Genetics
Abstract
Vacuolar H+-ATPase–dependent (V-ATPase–dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell–derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase–dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase–dependent signaling and protein degradation in the developing human central nervous system.
Authors
Takuo Hirose, Alfredo Cabrera-Socorro, David Chitayat, Thomas Lemonnier, Olivier Féraud, Carmen Cifuentes-Diaz, Nicolas Gervasi, Cedric Mombereau, Tanay Ghosh, Loredana Stoica, Jeanne d’Arc Al Bacha, Hiroshi Yamada, Marcel A. Lauterbach, Marc Guillon, Kiriko Kaneko, Joy W. Norris, Komudi Siriwardena, Susan Blasér, Jérémie Teillon, Roberto Mendoza-Londono, Marion Russeau, Julien Hadoux, Sadayoshi Ito, Pierre Corvol, Maria G. Matheus, Kenton R. Holden, Kohji Takei, Valentina Emiliani, Annelise Bennaceur-Griscelli, Charles E. Schwartz, Genevieve Nguyen, Matthias Groszer
Abstract
BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.
Authors
Christopher R. McEvoy, Huiling Xu, Kortnye Smith, Dariush Etemadmoghadam, Huei San Leong, David Y. Choong, David J. Byrne, Amir Iravani, Sophie Beck, Linda Mileshkin, Richard W. Tothill, David D. Bowtell, Bindi M. Bates, Violeta Nastevski, Judy Browning, Anthony H. Bell, Chloe Khoo, Jayesh Desai, Andrew P. Fellowes, Stephen B. Fox, Owen W.J. Prall
Abstract
Background. Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies, however, the entire spectrum of sphingolipid metabolism disorders remained elusive. Methods. A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder.Results. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous system, we identified a homozygous p.(Ala280Val) variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species which was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9-derived DEGS1 knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared to wild type cells which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. Conclusion. We report DEGS1 dysfunction as cause for a novel sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous system.Trial registration. Not applicable.Funding. RESOLVE: Project number 305707; SNF: Project 31003A_153390/1; Rare Disease Initiative Zurich.
Authors
Gergely Karsai, Florian Kraft, Natja Haag, G. Christoph Korenke, Benjamin Hänisch, Alaa Othman, Saranya Suriyanarayanan, Regula Steiner, Cordula Knopp, Michael Mull, Markus Bergmann, J. Michael Schröder, Joachim Weis, Miriam Elbracht, Matthias Begemann, Thorsten Hornemann, Ingo Kurth
Abstract
Prostate cancer (PCa) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation anti-androgen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable up-regulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of TGF-β pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 down-regulation induces TGF-β signaling, EMT, and cell motility, which is effectively blocked by TGF-β receptor I inhibitor Galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-β signaling, indicated by SMAD2 phosphorylation, in CRPC as compared to primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PCa cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-β signaling and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.
Authors
Bing Song, Su-Hong Park, Jonathan C. Zhao, Ka-wing Fong, Shangze Li, Yongik Lee, Yeqing A. Yang, Subhasree Sridhar, Xiaodong Lu, Sarki A. Abdulkadir, Robert L. Vessella, Colm Morrissey, Timothy M. Kuzel, William J. Catalona, Ximing J. Yang, Jindan Yu
Abstract
Recurrent broad-scale heterozygous deletions are frequently observed in human cancer. Here we tested the hypothesis that compound haploinsufficiency of neighboring genes at chromosome 8p promotes tumorigenesis. By targeting the mouse orthologs of human DOK2 and DUSP4 genes, which were co-deleted in approximately half of human lung adenocarcinomas, we found that compound-heterozygous deletion of Dok2 and Dusp4 in mice resulted in lung tumorigenesis with short latency and high incidence, and that their co-deletion synergistically activated MAPK signaling and promoted cell proliferation. Conversely, restoration of DOK2 and DUSP4 in lung cancer cells suppressed MAPK activation and cell proliferation. Importantly, in contrast to downregulation of DOK2 or DUSP4 alone, concomitant downregulation of DOK2 and DUSP4 was associated with poor survival in human lung adenocarcinoma. Therefore, our findings lend in vivo experimental support to the notion that compound haploinsufficiency, due to broad-scale chromosome deletions, constitutes a driving force in tumorigenesis.
Authors
Ming Chen, Jiangwen Zhang, Alice H. Berger, Moussa S. Diolombi, Christopher Ng, Jacqueline Fung, Roderick T. Bronson, Mireia Castillo-Martin, Tin Htwe Thin, Carlos Cordon-Cardo, Robin Plevin, Pier Paolo Pandolfi
Abstract
X-linked dominant incontinentia pigmenti (IP) and X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) are caused by loss-of-function and hypomorphic NEMO mutations, respectively. We describe a European mother with mild IP and a Japanese mother without IP, whose three boys with EDA-ID died of immunodeficiency. We identify the same private variant in an intron of IKBKG/NEMO, IVS4+866 C>T, which was inherited from and occurred de novo in the European and Japanese mothers, respectively. This mutation creates a new splicing donor site, giving rise to a 44-nucleotide pseudo-exon generating a frameshift. Its leakiness accounts for NF-κB activation being impaired, but not abolished in the boys’ cells. However, aberrant splicing rates differ between cell types, with WT NEMO mRNA and protein levels ranging from barely detectable in leukocytes to residual amounts in iPSC-derived macrophages, and higher levels in fibroblasts and iPSC-derived neuronal precursor cells. Finally, SRSF6 binds to the pseudo-exon, facilitating its inclusion. Moreover, SRSF6 knockdown or CLK inhibition restores WT NEMO expression and function in mutant cells. A recurrent deep intronic splicing mutation in IKBKG/NEMO underlies a purely quantitative NEMO defect in males that is most severe in leukocytes and can be rescued by the inhibition of SRSF6 or CLK.
Authors
Bertrand Boisson, Yoshitaka Honda, Masahiko Ajiro, Jacinta Bustamante, Matthieu Bendavid, Andrew R. Gennery, Yuri Kawasaki, Jose Ichishima, Mitsujiro Osawa, Hiroshi Nihira, Takeshi Shiba, Takayuki Tanaka, Maya Chrabieh, Benedetta Bigio, Hong Hur, Yuval Itan, Yupu Liang, Satoshi Okada, Kazushi Izawa, Ryuta Nishikomori, Osamu Ohara, Toshio Heike, Laurent Abel, Anne Puel, Megumu K. Saito, Jean-Laurent Casanova, Masatoshi Hagiwara, Takahiro Yasumi
Abstract
Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide–gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3–/–) mice to obtain triallelic Cnga3+/– Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
Authors
Markus Burkard, Susanne Kohl, Timm Krätzig, Naoyuki Tanimoto, Christina Brennenstuhl, Anne E. Bausch, Katrin Junger, Peggy Reuter, Vithiyanjali Sothilingam, Susanne C. Beck, Gesine Huber, Xi-Qin Ding, Anja K. Mayer, Britta Baumann, Nicole Weisschuh, Ditta Zobor, Gesa-Astrid Hahn, Ulrich Kellner, Sascha Venturelli, Elvir Becirovic, Peter Charbel Issa, Robert K. Koenekoop, Günther Rudolph, John Heckenlively, Paul Sieving, Richard G. Weleber, Christian Hamel, Xiangang Zong, Martin Biel, Robert Lukowski, Matthias W. Seeliger, Stylianos Michalakis, Bernd Wissinger, Peter Ruth
Abstract
Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene encoding C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20-30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH leading to creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant negative disease mechanisms affecting C1INH plasma levels in HAE type I patients and may pave the way for new treatments of HAE.
Authors
Didde Haslund, Laura Barrett Ryø, Sara Seidelin Majidi, Iben Kløvgaard Rose, Kristian Alsbjerg Skipper, Tue Fryland, Anja Bille Bohn, Claus Koch, Martin K. Thomsen, Yaseelan Palarasah, Thomas J. Corydon, Anette Bygum, Lene N. Nejsum, Jacob Giehm Mikkelsen
Abstract
We report the molecular, cellular, and clinical features of 5 patients from 3 kindreds with biallelic mutations in the autosomal LIG1 gene encoding DNA ligase 1. The patients exhibited hypogammaglobulinemia, lymphopenia, increased proportions of circulating γδT cells, and erythrocyte macrocytosis. Clinical severity ranged from a mild antibody deficiency to a combined immunodeficiency requiring hematopoietic stem cell transplantation. Using engineered LIG1-deficient cell lines, we demonstrated chemical and radiation defects associated with the mutant alleles, which variably impaired the DNA repair pathway. We further showed that these LIG1 mutant alleles are amorphic or hypomorphic, and exhibited variably decreased enzymatic activities, which lead to premature release of unligated adenylated DNA. The variability of the LIG1 genotypes in the patients was consistent with that of their immunological and clinical phenotypes. These data suggest that different forms of autosomal recessive, partial DNA ligase 1 deficiency underlie an immunodeficiency of variable severity.
Authors
Patrick Maffucci, Jose Chavez, Thomas J. Jurkiw, Patrick J. O’Brien, Jordan K. Abbott, Paul R. Reynolds, Austen Worth, Luigi D. Notarangelo, Kerstin Felgentreff, Patricia Cortes, Bertrand Boisson, Lin Radigan, Aurélie Cobat, Chitra Dinakar, Mohammad Ehlayel, Tawfeg Ben-Omran, Erwin W. Gelfand, Jean-Laurent Casanova, Charlotte Cunningham-Rundles
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
Copyright © 2019 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)