Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome

SPTBN1 mutations cause a neurodevelopmental syndrome characterized by intellectual disability, language and motor delays, autism, seizures and other features. The variants disrupt beta II-spectrin function and disturb cytoskeletal organization and dynamics. SPTBN1 encodes beta II-spectrin, the ubiquitously expressed beta-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal beta II-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 beta II-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 beta II-spectrin in the central nervous system

Developmental Changes in Expression of βIV Spectrin Splice Variants at Axon Initial Segments and Nodes of Ranvier

International audienceAxon initial segments (AIS) and nodes of Ranvier are highly specialized axonal membrane domains enriched in Na(+) channels. These Na(+) channel clusters play essential roles in action potential initiation and propagation. AIS and nodal Na(+) channel complexes are linked to the actin cytoskeleton through βIV spectrin. However, neuronal βIV spectrin exists as two main splice variants: a longer βIVΣ1 variant with canonical N-terminal actin and αII spectrin-binding domains, and a shorter βIVΣ6 variant lacking these domains. Here, we show that the predominant neuronal βIV spectrin splice variant detected in the developing brain switches from βIVΣ1 to βIVΣ6, and that this switch is correlated with expression changes in ankyrinG (ankG) splice variants. We show that βIVΣ1 is the predominant splice variant at nascent and developing AIS and nodes of Ranvier, but with increasing age and in adults βIVΣ6 becomes the main splice variant. Remarkably, super-resolution microscopy revealed that the spacing of spectrin tetramers between actin rings remains unchanged, but that shorter spectrin tetramers may also be present. Thus, during development βIV spectrin may undergo a switch in the splice variants found at AIS and nodes of Ranvier

Isoforms of Spectrin and Ankyrin Reflect the Functional Topography of the Mouse Kidney.

The kidney displays specialized regions devoted to filtration, selective reabsorption, and electrolyte and metabolite trafficking. The polarized membrane pumps, channels, and transporters responsible for these functions have been exhaustively studied. Less examined are the contributions of spectrin and its adapter ankyrin to this exquisite functional topography, despite their established contributions in other tissues to cellular organization. We have examined in the rodent kidney the expression and distribution of all spectrins and ankyrins by qPCR, Western blotting, immunofluorescent and immuno electron microscopy. Four of the seven spectrins (αΙΙ, βΙ, βΙΙ, and βΙΙΙ) are expressed in the kidney, as are two of the three ankyrins (G and B). The levels and distribution of these proteins vary widely over the nephron. αΙΙ/βΙΙ is the most abundant spectrin, found in glomerular endothelial cells; on the basolateral membrane and cytoplasmic vesicles in proximal tubule cells and in the thick ascending loop of Henle; and less so in the distal nephron. βΙΙΙ spectrin largely replaces βΙΙ spectrin in podocytes, Bowman's capsule, and throughout the distal tubule and collecting ducts. βΙ spectrin is only marginally expressed; its low abundance hinders a reliable determination of its distribution. Ankyrin G is the most abundant ankyrin, found in capillary endothelial cells and all tubular segments. Ankyrin B populates Bowman's capsule, podocytes, the ascending thick loop of Henle, and the distal convoluted tubule. Comparison to the distribution of renal protein 4.1 isoforms and various membrane proteins indicates a complex relationship between the spectrin scaffold, its adapters, and various membrane proteins. While some proteins (e.g. ankyrin B, βΙΙΙ spectrin, and aquaporin 2) tend to share a similar distribution, there is no simple mapping of different spectrins or ankyrins to most membrane proteins. The implications of this data are discussed

Ankyrin facilitates intracellular trafficking of α1-Na+-K+-ATPase in polarized cells

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of α1-Na+-K+-ATPase facilitates the entry of α1,β1-Na+-K+-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of α1-Na+-K+-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type α1-Na+-K+-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of α1-Na+-K+-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization

Spectrin and ankyrin mRNA in mouse kidney.

<p>Spectrin and ankyrin mRNA expression were measured by RT-PCR and qPCR with primers summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.t001" target="_blank">Table 1</a>. (A) Amplimers were detected for spectrins βΙ, βΙΙ, βΙΙΙ, and αΙΙ and for ankyrins R (Ank1), G (Ank2), and B (Ank3). NC is no-RNA control. (B) The levels of the various transcripts varied widely, as measured by qPCR. Relative abundance is presented. Results shown for three separate determinations. Error bars ±1SD from mean. (Inset) RT-PCR detected two alternative transcripts of βΙΙ spectrin (βΙΙΣ1 & βΙΙΣ2), but only one of two potential transcripts of βΙ spectrin.</p

Ankyrin B is expressed in the TAL and DCT.

<p>(A) Distribution of ankyrin B overlaps that of calbindin1, a marker of the distal convoluted tubule (DCT). (B) Distribution of ankyrin B overlaps that of NKCC2, a marker of the thick ascending loop of Henle (TAL).</p

Spectrins associate with internal organelles.

<p>ImmunoEM micrographs highlight a pool of αΙΙ/βΙΙ spectrin in association with a variety of organelles including canaliculi (arrow heads) and coated vesicles (arrows) in the cytoplasm and along the lateral and apical membranes of proximal and distal tubule cells and the collecting duct. The boxed areas are enlarged in the right column. PC, principal cell; IC, intercalated cell.</p

Cartoon depicting the distribution of renal spectrin and ankyrin and their relationship to other membrane proteins.

<p>The relative abundance of the proteins studied here is an estimate based on their localization and relative intensity of fluorescent staining. The distribution of the other proteins is derived from the published literature. While a variety of membrane and adapter proteins have been noted to interact with spectrin or ankyrin, no simple mapping of one protein to another is evident. The citations for the depicted distributions were as follows: AQP1,2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref028" target="_blank">28</a>]; NKCC2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref033" target="_blank">33</a>]; calbindin1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref031" target="_blank">31</a>]; protein 4.1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref002" target="_blank">2</a>]; RhBG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref061" target="_blank">61</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref062" target="_blank">62</a>]; NCX1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref063" target="_blank">63</a>]; ENaC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref064" target="_blank">64</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref065" target="_blank">65</a>]; IP3R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref066" target="_blank">66</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref067" target="_blank">67</a>]; NHE3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref068" target="_blank">68</a>]; NHE2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref068" target="_blank">68</a>]; KCC3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref069" target="_blank">69</a>]; KCC4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref069" target="_blank">69</a>]; ClC5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref070" target="_blank">70</a>]; ROMK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref071" target="_blank">71</a>]; NCC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref064" target="_blank">64</a>]; NCKX3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142687#pone.0142687.ref072" target="_blank">72</a>].</p