FIGURE SUMMARY
Title

Bi-allelic variants in SNF8 cause a disease spectrum ranging from severe developmental and epileptic encephalopathy to syndromic optic atrophy

Authors
Brugger, M., Lauri, A., Zhen, Y., Gramegna, L.L., Zott, B., Sekulić, N., Fasano, G., Kopajtich, R., Cordeddu, V., Radio, F.C., Mancini, C., Pizzi, S., Paradisi, G., Zanni, G., Vasco, G., Carrozzo, R., Palombo, F., Tonon, C., Lodi, R., La Morgia, C., Arelin, M., Blechschmidt, C., Finck, T., Sørensen, V., Kreiser, K., Strobl-Wildemann, G., Daum, H., Michaelson-Cohen, R., Ziccardi, L., Zampino, G., Prokisch, H., Abou Jamra, R., Fiorini, C., Arzberger, T., Winkelmann, J., Caporali, L., Carelli, V., Stenmark, H., Tartaglia, M., Wagner, M.
Source
Full text @ Am. J. Hum. Genet.

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Pedigrees of the families with bi-allelic SNF8 variants and T2-weighted brain MRI scans of affected individuals with bi-allelic variants in SNF8

(A) Pedigrees of the families A–F. Variants identified in the individuals are depicted underneath the corresponding symbol. In family A, the healthy male sibling (II-1) of individuals A1 and A2 was not compound heterozygous for the SNF8 variants. In family D, the healthy female sibling (II-1) was not compound heterozygous for the SNF8 variants. Solid symbol, affected individual; circles, females; squares, males; slashed symbols, deceased.

(B) Brain MRI scans of the affected individuals with pathogenic variants of SNF8. (i–xii) T2-scans of the severely affected individuals. Images of A1 at the age of two (i, ii) and six months (iii, iv), of subject A2 at the age of two months (v, vi), of subject B1 at the age of four (vii, viii) and 15 months (ix, x), and of subject C2 at the age of one month (xi, xii). All severely affected subjects (i–xii) show a pronounced and persistent white matter hyperintensity, an atrophy of the cerebrum and callosal hypoplasia. The brainstem appears comparatively normal and the cerebellum is less severely affected. Pachygyria was noted in individual A2 (v). Note the rapidly progressive enlargement of the lateral ventricles due to cerebral white matter loss in subjects A1 and B. (xiii–xx) MRI images of the less severely affected individuals. Individuals D1 (xiii–xv) and E1 (xvi–xviii), both recorded at the age of 16 years, 3D volumetric T1 showing cerebellar atrophy (xiii and xvi, red arrows), as well as severe volume reduction of the anterior optic pathway including optic nerves, optic chiasm, and optic tracts (xiv and xvii, axial magnification of the 3D volumetric T1, red arrows). Moreover, on axial T2 (xv) or FLAIR (xviii) images, slight hyperintensity of the parieto-occipital white matter was observed (red arrows); notably automatic software evaluation (freesurfer) demonstrated a total cerebral white matter volume reduction in both affected individuals. (xix and xx) MRI scan of individual F1 at the age of two years showing normal myelination and white matter volume in T2 (xix) and a slightly dysmorphic callosal body as well as slight cerebellar atrophy in a sagittal T1 scan (xx, red arrow).

Macroscopic and microscopic alterations in individual A2 deceased at the age of 3 months

(A) Lateral view on the formalin-fixed autopsy brain.

(B) Coronally cut slice of the formalin-fixed brain at the level of thalamus/hippocampus showing a massive reduction of the subcortical white matter and a severe atrophy of the corpus callosum. The gyri appear coarse.

(C–H) Frontal subcortical white matter at level of anterior cingulate gyrus; (C) age-matched control. (D–H) Consecutive sections in individual A2. (C and D) Hematoxylin-eosin (HE) stains. Compared to the age-matched control case (C), the astrocytes of individual A2 (D) are reactively altered showing enlarged eosinophilic cell bodies (inset displays zoomed marked area). The number of astrocytes is also increased in individual A2. (E) With an antibody against human leukocyte antigen-DR isotype (HLA-DR), numerous activated microglial cells (brown color) are visualized as a sign of a resorptive process. (F) The immunohistochemical detection of glial fibrillary acidic protein (GFAP; brown color) illustrates the large number of reactively changed astrocytes. (G) Myelin is dramatically reduced (blue color) in a luxol fast blue-periodic acid Schiff (LFB-PAS) stain, while in (H) the axons are relatively well preserved as demonstrated by immunohistochemistry for neurofilament (NF; brown color). Insets in (G) and (H) show overviews of the slides, rectangles mark zoomed areas. Highlights the same blood vessel in consecutive tissue sections. Scale bars in (C)–(H) correspond to 50 μm.

Representation of the 3D structure of SNF8 with the observed missense variants

(A) The surface rendering of human ESCRT II complex (PDB: 2ZME7). VPS36 is yellow, SNF8 is magenta, and 2 molecules of VPS25 are green and blue. The ribbon diagram with secondary structure elements is shown for SNF8, and the residues affected by the reported missense variants are shown as black spheres.

(B) Magnified view of the residues affected by the reported missense variants. The affected residues are shown in black and labeled accordingly. Hydrogen bonds involving affected residues are depicted as dashed black lines. The variants p.Pro79Leu and p.Gly191Asp introduce larger residues that lead to steric clashes with surrounding residues and likely affect the conformation and stability of SNF8. In contrast, the p.Arg208Leu variant replaces the long and charged arginine with a short and hydrophobic leucine, resulting in a loss of interaction with aspartic acid 179 and tryptophan 204, thus most likely also affecting the conformation and stability of SNF8. The most conservative amino acid substitution with the least impact is p.Val102Ile, in which the small hydrophobic valine is replaced by a slightly larger (one atom) hydrophobic isoleucine.

Effects of bi-allelic variants in SNF8 on SNF8 levels and ESCRT II complex subunits

Volcano plot of the proteomics analysis performed on cultured fibroblasts from individual A2 (A), D1 (B), and E1 (C). SNF8, VPS25, VPS36, and proteins with statistically different levels are highlighted in color. Vertical black lines indicate log2fold changes of −1 and 1. Horizontal black lines depict significance level of p = 7.14 × 10−6 (Bonferroni correction for 7,000 hypotheses representing the number of proteins identified). Note the significant reduced protein levels of ESCRT II complex subunits SNF8, VPS25, and VPS36 for individual A2.

Bi-allelic variants in SNF8 in individual A2 lead to loss of ESCRT II complex resulting in an autophagic phenotype in fibroblasts

(A) Fibroblasts derived from individual A2 (top) or control individuals (bottom) were analyzed by transmission electron microscopy. Arrows indicate large vesicular structures that contain cytoplasmic content, consistent with being autolysosomes. Scale bars: 1 μm.

(B) Fibroblasts were incubated with BSA-gold (10 nm) for 24 h and washed and then the gold was chased overnight to accumulate in lysosomal compartments. Cells were then studied by transmission electron microscopy. Images of untreated fibroblasts are found on the left (Ctrl) whereas images on the right side (24 h) represent fibroblasts stained with BSA-gold. In BJ control fibroblasts, most of the gold tracer is found in uniformly dense lysosomes. In patient-derived cells, the gold is found in the dense part of bigger lysosomal compartments, which mostly consist of an electron-lucent lumen. This phenotype is similar for the various patient-derived fibroblasts. Scale bars: 1 μm.

(C) Fibroblasts derived from individual A2 or control individuals were left untreated of incubated with Bafilomycin A1 and then fixed and processed for immunofluorescence confocal microscopy with antibodies against LC3 and LAMP1. Co-localization between the two markers after Bafilomycin A1 treatment is indicative of autolysosomes. Scale bar: 10 μm.

Snf8 loss of function in zebrafish leads to developmental defects that are rescued by expression of the wild-type SNF8 cDNA but not disease-associated variants, and causes reduced anterior brain area and ectopic proliferation

(A) Representative bright-field pictures of fish not injected (not inj.), zebrafish injected with snf8 MO with or without mRNA encoding wild-type SNF8, or the disease-associated SNF8Y167∗;G191D, SNF8P79L;V102I, SNF8V102I, or SNF8Y167∗ alleles at 48 hpf. The dashed vertical lines separate different images from different representative fields of view.

(B and C) Quantification of the percentage of fish showing normal (white) or aberrant (cyan) development upon injection of different snf8 MO doses (B) and of snf8 MO at 0.8 mM with or without the WT and mutant SNF8 mRNAs (C). In (B), n = 75 (not inj. on the left), 19 (snf8 MO 0.6 mM), 67 (snf8 MO 0.8 mM), and 104 (not inj. on the right). In (C) (left), n = 39 (not inj.), 8 (snf8 MO at 0.8 mM), 23 (snf8 MO + SNF8WT), 11 (snf8 MO + SNF8Y167∗;G191D), 18 (snf8 MO + SNF8P79L;V102I). In (C) (right), n = 165 (not inj.), 99 (snf8 MO at 0.8 mM), 98 (snf8 MO + SNF8WT), 55 (snf8 MO + SNF8V102I), 39 (snf8 MO + SNF8Y167∗).

(D–F) Representative confocal maximum intensity projections of fish fluorescently stained with antibodies against acetylated tubulin (cyan) and pH3 (magenta) from not injected (not inj.) (D), mild and severe cases of fish injected with snf8 MO (E), and fish co-injected with snf8 MO (at 0.8 mM) and SNF8WT (F). Fish injected with snf8 MO show a reduced anterior brain area and ectopic proliferative cells within the forebrain partially rescued by SNF8WT.

(G) Quantification of the overall brain area from the confocal z-projections.

(H) Number of embryos showing “normal” or “mildly or severely ectopic” proliferative cells (pH3+) within the anterior ventral forebrain. n = 15 (not inj. and snf8 MO) and 12 (snf8 MO + SNF8WT). Data are expressed as boxplot, for experiments including different batches mean ± SEM are shown.

Number of replicates in (B), one and two (left and right graphs, respectively). In (C) (left), number of replicates: three for not inj, snf8 MO, snf8 MO + SNF8WT, and snf8 MO + SNF8Y167∗;G191D; two for snf8 MO + SNF8P79L;V102I. In (C) (right), number of replicates: six (not inj, snf8 MO, and snf8 MO + SNF8WT); three (the other two mutants). Two-sided chi-square test is used to assess statistical significance in a 2×2 contingency table. In (B), not inj. vs. snf8 MO, ∗∗∗p = 0.0008 left and ∗∗∗∗p < 0.0001 right. In (C) (left), not inj. vs. snf8 MO, ∗∗∗p = 0.0005; snf8 MO vs. snf8 MO + SNF8WT, p = 0.0312; snf8 MO + SNF8WT vs. + SNF8Y167∗;G191D, ∗∗p = 0.0086; snf8 MO + SNF8WT vs. snf8 MO + SNF8P79L;V102I, not significant (ns, p = 0.06); snf8 MO vs. mutants, ns. In (C) (right): not inj. vs. MO, ∗∗∗∗p < 0.0001; snf8 MO vs. snf8 MO + SNF8WT, ∗∗∗p = 0.0008; snf8 MO + SNF8WT vs. snf8 MO + SNF8V102I, ∗∗p = 0.0021; snf8 MO + SNF8WT vs. snf8 MO + SNF8Y167∗, ∗∗p = 0.0022 (ns: not significant). In (G) non-parametric Kruskal-Wallis test with Dunn’s post hoc test is used for statistical assessment (not inj. vs. snf8 MO, p = 0.0141; snf8 MO vs. snf8 MO + SNF8WT, ∗∗p = 0.0032; ns = not significant). In (H), two-sided chi-square test is used: not inj. vs. snf8 MO, p = 0.0309; not inj. vs. snf8 MO + SNF8WT, p = 0.8695 (ns, not significant); and snf8 MO vs. snf8 MO + SNF8WT, p = 0.0621 (ns). Fb, forebrain; oe, olfactory epithelium; OC, optic chiasm indicated by a white arrow. Asterisks indicate the eyes.

Zebrafish embryos injected with snf8 MO exhibit altered optic nerve and optic chiasm morphology at 48 hpf partially rescued by SNF8WT

(A) Representative confocal maximum intensity projections of fish fluorescently stained with the antibody against acetylated tubulin (cyan) from not injected (not inj., up), mild, moderate, and severe cases of fish injected with snf8 MO (center) and fish co-injected with snf8 MO and mRNA encoding SNF8WT (bottom). The white asterisks indicate the position of the eye.

(B) Quantification of the percentage of fish showing normal (white) or mild, moderate, or severely aberrant (gradients of cyan) ON phenotypes as described in the main text. n = 15 (not inj.), 15 (snf8 MO), and 12 (snf8 MO + SNF8WT). Two-tailed chi-squared test is used to assess statistical significance of the occurrence of the phenotype (moderate + severe) in a 2×2 contingency table: not inj. vs. snf8 MO, ∗∗∗p < 0.0001; snf8 MO vs. snf8 MO + SNF8WT, p = 0.0330.

(C and D) Quantification of the optic nerve length, measured as ON extension between two eyes and thickness, measured on both side of ON. In (C), n = 15 (not inj.), 15 (snf8 MO), and 12 (snf8 MO + SNF8WT). One-way ANOVA with Tukey’s post hoc test is used to assess statistical significance: not inj. vs. snf8 MO, ∗∗∗∗p < 0.0001; snf8 MO vs. snf8 MO + SNF8WT, ∗∗p = 0.0024. In (D), n = 15 (not inj.), 12 (snf8 MO), and 12 (snf8 MO + SNF8WT). Two-way ANOVA with Tukey’s post hoc test (not inj. vs. snf8 MO, ∗∗∗∗p < 0.0001; snf8 MO vs. snf8 MO + SNF8WT, ∗∗∗∗p < 0.0001).

(E) Quantification of the dimension of the angle formed by the optic chiasm (OC) in the midline. n = 15 (not inj.), 9 (snf8 MO), and 12 (snf8 MO + SNF8WT). One-way ANOVA with Tukey’s post hoc test is used to assess statistical significance: not inj. vs. snf8 MO, ∗∗p = 0.0039; snf8 MO vs. snf8 MO + SNF8WT, p = 0.0463. In (C)–(E) data are expressed as boxplots with mean ± SEM.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Am. J. Hum. Genet.