Pathogenicity of de novo CACNA1D Ca2+ channel variants predicted from sequence co-variation.

Voltage-gated L-type Cav1.3 Ca2+ channels support numerous physiological functions including neuronal excitability, sinoatrial node pacemaking, hearing, and hormone secretion. De novo missense mutations in the gene of their pore-forming α1-subunit (CACNA1D) induce severe gating defects which lead to autism spectrum disorder and a more severe neurological disorder with and without endocrine symptoms. The number of CACNA1D variants reported is constantly rising, but their pathogenic potential often remains unclear, which complicates clinical decision-making. Since functional tests are time-consuming and not always available, bioinformatic tools further improving pathogenicity potential prediction of novel variants are needed. Here we employed evolutionary analysis considering sequences of the Cav1.3 α1-subunit throughout the animal kingdom to predict the pathogenicity of human disease-associated CACNA1D missense variants. Co-variation analyses of evolutionary information revealed residue-residue couplings and allowed to generate a score, which correctly predicted previously identified pathogenic variants, supported pathogenicity in variants previously classified as likely pathogenic and even led to the re-classification or re-examination of 18 out of 80 variants previously assessed with clinical and electrophysiological data. Based on the prediction score, we electrophysiologically tested one variant (V584I) and found significant gating changes associated with pathogenic risks. Thus, our co-variation model represents a valuable addition to complement the assessment of the pathogenicity of CACNA1D variants completely independent of clinical diagnoses, electrophysiology, structural or biophysical considerations, and solely based on evolutionary analyses.

Novel CACNA1A Variant p.Cys256Phe Disrupts Disulfide Bonds and Causes Spinocerebellar Ataxia.

BACKGROUND: Spinocerebellar ataxia (SCA) is a progressive, autosomal dominant neurodegenerative disorder typically associated with CAG repeat expansions. OBJECTIVE: We assessed the pathogenicity of the novel, heterozygous missense variant p.Cys256Phe (C256F) in the pore-forming α1-subunit of the Cav2.1 Ca2+ channel found in a 63-year-old woman with SCA with no CAG repeat expansion. METHODS: We examined the effect of the C256F variant on channel function using whole-cell patch-clamp recordings in transfected tsA-201 cells. RESULTS: The maximum Ca2+ current density was significantly reduced in the mutant compared to wild-type, which could not be explained by lower expression levels of mutant Cav2.1 α1- protein. Together with a significant increase in current inactivation, this is consistent with a loss of channel function. Molecular modeling predicted disruption of a conserved disulfide bond through the C256F variant. CONCLUSIONS: Our results support the pathogenicity of the C256F variant for the SCA phenotype and provide further insight into Cav2.1 structure and function.

Stabilization of negative activation voltages of Cav1.3 L-Type Ca2+-channels by alternative splicing.

-->Low voltage-activated Cav1.3 L-type Ca2+-channels are key regulators of neuronal excitability controlling neuronal development and different types of learning and memory. Their physiological functions are enabled by their negative activation voltage-range, which allows Cav1.3 to be active at subthreshold voltages. Alternative splicing in the C-terminus of their pore-forming α1-subunits gives rise to C-terminal long (Cav1.3L) and short (Cav1.3S) splice variants allowing Cav1.3S to activate at even more negative voltages than Cav1.3L. We discovered that inclusion of exons 8b, 11, and 32 in Cav1.3S further shifts activation (-3 to -4 mV) and inactivation (-4 to -6 mV) to more negative voltages as revealed by functional characterization in tsA-201 cells. We found transcripts of these exons in mouse chromaffin cells, the cochlea, and the brain. Our data further suggest that Cav1.3-containing exons 11 and 32 constitute a significant part of native channels in the brain. We therefore investigated the effect of these splice variants on human disease variants. Splicing did not prevent the gating defects of the previously reported human pathogenic variant S652L, which further shifted the voltage-dependence of activation of exon 11-containing channels by more than -12 mV. In contrast, we found no evidence for gating changes of the CACNA1D missense variant R498L, located in exon 11, which has recently been identified in a patient with an epileptic syndrome. Our data demonstrate that alternative splicing outside the C-terminus involving exons 11 and 32 contributes to channel fine-tuning by stabilizing negative activation and inactivation gating properties of wild-type and mutant Cav1.3 channels.

Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder.

Background: There is increasing evidence that de novo CACNA1D missense mutations inducing increased Cav1.3 L-type Ca2+-channel-function confer a high risk for neurodevelopmental disorders (autism spectrum disorder with and without neurological and endocrine symptoms). Electrophysiological studies demonstrating the presence or absence of typical gain-of-function gating changes could therefore serve as a tool to distinguish likely disease-causing from non-pathogenic de novo CACNA1D variants in affected individuals. We tested this hypothesis for mutation S652L, which has previously been reported in twins with a severe neurodevelopmental disorder in the Deciphering Developmental Disorder Study, but has not been classified as a novel disease mutation. Methods: For functional characterization, wild-type and mutant Cav1.3 channel complexes were expressed in tsA-201 cells and tested for typical gain-of-function gating changes using the whole-cell patch-clamp technique. Results: Mutation S652L significantly shifted the voltage-dependence of activation and steady-state inactivation to more negative potentials (~ 13-17 mV) and increased window currents at subthreshold voltages. Moreover, it slowed tail currents and increased Ca2+-levels during action potential-like stimulations, characteristic for gain-of-function changes. To provide evidence that only gain-of-function variants confer high disease risk, we also studied missense variant S652W reported in apparently healthy individuals. S652W shifted activation and inactivation to more positive voltages, compatible with a loss-of-function phenotype. Mutation S652L increased the sensitivity of Cav1.3 for inhibition by the dihydropyridine L-type Ca2+-channel blocker isradipine by 3-4-fold.Conclusions and limitationsOur data provide evidence that gain-of-function CACNA1D mutations, such as S652L, but not loss-of-function mutations, such as S652W, cause high risk for neurodevelopmental disorders including autism. This adds CACNA1D to the list of novel disease genes identified in the Deciphering Developmental Disorder Study. Although our study does not provide insight into the cellular mechanisms of pathological Cav1.3 signaling in neurons, we provide a unifying mechanism of gain-of-function CACNA1D mutations as a predictor for disease risk, which may allow the establishment of a more reliable diagnosis of affected individuals. Moreover, the increased sensitivity of S652L to isradipine encourages a therapeutic trial in the two affected individuals. This can address the important question to which extent symptoms are responsive to therapy with Ca2+-channel blockers.