DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory ...disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.
DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA ...methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3a
pluripotent cells in vitro, and is also evident in Dnmt3a
dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.
The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have ...developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.
Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid ...disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.
Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage ...response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.
Medical Education 2012: 46: 1179–1188
Context The adverse patient event is an inherent component of surgical practice, but many surgeons are unprepared for the profound emotional responses these ...events can evoke. This study explored surgeons’ reactions to adverse events and their impact on subsequent judgement and decision making.
Methods Using a constructivist grounded theory approach, we conducted 20 semi‐structured, 60‐minute interviews with surgeons across subspecialties, experience levels, and sexes to explore surgeons’ recollections of reactions to adverse events. Further interviews were conducted with six general surgeons to explore more immediate reactions after 28 adverse events. Data coding was both inductive, developing a new framework based on emergent themes, and deductive, using an existing framework for care providers’ reactions to adverse events.
Results Surgeons expressed feeling unique and alone in the depths of their reactions to adverse events and consistently described four phases of response, each containing cognitive and emotive components, following such events. The initial phase (the kick) involved feelings of failure (‘Am I good enough?’) experienced with a significant physiological response. This was shortly followed by a second phase (the fall), during which the surgeon experienced a sense of chaos and assessed the extent of his or her contribution to the event (‘Was it my fault?’). During the third phase (the recovery), the surgeon reflected on the adverse event (‘What can I learn?’) and experienced a sense of ‘moving on’. In the fourth phase (the long‐term impact), the surgeon experienced the prolonged and cumulative effects of these reactions on his or her own personal and professional identities. Surgeons also described an effect on their clinical judgement, both for the case in question (minimisation) and future cases (overcompensation).
Conclusions Surgeons progress through a series of four phases following adverse events that are potentially caused by or directly linked to surgeon error. The framework provided by this study has implications for teaching, surgeon wellness and surgeon error.
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IntroductionThere is little to no evidence in Canada on the barriers that youth face when accessing contraception. We seek to identify the contraception access, experiences, beliefs, attitudes, ...knowledge, and needs of youth in Canada, from the perspectives of youth and youth service providers.Methods and analysisThis prospective, mixed-methods, integrated knowledge mobilisation study, the Ask Us project, will involve a national sample of youth, healthcare and social service providers, and policy makers recruited via a novel relational mapping and outreach approach led by youth. Phase I will centre the voices of youth and their service providers through in-depth one-on-one interviews. We will explore the factors influencing youth access to contraception, theoretically guided by Levesque’s Access to Care framework. Phase II will focus on the cocreation and evaluation of knowledge translation products (youth stories) with youth, service providers, and policy makers.Ethics and disseminationEthical approval was received from the University of British Columbia’s Research Ethics Board (H21-01091). Full open-access publication of the work will be sought in an international peer-reviewed journal. Findings will be disseminated to youth and service providers through social media, newsletters, and communities of practice, and to policy makers through invited evidence briefs and face-to-face presentations.
Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical ...features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.
Large brain size is one of the defining characteristics of modern humans. Seckel syndrome (MIM 210600), a disorder of markedly reduced brain and body size, is associated with defective ATR-dependent ...DNA damage signaling. Only a single hypomorphic mutation of ATR has been identified in this genetically heterogeneous condition. We now report that mutations in the gene encoding pericentrin (PCNT)-resulting in the loss of pericentrin from the centrosome, where it has key functions anchoring both structural and regulatory proteins-also cause Seckel syndrome. Furthermore, we find that cells of individuals with Seckel syndrome due to mutations in PCNT (PCNT-Seckel) have defects in ATR-dependent checkpoint signaling, providing the first evidence linking a structural centrosomal protein with DNA damage signaling. These findings also suggest that other known microcephaly genes implicated in either DNA repair responses or centrosomal function may act in common developmental pathways determining human brain and body size.
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