Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)*

1. Christina T. Thiesler‡,§,^c, 
2. Samanta Cajic¶,^c, 
3. Dirk Hoffmann‡,‖,
4. Christian Thiel**, 
5. Laura van Diepen‡‡, 
6. René Hennig¶,§§, 
7. Malte Sgodda‡,¶¶,
8. Robert Weiβmann‡‡, 
9. Udo Reichl¶, 
10. Doris Steinemann‡,‖‖, 
11. Ulf Diekmann^a,
12. Nicolas M. B. Huber‡,§, 
13. Astrid Oberbeck‡,§, 
14. Tobias Cantz‡,¶¶,
15. Andreas W. Kuss‡‡, 
16. Christian Körner**, 
17. Axel Schambach‡,‖, 
18. Erdmann Rapp¶,§§and 
19. Falk F. R. Buettner‡,§,^b

+Author Affiliations

1. From the ‡REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany;
2. §Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany;
3. ¶Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany;
4. ‖Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany;
5. **Center for Child and Adolescent Medicine, Department Kinderheilkunde I, 69120 Heidelberg, Germany;
6. ‡‡Department of Human Genetics, University Medicine Greifswald and Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University, 17475 Greifswald, Germany;
7. §§glyXera GmbH, 39120 Magdeburg, Germany;
8. ¶¶Translational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, 30625 Hannover, Germany;
9. ‖‖Institute of Human Genetics, Hannover Medical School, 30625 Hannover, Germany;
10. ^aInstitute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

1. ↵^b To whom correspondence should be addressed: Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany. Tel.: +49(0)0511/532-8245; Fax: +49(0)511/532-8801; E-mail: buettner.falk@mh-hannover.de.

1. ↵^c These authors contributed equally to this study.

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 and in vitro differentiation 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 iPSCs in vitro. Knock-down of PMM2 by 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.

Footnotes

• Author contributions: F.F.B. designed research; C.T.T., S.C., D.H., C.T., L.v.D., R.H., M.S., U.D., N.M.H., and A.O. performed research; S.C., C.T., L.v.D., R.H., U.R., D.S., T.C., A.W.K., C.K., A.S., and E.R. contributed new reagents or analytic tools; C.T.T., S.C., C.T., L.v.D., R.H., R.W., D.S., E.R., and F.F.B. analyzed data; C.T.T. and F.F.B. wrote the paper.