Phenotypic expansion of CACNA1C-associated disorders to include isolated neurological manifestations

Abstract

Purpose: CACNA1C encodes the alpha-1-subunit of a voltage-dependent L-type calcium channel expressed in human heart and brain. Heterozygous variants in CACNA1C have previously been reported in association with Timothy syndrome and long QT syndrome. Several case reports have suggested that CACNA1C variation may also be associated with a primarily neurological phenotype.

Methods: We describe 25 individuals from 22 families with heterozygous variants in CACNA1C, who present with predominantly neurological manifestations.

Results: Fourteen individuals have de novo, nontruncating variants and present variably with developmental delays, intellectual disability, autism, hypotonia, ataxia, and epilepsy. Functional studies of a subgroup of missense variants via patch clamp experiments demonstrated differential effects on channel function in vitro, including loss of function (p.Leu1408Val), neutral effect (p.Leu614Arg), and gain of function (p.Leu657Phe, p.Leu614Pro). The remaining 11 individuals from eight families have truncating variants in CACNA1C. The majority of these individuals have expressive language deficits, and half have autism.

Conclusion: We expand the phenotype associated with CACNA1C variants to include neurodevelopmental abnormalities and epilepsy, in the absence of classic features of Timothy syndrome or long QT syndrome.

Figure 1

a: Spectrum of dysmorphology seen in individuals with CACNA1C non-truncating variants. A) Individual P1 at age 23 years. Note pes planovalgus B) Individual P5 at age 4 years C) Individual P10. Note clubfoot in infancy, and small appearing feet with prominent heels, adducted thumb, and camptodactyly later in childhood. D) Individual P12 at age 6 years of age. Note strabismus and kyphoscoliosis. E) Individual 13 at age 6 years old. b: Spectrum of dysmorphology seen in individuals with CACNA1C truncating variants: A) Individual 15 at 15 years old. B) Individual 16a at 6 years old. Note large ears. C) Individual P18. Note mildly broad thumbs, hypoplastic 5^th fingernails, broad halluces, and hypoplastic fifth toes. D) Individual P19 at age.

Figure 2:. Characterization of Cav1.2 missense variants (L614P, L657F, L1408V, and L614R):

A), Representative whole-cell Ca²⁺ current traces after heterologous expression of Cavβ3, Cavα2δ1 and either Cav1.2 WT or Cav1.2 variants L614P, L657F, and L1408V in HEK293 cells. Solutions and voltage protocols used to elicit Ca²⁺ currents are described in the methods section. B), Averaged current-voltage (I-V) curves from whole-cell patch clamp measurements of HEK293 cells expressing Cavβ3, Cavα2δ1 and either Cav1.2 WT (n=14) or Cav1.2 variants L614P (n=11), L657F (n=8), and L1408V (n=10). C), Current density (pA/pF) analysis of the peak current of Cav1.2 WT (n=14) or Cav1.2 variants L614P (n=11), L657F (n=8), and L1408V (n=10) from cell recordings in A and B. Individual cells (open circles) are overlapped with the average current density (pA/pF), depicted as bars in the graph. Statistical analyses were carried out with a one-way ANOVA test. * p < 0.05 vs. Cav1.2 WT. D), Representative Western blot of whole-cell lysate from HEK293 cells heterologously expressed with Cavβ3, Cavα2δ1 and either Cav1.2 WT or Cav1.2 variants L614P, L657F, and L1408V. Actin was used as loading control. Antibodies used for immunoblotting of proteins are described in the methods section. E), Densitometry analysis of Cav1.2 WT (n=9) or Cav1.2 variants (L614P (n=9), L657F (n=9), and L1408V (n=9)) protein band intensities relative to the respective actin band intensities. Individual cells (open circles) are overlapped with the average relative Cav1.2 band intensities (240 kDa), depicted as bars in the graph. Statistical analyses were carried out with a one-way ANOVA test. * p < 0.05 vs. Cav1.2 WT. F) Averaged current-voltage (I-V) curves from whole-cell patch clamp measurements of HEK293 cells expressing CACNB2, CACNA2D, EGFP and either Cav1.2 WT (n=14) or Cav1.2 variant L614R (n=9). Differences in current density do not meet statistical significance.

Figure 3:. Location of CACNA1C variants in 25 individuals.

Blue dots indicate missense variants or in-frame deletion, and red dots indicate truncating variants. Protein alterations are spread throughout the protein, although there is a clustering of missense variants in one of the linker segments of domain II (exon 13) and domain IV (exon 35), suggesting the potential significance of these regions of the protein in the function of CACNA1C in the central nervous system. Figure was created using open-source tool, Protter.