Timothy Syndrome

Role of Splicing

CACNA1c spans over 500 kb in the human genome and undergoes extensive alternative splicing. ^, The splicing of this gene is complex and only partially understood. Nonetheless, it may have a role in the definition of the variability of the clinical manifestations of the disease. There are at least 42 splicing variants (also called isoforms) and there is tissue-specificity of splicing control; for example, the isoforms found in the heart are different from those found in the brain. ^, Furthermore, there is interindividual variability of isoform abundance. Wang et al., by analyzing 65 left ventricular (LV) human heart tissues, detected possible individual variability of inclusion of two key alternative exons, 8 and 8A, that harbor the typical TS mutations.

No experimental data directly address the clinical relevance of such individual splicing variability in TS, but experimental studies show that alternative splicing may affect the channel function. Bartels et al. has shown that the alternatively spliced exons 1 and 8 affect the current density with increased current densities when channels are produced with 1/8A or 1/8 variants compared with the 1A/8A and 1A/8 variants. This behavior is likely because of the increased number of channels expressed in the plasma membrane rather than biophysical differences because the single channel properties were not different. It is therefore tempting to speculate that individual variability of splicing machinery or environmental factors affecting this process can have a role in determining the clinical manifestations of TS.

Ca _V 1.2 alternative splicing leading to isoforms with different biophysical properties is also influenced by the presence of pathologic conditions such as heart failure and hypertension. Less is known about the splicing profile of Ca _V 1.2 in the brain, and even less is known about other tissues. The available evidence supports the presence of regional differences of Ca _V 1.2 isoform expression in the brain that are possibly associated with psychiatric disorders and, interestingly, autism spectrum disorder (ASD), often observed in TS patients.

Calcium Channel Inactivation

Upon square- or AP-shaped voltage pulses, I _Ca activates rapidly (2–3 ms), which is then followed by a slow decay. The I _Ca decay (inactivation) shows a biphasic behavior that has been linked to the separate processes of Ca ²⁺ (fast decay) and voltage (slow decay) dependent inactivation. The Ca ²⁺ mediated component of inactivation (CDI) is mainly modulated by the intracellular (cytosolic) concentration of calcium ([Ca ²⁺ ]i) and by the Ca ²⁺ /calmodulin complex that binds to the C-terminus of the channel. ^, The majority of Ca _V 1.2 mutations associated with TS impair the voltage-dependent inactivation (VDI) process. To take place, VDI requires a sequence of events triggered by the membrane voltage shift during depolarization that induces a movement of the positively charged S4 transmembrane segments and of the DI-DII loop leading to pore closure. The pore-forming transmembrane segments S6, the intracellular linkers between the other domains, and the carboxyl terminus also participate in the VDI process. ^, Furthermore, the binding of the β ₁ -subunit to the DI-II linker speeds VDI by moving it toward the pore upon cell depolarization. All these events that control VDI have a role in the pathophysiology of TS mutations.

Position of Mutants and Structure-Function Correlations

Among the various components of the calcium channel macromolecular complex, only α ₁ -subunit mutations have been causally associated with TS. In 2004, as a result of an international collaborative study, we identified 17 cases with a distinguishing phenotype (see clinical session later) and demonstrated the presence of a single CACNA1c mutation in all of them. This mutation, G406R , was present in the alternatively spliced exon 8A located at the end of transmembrane segment S6 of domain I. A few months later Igor Splawski identified two CACNA1c mutations in the alternatively spliced exon 8: G406R (homologous to the mutation identified in exon 8A) and G402S. Interestingly both patients displayed the entire spectrum of clinical manifestations (see clinical session later) but without syndactyly. For this reason, the disorder was classified as a new variant known as TS2.

In the subsequent years the number of mutations associated with typical TS and atypical phenotypes has progressively grown ( Table 98.1 ). Currently, among the known Ca _V 1.2 mutations, seven are associated with typical TS: G402S (exon 8), G402R (exon 8), S405R (exon 8), G406R (exon 8), G406R (exon 8A), I1166T , and A1473G . The G406R in exon 8A is the mutation identified in the large majority (>90%) of patients. It is very interesting to observe that these mutants are located in similar positions at the terminal portion of transmembrane segments lying close to the inner pore ^, and particularly the S6 transmembrane segments ( Fig. 98.1 ). Mutations at these positions may induce conformational abnormalities of the interdomain linkers that are thought to be relevant for VDI because they control the channel closure with a “ball and chain” mechanism. ^, Interestingly, the other Ca _V 1.2 mutations that cause QT prolongation (gain of function) are invariably located in the intracellular portion of the protein (see Table 98.1 ) even when they do not recapitulate the full picture of TS. Other reported mutations cause complex multiorgan phenotypes, including growth failure, developmental delay, joint contractures, seizures, and primary pulmonary hypertension in the absence of QT prolongation. On the bases of the current evidence, the intracellular position of LQT8 mutations is remarkably constant even if robust structure-function explanations are lacking.