title: Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG).
abstract: PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors andin vitrodifferentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCsin vitro Knock-down ofPMM2by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; §Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Christina T
authors.initials: CT
authors.lastname: Thiesler
authors.affiliation: ¶Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany;
authors.forename: Samanta
authors.initials: S
authors.lastname: Cajic
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ‖Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Dirk
authors.initials: D
authors.lastname: Hoffmann
authors.affiliation: **Center for Child and Adolescent Medicine, Department Kinderheilkunde I, 69120 Heidelberg, Germany;
authors.forename: Christian
authors.initials: C
authors.lastname: Thiel
authors.affiliation: ‡‡Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University, 17475 Greifswald, Germany;
authors.forename: Laura
authors.initials: L
authors.lastname: van Diepen
authors.affiliation: ¶Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; §§glyXera GmbH, 39120 Magdeburg, Germany;
authors.forename: René
authors.initials: R
authors.lastname: Hennig
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ¶¶Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Malte
authors.initials: M
authors.lastname: Sgodda
authors.affiliation: ‡‡Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University, 17475 Greifswald, Germany;
authors.forename: Robert
authors.initials: R
authors.lastname: Weiβmann
authors.affiliation: ¶Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany;
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ‖‖Institute of Human Genetics, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Doris
authors.initials: D
authors.lastname: Steinemann
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Diekmann
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; §Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Nicolas M B
authors.initials: NM
authors.lastname: Huber
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; §Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Astrid
authors.initials: A
authors.lastname: Oberbeck
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ¶¶Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.affiliation: ‡‡Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University, 17475 Greifswald, Germany;
authors.forename: Andreas W
authors.initials: AW
authors.lastname: Kuss
authors.affiliation: **Center for Child and Adolescent Medicine, Department Kinderheilkunde I, 69120 Heidelberg, Germany;
authors.forename: Christian
authors.initials: C
authors.lastname: Körner
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ‖Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany;
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: ¶Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; §§glyXera GmbH, 39120 Magdeburg, Germany;
authors.forename: Erdmann
authors.initials: E
authors.lastname: Rapp
authors.affiliation: From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; §Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany; buettner.falk@mh-hannover.de.
authors.forename: Falk F R
authors.initials: FF
authors.lastname: Buettner
mesh_terms: D002478:Cells, Cultured; D018981:Congenital Disorders of Glycosylation / Q000378:metabolism / Q000473:pathology*; D020869:Gene Expression Profiling / Q000379:methods; D054794:Glycomics / Q000379:methods*; D006031:Glycosylation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D008954:Models, Biological; D017875:Phosphotransferases (Phosphomutases) / Q000172:deficiency* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D017875:Phosphotransferases (Phosphomutases)