CACNA1C pore-forming, alpha-1C subunit of the voltage-gated calcium channel gives rise to L-type calcium currents mediates influx of calcium ions into the cytoplasm, and thereby triggers calcium release from the sarcoplasm1,2. CaV1.2–the predominant L-type calcium channel in the cardio-vascular system and in the brain has an intermediate voltage-dependence of activation3. It is composed of four homologous but non-identical domains (repeats I, II, III, IV), each consisting of six membrane-spanning helices (S1-S6). Helices S1 through S4 of each repeat form voltage-sensing domains (VSD); helices S5, S6 and the connecting P-loop of all four repeats together form the channel pore16.
CACNA1C variants are relatively rare and maybe related to high clinical heterogeneity, not only in arrhythmic conditions but also in structural progressive myocardial disease and CHD. Previous studies are mainly divided into two groups: those discussing basic science studies and animal models (which were not the topic of this study) and those describing heterogeneous cohorts of patients. To the best of our knowledge, this is the first comprehensive study which includes previously described variants and a cohort of 9 patients from our centre. Variants were classified according to their localization, in order to identify genotype-phenotype correlation and potential risk stratification in relation to survival and long-term results in each category.
Different survival rates are identified according to ACMG variants pathogenicity score and variants localization, as explained by Kaplan-Meier curves. In fact, Fig. 3a considers the different variant localisation domains (N-terminal, Extracellular, Transmembrane, Cytoplasmic and C-terminal) with a p- value of 0.028.
While Fig. 3b includes Kaplan Meier analysis for survival expressed in years depending on the degree of variant pathogenicity according to ACMG score (Log rank = 0.000). Pathogenic and likely pathogenic variants are shown in a single plot (ACMG score = 4–5), the second plot represents Variant of Uncertain Significance (VUS) with ACMG score = 3, and the last plot represents Not Applicable (NA) variants for the lack of data (ACMG score < 3). There is significance difference between the three plots with a log rank of 0.000. This means that validity of the ACMG score is supported by the present analysis, with the score of pathogenicity (ACMG score 4–5) that result associated to high mortality.
This study might help in determining the prognosis especially in terms of active management for early therapeutic intervention, close follow-up and potential benefit from early implantation of ICD/PMK.
The last Kaplan Meier curve (Fig. 3c) compared survival between combined Cytoplasmic domains and Transmembrane variants versus other domains (N-Terminal, Extracellular and C-terminal). There was significance between the two curves in terms of mortality with the first showing highest mortality compared to the latter domains (p-value = 0.005).
Univariate analysis considering localisation domain (table 1) showed, using N-terminal domain as reference, that arrhythmic event was significantly related to C-terminal and Extracellular domain (p = 0–012 and p = 0.044, respectively), LQTS was correlated to Cytoplasmic domain (p = 0.019). Cytoplasmic domain is important for Calcium ion selectivity and permeability and is performed by four internal repeats that contains five hydrophobic transmembrane segments (S1, S2, S3, S5, and S6) and one positively charged transmembrane segment (S4). S4 segments represent the voltage-sensor and are characterized by a series of positively charged amino acids at every third position. LQT electrophysiological phenotype is characterized by loss of current density and gain-of-function shift in activation leading to increased steady-state current4.
Brugada syndrome is significantly related to C-terminal domain (p = 0–002). Calcium binding region is localized in C-terminal domain. Variants might cause loss of a low-affinity interaction with CALM1 or loss of channel inactivation by Ca (2+) and calmodulin17.
Multivariate analysis has shown that HCM and ACMG variants score pathogenicity (4–5) are independent predictors of mortality. The p-value is 0.031 for HCM and 0.04 for variants pathogenicity (supplementary Table 3). There is no significance considering variants localisation domain. This suggestes that the differences among different regions in Figs. 3 and 5 is possibly due to a higher incidence of HCM in CACNA1C variants located in the transmembrane and cytosolic domains, as showed in Fig. 2. In order to exclude other known genetic factors, we analysed with exome sequencing our patient with the classic CACNA1C variant without identifying any pathogenic variants of genes related to HCM. This data strengthens the potential role of this gene on structural myocardial changes. A similar finding has been reported previously in literature 8 where authors have demonstrated that exome sequencing identified only one variant that was related to a complex phenotype in a family where different members showed LQTS, HCM and CHDs.
In order to give a comprehensive vision of genotype phenotype correlation, we summarized in Fig. 4 the topographic representation of different variants domains and associated major clinical features and outcome.
Another aspect that need to be considered is the mosaicism. Mosaicism refers to the co-existence, in an individual, of cells with different genotypes, although derived from a single zygote, so that some cells may present with the gene mutation (and the resulting loss/gain of function), and others do not18. The incidence of mosaicism in the human is underestimated, especially in the low-grade mosaicism. While somatic mosaicism has been implicated in over 30, monogenic disorders, mosaicism is rarely reported in LQTS19. In this review we identified five reports3, 20, 21, 22, 23 with parental mosaicism. It makes a relatively high percentage for such a small cohort, reaching 4–5% of the total analysed series. These observations have important consequences for genetic counselling, as previously identified de novo mutations may represent parental mosaicism. A shared partial phenotype should not be dismissed as a benign or insignificant finding but should be evaluated further to rule out the possibility of parental mosaicism concealing a potentially fatal heritable disease.
Another previously and relatively widely reported aspect related to CACNA1C variants is the association with multisystemic disorder, well recognized as Timothy syndrome. It is characterized by cardiac involvement (LQTS, HCM, CHD), hand/foot variable syndactyly, facial dysmorphic features (depressed nasal bridge, low-set ears, thin vermilion border of the upper lip, and round face), and neurodevelopmental features including global developmental delays and autism spectrum disorders. Another less investigated aspect is immunodeficiency and recurrent infections 24–27.