Whole Exome Sequencing Identifies a Heterozygous Variant in the Cav1.3 Gene CACNA1D Associated with Familial Sinus Node Dysfunction and Focal Idiopathic Epilepsy

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  Figure 1. Identification of the heterozygous CACNA1D variant R390H. (a) Pedigree of the family with a combined clinical phenotype of cardiac and extra-cardiac symptoms with a heterozygous CACNA1D variant (p.Arg930His; shortly: R930H) and autosomal dominant syndromic form of SND and epilepsy. Men are denoted by squares, woman by circles. Filled symbols represent clinically affected family members; N indicates family members without a diagnosis of SND or epilepsy, + presence of the heterozygous variant R930H, − absence of the heterozygous variant R930H, 0 not tested. The proband is indicated by an arrow. Characteristics of cardiac and neurologic phenotypes are indicated below (SND, sinus node dysfunction; AVB, atrio-ventricular block; Epil., epilepsy; sEpil., suspected epilepsy; ADHD, attention deficit hyperactivity disorder). (b) ECG of proband (III:1) at age 5 years with documented sinus arrest, and (c) EEG of 3 CACNA1D variant carriers demonstrating occurrence of sharp waves with different localization. Above: EEG of family member III:I at the age of five shows occipital sharp waves (P3-O1) and bulbus artifacts (Fp1 and Fp2). Middle: EEG of family member III:2 at the age of 10 with…

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  Figure 2. Conservation and localization of the heterozygous CACNA1D variant R390H. (a) Electropherogram of the proband (III:1) with the heterozygous CACNA1D nucleotide variant c.2789G>A (NM_000720.3). The peaks represent each base in the gDNA sequence (red, thymine; green, adenine; black, guanine and blue, cytosine). (b) Multiple sequence alignment of the human CACNA1D protein region encompassing arginine 930 with orthologous protein sequences. Identical amino acids are indicated by an asterisk and highly conserved amino acids by a colon in the lower lane. The mutated amino acid residue is indicated in bold. (c) The predicted topology of the Cav1.3 channel and structure of the human transcript variants used in functional studies of Cav1.3. Cav1.3 α1-subunit comprised of four structurally homologous domains (I, II, III, IV), each containing six transmembrane spanning domains (S1–S6) together with a pore region between transmembrane helices S5 and S6. Both human Cav1.3 constructs used in expression studies (Cav1.3L and Cav1.3S) contain the alternatively spliced exon 8A in domain I (Supplementary File: Figure S1). Alternative splicing in exon 43 located in the C-terminus results in a…

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  Figure 3. The R930H variant induces transcript variant dependent gating changes of Cav1.3 channels. (a) Representative current traces of wild-type Cav1.3S and mutant Cav1.3S R930H (R930HS) or (b) wild-type Cav1.3L and mutant Cav1.3L R930H (R930HL) upon a depolarization step to the potential of their maximal inward currents. (c,d) Current-voltage relationships of the ICa (mean ± S.E.M.) of (c) wild-type versus mutant short and (d) long Cav1.3 transcript variants. Note that the R930H variant enhanced current densities in the short splice variant. In contrast, current densities were reduced in the mutant long splice transcript variant. (e) Boxplots of the peak current densities obtained from depolarization to the respective voltage of the peak current (displayed as median (dotted lines), 25/75 percentile (whiskers) and mean (black lines)); median and 25/75 percentiles. In pA/pF: wild-type Cav1.3S: −6.8 ± (−10.5/−5.7), n = 32 and R930HS: −11.3 ± (−23.7/−7.7), n = 39, p = 0.0016; wild-type Cav1.3L: −11.2 ± (−19.1/−6.4), n = 55 and R930HL: −9.5 ± (−10.4/−7.2), n = 32, p = 0.14: **: p < 0.01. Statistics were performed using the Mann–Whitney test for both splice variants.

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  Figure 4. R930H does not alter activation and inactivation parameters of Cav1.3S and Cav1.3L. (a) Voltage-dependence of activation of the wild-type and mutant Cav1.3S and (b) Cav1.3L variants. (c) Voltage-dependence of inactivation of wild-type Cav1.3S versus R930HS and (d) wild-type Cav1.3L versus R930HL. (e) Recovery from inactivation of wild-type Cav1.3S versus R930HS (τ Cav1.3S: 270.1 ± 14.4 ms, n = 10; τ R930HS: 261.9 ± 18.2 ms, n = 12; not significant, unpaired Student’s t-test) and (f) wild-type Cav1.3L versus R930HL (τ Cav1.3L: 166.1 ± 20.56 ms, n = 5; τ R930HL: 180.6 ± 36.4 ms, n = 5; not significant, unpaired Student’s t-test. Consistent with the literature, recovery from inactivation was faster in the Cav1.3L variant (τ Cav1.3L vs. τ Cav1.3S p = 0.0011, unpaired Student’s t-test) [20]. However, this parameter was not affected by the R930H variant. Recovery from inactivation was determined by 10 ms test pulses to Vmax at different time points after a 5-s conditioning pulse to the potential of the peak currents followed by a test pulse, as described in the Materials and Methods section. Data were fitted using a mono-exponential function. Data in (a–d) are shown as mean ± S.E.M.…