Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction (original) (raw)

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Acknowledgements

We thank Y. Itan and B. Boisson for sequencing, T. Meerloo and A. Schmitt for electron microscopy support, the Sanford Burnham Institute for iPSC reprogramming, and A.M. Cuervo and M. Farquhar for comments and suggestions. Analysis was performed by the University of California San Diego Glycotechnology Core and the University of California San Diego Microscopy Imaging Core. This work was supported by US National Institutes of Health grants P01HD070494 and R01NS048453 and the Howard Hughes Medical Institute (to J.G.G.), US National Institutes of Health grant K99NS089859-01 (to N.A.), Broad Institute grant U54HG003067, Yale Center for Mendelian Disorders grant U54HG006504 (to M.G.), INSERM, Paris Descartes University, the St. Giles Foundation, and the Candidoser Association and Howard Hughes Medical Institute (to J.-L.C.), the Scientific and Technology Research Council of Turkey (grant TÜBİTAK-SBAG, 111S217, grant TÜBİTAK-BİLGEM-UEKAE, K030-T439) and the Turkey Republic Ministry of Development (grant TRMOD, 108S420) (to A.D.).

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Authors and Affiliations

1. Laboratory for Pediatric Brain Disease, The Rockefeller University, New York, New York, USA
   Naiara Akizu, Xin Wang, Rasim Ozgur Rosti, Esra Dikoglu, Basak Rosti, Keith K Vaux, Eric M Scott, Jennifer L Silhavy, Jana Schroth, Brett Copeland, Ashleigh E Schaffer & Joseph G Gleeson
2. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
   Naiara Akizu, Xin Wang, Rasim Ozgur Rosti, Esra Dikoglu, Basak Rosti, Keith K Vaux, Eric M Scott, Jennifer L Silhavy, Jana Schroth, Brett Copeland, Ashleigh E Schaffer, Jean-Laurent Casanova & Joseph G Gleeson
3. Dorris Neuroscience Center, Scripps Research Institute, La Jolla, California, USA
   Naiara Akizu & Ulrich Müller
4. Institut Imagine, INSERM U1163, Hôpital Necker Enfants Malades, Paris, France
   Vincent Cantagrel
5. Human Genetics and Genome Research Division, Clinical Genetics Department, National Research Centre, Cairo, Egypt
   Maha S Zaki, Ghada M Abdel-Salam, Samira Ismail, Mona Aglan & Samia Temtamy
6. Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates
   Lihadh Al-Gazali
7. Département de Génétique, Assistance Publique–Hôpitaux de Paris, Hôpital Armand Trousseau, UF Génétique du Développement, Neuropathologie, Paris, France
   Antoinette Bernabe Gelot
8. Institut de Neurobiologie de la Méditerranée (INMED) INSERM U901, Marseille, France
   Antoinette Bernabe Gelot
9. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA.,
   Philip L S M Gordts & Jeffrey D Esko
10. Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA.,
    Matthew D Buschman & Seth J Field
11. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
    Gennaro Napolitano
12. Pediatric Metabolism, Institute of Child Health, Hacettepe University, Ankara, Turkey
    R Koksal Ozgul
13. Tübitak Bilgem Uekae, Gebze/Kocaeli, Turkey
    Mahmut Samil Sagıroglu
14. Wah Medical College, Wah, Pakistan
    Matloob Azam
15. Department of Pediatric Neurology, Children's Hospital, Cairo University, Cairo, Egypt
    Laila Selim, Iman G Mahmoud, Sawsan Abdel-Hadi & Amera El Badawy
16. Pediatric Neurology Department, Faculty of Medicine, Sohag University, Sohag, Egypt
    Abdelrahim A Sadek
17. Mashhad Medical Genetic Counseling Center, Mashhad, Iran
    Faezeh Mojahedi
18. Medical Genetics Department, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey
    Hulya Kayserili
19. Division of Child Neurology, Department of Pediatrics, University of Jordan, Amman, Jordan
    Amira Masri
20. Kuwait Medical Genetics Centre, Maternity Hospital, Safat, Kuwait
    Laila Bastaki
21. Department of Pediatric Neurology, Necker Enfants Malades Hospital, Paris Descartes University, Paris, France
    Isabelle Desguerre
22. Génétique Humaine des Maladies Infectieuses, INSERM U1163, Université Paris Descartes, Institut Imagine, Paris, France
    Jean-Laurent Casanova
23. St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, New York, USA
    Jean-Laurent Casanova
24. Pediatric Metabolism, Hacettepe University Faculty of Medicine, Ankara, Turkey
    Ali Dursun
25. Department of Neurosurgery, Yale University, School of Medicine, New Haven, Connecticut, USA
    Murat Gunel
26. Department of Neurobiology, Yale University, School of Medicine, New Haven, Connecticut, USA
    Murat Gunel
27. Department of Genetics, Yale University, School of Medicine, New Haven, Connecticut, USA
    Murat Gunel
28. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA
    Stacey B Gabriel
29. Reference Center of Inherited Metabolic Diseases, Paris Descartes University, Necker Enfants Malades Hospital, Assistance Publique–Hôpitaux de Paris, Paris, France
    Pascale de Lonlay
30. New York Genome Center, New York, New York, USA
    Joseph G Gleeson

Authors

1. Naiara Akizu
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2. Vincent Cantagrel
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3. Maha S Zaki
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4. Lihadh Al-Gazali
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5. Xin Wang
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6. Rasim Ozgur Rosti
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7. Esra Dikoglu
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8. Antoinette Bernabe Gelot
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9. Basak Rosti
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10. Keith K Vaux
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11. Eric M Scott
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12. Jennifer L Silhavy
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13. Jana Schroth
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14. Brett Copeland
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15. Ashleigh E Schaffer
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16. Philip L S M Gordts
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17. Jeffrey D Esko
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18. Matthew D Buschman
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19. Seth J Field
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20. Gennaro Napolitano
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21. Ghada M Abdel-Salam
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22. R Koksal Ozgul
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23. Mahmut Samil Sagıroglu
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24. Matloob Azam
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25. Samira Ismail
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26. Mona Aglan
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27. Laila Selim
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28. Iman G Mahmoud
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29. Sawsan Abdel-Hadi
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30. Amera El Badawy
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31. Abdelrahim A Sadek
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32. Faezeh Mojahedi
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33. Hulya Kayserili
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34. Amira Masri
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35. Laila Bastaki
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36. Samia Temtamy
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37. Ulrich Müller
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38. Isabelle Desguerre
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39. Jean-Laurent Casanova
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40. Ali Dursun
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41. Murat Gunel
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42. Stacey B Gabriel
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43. Pascale de Lonlay
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44. Joseph G Gleeson
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Contributions

Patient recruitment and phenotyping: M.S.Z., L.A.-G., R.O.R., E.D., A.B.G., R.K.O., M.S.S., M. Azam, L.S., I.G.M., S.A.-H., M. Aglan, G.M.A.-S., S.I., A.E.B., A.A.S., F.M., H.K., A.M., L.B., S.T., I.D., A.D., K.K.V. and J.G.G. Genetic sequencing and interpretation: N.A., V.C., X.W., J.L.S., J.S., E.M.S., B.C., J.-L.C., M.G., S.B.G., P.d.L. and A.D. Cell biology: N.A., V.C., J.D.E., M.D.B., S.J.F., G.N., P.L.S.M.G. and U.M. Zebrafish: B.R., N.A. and X.W. Cell culture: A.E.S. and N.A. Histology: A.B.G. and I.D.

Corresponding author

Correspondence toJoseph G Gleeson.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Linkage and homozygosity plots and analysis of founder mutation.

(a) Parametric multipoint linkage analysis results from parents and four children genotyped from family 468, with a single peak at chromosome 6. (b) Homozygosity plots showing homozygous blocks (red) in affected individuals from families that were subjected to exome sequencing. (c) A 1.5-Mb haplotype block shared by three affected subjects containing the same c.1132C>T SNX14 mutation. The green arrow depicts the location of the SNX14 mutation.

Supplementary Figure 2 Pedigrees with SNX14 mutations and additional representative MRIs and clinical photographs.

(a) Pedigrees of each of the 12 families harboring homozygous truncating mutations in SNX14. Double bar, consanguinity; filled symbol, affected; diagonal line, deceased. The location of each mutation is depicted. (b) MRIs and facial photographs from families 1902, 3087, ABD, 468, HMF and 1971 showing neuroradiographic and facies consistent with those of the other _SNX14-_mutated patients. Consent to publish images of the subjects was obtained.

Supplementary Figure 3 Neuropathological postmortem evaluation of ABD-II-2.

(a) Histological and ultrastructural evaluation of cerebellar tissue showing fragmented calbindin and neurofilament staining and the presence of lipid-like droplets in Purkinje cells. (b) Histological evaluation of the neocortex showing mild neuronal loss in superficial layers and reduced myelinated axonal tracts with vacuolization. The control corresponds biobank identification number Neuropathologie du developpement _BB-0033-00082/C 2009-935. (c) Ultrastructural analysis of ABD-II-2 spinal cord postmortem tissue, showing cytoplasmic membranous bodies (red arrowheads) and autophagosomes (green arrows).

Supplementary Figure 4 SNX14 colocalizes with LAMP2.

Retinal pigment epithelial (RPE) cells transfected with dsRED-tagged SNX14 and then fixed and immunostained for the lysosomal glycoprotein LAMP2, showing overlapping distribution, or for the early endosome marker EEA1, showing no codistribution. Scale bars, 20 μm.

Supplementary Figure 5 No differences in reprograming and differentiation to neural progenitors between affected and unaffected cells.

(a) Affected (A, i.e., patient) fibroblasts, compared with unaffected (U), matched family fibroblasts, show slightly reduced levels of SNX14 mRNA as detected by RT-PCR and undetectable levels of SNX14 protein as detected by immunoblot analysis. GAPDH was used as a loading control. (b) Reprogramming of fibroblasts to iPSCs was indistinguishable in affected and unaffected cells. Scale bars, 400 μm. (c) Conversion of iPSCs to embryoid bodies was indistinguishable in affected and unaffected cells. Scale bars, 400 μm. (d) Differentiation of iPSCs to neural progenitors (Pax6 in green, nestin in red) was indistinguishable in affected and unaffected cells.

Supplementary Figure 6 _SNX14_-mutated neural progenitors show enlarged lysosomes.

(a) Flow cytometry analysis of NPCs labeled with Lysotracker Green DND-26. Affected NPCs showed more intense labeling with Lysotracker Green DND-26. The graph shows the percentage of cells outside the gated area defined by control cells. Mean ± s.d. from two clones from family 1382 and one clone from family 468 each analyzed in duplicate. *P < 0.05 (two-tailed t test). (b) LAMP1 staining in affected (A, red) and unaffected (U, black) NPCs confirms enlarged lysosomes in affected cells under fed and starved conditions. (c) Lysotracker Green DND-26 staining of engorged lysosomes in affected NPCs under nutrient deprivation for 1.5 h. Scale bars, 5 μm. The dot plot shows the relative area for individual Lysotracker-positive lysosomes (n = 123 and 161 lysosomes from 2 families unaffected and affected, respectively). Graph bars represent the average number of Lysotracker-positive lysosomes per cell (n = 7 and 8 cells from 2 families unaffected and affected, respectively). ***P < 0.005 (two-tailed t test). (d) Unaffected (U) and affected (A) neural progenitors were incubated with Bodipy FL Pepstatin A (green) to test for cathepsin D activity (DNA in blue).

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Supplementary Figure 7 SNX14 is involved in autophagic regulation.

(a) Cell fractionation from human NPCs grown under starvation conditions to induce autophagy. SNX14 is enriched in the compartment with the strongest signal for LC3-II (lane 4) and in endosomal/lysosomal compartments (red). (b) SNX14 dynamically localizes to vesicles positive for LC3 upon induction of autophagy (2-h treatment with EBSS) in wild-type human NPCs expressing dsRED-SNX14. (c) Immunoblot analysis of LAMP1, BECLIN1, cathepsin D and p62, with quantification by densitometry. α-tubulin was used as a loading control. The graph presents means ± s.d. (n = 3 clones). *P < 0.05 (two-tailed t test).

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Supplementary Figure 8 Zebrafish snx14 shows brain expression.

In situ hybridization of the snx14 zebrafish gene at 24 h.p.f., showing ubiquitous expression, and at 48 h.p.f., showing predominantly neural expression in (1) a coronal image section through the rostral tectum; (2) a coronal section through the caudal tectum; and (3) a parasagittal section.

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Akizu, N., Cantagrel, V., Zaki, M. et al. Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.Nat Genet 47, 528–534 (2015). https://doi.org/10.1038/ng.3256

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• Received: 24 October 2014
• Accepted: 02 March 2015
• Published: 06 April 2015
• Issue Date: May 2015
• DOI: https://doi.org/10.1038/ng.3256