Helene Halkjær Jensen

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.

Calmodulin is an intracellular Ca2+ sensor that regulates numerous cellular processes through binding effector proteins and changing their activity. Humans have three calmodulin paralogs, CALM1, CALM2 and CALM3, encoding identical proteins, and missense variants in all three are included in the ACMG recommendations for reporting of secondary findings related to early onset sudden cardiac death caused by arrhythmias long QT syndrome and CPVT. Recently, a subset of individuals with de novo variants in calmodulin genes were described who also presented with neurologic phenotypes. Here we report two individuals with the same de novo variant c.419A > T in CALM1 or CALM2 that may generate a 5′ splice donor gain and/or a missense change p.E140V. These individuals share hypotonia, motor delay, intellectual disability, and abnormal electroencephalograms but lack cardiac arrhythmia/electrocardiogram abnormalities of previously described CALM-associated disease, suggesting the variant causes phenotypic expansion beyond the known Mendelian phenotypes. RNA-seq of the CALM1 proband blood’s sample revealed that most transcripts from the variant allele showed usage of the new splice site or intron retention, without NMD, resulting in frameshifted C-terminal truncations, while a minority resulted in production of the p.E140V missense protein. We modeled the CALM1/2 p.E140V variant as well as a known arrhythmia variant, CALM1 p.E141G, using the C. elegans ortholog cmd-1. We found that cmd-1 E140V displayed both qualitative and quantitative differences in phenotype from E141G, indicating distinct genetic mechanisms. Together, these findings support CALM1/2 phenotypic expansion, with c.419A > T p.E140V resulting in neurologic, but not arrhythmia phenotypes.

Calmodulin is a vital cellular calcium sensor expressed by the genes CALM1-3 . Ultra-rare loss-of-function missense variants in the C-terminal lobe (C-lobe) of calmodulin are linked to cardiac arrhythmia, primarily long QT syndrome. Emerging evidence suggests calmodulin variants also contribute to neurological disorders. This study explores the allelic spectrum and functional consequences of calmodulin variants in schizophrenia. In a large-scale sequencing effort involving 24,248 schizophrenia patients and 97,322 control individuals, 25 unique calmodulin variants were found in 27 carriers, all ultra-rare. Seven carriers were schizophrenia patients, and 20 were controls. All patient variants affected the C-lobe, while only five of 20 control variants were in the C-lobe, linking C-lobe variants to increased schizophrenia risk. Functional analyses identified two variant classes in patients: Loss-of-function variants, reducing calcium affinity and impairing Ca _V 1.2 interaction, and gain-of-function variants, enhancing calcium affinity without affecting Ca _V 1.2 gating. Together, this study suggests that consequences of calmodulin variants include increased schizophrenia risk.

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.

CED-1 (cell death abnormal) is a transmembrane receptor involved in the recognition of “eat-me” signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in Caenorhabditis elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter include the adapter protein CED-6/GULP and the ATP-binding cassette family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the Adaptor protein 2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

Digital tools and platforms offer new solutions to design and conduct university teaching. This case illustrates how such digital solutions may be utilized in problem-based learning programmes within life science educations. Specifically, the case evaluated the use of live-streamed and recorded lectures, the incorporation of digital formative assessment in lectures, and the use of a digital platform to support experimental project work in a research laboratory. We find that digital solutions provide flexibility for both lecturers and students, advantageous options for collecting and sharing information, and for engaging students in their learning process. However, digital tools cannot replace all aspects of traditional in-person teaching, such as social interactions. Rather, when blended with in-person teaching, digital solutions have a large potential for supporting new forms of and approaches to both theoretical and experimental university teaching.

In 2003, an Australian woman was convicted by a jury of smothering and killing her four children over a 10-year period. Each child died suddenly and unexpectedly during a sleep period, at ages ranging from 19 days to 18 months. In 2019 we were asked to investigate if a genetic cause could explain the children’s deaths as part of an inquiry into the mother’s convictions. Whole genomes or exomes of the mother and her four children were sequenced. Functional analysis of a novel CALM2 variant was performed by measuring Ca²⁺-binding affinity, interaction with calcium channels and channel function. We found two children had a novel calmodulin variant (CALM2 G114R) that was inherited maternally. Three genes (CALM1-3) encode identical calmodulin proteins. A variant in the corresponding residue of CALM3 (G114W) was recently reported in a child who died suddenly at age 4 and a sibling who suffered a cardiac arrest at age 5. We show that CALM2 G114R impairs calmodulin's ability to bind calcium and regulate two pivotal calcium channels (Ca_V1.2 and RyR2) involved in cardiac excitation contraction coupling. The deleterious effects of G114R are similar to those produced by G114W and N98S, which are considered arrhythmogenic and cause sudden cardiac death in children. A novel functional calmodulin variant (G114R) predicted to cause idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, or mild long QT syndrome was present in two children. A fatal arrhythmic event may have been triggered by their intercurrent infections. Thus, calmodulinopathy emerges as a reasonable explanation for a natural cause of their deaths.