Calmodulin Mutations Associated With Recurrent Cardiac Arrest in Infants

Abstract

Background Life-threatening disorders of heart rhythm may arise during infancy and can result in the sudden and tragic death of a child. We performed exome sequencing on two unrelated infants presenting with recurrent cardiac arrest to discover a genetic cause. Methods and Results We ascertained two unrelated infants (probands) with recurrent cardiac arrest and dramatically prolonged QTc interval who were both born to healthy parents. The two parent-child trios were investigated using exome sequencing to search for de novo genetic variants. We then performed follow-up candidate gene screening on an independent cohort of 82 subjects with congenital long-QT syndrome without an identified genetic cause. Biochemical studies were performed to determine the functional consequences of mutations discovered in two genes encoding calmodulin. We discovered three heterozygous de novo mutations in either CALM1 or CALM2, two of the three human genes encoding calmodulin, in the two probands and in two additional subjects with recurrent cardiac arrest. All mutation carriers were infants who exhibited life-threatening ventricular arrhythmias combined variably with epilepsy and delayed neurodevelopment. Mutations altered residues in or adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain. Recombinant mutant calmodulins exhibited several fold reductions in calcium binding affinity. Conclusions Human calmodulin mutations disrupt calcium ion binding to the protein and are associated with a life-threatening condition in early infancy. Defects in calmodulin function will disrupt important calcium signaling events in heart affecting membrane ion channels, a plausible molecular mechanism for potentially deadly disturbances in heart rhythm during infancy.

A framework for exhaustively mapping functional missense variants

“…We applied the higher‐throughput TileSeq approach, coupled with yeast complementation, to a diverse set of genes: SUMO1, for which heterozygous null variants are associated with cleft palate (Andreou et al , 2007); thiamine pyrophosphokinase 1 (TPK1), associated with vitamin B1 metabolism dysfunction (Mayr et al , 2011); and CALM1, CALM2, and CALM3, associated with cardiac arrhythmias (long‐QT syndrome (Crotti et al , 2013) and catecholaminergic polymorphic ventricular tachycardia (Nyegaard et al , 2012)). Because the three calmodulin genes encode the same polypeptide sequence, performing DMS for CALM1 also provided maps for CALM2 and CALM3.…”

“…Because the inheritance pattern of calmodulin disorders is typically dominant (Crotti et al , 2013), we did not consider diploid genotypes but simply evaluated the ability of the (refined) DMS scores to distinguish disease from non‐disease variants (Fig 5B). DMS scores performed well according to precision‐recall analysis, with an area under the precision‐recall curve (AUC) of 0.72, exceeding both PROVEAN (AUC = 0.48) and PolyPhen‐2 (AUC = 0.47) (Fig 5C).…”

Conserved properties of individual Ca 2+ -binding sites in calmodulin

“…For instance, although CaM is essential in yeast, CaM gene knockout can be rescued with a vertebrate CaM, which is only 60% identical (62,63). Additionally, it appears that yeast can survive with Ca 2+ binding knocked out in all four EF-hands (34), whereas single mutations in just one of the three identical copies of CaM present in humans can cause major diseases (4)(5)(6). Yeast is therefore more robust to changes in CaM sequence than vertebrates, and this robustness probably translates into the higher evolutionary rate within ascomycetes.…”

“…In humans, mutations at phenylalanine sites result in cardiomyopathies. The mutation F89L has been linked to intraventricular fibrillation, and F141L has been linked to long QT syndrome (5,6). Leucine is substituted with very low frequencies, 0.005 or 0.010 for F89 and A cartoon of an EF-hand peptide chain threads through a semitransparent representation of its molecular surface.…”

“…In humans it binds to more than 300 targets (1)(2)(3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4)(5)(6)(7)(8), as can altered gene expression (9). Although CaM has been extensively studied, many details about its function are still poorly understood.…”