A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming Ca(V)2.1 alpha(1) subunit and the auxiliary alpha 2 delta-2 and beta(4) subunits. beta(4) is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat beta(4b) cDNA. Heterologously expressed wild-type beta(4b) associated with GFP-Ca(V)1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the beta(4b)-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with Ca(V)2.1 in tsA201 cells, beta(4b) and beta(4b)-L125P augmented the calcium current amplitudes, however, beta(4b)-L125P failed to stably complex with alpha(1) subunits. These results indicate that p.Leu125Pro disrupts the stable association of beta(4b) with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype beta(4b) was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of beta(4b) in cultured myotubes and hippocampal neurons. While binding of beta(4b) to the known interaction partner PPP2R5D (B56 delta) was not affected, complex formation between beta(4b)-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of beta(4b). Author summary Neurodevelopmental disorders encompass a broad spectrum of neurological and psychiatric conditions and are caused by mutations in many different genes. For example, mutations in genes encoding voltage-gated calcium channels have been linked to various diseases of the nervous system in humans and mice. Voltage-gated calcium channels are critical regulators of the synaptic communication between neurons, of processes involved in learning and memory, and of activity-dependent gene expression. Here we report a disease-associated mutation on both copies of the CACNB4 gene encoding an auxiliary beta(4) subunit of the chief presynaptic calcium channel in the brain. Two siblings with a severe neurodevelopmental disorder carry the homozygous CACNB4 mutation causing an amino acid substitution known to disrupt the folding of the calcium channel beta(4) subunit. We demonstrate that this amino acid change abolished the incorporation of the beta(4) subunit into channel complexes in the synapse, as well as beta(4)'s ability to translocate into the cell nucleus, and to complex with alpha(1) channel subunits and a neuronal scaffolding protein. The combined evidence from our genetic and functional analysis suggests that dysfunction of both beta(4) subunit channel and non-channel functions underlies the severe neurological phenotype in the two siblings. We therefore identified CACNB4 as a neurodevelopmental disease gene.