIP-seq signals, we found the majority (∼80%) was shared by ≥ 2 cell types. Integration with RNA-seq data showed that REST binding was generally correlated with low gene expression. Close examination revealed that multiple contexts were correlated with reduced expression of REST targets, e.g., the presence of a cognate RE1 motif and cellular specificity of REST binding. These contexts were shown to play a role in differential corepressor recruitment. Furthermore, transcriptional outcome was highly influenced by REST cofactors, e.g., SIN3 and EZH2 co-occupancy marked higher and lower expression of REST targets, respectively. Unexpectedly, the REST cistrome in differentiated neurons exhibited unique features not observed in non-neuronal cells, e.g., the lack of RE1 motifs and an association with active gene expression. Finally, our analysis demonstrated how REST could differentially regulate a transcription network constituted of miRNAs, REST complex and neuronal factors. Overall, our findings of contexts playing critical roles in REST occupancy and regulatory outcome provide insights into the molecular interactions underlying REST's diverse functions, and point to novel roles of REST in differentiated neurons.

ETS factors reprogram the androgen receptor cistrome and prime prostate tumorigenesis in response to PTEN loss.

Studies of ETS-mediated prostate oncogenesis have been hampered by a lack of suitable experimental systems. Here we describe a new conditional mouse model that shows robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, the mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG markedly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included the restoration of AR transcriptional output and upregulation of genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETS variant 1 (ETV1) positively regulated the AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, expression of ERG and ETV1 correlated with higher AR transcriptional output in PTEN-deficient prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.

An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting.

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.