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, 77 (9), 816-22

CACNA1D De Novo Mutations in Autism Spectrum Disorders Activate Cav1.3 L-type Calcium Channels

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CACNA1D De Novo Mutations in Autism Spectrum Disorders Activate Cav1.3 L-type Calcium Channels

Alexandra Pinggera et al. Biol Psychiatry.

Abstract

Background: Cav1.3 voltage-gated L-type calcium channels (LTCCs) are part of postsynaptic neuronal signaling networks. They play a key role in brain function, including fear memory and emotional and drug-taking behaviors. A whole-exome sequencing study identified a de novo mutation, p.A749G, in Cav1.3 α1-subunits (CACNA1D), the second main LTCC in the brain, as 1 of 62 high risk-conferring mutations in a cohort of patients with autism and intellectual disability. We screened all published genetic information available from whole-exome sequencing studies and identified a second de novo CACNA1D mutation, p.G407R. Both mutations are present only in the probands and not in their unaffected parents or siblings.

Methods: We functionally expressed both mutations in tsA-201 cells to study their functional consequences using whole-cell patch-clamp.

Results: The mutations p.A749G and p.G407R caused dramatic changes in channel gating by shifting (~15 mV) the voltage dependence for steady-state activation and inactivation to more negative voltages (p.A749G) or by pronounced slowing of current inactivation during depolarizing stimuli (p.G407R). In both cases, these changes are compatible with a gain-of-function phenotype.

Conclusions: Our data, together with the discovery that Cav1.3 gain-of-function causes primary aldosteronism with seizures, neurologic abnormalities, and intellectual disability, suggest that Cav1.3 gain-of-function mutations confer a major part of the risk for autism in the two probands and may even cause the disease. Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals. Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism.

Keywords: Autism spectrum disorders; Calcium channel blockers; Human genetics; L-type calcium channels; Neuropsychiatric disorders; Whole-exome sequencing.

Figures

Figure 1
Figure 1
Biophysical properties of A749G expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and A749G and A749G mutants coexpressed together with wild-type (WT + A749G, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (six independent transfections) to account for differences in expression levels between transfections. A749G cotransfected with wild-type (WT + A749G) resulted in significantly increased peak current amplitudes (for statistics and numbers, see Results). Statistics for gating parameters are summarized in Table 1. (B) Steady-state activation (circles) and inactivation (squares) curves for wild-type and A749G were obtained as described in Methods and Materials. Means ± SEM are illustrated. Wild-type, n = 29 (nine transfections); A749G, n = 27 (six transfections). Steady-state activation parameters for WT + A749G are given in Table 1.
Figure 2
Figure 2
Functional consequences of A749G and G407R expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and G407R and G407R mutants coexpressed together with wild-type (WT + G407R, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (n = 4) to account for differences in expression levels between experiments. Transfections with high expression levels of wild-type were selected for analysis to provide sufficient current amplitudes for G407R channels. Peak current amplitudes of G407R mutants and G407R cotransfected with wild-type were significantly reduced (mean ± SEM [pA/pF]; wild-type, −24.6 ± 4.18, n = 15; G407R, −8.12 ± .70, n = 13; WT + G407R, −7.63 ± .87, n = 9; both p < .0001 vs. wild-type, Mann-Whitney test). Statistics for gating parameters obtained from all transfections are summarized in Table 1.(B) Inactivation of wild-type, A749G, and G407R mutants during 5-sec depolarizations from a holding potential of −80 mV to maximum voltage. Peak inward calcium currents for overlapping wild-type and A749G traces are indicated by arrows for clarity. Traces represent the means ± SEM (only ± SEM is illustrated) from parallel recordings of wild-type and mutant channels performed on the same day in four (G407R) or two (A749G) independent transfections. The pronounced slowing of inactivation is evident from the respective normalized current traces shown in the inset. See text for numbers and % current persisting after 5-sec depolarizations. WT, wild-type.

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