Congenital long QT syndrome (LQTS) is an inherited channelopathy associated with life-threatening arrhythmias. LQTS type 2 (LQT2) is caused by mutations in KCNH2, which encodes the potassium channel hERG. We hypothesized that modifier genes are partly responsible for the variable phenotype severity observed in some LQT2 families. Here, we identified contributors to variable expressivity in an LQT2 family by using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) and whole exome sequencing in a synergistic manner. We found that iPSC-CMs recapitulated the clinical genotype-phenotype discordance in vitro. Importantly, iPSC-CMs derived from the severely affected LQT2 patients displayed prolonged action potentials compared with cells from mildly affected first-degree relatives. The iPSC-CMs derived from all patients with hERG R752W mutation displayed lower IKr amplitude. Interestingly, iPSC-CMs from severely affected mutation-positive individuals exhibited greater L-type Ca2+ current. Whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2, providing biologically plausible explanations for this variable expressivity. Genome editing to correct a REM2 variant reversed the enhanced L-type Ca2+ current and prolonged action potential observed in iPSC-CMs from severely affected individuals. Thus, our findings showcase the power of combining complementary physiological and genomic analyses to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. Furthermore, we propose that this strategy can be deployed to unravel myriad confounding pathologies displaying variable expressivity.
Sam Chai, Xiaoping Wan, Angelina Ramirez-Navarro, Paul J. Tesar, Elizabeth S. Kaufman, Eckhard Ficker, Alfred L. George Jr., Isabelle Deschênes
A modifier variant can abrogate risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin-homology-domain of GAB1, an essential scaffold in the MET/HGF pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescues the GAB1 associated morphant phenotype. In mouse, GAB1 and METTL13 co-localize in auditory sensory neurons, and METTL13 co-immunoprecipitates with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET-signaling provides potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET-signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET-signaling is likely to have broad clinical implications.
Rizwan Yousaf, Zubair M. Ahmed, Arnaud P.J. Giese, Robert J. Morell, Ayala Lagziel, Alain Dabdoub, Edward R. Wilcox, Sheikh Riazuddin, Thomas B. Friedman, Saima Riazuddin
Profound hyperphagia is a major disabling feature of Prader-Willi syndrome (PWS). Characterization of the mechanisms that underlie PWS-associated hyperphagia has been slowed by the paucity of animal models with increased food intake or obesity. Mice with a microdeletion encompassing the Snord116 cluster of noncoding RNAs encoded within the Prader-Willi minimal deletion critical region have previously been reported to show growth retardation and hyperphagia. Here, consistent with previous reports, we observed growth retardation in Snord116+/–P mice with a congenital paternal Snord116 deletion. However, these mice neither displayed increased food intake nor had reduced hypothalamic expression of the proprotein convertase 1 gene PCSK1 or its upstream regulator NHLH2, which have recently been suggested to be key mediators of PWS pathogenesis. Specifically, we disrupted Snord116 expression in the mediobasal hypothalamus in Snord116fl mice via bilateral stereotaxic injections of a Cre-expressing adeno-associated virus (AAV). While the Cre-injected mice had no change in measured energy expenditure, they became hyperphagic between 9 and 10 weeks after injection, with a subset of animals developing marked obesity. In conclusion, we show that selective disruption of Snord116 expression in the mediobasal hypothalamus models the hyperphagia of PWS.
Joseph Polex-Wolf, Brian Y.H. Lam, Rachel Larder, John Tadross, Debra Rimmington, Fàtima Bosch, Verónica Jiménez Cenzano, Eduard Ayuso, Marcella K.L. Ma, Kara Rainbow, Anthony P. Coll, Stephen O’Rahilly, Giles S.H. Yeo
A critical event in the adaptation to extrauterine life is relaxation of the pulmonary vasculature at birth, allowing for a rapid increase in pulmonary blood flow that is essential for efficient gas exchange. Failure of this transition leads to pulmonary hypertension (PH), a major cause of newborn mortality associated with preterm birth, infection, hypoxia, and malformations including congenital diaphragmatic hernia (CDH). While individual vasoconstrictor and dilator genes have been identified, the coordination of their expression is not well understood. Here, we found that lung mesenchyme–specific deletion of CDH-implicated genes encoding pre–B cell leukemia transcription factors (Pbx) led to lethal PH in mice shortly after birth. Loss of Pbx genes resulted in the misexpression of both vasoconstrictors and vasodilators in multiple pathways that converge to increase phosphorylation of myosin in vascular smooth muscle (VSM) cells, causing persistent constriction. While targeting endothelin and angiotensin, which are upstream regulators that promote VSM contraction, was not effective, treatment with the Rho-kinase inhibitor Y-27632 reduced vessel constriction and PH in Pbx-mutant mice. These results demonstrate a lung-intrinsic, herniation-independent cause of PH in CDH. More broadly, our findings indicate that neonatal PH can result from perturbation of multiple pathways and suggest that targeting the downstream common effectors may be a more effective treatment for neonatal PH.
David J. McCulley, Mark D. Wienhold, Elizabeth A. Hines, Timothy A. Hacker, Allison Rogers, Ryan J. Pewowaruk, Rediet Zewdu, Naomi C. Chesler, Licia Selleri, Xin Sun
Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
Alexia T. Kedves, Scott Gleim, Xiaoyou Liang, Dennis M. Bonal, Frederic Sigoillot, Fred Harbinski, Sneha Sanghavi, Christina Benander, Elizabeth George, Prafulla C. Gokhale, Quang-De Nguyen, Paul T. Kirschmeier, Robert J. Distel, Jeremy Jenkins, Michael S. Goldberg, William C. Forrester
Dysregulated adipocyte physiology leads to imbalanced energy storage, obesity, and associated diseases, imposing a costly burden on current health care. Cannabinoid receptor type-1 (CB1) plays a crucial role in controlling energy metabolism through central and peripheral mechanisms. In this work, adipocyte-specific inducible deletion of the CB1 gene (Ati-CB1–KO) was sufficient to protect adult mice from diet-induced obesity and associated metabolic alterations and to reverse the phenotype in already obese mice. Compared with controls, Ati-CB1–KO mice showed decreased body weight, reduced total adiposity, improved insulin sensitivity, enhanced energy expenditure, and fat depot–specific cellular remodeling toward lowered energy storage capacity and browning of white adipocytes. These changes were associated with an increase in alternatively activated macrophages concomitant with enhanced sympathetic tone in adipose tissue. Remarkably, these alterations preceded the appearance of differences in body weight, highlighting the causal relation between the loss of CB1 and the triggering of metabolic reprogramming in adipose tissues. Finally, the lean phenotype of Ati-CB1–KO mice and the increase in alternatively activated macrophages in adipose tissue were also present at thermoneutral conditions. Our data provide compelling evidence for a crosstalk among adipocytes, immune cells, and the sympathetic nervous system (SNS), wherein CB1 plays a key regulatory role.
Inigo Ruiz de Azua, Giacomo Mancini, Raj Kamal Srivastava, Alejandro Aparisi Rey, Pierre Cardinal, Laura Tedesco, Cristina Maria Zingaretti, Antonia Sassmann, Carmelo Quarta, Claudia Schwitter, Andrea Conrad, Nina Wettschureck, V. Kiran Vemuri, Alexandros Makriyannis, Jens Hartwig, Maria Mendez-Lago, Laura Bindila, Krisztina Monory, Antonio Giordano, Saverio Cinti, Giovanni Marsicano, Stefan Offermanns, Enzo Nisoli, Uberto Pagotto, Daniela Cota, Beat Lutz
Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond–like phenotype.
Raphael Carapito, Martina Konantz, Catherine Paillard, Zhichao Miao, Angélique Pichot, Magalie S. Leduc, Yaping Yang, Katie L. Bergstrom, Donald H. Mahoney, Deborah L. Shardy, Ghada Alsaleh, Lydie Naegely, Aline Kolmer, Nicodème Paul, Antoine Hanauer, Véronique Rolli, Joëlle S. Müller, Elisa Alghisi, Loïc Sauteur, Cécile Macquin, Aurore Morlon, Consuelo Sebastia Sancho, Patrizia Amati-Bonneau, Vincent Procaccio, Anne-Laure Mosca-Boidron, Nathalie Marle, Naël Osmani, Olivier Lefebvre, Jacky G. Goetz, Sule Unal, Nurten A. Akarsu, Mirjana Radosavljevic, Marie-Pierre Chenard, Fanny Rialland, Audrey Grain, Marie-Christine Béné, Marion Eveillard, Marie Vincent, Julien Guy, Laurence Faivre, Christel Thauvin-Robinet, Julien Thevenon, Kasiani Myers, Mark D. Fleming, Akiko Shimamura, Elodie Bottollier-Lemallaz, Eric Westhof, Claudia Lengerke, Bertrand Isidor, Seiamak Bahram
The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient’s primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation’s aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.
Davor Lessel, Danyi Wu, Carlos Trujillo, Thomas Ramezani, Ivana Lessel, Mohammad K. Alwasiyah, Bidisha Saha, Fuki M. Hisama, Katrin Rading, Ingrid Goebel, Petra Schütz, Günter Speit, Josef Högel, Holger Thiele, Gudrun Nürnberg, Peter Nürnberg, Matthias Hammerschmidt, Yan Zhu, David R. Tong, Chen Katz, George M. Martin, Junko Oshima, Carol Prives, Christian Kubisch
Acute myelogenous leukemia (AML) frequently relapses after complete remission (CR), necessitating improved detection and phenotypic characterization of treatment-resistant residual disease. In this work, we have optimized droplet digital PCR to broadly measure mutated alleles of recurrently mutated genes in CR marrows of AML patients at levels as low as 0.002% variant allele frequency. Most gene mutations persisted in CR, albeit at highly variable and gene-dependent levels. The majority of AML cases demonstrated residual aberrant oligoclonal hematopoiesis. Importantly, we detected very rare cells (as few as 1 in 15,000) that were genomically similar to the dominant blast populations at diagnosis and were fully clonally represented at relapse, identifying these rare cells as one common source of AML relapse. Clinically, the mutant allele burden was associated with overall survival in AML, and our findings narrow the repertoire of gene mutations useful in minimal residual disease–based prognostication in AML. Overall, this work delineates rare cell populations that cause AML relapse, with direct implications for AML research directions and strategies to improve AML therapies and outcome.
Brian Parkin, Angelina Londoño-Joshi, Qing Kang, Muneesh Tewari, Andrew D. Rhim, Sami N. Malek
Varicella zoster virus (VZV) typically causes chickenpox upon primary infection. In rare cases, VZV can give rise to life-threatening disease in otherwise healthy people, but the immunological basis for this remains unexplained. We report 4 cases of acute severe VZV infection affecting the central nervous system or the lungs in unrelated, otherwise healthy children who are heterozygous for rare missense mutations in POLR3A (one patient), POLR3C (one patient), or both (two patients). POLR3A and POLR3C encode subunits of RNA polymerase III. Leukocytes from all 4 patients tested exhibited poor IFN induction in response to synthetic or VZV-derived DNA. Moreover, leukocytes from 3 of the patients displayed defective IFN production upon VZV infection and reduced control of VZV replication. These phenotypes were rescued by transduction with relevant WT alleles. This work demonstrates that monogenic or digenic POLR3A and POLR3C deficiencies confer increased susceptibility to severe VZV disease in otherwise healthy children, providing evidence for an essential role of a DNA sensor in human immunity.
Benson Ogunjimi, Shen-Ying Zhang, Katrine B. Sørensen, Kristian A. Skipper, Madalina Carter-Timofte, Gaspard Kerner, Stefanie Luecke, Thaneas Prabakaran, Yujia Cai, Josephina Meester, Esther Bartholomeus, Nikhita Ajit Bolar, Geert Vandeweyer, Charlotte Claes, Yasmine Sillis, Lazaro Lorenzo, Raffaele A. Fiorenza, Soraya Boucherit, Charlotte Dielman, Steven Heynderickx, George Elias, Andrea Kurotova, Ann Vander Auwera, Lieve Verstraete, Lieven Lagae, Helene Verhelst, Anna Jansen, Jose Ramet, Arvid Suls, Evelien Smits, Berten Ceulemans, Lut Van Laer, Genevieve Plat Wilson, Jonas Kreth, Capucine Picard, Horst Von Bernuth, Joël Fluss, Stephane Chabrier, Laurent Abel, Geert Mortier, Sebastien Fribourg, Jacob Giehm Mikkelsen, Jean-Laurent Casanova, Søren R. Paludan, Trine H. Mogensen