Abstract
SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system.
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Data availability
The whole-genome and -exome sequencing or transcriptomic data will not be made publicly available as they contain information that could compromise research participant privacy/consent. Source data are provided with this paper. Information on the DNA- and RNA-sequencing raw data and other analyses supporting the findings of this study is available from the corresponding authors upon request.
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Acknowledgements
We thank all the families who participated in this study. We thank M. Rasband and K.-A. Nave for the gift of the βII-spectrin conditional null and the Nex-Cre mice, respectively. We thank N. V. Riddick for her assistance with the behavioral studies. We thank B. Koller and K. Mohlke for their insightful comments on this manuscript and J. Bear for helpful discussions. M.A.C., L.E.S.-R. and E.W.K. were supported by the Center for Individualized Medicine at the Mayo Clinic. D.N.L. was supported by the University of North Carolina at Chapel Hill (UNC-CH) School of Medicine as a Simmons Scholar, by the National Ataxia Foundation and by the US National Institutes of Health (NIH) grant no. R01NS110810. E.E.E. was supported by the NIH grant no. MH101221. Microscopy was performed at the UNC-CH Neuroscience Microscopy Core Facility, supported, in part, by funding from the NIH-NINDS Neuroscience Center Grant no. P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant no. U54 HD079124, which also supported the behavioral studies. Research reported in this manuscript was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under Award Number U01HG007672 (Duke University to V. Shashi). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support.
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M.A.C. and D.N.L. conceived and planned the study with input from Q.K.-G.T. and R.C.S. M.A.C. managed the collection, analysis and interpretation of patient clinical data with Q.K.-G.T., R.C.S. and D.N.L. D.N.L. designed the cell biology, histology and biochemistry studies; performed these; and analyzed the data together with B.A.C., K.A.B., S.D., D.A., R.J.E., S. Afriyie., J.C.B. and L.F.R. A.A.B., L.J.M. and A.S.B. generated and characterized the iPSCs. S.T., M.T.Z., B.T. and D.N.L. performed the structural modeling. K.M.H. and S.S.M. performed the mouse behavioral studies. M.C.S. contributed reagents. M.A.C. and D.N.L. wrote the manuscript with contributions from B.A.C., R.C.S., S.S.M., M.T.Z. and B.T. E.W.K. and D.N.L. supervised the study. All other authors including Q.K.-G.T. and R.C.S. contributed clinical data. All authors approved the final manuscript.
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E.T., R.E.P., Y.S., E.A.N. and A.B. are employees of GeneDx, Inc.
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Extended data
Extended Data Fig. 1 Expression of SPTBN1 variants alters protein expression, cellular distribution and morphology.
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.
Extended Data Fig. 2 SPTBN1 variants alter interaction with critical cytoskeleton partners.
a, Immunofluorescence images, representative of three independent experiments, show HEK 293 T/17 cells transfected with mCherry-αIISp and with either WT or mutant GFP-βIISp plasmids. Cells were stained for actin (phalloidin) and DAPI. Scale bar, 10 μm. b, Immunofluorescence images of DIV8 mouse βIISp-WT (top) and βIISp-Het (bottom) cortical neurons transfected with indicated GFP-βIISp plasmids. Scale bar, 10 μm. GFP-positive aggregates are detected in neurons expressing these subsets of CH domain variants regardless of the level of endogenous βII-spectrin. Images are representative of n = 15 neurons per transfection derived from three independent experiments. c, Western blot from a binding assay to assess interaction between mCherry-αIISp and GFP-βII-spectrin proteins representative of n = 3 biological replicates from three independent experiments. Lysates from HEK 293 T/17 cells expressing mCherry-αII-spectrin were incubated with GFP-βII-spectrin proteins coupled to GFP beads. The presence of mCherry-αII-spectrin in eluates from GFP beads was evaluated by blotting with anti-GFP and anti-mCherry antibodies. d, Coomassie blue staining showing the presence of purified full-length βII-spectrin and F-actin in the supernatant (S) and pellet (P) fractions from an actin co-sedimentation assay. Blot is representative of three independent experiments each with n = 1 biological replicate. e, Co-IP assay in HEK 293 T/17 cells to assess interaction between 220-kDa ankyrin-B (AnkB)-2HA and GFP-βII-spectrin proteins. The presence of 220-kDa AnkB-3xHA and GFP-βII-spectrin proteins in initial lysates and eluates from beads coupled to rabbit IgG isotype control of a rabbit anti-GFP antibody was detected by blotting with anti-GFP and anti-HA antibodies. Blot is representative of four independent experiments, each with n = 1 biological replicate. Western blot images were cropped from Source Data Extended Data Fig. 2.
Extended Data Fig. 3 Modeling effects of SPTBN1 variants.
a-c, Potential coevolution of the closed conformation of the tandem calponin homology domains (CH1-CH2) of βII-spectrin (SPTBN1) (CH1 domain (teal), CH2 domain (red)), actinin-4 (ACTN4) (brown), and utrophin (UTRN) (purple)47. d,e, Top hits from docking simulations of βII-spectrin’s CH1 (d) and CH248 (e) onto F-actin (gray). Domains in dark blue correspond to cryo-EM structure of the CH1 domain of βIII-spectrin bound to F-actin49. f, Correct length of simulated interdomain linker (dark blue) in agreement with the orientation of the docked CH2 domain (red). g,h, Spatial distributions of the missense variants in βII-spectrin implicate disease mechanisms. g, Linear conformation of the entire 3D protein model is shown with the calponin homology (CH) domains (CH1 and CH2) in the N-terminus (red), the spectrin repeats (SR) (green) and the pleckstrin homology (PH) domain in the C-terminus (purple). h, The 17 SR domains are superimposed with a minimal cartoon representation to emphasize the consistency of the 3D architecture despite high sequence diversity. The positions of the amino acid residues representing the missense variants are marked by gold-colored spheres.
Extended Data Fig. 4 Effects of SPTBN1 variants on axonal growth.
a, Images of DIV8 βII-SpWT, βII-SpHet, βII-SpKO, and GFP-βIISp rescued βII-SpKO neurons transfected at DIV3 with mCherry. Staining with an antibody specific for AnkG was used to label the AIS (yellow arrowhead) and to identify axonal processes. Scale bar, 30 μm. Images are representative of three independent experiments.
Extended Data Fig. 5 Effects of SPTBN1 variants on dendrites.
a, Images of DIV18 βII-SpWT, βII-SpHet, βII-SpKO, and GFP-βIISp rescued βII-SpKO neurons stained with an anti-GFP antibody. Scale bar, 30 μm. b,c, Quantification of length of primary dendrites (b) and of total number of primary and secondary dendrites (c) of βII-SpWT, βII-SpHet, βII-SpKO, and rescued βII-SpKO DIV18 neurons (n = 6-16 neurons/genotype) compiled from three independent experiments. Data represent mean ± SEM. One-way ANOVA with Dunnett’s post hoc analysis test for multiple comparisons, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See statistics summary in Source Data Extended Data Fig. 5.
Extended Data Fig. 6 Effects of βII-spectrin deficiency on neuronal morphology and brain development.
a, Image, representative from three independent experiments show DIV8 βII-SpKO cortical neurons rescued with WT GFP-βIISp or with GFP-βIISp bearing variants within the distal portion of the CH2 domain. Neurons were stained for actin (phalloidin) and endogenous αII-spectrin. Yellow dotted lines demark the cell edge. Scale bar, 5 μm. b, Images of PND25 βII-SpNexWT and βII-SpNexKO brains stained for neurofilament to label axons and DAPI. Staining for βII-spectrin show specific loss of the protein in axons from callosal projection neurons from βII-SpNexKO mice. Scale bar, 50 μm. White dotted lines denote the position and boundaries of the corpus callosum (CC). Brains were collected from two separate litters and processed for staining and imaging as part of one independent experiment.
Extended Data Fig. 7 Developmental and behavioral phenotypes of βII-spectrin deficient mice.
a, Images of male PND25 wildtype (βII-SpNexWT) mice and mice lacking βII-spectrin only in cortical and hippocampal projection neurons (βII-SpNexKO) driven by Nex-Cre. b-e, Magnitude of acoustic startle responses (b,d) and percent of prepulse inhibition (c,e) in βII-SpWT mice and mice with partial (βII-SpHet) and complete (βII-SpKO) loss of βII-spectrin in neural progenitors driven by Nestin-Cre. Trials included no stimulus (NoS) trials and acoustic startle stimulus (AS; 120 dB) alone trials. Data in b and c represent mean ± SEM (n = 15 βII-SpWT and n = 5 βII-SpKO male mice). Data in d and e represent mean ± SEM (n = 12 male mice/genotype). Fisher’s PLSD tests following repeated measures ANOVA. b,*P < 0.05, **P < 0.01. c-e, P > 0.05. f, Latency to fall from an accelerating rotarod. Trials 4 and 5 were given 48 h after the first three trials. g,h, Latencies to find the hidden escape platform during acquisition (g) and reversal (h) learning phases of the Morris water maze test for βII-SpWT and βII-SpHet mice. Data represent mean ± SEM of four trials per day. Fisher’s PLSD tests following repeated measures ANOVA. f-h, P > 0.05. i,j, Mice were given a one-minute probe trial without the platform following the acquisition and reversal phases of the Morris water maze test. Target indicates the site where the platform had been located in each phase. Measures were taken of swim path crossings over the target location or corresponding areas in the other quadrants. Within-genotype repeated measures ANOVA, effect of quadrant (the repeated measure), **P = 0.0012, ****P < 0.0001. k,l, Preference for social novelty during a three-chamber choice task. Within-genotype repeated measures ANOVA, *P = 0.0145, **P = 0.0052. Data in f-l represent mean ± SEM (n = 12 male mice/genotype). m, Lack of significant genotype effects on anxiety-like behavior in the elevated plus maze, marble-burying assay, and open field; sensory ability in the buried food test for olfactory function and hot plate test for thermal sensitivity; and vision and swimming ability in the Morris water maze. Data represent mean ± SEM (n = 12 male mice/genotype). Within-genotype repeated measures ANOVA, P > 0.05.
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Cousin, M.A., Creighton, B.A., Breau, K.A. et al. Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nat Genet 53, 1006–1021 (2021). https://doi.org/10.1038/s41588-021-00886-z
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DOI: https://doi.org/10.1038/s41588-021-00886-z
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