Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness

Journal name:
Nature Genetics
Volume:
44,
Pages:
797–802
Year published:
DOI:
doi:10.1038/ng.2325
Received
Accepted
Published online

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.

At a glance

Figures

  1. Key phospholipids and a schematic representation of SERAC1.
    Figure 1: Key phospholipids and a schematic representation of SERAC1.

    (a) Structural formulas of key phospholipids. (b) Schematic representation of human SERAC1 showing the positions of all mutations identified. The black box represents the lipase/esterase domain. Del, deletion.

  2. SERAC1 and its role in phosphatidylglycerol remodeling and cholesterol trafficking.
    Figure 2: SERAC1 and its role in phosphatidylglycerol remodeling and cholesterol trafficking.

    (a) Representative high-performance liquid chromatography (HPLC) tandem mass spectrometry spectra of bis(monoacylglycerol)phosphate (BMP) and phosphatidylglycerol (PG) in fibroblasts. In patients with MEGDEL syndrome, phosphatidylglycerol-34:1 accumulates, phosphatidylglycerol-36:1 is deficient and bis(monoacylglycerol)phosphate concentrations are low. (b) Box and whisker plots (minimum and maximum) of phosphatidylglycerol-34:1 and phosphatidylglycerol-36:1 concentrations, the ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1, total concentrations of bis(monoacylglycerol)phosphate and total concentrations of cardiolipin (CL) in controls (C, n = 10) and patients (P, n = 5). (c) Filipin staining of fibroblasts from patients with MEGDEL syndrome, a patient with Niemann-Pick disease type C (NPC) as positive control and a healthy control showing background fluorescence. Scale bar, 5 μm. (d) The role of SERAC1 in phosphatidylglycerol remodeling. CLS, cardiolipin synthase; IS, internal standard; TAZ, tafazzin.

  3. Cardiolipin species composition in patients and controls.
    Figure 3: Cardiolipin species composition in patients and controls.

    Box and whisker plots (minimum and maximum) showing the concentrations of the cardiolipin (CL) species cardiolipin-66:3, cardiolipin-66:4, cardiolipin-68:3, cardiolipin-68:4 and cardiolipin-68:5 in control (C, n = 10) and patient (P, n = 5) fibroblasts. These specific cardiolipin species are significantly more abundant in patients than in controls. No significant differences were found in the other cardiolipin species: cardiolipin-70:7 through cardiolipin-70:5, cardiolipin-72:8 through cardiolipin-72:5 and cardiolipin-74:8 through cardiolipin-74:6 (Supplementary Fig. 7).

  4. Subcellular localization of SERAC1.
    Figure 4: Subcellular localization of SERAC1.

    (a,b) Immunodetection of SERAC1 in total cell lysate (TC), cytoplasmic fraction (CF), crude mitochondrial fraction (CMF) and ER of HeLa (a) or HEK293 (b) cells showing SERAC1 enrichment in the CMF and ER. Calnexin (MAM-ER marker), NDUFS3 and COXII (inner mitochondrial membrane markers) and creatine kinase-B (CK-B; cytoplasmic marker) were used for comparison. (c) Immunodetection of SERAC1 in CMFs subjected to no or mild digitonin treatment and incubated with or without proteinase K (Prot K) in the absence or presence of Triton X-100 showing SERAC1 association with the mitochondria, ER and MAMs of HEK293 cells. Calnexin, TOM20 (mitochondrial outer membrane marker) and NDUFS3 were used for comparison. (d) Partial colocalization of SERAC1 with the mitochondrial matrix marker MRPL12 in control fibroblasts showing SERAC1 in close proximity to mitochondria. Scale bar, upper two rows, 5 μm; bottom row, 2.5 μm. The bottom row shows the images at the highest magnification. (e) Ratio of phosphatidylglycerol (PG)-34:1 to phosphatidylglycerol-36:1 in the CMF of patient (P, n = 3) and control fibroblasts (C, n = 3).

  5. The ratio of phosphatidylglycerol (PG)-34:1 to phosphatidylglycerol-36:1 in patient fibroblasts complemented with wild-type SERAC1.
    Figure 5: The ratio of phosphatidylglycerol (PG)-34:1 to phosphatidylglycerol-36:1 in patient fibroblasts complemented with wild-type SERAC1.

    Fibroblasts from patients were infected with a mock vector (n = 4) or an expression vector containing wild-type SERAC1 (+SERAC1, n = 4). Complementation of SERAC1 (for western blot see Supplementary Fig. 13) restores the ratio of phosphatidylglycerol-34:1) to phosphatidylglycerol-36:1.

Accession codes

Referenced accessions

NCBI Reference Sequence

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Author information

  1. These authors jointly directed this work.

    • Ron A Wevers,
    • Eva Morava &
    • Arjan P M de Brouwer

Affiliations

  1. Department of Pediatrics, Radboud University Nijmegen Medical Centre (RUNMC), Nijmegen, The Netherlands.

    • Saskia B Wortmann,
    • Thatjana Gardeitchik,
    • G Herma Renkema,
    • Richard J Rodenburg,
    • Leo G J Nijtmans,
    • Joachim M Gerhold,
    • Jan A M Smeitink,
    • Johannes N Spelbrink &
    • Eva Morava
  2. Institute of Genetic and Metabolic Disease (IGMD), RUNMC, Nijmegen, The Netherlands.

    • Saskia B Wortmann,
    • Thatjana Gardeitchik,
    • Lisenka E L M Vissers,
    • G Herma Renkema,
    • Richard J Rodenburg,
    • Leo G J Nijtmans,
    • Joachim M Gerhold,
    • Jan A M Smeitink,
    • Dirk J Lefeber,
    • Johannes N Spelbrink,
    • Ron A Wevers &
    • Eva Morava
  3. Department of Clinical Chemistry and Pediatrics, Laboratory Genetic Metabolic Disease, Academic Medical Center, Amsterdam, The Netherlands.

    • Frédéric M Vaz &
    • Wim Kulik
  4. Laboratory of Genetic, Endocrine and Metabolic Diseases (LGEM), Department of Laboratory Medicine, RUNMC, Nijmegen, The Netherlands.

    • Thatjana Gardeitchik,
    • Leo A J Kluijtmans,
    • Richard J Rodenburg,
    • Dirk J Lefeber &
    • Ron A Wevers
  5. Department of Human Genetics, RUNMC, Nijmegen, The Netherlands.

    • Lisenka E L M Vissers,
    • Janneke H M Schuurs-Hoeijmakers,
    • Christian Gilissen,
    • Hans van Bokhoven,
    • Joris A Veltman &
    • Arjan P M de Brouwer
  6. Nijmegen Centre for Molecular Life Sciences, RUNMC, Nijmegen, The Netherlands.

    • Lisenka E L M Vissers &
    • Hans van Bokhoven
  7. Centre for Systems Biology and Bioenergetics, RUNMC, Nijmegen, The Netherlands.

    • G Herma Renkema
  8. Department of Pathology, RUNMC, Nijmegen, The Netherlands.

    • Martin Lammens
  9. Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Bioinformatics Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

    • Christin Christin
  10. Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.

    • Anne Grünewald &
    • Christine Klein
  11. Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands.

    • Tamas Kozicz &
    • Hans van Bokhoven
  12. Department of Animal Physiology, Radboud University Nijmegen, Nijmegen, The Netherlands.

    • Tamas Kozicz
  13. Department of Metabolic Diseases, Wilhelmina Children's Hospital Utrecht, University Medical Center Utrecht, Utrecht, The Netherlands.

    • Peter M van Hasselt
  14. Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.

    • Magdalena Harakalova &
    • Wigard Kloosterman
  15. Department of Pediatrics, University Hospital Centre Zagreb and School of Medicine, Zagreb, Croatia.

    • Ivo Barić
  16. Department of Metabolic Diseases, Children's Memorial Health Institute, Warsaw, Poland.

    • Ewa Pronicka
  17. Department of Pediatrics, Ege University Faculty of Medicine, Izmir, Turkey.

    • Sema Kalkan Ucar
  18. Department of Pediatric Neurology, Karolinska University Hospital, Stockholm, Sweden.

    • Karin Naess
  19. Department of Neurology, All India Institute of Medical Science, Delhi, India.

    • Kapil K Singhal
  20. Medical Genetics Clinic, Children's University Hospital, Riga, Latvia.

    • Zita Krumina
  21. Department of Neurology, RUNMC, Nijmegen, The Netherlands.

    • Dirk J Lefeber
  22. Institute of Biomedical Technology and Tampere University Hospital, Pirkanmaa Hospital District, University of Tampere, Tampere, Finland.

    • Johannes N Spelbrink

Contributions

S.B.W., E.M., R.A.W. and A.P.M.d.B. designed and supervised the study. S.B.W. and E.M. characterized MEGDEL syndrome and collected clinical data and patient tissues. L.A.J.K. performed and supervised the metabolic metabolite screening. P.M.v.H., I.B., E.P., S.K.U., K.N., K.K.S., Z.K. and J.A.M.S. diagnosed and referred patients. L.E.L.M.V., C.G., J.H.M.S.-H., J.A.V., M.H., W. Kloosterman, H.v.B. and A.P.M.d.B. performed the genetic studies and identified the causative genetic defect. G.H.R., R.J.R., T.G. and A.P.M.d.B. performed the lentiviral complementation. F.M.V., C.C. and W. Kulik performed the phospholipid spectra analysis and interpreted the data together with S.B.W., E.M., R.A.W. and A.P.M.d.B. D.J.L. performed and interpreted the filipin staining. A.G. and C.K. performed and interpreted the studies on autophagy and mitophagy. T.G., L.G.J.N., T.K., J.M.G. and J.N.S. performed colocalization and western blotting studies on SERAC1. T.K. performed the studies on fusion-fission. R.J.R. performed and interpreted oxidative phosphorylation measurements. M.L. captured and interpreted electron microscopic pictures of muscle tissue. F.M.V. and E.M. contributed to the draft manuscript. S.B.W., R.A.W. and A.P.M.d.B. prepared the final manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Text and Figures (4 MB)

    Supplementary Note, Supplementary Figures 1–13 and Supplementary Tables 1–6.

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