Genetics
Abstract
Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and a SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type 3 and 4, respectively. Here, we demonstrate that classical non-homologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7 and HELLS deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells mutated for the other ICF genes DNMT3B and ZBTB24 (responsible for ICF type 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.
Authors
Motoko Unoki, Hironori Funabiki, Guillaume Velasco, Claire Francastel, Hiroyuki Sasaki
Abstract
Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.
Authors
Daniela A. Braun, Svjetlana Lovric, David Schapiro, Ronen Schneider, Jonathan Marquez, Maria Asif, Muhammad Sajid Hussain, Ankana Daga, Eugen Widmeier, Jia Rao, Shazia Ashraf, Weizhen Tan, C. Patrick Lusk, Amy Kolb, Tilman Jobst-Schwan, Johanna Magdalena Schmidt, Charlotte A. Hoogstraten, Kaitlyn Eddy, Thomas M. Kitzler, Shirlee Shril, Abubakar Moawia, Kathrin Schrage, Arwa Ishaq A. Khayyat, Jennifer A. Lawson, Heon Yung Gee, Jillian K. Warejko, Tobias Hermle, Amar J. Majmundar, Hannah Hugo, Birgit Budde, Susanne Motameny, Janine Altmüller, Angelika Anna Noegel, Hanan M. Fathy, Daniel P. Gale, Syeda Seema Waseem, Ayaz Khan, Larissa Kerecuk, Seema Hashmi, Nilufar Mohebbi, Robert Ettenger, Erkin Serdaroğlu, Khalid A. Alhasan, Mais Hashem, Sara Goncalves, Gema Ariceta, Mercedes Ubetagoyena, Wolfram Antonin, Shahid Mahmood Baig, Fowzan S. Alkuraya, Qian Shen, Hong Xu, Corinne Antignac, Richard P. Lifton, Shrikant Mane, Peter Nürnberg, Mustafa K. Khokha, Friedhelm Hildebrandt
Abstract
BACKGROUND. Understanding the integrated immunogenomic landscape of advanced prostate cancer (APC) could impact stratified treatment selection. METHODS. Defective mismatch repair (dMMR) status was determined by either loss of mismatch repair protein expression on IHC or microsatellite instability (MSI) by PCR in 127 APC biopsies from 124 patients (Royal Marsden [RMH] cohort); MSI by targeted panel next-generation sequencing (MSINGS) was then evaluated in the same cohort and in 254 APC samples from the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF). Whole exome sequencing (WES) data from this latter cohort were analyzed for pathogenic MMR gene variants, mutational load, and mutational signatures. Transcriptomic data, available for 168 samples, was also performed. RESULTS. Overall, 8.1% of patients in the RMH cohort had some evidence of dMMR, which associated with decreased overall survival. Higher MSINGS scores associated with dMMR, and these APCs were enriched for higher T cell infiltration and PD-L1 protein expression. Exome MSINGS scores strongly correlated with targeted panel MSINGS scores (r = 0.73, P < 0.0001), and higher MSINGS scores associated with dMMR mutational signatures in APC exomes. dMMR mutational signatures also associated with MMR gene mutations and increased immune cell, immune checkpoint, and T cell–associated transcripts. APC with dMMR mutational signatures overexpressed a variety of immune transcripts, including CD200R1, BTLA, PD-L1, PD-L2, ADORA2A, PIK3CG, and TIGIT. CONCLUSION. These data could impact immune target selection, combination therapeutic strategy selection, and selection of predictive biomarkers for immunotherapy in APC. FUNDING. We acknowledge funding support from Movember, Prostate Cancer UK, The Prostate Cancer Foundation, SU2C, and Cancer Research UK.
Authors
Daniel Nava Rodrigues, Pasquale Rescigno, David Liu, Wei Yuan, Suzanne Carreira, Maryou B. Lambros, George Seed, Joaquin Mateo, Ruth Riisnaes, Stephanie Mullane, Claire Margolis, Diana Miao, Susana Miranda, David Dolling, Matthew Clarke, Claudia Bertan, Mateus Crespo, Gunther Boysen, Ana Ferreira, Adam Sharp, Ines Figueiredo, Daniel Keliher, Saud Aldubayan, Kelly P. Burke, Semini Sumanasuriya, Mariane Sousa Fontes, Diletta Bianchini, Zafeiris Zafeiriou, Larissa Sena Teixeira Mendes, Kent Mouw, Michael T. Schweizer, Colin C. Pritchard, Stephen Salipante, Mary-Ellen Taplin, Himisha Beltran, Mark A. Rubin, Marcin Cieslik, Dan Robinson, Elizabeth Heath, Nikolaus Schultz, Joshua Armenia, Wassim Abida, Howard Scher, Christopher Lord, Alan D’Andrea, Charles L. Sawyers, Arul M. Chinnaiyan, Andrea Alimonti, Peter S. Nelson, Charles G. Drake, Eliezer M. Van Allen, Johann S. de Bono
Abstract
The mechanisms that drive T cell aging are not understood. We report children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell aging phenotypes seen in adults five decades older including depleted naïve T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting newborn screening may identify individuals with germline telomere maintenance defects. Telomerase null mice with short TL showed defects throughout T cell development including increased apoptosis of stimulated thymocytes, their intra-thymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells up-regulated DNA damage and intrinsic apoptosis pathways, while older adult T cells up-regulated extrinsic apoptosis pathways and PD-1 expression. T cells from mice with short TL also showed an active DNA damage response, in contrast to old wild-type mice, despite their shared propensity to apoptosis. Our data suggest there are telomere length-dependent and telomere length-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.
Authors
Christa L. Wagner, Vidya Sagar Hanumanthu, C. Conover Talbot Jr., Roshini S. Abraham, David Hamm, Dustin L. Gable, Christopher G. Kanakry, Carolyn D. Applegate, Janet Siliciano, J. Brooks Jackson, Stephen V. Desiderio, Jonathan K. Alder, Leo Luznik, Mary Armanios
Abstract
Quantitative abnormalities of the von Willebrand factor–factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII. However, the mechanistic basis and pathophysiological consequences of this association are unknown. We have demonstrated that stabilin-2–expressing cells bind and internalize human VWF and FVIII in a VWF-dependent manner, and stabilin-2–deficient mice displayed prolonged human VWF-FVIII half-life compared with controls. The stabilin-2 variant p.E2377K significantly decreased stabilin-2 expression and impaired VWF endocytosis in a heterologous expression system, and common STAB2 variants associated with plasma VWF levels in type 1 von Willebrand disease patients. STAB2-deficient mice displayed a decreased immunogenic response to human VWF-FVIII complex, while coinfusion of human VWF-FVIII with the stabilin-2 ligand hyaluronic acid attenuated the immune response to exogenous FVIII. Collectively, these data suggest that stabilin-2 functions as both a clearance and an immunoregulatory receptor for VWF-FVIII, making stabilin-2 a novel molecular target for modification of the half-life of VWF-FVIII and the immune response to VWF-FVIII concentrates.
Authors
Laura L. Swystun, Jesse D. Lai, Colleen Notley, Ilinca Georgescu, A. Simonne Paine, Jeff Mewburn, Kate Nesbitt, Kai Schledzewski, Cyrill Géraud, Julia Kzhyshkowska, Sergij Goerdt, Wilma Hopman, Robert R. Montgomery, Paula D. James, David Lillicrap
Abstract
Mutations underlie all cancers, and their identification and study are the foundation of cancer biology. We describe what we believe to be a novel approach to mutagenesis and cancer studies based on the DNA polymerase ε (POLE) ultramutator phenotype recently described in human cancers, in which a single amino acid substitution (most commonly P286R) in the proofreading domain results in error-prone DNA replication. We engineered a conditional PoleP286R allele in mice. PoleP286R/+ embryonic fibroblasts exhibited a striking mutator phenotype and immortalized more efficiently. PoleP286R/+ mice were born at Mendelian ratios but rapidly developed lethal cancers of diverse lineages, yielding the most cancer-prone monoallelic model described to date, to our knowledge. Comprehensive whole-genome sequencing analyses showed that the cancers were driven by high base substitution rates in the range of human cancers, overcoming a major limitation of previous murine cancer models. These data establish polymerase-mediated ultramutagenesis as an efficient in vivo approach for the generation of diverse animal cancer models that recapitulate the high mutational loads inherent to human cancers.
Authors
Hao-Dong Li, Ileana Cuevas, Musi Zhang, Changzheng Lu, Md Maksudul Alam, Yang-Xin Fu, M. James You, Esra A. Akbay, He Zhang, Diego H. Castrillon
Abstract
Bi-allelic loss-of-function mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only one patient. We report 24 p40phox-deficient patients from 12 additional families in eight countries. These patients display eight different in-frame or out-of-frame mutations of NCF4, homozygous in 11 families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be loss-of-function, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was subnormal in the patients’ neutrophils, whereas PMA-induced DHR oxidation, which is widely used as a diagnostic test for CGD, was normal in some of the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also subnormal, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients. The patients described here suffer from hyperinflammation and peripheral infections, but they do not display any of the invasive bacterial and fungal infections seen in CGD. In conclusion, inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD
Authors
Annemarie van de Geer, Alejandro Nieto-Patlán, Douglas B. Kuhns, Anton T.J. Tool, Andrés A. Arias, Matthieu Bouaziz, Martin de Boer, José Luis Franco, Roel P. Gazendam, John L. van Hamme, Michel van Houdt, Karin van Leeuwen, Paul J.H. Verkuijlen, Timo K. van den Berg, Juan F. Alzate, Carlos A. Arango-Franco, Vritika Batura, Andrea R. Bernasconi, Barbara Boardman, Claire Booth, Siobhan O. Burns, Felipe Cabarcas, Nadine Cerf Bensussan, Fabienne Charbit-Henrion, Anniek Corveleyn, Caroline Deswarte, María Esnaola Azcoiti, Dirk Foell, John I. Gallin, Carlos Garcés, Margarida Guedes, Claas H. Hinze, Steven M. Holland, Stephen M. Hughes, Patricio Ibañez, Harry L. Malech, Isabelle Meyts, Marcela Moncada-Velez, Kunihiko Moriya, Esmeralda Neves, Matias Oleastro, Laura Perez, Vimel Rattina, Carmen Oleaga-Quintas, Neil Warner, Aleixo M. Muise, Jeanet Serafin López, Eunice Trindade, Julia Vasconselos, Severine Vermeire, Helmut Wittkowski, Austen Worth, Laurent Abel, Mary C. Dinauer, Peter D. Arkwright, Dirk Roos, Jean-Laurent Casanova, Taco W. Kuijpers, Jacinta Bustamante
Abstract
BACKGROUND. The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism and behaviour, and it is essential to health. Whereas time-of-day adapted therapy is increasingly reported to be highly successful, it needs to be personalized since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors including genetic predisposition, age, gender, environmental light levels and season. An easy and convenient diagnostic tool is currently missing. METHODS. To establish a validated test, we followed a three-stage biomarker development strategy: (i) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; (ii) these biomarkers were migrated to a clinically relevant gene expression-profiling platform (NanoString), and (iii) externally validated using an independent study with 28 early or late chronotypes. RESULTS. We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard dim light melatonin onset method at smaller monetary, time and sample number cost. CONCLUSION. The BodyTime assay provides a new diagnostic tool for personalization of healthcare according to the patient’s circadian clock. FUNDING. This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.
Authors
Nicole Wittenbrink, Bharath Ananthasubramaniam, Mirjam Münch, Barbara Koller, Bert Maier, Charlotte Weschke, Frederik Bes, Jan de Zeeuw, Claudia Nowozin, Amely Wahnschaffe, Sophia Wisniewski, Mandy Zaleska, Osnat Bartok, Reut Ashwal-Fluss, Hedwig Lammert, Hanspeter Herzel, Michael Hummel, Sebastian Kadener, Dieter Kunz, Achim Kramer
Abstract
Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.
Authors
David Boutboul, Hye Sun Kuehn, Zoé Van de Wyngaert, Julie E. Niemela, Isabelle Callebaut, Jennifer Stoddard, Christelle Lenoir, Vincent Barlogis, Catherine Farnarier, Frédéric Vely, Nao Yoshida, Seiji Kojima, Hirokazu Kanegane, Akihiro Hoshino, Fabian Hauck, Ludovic Lhermitte, Vahid Asnafi, Philip Roehrs, Shaoying Chen, James W. Verbsky, Katherine R. Calvo, Ammar Husami, Kejian Zhang, Joseph Roberts, David Amrol, John Sleaseman, Amy P. Hsu, Steven M. Holland, Rebecca Marsh, Alain Fischer, Thomas A. Fleisher, Capucine Picard, Sylvain Latour, Sergio D. Rosenzweig
Abstract
Epigenetic modifications control cancer development and clonal evolution in various cancer types. Here, we show that loss of the male-specific histone demethylase lysine-specific demethylase 5D (KDM5D) encoded on the Y chromosome epigenetically modifies histone methylation marks and alters gene expression, resulting in aggressive prostate cancer. Fluorescent in situ hybridization demonstrated that segmental or total deletion of the Y chromosome in prostate cancer cells is one of the causes of decreased KDM5D mRNA expression. The result of ChIP-sequencing analysis revealed that KDM5D preferably binds to promoter regions with coenrichment of the motifs of crucial transcription factors that regulate the cell cycle. Loss of KDM5D expression with dysregulated H3K4me3 transcriptional marks was associated with acceleration of the cell cycle and mitotic entry, leading to increased DNA-replication stress. Analysis of multiple clinical data sets reproducibly showed that loss of expression of KDM5D confers a poorer prognosis. Notably, we also found stress-induced DNA damage on the serine/threonine protein kinase ATR with loss of KDM5D. In KDM5D-deficient cells, blocking ATR activity with an ATR inhibitor enhanced DNA damage, which led to subsequent apoptosis. These data start to elucidate the biological characteristics resulting from loss of KDM5D and also provide clues for a potential novel therapeutic approach for this subset of aggressive prostate cancer.
Authors
Kazumasa Komura, Yuki Yoshikawa, Teppei Shimamura, Goutam Chakraborty, Travis A. Gerke, Kunihiko Hinohara, Kalyani Chadalavada, Seong Ho Jeong, Joshua Armenia, Shin-Yi Du, Ying Z. Mazzu, Kohei Taniguchi, Naokazu Ibuki, Clifford A. Meyer, Gouri J. Nanjangud, Teruo Inamoto, Gwo-Shu Mary Lee, Lorelei A. Mucci, Haruhito Azuma, Christopher J. Sweeney, Philip W. Kantoff
Copyright © 2018 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)