Abstract
Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain–containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.
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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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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.
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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).
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).
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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Supplementary Table 4
Detailed clinical table. (XLSX 18 kb)
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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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DOI: https://doi.org/10.1038/ng.3256
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