DEGS1-associated aberrant sphingolipid metabolism impairs nervous system function in humans
Gergely Karsai, … , Thorsten Hornemann, Ingo Kurth
Gergely Karsai, … , Thorsten Hornemann, Ingo Kurth
Published March 1, 2019; First published January 8, 2019
Citation Information: J Clin Invest. 2019;129(3):1229-1239. https://doi.org/10.1172/JCI124159.
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Categories: Clinical Medicine Genetics Metabolism

DEGS1-associated aberrant sphingolipid metabolism impairs nervous system function in humans

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 remains 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 systems, 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 that 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 with 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 the cause of a sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous systems. TRIAL REGISTRATION. Not applicable. FUNDING. Seventh Framework Program of the European Commission, Swiss National Foundation, 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

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Figure 2

Characterization of mutant DEGS1 protein.

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Characterization of mutant DEGS1 protein. The DEGS1 p.Ala280Val mutation...
The DEGS1 p.Ala280Val mutation decreases protein stability. (A) IGV plots of direct cDNA nanopore sequencing results. The upper part of each plot shows the coverage plot (cp, gray) and the lower part the single reads. (B–E) Cellular distribution of DEGS1 WT and the DEGS1 mutant (p.Ala280Val). EGFP-tagged (B) WT and (C) mutant DEGS1 colocalize with the endoplasmic reticulum marker protein disulfide isomerase (PDI). The reticular staining pattern of PDI in untransfected cells (asterisk in C) seems undisturbed in mut DEGS1–overexpressing cells (arrow in C). (D and E) Only minor overlap of immunofluorescence signals is observed for EGFP-tagged WT– and mut DEGS1–overexpressing cells with the mitochondrion inner membrane marker Tim23. Scale bar: 10 μm; insets show 2-fold magnification. (F and G) DEGS1 expression was analyzed by Western blot in HAP1 WT and HAP1 DEGS1–/– cells or fibroblasts from a healthy control and the index patient. (G) Reduced DEGS1 protein levels in patient fibroblasts, quantified from the blot in F (normalized to α-tubulin and the control sample). (H) DEGS1-EGFP expression in HAP1 WT cells transfected with pEF1α-WT DEGS1-EGFP or mut DEGS1-EGFP after treatment with cycloheximide (CHX) and MG-132 for the indicated times. (I and J) Quantification of WT and mut DEGS1-EGFP protein amounts from H normalized to α-tubulin and DEGS1-EGFP (I) or WT DEGS1-EGFP and mut DEGS1-EGFP (J).