Search

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects’ cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycleprogression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitinligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIPrelocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX andRPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-inducedDNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP thereforelimit cellular proliferation, providing a potential mechanism for microcephaly and dwarfismphenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damageresponse to replication-blocking DNA lesions.

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.

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.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Correction to Martin et al. available at: Genes & Development 30 (19): 2158 (http://genesdev.cshlp.org/content/31/9/953.full.pdf+html). Compaction of chromosomes is essential for accurate segregation of the genome duringmitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here,we report that biallelic mutations inNCAPD2,NCAPH, orNCAPD3, 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. This work was supported by funding from the Medical Research Council, the Lister Institute for Preventative Medicine, and the European Research Council (ERC; 281847 to A.P.J.); a Biotechnology and Biological Sciences Research Council grant (BB/ K017632/1 to P.V); a Sir Henry Dale Fellowship (grant 102560/ Z/13/Z to A.J.W.); Medical Research Scotland (to L.S.B.); the Potentials Foundation (to C.A.W.); and the Indian Council of Medical Research (BMS 54/2/2013 to S.R.P). The Deciphering Developmental Disorders Study presents independent research commissioned by the Health Innovation Challenge Fund (grant no. HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant no. WT098051). The views expressed here are those of the authors and not necessarily those of the Wellcome Trust or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83) granted by the Cambridge South Research Ethics Committee, and GEN/ 284/12 granted by the Republic of Ireland. We acknowledge the support of the National Institute for Health Research through the Comprehensive Clinical Research Network.

Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells., Graphical Abstract Highlights ► Ribonucleotides are the most common nucleotide base lesion in the mouse genome ► RNase H2 is a key genome surveillance enzyme required for removal of nucleotides ► RNase H2 is essential for mammalian development ► Without RNase H2, cells exhibit genome instability and p53 pathway activation, DNA polymerases can incorporate more than a million ribonucleotides into replicating mouse DNA in each cell, making ribonucleotides the most abundant kind of DNA lesion. RNase H2, which removes this “damage,” has an essential role in genome surveillance and is required for embryonic development.

Contains fulltext : 97141.pdf (Publisher’s version ) (Closed access) Meier-Gorlin syndrome (ear, patella and short-stature syndrome) is an autosomal recessive primordial dwarfism syndrome characterized by absent or hypoplastic patellae and markedly small ears(1)(3). Both pre- and post-natal growth are impaired in this disorder, and although microcephaly is often evident, intellect is usually normal in this syndrome. We report here that individuals with this disorder show marked locus heterogeneity, and we identify mutations in five separate genes: ORC1, ORC4, ORC6, CDT1 and CDC6. All of these genes encode components of the pre-replication complex, implicating defects in replication licensing as the cause of a genetic syndrome with distinct developmental abnormalities.

Enzymes that degrade nucleic acids are emerging as important players in the pathogenesis of inflammatory disease. This is exemplified by the recent identification of four genes that cause the childhood inflammatory disorder, Aicardi-Goutières syndrome (AGS). This is an autosomal recessive neurological condition whose clinical and immunological features parallel those of congenital viral infection. The four AGS genes encode two nucleases: TREX1 and the hetero-trimeric Ribonuclease H2 (RNase H2) complex. The biochemical activity of these enzymes was initially characterised 30 years ago but a role in neurological inflammation was entirely unanticipated until they were found to be mutated in AGS. This has led to a hypothesis that accumulation of intracellular nucleic acids occurs as a consequence of mutation in these enzymes and triggers an inflammatory response through activation of innate immune pattern recognition receptors.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:22:37Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:33:38Z : No. of bitstreams: 1 2-s2.0-35349019691.pdf: 4144234 bytes, checksum: 9b86846640bd2e66375c65e289357bd0 (MD5) Made available in DSpace on 2014-05-27T11:22:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-10-24 Aicardi-Goutières syndrome (AGS) is a genetic encephalopathy whose clinical features mimic those of acquired in utero viral infection. AGS exhibits locus heterogeneity, with mutations identified in genes encoding the 3′→5′ exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To define the molecular spectrum of AGS, we performed mutation screening in patients, from 127 pedigrees, with a clinical diagnosis of the disease. Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In five families, we identified an RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no mutations were found. Null mutations were common in TREX1, although a specific missense mutation was observed frequently in patients from northern Europe. Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We identified an RNASEH2C founder mutation in 13 Pakistani families. We also collected clinical data from 123 mutation-positive patients. Two clinical presentations could be delineated: an early-onset neonatal form, highly reminiscent of congenital infection seen particularly with TREX1 mutations, and a later-onset presentation, sometimes occurring after several months of normal development and occasionally associated with remarkably preserved neurological function, most frequently due to RNASEH2B mutations. Mortality was correlated with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations versus 8.0% RNASEH2B mutation-positive patients were known to have died (P = .001). Our analysis defines the phenotypic spectrum of AGS and suggests a coherent mutation-screening strategy in this heterogeneous disorder. Additionally, our data indicate that at least one further AGS-causing gene remains to be identified. © 2007 by The American Society of Human Genetics. All rights reserved. Leeds Institute of Molecular Medicine, Leeds DNA Laboratory Department of Clinical Genetics St. James's University Hospital, Leeds Cancer Research UK Mutation Detection Facility, Leeds Department of Paediatric Neurology Leeds General Infirmary, Leeds Department of Paediatric Neurology Erasme Hospital, Brussels Children's Hospital Queen Fabiola, Brussels Service de Neuropédiatrie Hôpital Trousseau Department of Paediatric Neurology Hôpital Trousseau Pediatric Neurology Department Hôpital Bicêtre Institut de Myologie Groupe Hospitalier Pitié-Salpêtrière Service de Virologie Hôpital Cochin-St. Vincent de Paul, Paris Department of Clinical Biochemistry Hospital Sant Joan de Déu-Ciberer, Barcelona Department of Barcelona Pediatric Neurology Hospital Sant Joan de Déu-Ciberer, Barcelona Department of Paediatrics St. Luke's Hospital, Guardamangia Department of Molecular and Human Genetics Baylor College of Medicine, Houston Serive de Neurologie Centre Hospitalier, Pau Department of Paediatrics Children's Hospital, Sheffield Developmental and Metabolic Neurology Branch National Institute of Neurological Disorders and Stroke National Institutes of Health, Bethesda Department of Human Genetics Rheinisch-Westfälische Technische Hochschule Aachen University, Aachen Unit of Molecular Medicine Bambino Gesù Children's Research Hospital, Rome Istituto di Ricerca e Cura a Carattere Scientifico (IRCCS) Casa Sollievo Della Sofferenza Mendel Institute, Rome Muscular and Neurodegenerative Disease Unit G. Gaslini Institute, Genova Department of Clinical Genetics Churchill Hospital, Oxford Division of Clinical Chemistry and Biochemistry University Children's Hospital, Zurich Clinical Genetics Unit Birmingham Women's Hospital, Birmingham Department of Paediatrics Sandwell and West Birmingham NHS Trust, Birmingham Neurology Department Birmingham Children's Hospital, Birmingham Department of Human Genetics Radboud University, Nijmegen Department of Pediatric Neurology Radboud University, Nijmegen Department of Paediatric Neurology Royal Children's Hospital, Brisbane, QLD Genetic Health Queensland Royal Children's Hospital, Brisbane, QLD Serviço de Aconselhamento Genético Universidade Estadual de São Paulo, Botucatu Academic Unit of Medical Genetics St. Mary's Hospital, Manchester Kinderkrankenhaus Auf der Bult, Hannover Department of Paediatrics Bradford National Health Service (NHS) Trust, Bradford Developmental Neurology Department Fondazione Istituto Neurologico C. Besta, Milan Grampian Clinical Genetics Centre, Aberdeen Department of Neonatology University Hospital, Ghent Department of Pediatrics University Hospital, Ghent Department of Clinical Genetics Maastricht University Hospital, Maastricht Department of Neurology Maastricht University Hospital, Maastricht Department of Paediatrics and Imaging Sciences Imperial College Great Ormond Street Hospital, London St. Mary's NHS Trust Great Ormond Street Hospital, London Department of Ophthalmology Great Ormond Street Hospital, London North East Thames Regional Genetics Service Great Ormond Street Hospital, London Evelina Children's Hospital Guy's and St. Thomas' NHS Trust, London Department of Paediatrics Université Laval Medical School, Québec Clinical Genetics Unit Hospital de Cruces, Baracaldo Service de Génétique Médicale Centre Hospitalier Universitaire Pellegrin Enfants, Bordeaux Unité de Neurologie de l'Enfant et de l'Adolescent Centre Hospitalier Universitaire Pellegrin Enfants, Bordeaux National Centre for Medical Genetics Our Lady's Hospital, Dublin Department of Paediatric Neurology Children's University Hospital, Dublin Department of Medical Genetics Rikshospitalet-Radiumhospitalet, Oslo Department of Paediatrics Rikshospitalet-Radiumhospitalet, Oslo Rikshospitalet-Radiumhospitalet, Oslo Department of Pediatrics Academic Medical Center, Amsterdam Department of Child Neurology Vrije Universiteit Medical Center, Amsterdam Medical Research Council Human Genetics Unit Western General Hospital, Edinburgh Department of Clinical Genetics Leiden University Medical Center, Leiden Division of Pediatric Neurology Oregon Health and Science University, Portland, OR Pediatric Neurology Klinikum Aschaffenburg, Aschaffenburg Department of Neurology Royal Children's Hospital, Parkville, Vic. Division of Clinical Genetics Department for Medical Genetics Medical University Innsbruck, Innsbruck Department of Pediatrics Division of Pediatric Neurology and Inborn Errors of Metabolism Children's Hospital Innsbruck, Innsbruck Klinik für Kinder und Jugendliche, Konstanz Paediatric Neurology University Hospitals of Gasthuisberg, Leuven Department of Child Neurology and Psychiatry IRCCS Casimiro Mondino Institute of Neurology, Pavia Department of Neurogenetics School of Medicine of Ribeirao Preto, Ribeirao Preto Greenwood Genetic Center, Greenwood, SC Raphael Recanati Genetic Institute Rabin Medical Center, Petach-Tikva Department of Paediatrics Crosshouse Hospital, Ayr Fraser of Allander Neurosciences Unit Royal Hospital for Sick Children, Glasgow Duncan Guthrie Institute of Medical Genetics Royal Hospital for Sick Children, Glasgow Division of Medical Genetics Montreal Children's Hospital, Montreal Department of Paediatric Neurology University Hospitals of Leicester NHS Trust, Leicester University Hospital of Aarhus, Aarhus Division of Pediatric Dermatology British Columbia's Children's Hospital, Vancouver, BC Institut de Pathologie et de Génétique, Gosselies Pediatric Neurology Department Guide Chauliac Hospital, Montpellier Servicio de Pediatría Hospital Universitario Doctor Peset, Valencia Genetic Institute Ha'Emek Medical Center, Afula Rappaport Faculty of Medicine Technion, Haifa Neuropediatrics Unit Complejo Hospitalario de Jean, Jean Department of Paediatrics Manor Hospital, Walsall Division of Neuropediatrics University Hospital, Freiburg Genetic Health Services Victoria Royal Children's Hospital, Vic. Service de Génétique Hôpital Debrousse, Lyon Lancashire Teaching Hospitals Trust, Preston Neonatal Intensive Care Unit Arcispedale Santa Maria Nuova, Reggio Emilia Center for Medical Genetics, Antwerp Department of Neurology Children's National Medical Center, Washington, DC Department of Neuropediatrics Humboldt University, Berlin Leeds Institute of Molecular Medicine St. James's University Hospital Wellcome Trust Brenner Building, Leeds LS9 7TF Serviço de Aconselhamento Genético Universidade Estadual de São Paulo, Botucatu

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity., La lista completa de autores que integran el documento puede consultarse en el archivo, Instituto de Física de Líquidos y Sistemas Biológicos

Contains fulltext : 50027.pdf (Publisher’s version ) (Closed access) Aicardi-Goutieres syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection. Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype. Here, we show that TREX1, encoding the major mammalian 3' --> 5' DNA exonuclease, is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1(-/-) mouse leads to an inflammatory phenotype. Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.

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.

Primary microcephaly is an autosomal recessive disorder characterized by marked reduction in human brain size. Microcephalin (MCPH1), one of the genes mutated in primary microcephaly, plays an important role in DNA damage checkpoint control and mitotic entry. Additionally, MCPH1 ensures the proper temporal activation of chromosome condensation during mitosis, by acting as a negative regulator of the condensin II complex. We previously found that deletion of the of the MCPH1 N terminus leads to the premature chromosome condensation (PCC) phenotype. In the present study, we unexpectedly observed that a truncated form of MCPH1 appears to be expressed in MCPH1(S25X/S25X) patient cells. This likely results from utilization of an alternative translational start codon, which would produce a mutant MCPH1 protein with a small deletion of its N-terminal BRCT domain. Furthermore, missense mutations in the MCPH1 cluster at its N terminus, suggesting that intact function of this BRCT protein-interaction domain is required both for coordinating chromosome condensation and human brain development. Subsequently, we identified the SET nuclear oncogene as a direct binding partner of the MCPH1 N-terminal BRCT domain. Cells with SET knockdown exhibited abnormal condensed chromosomes similar to those observed in MCPH1-deficient mouse embryonic fibroblasts. Condensin II knockdown rescued the abnormal chromosome condensation phenotype in SET-depleted cells. In addition, MCPH1 V50G/I51V missense mutations, impair binding to SET and fail to fully rescue the abnormal chromosome condensation phenotype in Mcph1(-/-) mouse embryonic fibroblasts. Collectively, our findings suggest that SET is an important regulator of chromosome condensation/decondensation and that disruption of the MCPH1-SET interaction might be important for the pathogenesis of primary microcephaly.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive neurological disorder, the clinical and immunological features of which parallel those of congenital viral infection. Here we define the composition of the human ribonuclease H2 enzyme complex and show that AGS can result from mutations in the genes encoding any one of its three subunits. Our findings demonstrate a role for ribonuclease H in human neurological disease and suggest an unanticipated relationship between ribonuclease H2 and the antiviral immune response that warrants further investigation.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

Persistently activated Stat3 is found in many different cancers, including ≈60% of breast tumors. Here, we demonstrate that a constitutively activated Stat3 transforms immortalized human mammary epithelial cells and that this oncogenic event requires the activity of matrix metalloproteinase-9 (MMP-9). By immunohistochemical analysis, we observe a positive correlation between strong MMP-9 expression and tyrosine phosphorylated Stat3 in primary breast cancer specimens. These results demonstrate a relationship between activated Stat3 and MMP-9 in breast oncogenesis.

In recent years, a number of proteins have been identified that can modify the activities of the Wilms' Tumor 1 (WT1) proteins. One of these modifiers is the p53 protein. To investigate a genetic interaction between the p53 gene and the wt1 gene, we have crossed their respective knockout mice. The absence of p53 appears to have no gross effect on the phenotype of wt1-null mice. Both wt1-null and double-null embryos develop pericardial bleeding and die in utero. In adult p53-null mice, wt1-heterozygosity (wt1het) predisposes to an earlier onset of lymphomagenesis and the development of kidney abnormalities resembling oncocytoma in humans. wt1-heterozygosity alone predisposes to the development of glomerular sclerosis.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients’ extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which environmental exposures like virus infection and UV-irradiation trigger activation of the innate and adaptive immune system in genetically predisposed individuals. Heterozygous mutations of the 3' repair exonuclease 1 (TREX1) are associated with SLE. Biallelic mutations in TREX1 and the three subunits of ribonuclease H2 (RNASEH2A-C) cause Aicardi-Goutieres syndrome, an inflammatory encephalopathy with clinical overlap with SLE. We therefore investigated the role of RNase H2 in SLE pathogenesis. RNase H2 is responsible for the removal of misincorporated ribonucleotides from DNA and is indispensable for genome integrity. We demonstrated a genetic association for rare RNase H2 sequence variants with SLE. RNase H2-deficient fibroblasts of AGS and SLE patients accumulated ribonucleotides in genomic DNA resulting in chronic low-level DNA damage, constitutive p53 phosphorylation and senescence. Patient fibroblasts proliferated slower than fibroblasts from healthy individuals and showed impairment of cell cycle progression. In addition, patient fibroblasts exhibited constitutive up-regulation of interferon-stimulated genes and an enhanced type I interferon response to the nucleic acid poly(I:C) and UV-irradiation. UV-irradiation induced enhanced cyclobutane pyrimidine dimer formation in ribonucleotide-containing DNA. This suggests that innate immune activation may be caused by immune recognition of DNA metabolites of DNA damage repair and may also explain photosensitivity in SLE patients with RNase H2 mutation. In summary, our findings implicate RNase H2 in the pathogenesis of SLE, and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.