A New Homozygous CACNB2 Mutation has Functional Relevance and Supports a Role for Calcium Channels in Autism Spectrum Disorder

Abstract

BackgroundDiagnostic yield in patients with autism spectrum disorder (ASD) has improved over the last years, thanks to the introduction of whole genome arrays and next generation sequencing, but etiology is still unknown for the majority of cases. Among distinct cellular pathways, evidence implicating dysregulation of cellular calcium homeostasis in ASD pathogenesis has been accumulating, and speci c mutations in voltagegated calcium channels found in patients with autism were shown to be functionally relevant. MethodsWhole exome sequencing and Sanger sequencing were performed to identify and con rm variants in a girl with ASD, global developmental delay and precocious puberty, born of rst-degree cousins. Sitedirected mutagenesis was used to generate a human Ca V β 2d calcium channel subunit carrying a CACNB2 mutation. Whole-cell patch-clamp recordings were performed to reveal functional effects of mutant Ca V β 2d on Ba 2+ -currents mediated by L-type (Ca V 1.2) calcium channels in transiently transfected HEK-293 cells. ResultsIn an ASD patient, we identi ed a rare homozygous variant (p.Arg70Cys) in the CACNB2 gene coding for the auxiliary Ca V β 2 subunit of voltage-gated calcium channels. In a recombinant system, the Ca V β 2 variant, which was not previously associated to ASD, was found to alter Ca V 1.2 calcium channel function by signi cantly affecting activation and inactivation of whole-cell Ba 2+ -currents. LimitationsAlthough the evidence of CACNB2 involvement in ASD is slowly accumulating, the number of reported patients is very limited. Deep clinical phenotyping and functional studies in larger sets of subjects will be instrumental to fully understand the penetrance and outcome of CACNB2 variants. ConclusionsThe p.Arg70Cys variant in CACNB2 shows functional consequences similar to other ASD-associated Ca V β 2 mutations. These results support the idea of CACNB2 variations contributing to the development of ASD and hint to a rare form of Mendelian recessive autism with possible speci c comorbidities.

Inhibitory effects on L- and N-type calcium channels by a novel CaVβ1 variant identified in a patient with autism spectrum disorder

“…We previously described different mutations of the Ca V β 2 isoform which led to an increase of Ca V 1.2 activity both at the single-channel and the whole-cell level (Breitenkamp et al 2014;Despang et al 2020). In a more recent study, we could show that another mutation in CACNB2 led to a reduced inactivation of whole-cell currents, which also indicates increased Ca V 1.2 activity (Graziano et al 2021). Taken together, the increase in Ca V 1.2 activity appears to be a common feature among Ca V β 2 variants found in ASD patients.…”

“…Whole-cell recordings were performed as in our previous studies (Breitenkamp et al 2014;Despang et al 2020;Graziano et al 2021). Recordings of EGFP-positive cells were obtained 48-72 h after transfection.…”

“…(2) A mutant Ca V 1.2 protein characterized by impaired current inactivation has been shown to underly the Timothy syndrome, a monogenetic "model disease" for ASD (Splawski et al 2004;Breitenkamp et al 2015). (3) In our previous studies, we identified functional effects of Ca V β 2 variants when co-expressed with Ca V 1.2 (Breitenkamp et al 2014;Despang et al 2020;Graziano et al 2021). For the following reasons, Ca V 2.2 was chosen as a second VGCC pore subunit for our analysis of the Ca V β 1b_R296C variant: Ca V 2.2 is expressed predominantly in neurons where most of the central nervous system's synapses rely on Ca V 2.2 and Ca V 2.1 for fast synaptic transmission (Wheeler et al 1994;Catterall et al 2021).…”

“…Mutated and WT Ca V β 2 subunits interacted with the inhibitory RGK-protein Gem but differed in the extent and characteristics of being modulated by Gem. More recently, we described a homozygous N-terminal Ca V β 2 mutant at position 70 (p.R70C) that also showed an increased activity in terms of a reduced inactivation of L-type calcium currents at the whole-cell level (Graziano et al 2021 ).…”

De novo variants in CACNA1E found in patients with intellectual disability, developmental regression and social cognition deficit but no seizures

“…A large multiplex gene network analysis identified CACNA1E as candidate gene for epilepsy and autism [ 13 ]. Subsequently, a genetic association between calcium channels in general and ASD was further suggested by Liao and Li [ 14 ] and Graziano et al [ 15 ].…”