Ivy E. Dick

Calmodulinopathies are caused by mutations in calmodulin (CaM) and result in debilitating cardiac arrythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In addition, many patients exhibit neurological comorbidities, including developmental delay and autism spectrum disorder. Prior studies have identified the impairment of Ca2+/CaM-dependent inactivation (CDI) of CaV1.2 channels as a major pathogenic mechanism leading to the LQTS phenotype in these patients. However, the impact of these mutations on other voltage-gated calcium channels (VGCCs) has yet to be fully explored. Here, we examine the potential for pathological CaM variants to impair the Ca2+/CaM-dependent regulation of CaV1.3 and CaV2.1, both essential for neuronal function. We find that pathogenic mutations in CaM can impair the CDI of CaV1.3, with overlapping yet distinct disruption of the Ca2+-dependent facilitation (CDF) of CaV2.1 channels. Moreover, while the majority of CaM variants demonstrated the ability to bind the IQ region of each channel, differences were noted between CaV1.3 and CaV2.1, demonstrating unique CaM interactions across the two channel subtypes. Further, C-lobe CaM variants display a reduced ability to sense Ca2+ when in complex with the CaV IQ domains, explaining the Ca2+/CaM regulation deficits. Overall, these results support the possibility that disrupted Ca2+/CaM regulation of multiple VGCCs may contribute to the pathogenesis of calmodulinopathies.

Timothy Syndrome is a multisystemic genetic disorder, classically characterised by prolonged QT interval and subsequent cardiac arrhythmias, neurodevelopmental disorders including developmental delay and autism, and syndactyly or hip dysplasia. It is caused by variants in the CACNA1C gene, which encodes the widely expressed Ca _v 1.2 voltage-gated calcium channel. Since it’s characterisation in 2004, the spread of variants in CACNA1C associated with Timothy Syndrome has expanded. With advances in sequencing and the inclusion of CACNA1C in genomic screening, further variants have been identified presenting with incomplete features of Timothy Syndrome or further aligned phenotypes which are inconsistent with the original description. In the absence of a formal nomenclature, these presentations have been reported in a proliferation of ill-defined terms, e.g. Atypical Timothy Syndrome. At the same time, advances in knowledge and therapeutics have improved morbidity and life expectancy for these individuals when appropriately identified and managed. Here, we present guidelines for the diagnosis of individuals presenting with variants in CACNA1C , developed by an international panel of experts through Delphi consensus with the involvement of the CACNA1C community. We formalise the language around syndromic presentations linked to CACNA1C variants, reassert and demarcate the classical Timothy Syndrome phenotype, and define a new syndrome, CACNA1C-Related Disorder. Finally, we present minimum expected standards of clinical care for individuals with CACNA1C-Related Disorder or Timothy Syndrome, with implications for long-term management and improved outcomes for affected individuals.

Voltage-gated Ca2+ channels (VGCCs) are highly expressed throughout numerous biological systems and play critical roles in synaptic transmission, cardiac excitation, and muscle contraction. To perform these various functions, VGCCs are highly regulated. Inactivation comprises a critical mechanism controlling the entry of Ca2+ through these channels and constitutes an important means to regulate cellular excitability, shape action potentials, control intracellular Ca2+ levels, and contribute to long-term potentiation and depression. For CaV1 and CaV2 channel families, inactivation proceeds via two distinct processes. Voltage-dependent inactivation (VDI) reduces Ca2+ entry through the channel in response to sustained or repetitive depolarization, while Ca2+-dependent inactivation (CDI) occurs in response to elevations in intracellular Ca2+ levels. These processes are critical for physiological function and undergo exquisite fine-tuning through multiple mechanisms. Here, we review known determinants and modulatory features of these two critical forms of channel regulation and their role in normal physiology and pathophysiology.

Calmodulinopathies are caused by mutations in calmodulin (CaM), and result in debilitating cardiac arrythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In addition, many patients exhibit neurological comorbidities, including developmental delay and autism spectrum disorder. Until now, most work into these mutations has focused on cardiac effects, identifying impairment of Ca ²⁺ /CaM-dependent inactivation (CDI) of Ca _V 1.2 channels as a major pathogenic mechanism. However, the impact of these mutations on neurological function has yet to be fully explored. CaM regulation of voltage-gated calcium channels (VGCCs) is a critical element of neuronal function, implicating multiple VGCC subtypes in the neurological pathogenesis of calmodulinopathies. Here, we explore the potential for pathological CaM variants to impair the Ca ²⁺ /CaM-dependent regulation of Ca _V 1.3 and Ca _V 2.1, both essential for neuronal function. We find that mutations in CaM can impair the CDI of Ca _V 1.3 and reduce the Ca ²⁺ -dependent facilitation (CDF) of Ca _V 2.1 channels. We find that mutations associated with significant neurological symptoms exhibit marked effects on Ca _V 1.3 CDI, with overlapping but distinct impacts on Ca _V 2.1 CDF. Moreover, while the majority of CaM variants demonstrated the ability to bind the IQ region of each channel, distinct differences were noted between Ca _V 1.3 and Ca _V 2.1, demonstrating distinct CaM interactions across the two channel subtypes. Further, C-domain CaM variants display a reduced ability to sense Ca ²⁺ when in complex with the Ca _V IQ domains, explaining the Ca ²⁺ /CaM regulation deficits. Overall, these results support the possibility that disrupted Ca ²⁺ /CaM regulation of VGCCs may contribute to neurological pathogenesis of calmodulinopathies.

Background: Timothy syndrome (OMIM #601005) is a rare disease caused by variants in the gene CACNA1C. Initially, Timothy syndrome was characterized by a cardiac presentation of long QT syndrome and syndactyly of the fingers and/or toes, all associated with the CACNA1C variant, Gly406Arg. However, subsequent identification of diverse variants in CACNA1C has expanded the clinical spectrum, revealing various cardiac and extra-cardiac manifestations. It remains underexplored whether individuals with the canonical Gly406Arg variants in mutually exclusive exon 8A (Timothy syndrome 1) or exon 8 (Timothy syndrome 2) exhibit overlapping symptoms. Moreover, case reports have indicated that some CACNA1C variants may produce a cardiac-selective form of Timothy syndrome often referred to as non-syndromic long QT type 8 or cardiac-only Timothy syndrome, however few reports follow up on these patients to confirm the cardiac selectivity of the phenotype over time. Methods: A survey was administered to the parents of patients with Timothy syndrome, querying a broad range of symptoms and clinical features. Study participants were organized into 5 separate categories based on genotype and initial diagnosis, enabling comparison between groups of patients which have been described differentially in the literature. Results: Our findings reveal that Timothy syndrome patients commonly exhibit both cardiac and extra-cardiac features, with long QT syndrome, neurodevelopmental impairments, hypoglycemia, and respiratory issues being frequently reported. Notably, the incidence of these features was similar across all patient categories, including those diagnosed with non-syndromic long QT type 8, suggesting that the ‘non-syndromic’ classification may be incomplete. Conclusions: This study represents the first Natural History Study of Timothy syndrome, offering a comprehensive overview of the disease’s clinical manifestations. We demonstrate that both cardiac and extra-cardiac features are prevalent across all patient groups, underscoring the syndromic nature of CACNA1C variants. While the critical role of long QT syndrome and cardiac arrhythmias in Timothy syndrome has been well recognized, our findings indicate that hypoglycemia and respiratory dysfunction also pose significant life-threatening risks, emphasizing the need for comprehensive therapeutic management of affected individuals.

Calmodulin acts as a vital calcium sensor in cells, crucial for relaying calcium signals to different protein partners. While rare missense variants in calmodulin are linked to cardiac arrhythmia, particularly long QT syndrome (LQTS), their role in schizophrenia remains unexplored. We investigated missense variants in the calmodulin-encoding genes CALM1-3 in a large-scale sequencing effort involving 24,248 schizophrenia patients and 97,322 controls. Seven carriers were found among cases and twenty among controls. Notably, all schizophrenia variants affected the C-terminal lobe of the protein, compared to only five in controls, linking calmodulin C-lobe missense variants and schizophrenia risk (odds ratio 5.62, P =0.043). Functional analyses revealed two classes of calmodulin variants in schizophrenia: 1) loss-of-function variants that reduce calcium affinity and impair the interaction with voltage-gated calcium channel 1.2 (Ca _V 1.2), akin to LQTS variants but with smaller effect size, and 2) gain-of-function variants that unexpectedly enhance calcium affinity with no impact on Ca _V 1.2 gating. This study for the first time statistically and functionally links calmodulin missense variants to a neurological disorder, expanding the phenotypic spectrum of calmodulinopathies to include schizophrenia. Significance: Neurons use calmodulin to monitor calcium signals and modulate hundreds of target proteins, thereby regulating key processes such as neuronal firing and memory and learning. Here, we link genetic variants in calmodulin to schizophrenia risk. Moreover, these genetic variants have divergent consequences for calmodulin protein function. Our results expand the current understanding of calmodulin variants, which are primarily reported in cardiac arrhythmia patients, and generally have a strong loss-of-function effect on the protein. We here provide the first identification and characterization of calmodulin variants in non-cardiac patients. This broadens our view of the physiological and functional consequences of human calmodulin variants and presents novel mechanistic entries to understanding the molecular mechanisms of schizophrenia.

Timothy syndrome (OMIM #601005) is a rare disease caused by variants in the gene CACNA1C . Timothy syndrome patients were first identified as having a cardiac presentation of Long QT and syndactyly of the fingers and/or toes, and an identical variant in CACNA1C , Gly406Arg. However, since this original identification, more individuals harboring diverse variants in CACNA1C have been identified and have presented with various cardiac and extra-cardiac symptoms. Furthermore, it has remained underexplored whether individuals harboring canonical Gly406Arg variants in mutually exclusive exon 8A (Timothy syndrome 1) or exon 8 (Timothy syndrome 2) have additional symptoms. Here, we describe the first Natural History Study for Timothy syndrome, providing a thorough resource describing the current understanding of disease manifestation in Timothy syndrome patients. Parents of Timothy syndrome children were queried regarding a wide-ranging set of symptoms and features via a survey. Importantly, we find that in addition to cardiac concerns, Timothy syndrome patients commonly share extra-cardiac features including neurodevelopmental impairments, hypoglycemia, and respiratory problems. Our work expands the current understanding of the disorder to better inform the care of Timothy syndrome patients.

Generating animal models for individual patients within clinically-useful timeframes holds great potential toward enabling personalized medicine approaches for genetic epilepsies. The ability to rapidly incorporate patient-specific genomic variants into model animals recapitulating elements of the patient’s clinical manifestations would enable applications ranging from validation and characterization of pathogenic variants to personalized models for tailoring pharmacotherapy to individual patients. Here, we demonstrate generation of an animal model of an individual epilepsy patient with an ultra-rare variant of the NMDA receptor subunit GRIN2A, without the need for germline transmission and breeding. Using in utero prime editing in the brain of wild-type mice, our approach yielded high in vivo editing precision and induced frequent, spontaneous seizures which mirrored specific elements of the patient’s clinical presentation. Leveraging the speed and versatility of this approach, we introduce PegAssist, a generalizable workflow to generate bedside-to-bench animal models of individual patients within weeks. The capability to produce individualized animal models rapidly and cost-effectively will reduce barriers to access for precision medicine, and will accelerate drug development by offering versatile in vivo platforms to identify compounds with efficacy against rare neurological conditions.

Calcium ions (Ca²⁺) are the basis of a unique and potent array of cellular responses. Calmodulin (CaM) is a small but vital protein that is able to rapidly transmit information about changes in Ca²⁺ concentrations to its regulatory targets. CaM plays a critical role in cellular Ca²⁺ signaling, and interacts with a myriad of target proteins. Ca²⁺-dependent modulation by CaM is a major component of a diverse array of processes, ranging from gene expression in neurons to the shaping of the cardiac action potential in heart cells. Furthermore, the protein sequence of CaM is highly evolutionarily conserved, and identical CaM proteins are encoded by three independent genes (CALM1-3) in humans. Mutations within any of these three genes may lead to severe cardiac deficits including severe long QT syndrome (LQTS) and/or catecholaminergic polymorphic ventricular tachycardia (CPVT). Research into disease-associated CaM variants has identified several proteins modulated by CaM that are likely to underlie the pathogenesis of these calmodulinopathies, including the cardiac L-type Ca²⁺ channel (LTCC) Ca_V1.2, and the sarcoplasmic reticulum Ca²⁺ release channel, ryanodine receptor 2 (RyR2). Here, we review the research that has been done to identify calmodulinopathic CaM mutations and evaluate the mechanisms underlying their role in disease.