Infanticide vs. inherited cardiac arrhythmias

Abstract

Aims: 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.

Methods and results: 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 (CaV1.2 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.

Conclusion: 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.

Keywords: BSN; CALM2; Calmodulinopathy; Infanticide; Sudden unexpected death.

Graphical abstract

Figure 1

H&E staining of cardiac sections from Child 4’s autopsy. Inflammatory infiltrates are seen in the myocardium associated in places with myocyte necrosis (magnification: 100–400×).

Figure 2

Pedigrees of kindreds with G114R and G114W CALM mutations and Sanger sequencing. (A) Family pedigree (kindred A) showing inheritance of the CALM2 gene and the protein variation glycine (G) at position 114 to arginine (R). (B) Pedigree showing inheritance of the CALM3 gene and protein variant G114W (tryptophan; W) in kindred B. Shaded symbols represent affected individuals with SUD/SCD/CA. WT=wildtype. (C) Sanger sequencing from the mother’s buccal swab highlighting the C→T variant.

Figure 3

G114R and G114W mutations impair the calcium binding strength of CaM. (A) Three-dimensional structure of CaM (PDB: 1cll). Ca²⁺-ions are displayed as black spheres. Mutations at green residues have been associated with SUD/CA during sleep in other patients. (B) Ca²⁺-binding curves (left) of the C-terminal domain of CaM variants N98S, G114R, and G114W, showing reduced Ca²⁺ affinity (right), a likely proarrhythmic substrate. Data displayed as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 using one-way ANOVA with Dunnett’s post hoc test against CaM-WT. CA, cardiac arrest; CaM, calmodulin; FI_norm, normalized fluorescence intensity; IVF, idiopathic ventricular fibrillation; K_A, association constant; LQTS, long QT syndrome; SCD, sudden cardiac death; SUD, sudden unexplained death.

Figure 4

G114R and G114W mutations impair the binding of CaM to calcium channels. (A–D) Ca²⁺-dependent binding of CaM to the IQ-domain of Ca_V1.2. Reduced binding of CaM variants N98S, G114R, and G114W unbalances the fine regulation of Ca_V1.2 by CaM at low and intermediate Ca²⁺ concentrations, predisposing to an altered Ca²⁺ inflow through Ca_V1.2. (E–H) Similarly, the binding to the RyR2 CaMBD2 domain was decreased at low and intermediate Ca²⁺ concentrations, suggesting an altered Ca²⁺ outflow from the sarcoplasmic reticulum. *P≤0.05, **P≤0.01, and ***P≤0.001 using one-way ANOVA with Dunnett’s post hoc test against CaM-WT values. CaM, calmodulin.

Figure 5

G114R and G114W mutations cause delayed closure of calcium channels Ca_V1.2 and RyR2. (A) Exemplar traces of electrophysiological recordings of Ca_V1.2 opening (downward deflection) and subsequent CaM-mediated calcium-dependent inactivation (CDI) (return towards baseline) during a 30 mV voltage clamp in the presence of Ca²⁺ (red) or Ba²⁺ (black). (B) CDI as quantified by the metric f₃₀₀, measured at 30 mV. All three CaM mutations significantly reduce CDI, thus increasing the Ca²⁺ inflow through Ca_V1.2 and potentially leading to a pathogenic QT prolongation. ***P < 0.001 using Student’s t-test. Quantified RyR2 channel opening (C) and closure (D) propensity as well as the amount of Ca²⁺ released (E) during an open event in store-overload induced calcium release (SOICR) assay. The CaM variant G114W significantly reduced the activation threshold, and the variants G114R and G114W significantly altered the termination threshold and fractional calcium release, potentially leading to an arrhythmogenic enhanced intracellular Ca²⁺ level. Data are displayed as mean ± SEM. *P ≤ 0.05 and **P ≤ 0.01 using one-way ANOVA with post hoc test. CaM, calmodulin; Ca_V1.2, voltage-gated calcium channel isoform 1.2; RyR2, ryanodine receptor type 2; WT, wild type.