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 2 unrelated infants presenting with recurrent cardiac arrest to discover a genetic cause.

Methods and results: We ascertained 2 unrelated infants (probands) with recurrent cardiac arrest and dramatically prolonged QTc interval who were both born to healthy parents. The 2 parent-child trios were investigated with the use of 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 2 genes encoding calmodulin. We discovered 3 heterozygous de novo mutations in either CALM1 or CALM2, 2 of the 3 human genes encoding calmodulin, in the 2 probands and in 2 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.

Figure 1

Clinical phenotypes and pedigrees. Representative electrocardiographic recordings from two probands with early onset, life-threatening cardiac arrhythmias. Upper trace represents baseline ECG for both probands. Middle trace illustrates T-wave alternans. Lower trace for proband 1 illustrates onset of ventricular fibrillation following a period of T-wave alternans. Lower trace for proband 2 illustrates 2:1 AV block (arrows mark p-waves coincident with atrial depolarization).

Figure 2

Exome variant filtering strategy. Sequential filters were applied to the pool of variants discovered by exome sequencing in the two probands. Total coding variants include all synonymous, nonsynonymous, nonsense, frameshift inducing insertions or deletions, and canonical splice site variants within captured exons, but exclude variants in 5’ and 3’ untranslated regions and introns. De novo variants are those found in proband but not in either parent. Novel variants were defined as those absent in public databases (dbSNP, 1000genomes). Novel variants in Proband 1 were also absent in 1202 exomes generated in the Institute of Human Genetics Helmholtz Zentrum München).

Figure 3

De novo calmodulin gene mutations in infants with severe cardiac arrhythmias. A, Nucleotide sequence traces indicating heterozygous calmodulin gene mutations in Proband 1 (same mutation as in Case 3), Proband 2 and Case 4. B, Amino acid sequence alignments for calmodulins from different species with location of missense mutations.

Figure 4

Expression of calmodulin genes in human heart. A, Chromosomal locations of the three human calmodulin genes indicated on ideograms representing G-banded chromosomes. B, Relative expression of mRNA for CALM1, CALM2, and CALM3 in normal human heart (left ventricle) normalized to expression of β-actin as determined by real-time quantitative RT-PCR using gene-specific Taqman probes. Human heart samples include fetal (n = 4), infant (n = 4), and adult (n = 8) developmental stages. Data plotted are mean ± SEM. Differences in expression among the three genes were significant (p<0.05; one-way ANOVA) in fetal, infant and adult hearts.

Figure 5

Impaired Ca²⁺ binding by mutant calmodulin C-domains. A, Schematic model of the Ca²⁺ binding loops in the C-terminal EF-hand domains of calmodulin showing location of the mutations. B, Titration curves for Ca²⁺ binding to recombinant WT (black circles) and mutant calmodulins (F142L, blue triangles; D96V, grey diamonds; D130G, red squares). C, Calcium ion binding affinities for WT and mutant calmodulins.