Extended Data Fig. 1: Expression of SPTBN1 variants alters protein expression, cellular distribution and morphology.
From: Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome
a, Western blot of total lysates from HEK 293 T/17 cells co-transfected with GFP-βIISp and mCherry plasmids and blotted with anti-GFP and anti-mCherry antibodies. Results are representative of three independent experiments. b, Western blot of Triton-X100 soluble and insoluble fractions from HEK 293 T/17 cell lysates transfected with GFP-βIISp plasmids and blotted with anti-GFP antibody. Images are representative of three independent experiments. c, Partition of indicated GFP-βIISp proteins expressed in HEK 293 T/17 cells between Triton-X100 soluble and insoluble fractions relative to total GFP-βIISp levels. Data in c were collected from n = 3 biological replicates in three independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons, ***P = 0.001, ****P < 0.0001. d, Western blot of total lysates from primary mouse cortical neurons from βIISp-KO mice transduced with lentivirus expressing RFP-PP-βIISp proteins driven by the neuronal-specific synapsin I promoter and blotted with anti-RFP and anti-βIII-tubulin antibodies. Red arrowheads and boxes mark the presence of an additional 70-kDa GFP-positive fragment in HEK 293 T/17 (a) and mouse neuron (d) lysates expressing variants that result in GFP-positive aggregates. Blots are representative of three separate experiments. e, Western blot of total lysates from human iPSC lines reprogrammed from PBMCs carrying the indicated variants and blotted with anti-βII-spectrin and anti-α-tubulin antibodies. A red asterisk indicates the presence of a truncated 205-kDa βII-spectrin fragment in lysates from iPSCs reprogrammed from P27 (p.W1787*, c.5361 G > A). Blots are representative of four independent experiments. Western blot images were cropped from Source Data Extended Data Fig. 1. f, Analysis of sequencing reads from RNA-seq of blood RNA obtained from P27 (p.W1787*, c.5361 G > A) indicate allelic expression bias, suggesting some level of nonsense mediated decay of the SPTBN1 allele transcript harboring the nonsense variant, and increased abundance of the major c.5361 G SPTBN1 allele. g, Quantification of the percent of GFP-positive HEK 293 T/17 cells with GFP aggregates for each of the indicated variants. Data were collected from n = 20 cells/genotype pooled from three independent experiments and the following number of transfection replicates: WT (n = 10), T59I (n = 3), I59Q_160Δ (n = 5), C183* (n = 6), Y190_R216Δ (n = 3), G205D (n = 4), G205S (n = 6), L247H (n = 5), L250R (n = 7), D255E (n = 5), T268A (n = 4), T268N (n = 4), T268S (n = 6), V271M (n = 4), H275R (n = 6), F344L (n = 4), R411W (n = 3), E491Q (n = 4), A850G (n = 3), E892* (n = 3), R1003W (n = 9), A1086T (n = 4), E1110D (n = 4), G1398S (n = 3), W1787* (n = 3), E1886Q (n = 4). h,i, Quantification of cell length (h) and filopodia density normalized to cell length (i) of GFP-positive HEK 293 T/17 cells expressing the indicated variants. Data in h and i were collected from WT (n = 23), T59I (n = 13), I59Q_160Δ (n = 12), C183* (n = 12), Y190_R216Δ (n = 26), G205D (n = 11), G205S (n = 11), L247H (n = 22), L250R (n = 14), D255E (n = 18), T268A (n = 13), T268N (n = 15), T268S (n = 12), V271M (n = 10), H275R (n = 13), F344L (n = 12), R411W (n = 10), E491Q (n = 11), A850G (n = 11), E892* (n = 12), R1003W (n = 15), A1086T (n = 10), E1110D (n = 12), G1398S (n = 10), W1787* (n = 12), and E1886Q (n = 12) cells pooled from six independent experiments. All data represent mean ± SEM. One-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons. (g) ****P < 0.0001, ns P > 0.05. (h) *P = 0.0119 (T268S), *P = 0.0376 (H275R), *P = 0.0184 (R411W), *P = 0.0492 (A850G); **P = 0.0029 (T59I), **P = 0.0083 (V271M); ***P = 0.0009 (E1110D), ***P = 0.0005 (E1886Q); ****P < 0.0001. (i) *P = 0.0141 (G205D); **P = 0.0079 (C183*), **P = 0.0023 (Y190_R216Δ), **P = 0.0027 (G205S), **P = 0.0083 (R411W); ***P = 0.0006 (E491Q), ***P = 0.0002 (E1886Q); ****P < 0.0001. See statistics summary in Source Data Extended Data Fig. 5.
