Regulation of Liprin-α phase separation by CASK is disrupted by a mutation in its CaM kinase domain.

CASK is a unique membrane-associated guanylate kinase (MAGUK) because of its Ca2+/calmodulin-dependent kinase (CaMK) domain. We describe four male patients with a severe neurodevelopmental disorder with microcephaly carrying missense variants affecting the CaMK domain. One boy who carried the p.E115K variant and died at an early age showed pontocerebellar hypoplasia (PCH) in addition to microcephaly, thus exhibiting the classical MICPCH phenotype observed in individuals with CASK loss-of-function variants. All four variants selectively weaken the interaction of CASK with Liprin-α2, a component of the presynaptic active zone. Liprin-α proteins form spherical phase-separated condensates, which we observe here in Liprin-α2 overexpressing HEK293T cells. Large Liprin-α2 clusters were also observed in transfected primary-cultured neurons. Cluster formation of Liprin-α2 is reversed in the presence of CASK; this is associated with altered phosphorylation of Liprin-α2. The p.E115K variant fails to interfere with condensate formation. As the individual carrying this variant had the severe MICPCH disorder, we suggest that regulation of Liprin-α2-mediated phase condensate formation is a new functional feature of CASK which must be maintained to prevent PCH.

BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy.

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin-5 for fusion of endoplasmic reticulum-derived vesicles with the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER-to-Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild-type, among them ERGIC-53. The BET1/ERGIC-53 interaction was validated by endogenous co-immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC-53 was observed in P1 and P2's derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC-53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD.