Genetic Mosaicism in Calmodulinopathy

Abstract

Background: CaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families.

Methods: CaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation.

Results: Genetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca²⁺ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca²⁺-dependent inactivation of L-type Ca²⁺ channels and prolonged action potential duration.

Conclusions: We report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca²⁺ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.

Keywords: arrhythmia; calmodulin; genotype; long QT syndrome; mosaicism.

Figure 1.

Abnormal electrocardiographic traces from probands. A. Twelve-lead ECG traces obtained at birth for Proband 1 illustrating sinus bradycardia, a prolonged QTc interval (725 ms), and 2:1 AV block. B. An episode of T-wave alternans recorded from Proband 1 is illustrated. C. ECG rhythm during resuscitation of Proband 1 from VF cardiac arrest on the 23^rd day of life. D. ECG trace recorded at age 2 weeks from Proband 2 showing a QTc of 660 ms. E. ECG trace recorded at age 18 months from Proband 3 showing a QTc of 630 ms and inverted T waves throughout the precordial leads. F. ECG trace from Proband 4 recorded at age 4 days showing a QTc of 505 ms and 2:1 AV block.

Figure 2.

Pedigrees of calmodulinopathy families and discovery of novel CaM mutations. A-D. Pedigrees representing the family structures of Probands 1–4. Males are represented as square symbols, females as circles, miscarriages as triangles, elective abortions as triangles with diagonal lines. The QTc or phenotype, and CALM3 genotype are given below each pedigree symbol. In Family D, age of sudden death (SD) is given. Individuals unavailable for genotyping are labeled as ‘No DNA’. E. Nucleotide sequencing traces showing heterozygous mutations in CALM3 for Proband 1 (Family A, II.1) and the mosaic mother (I.2), and heterozygous mutation in CALM1 for Proband 2 (Family C, II.1) compared to a normal genotype in the father (I.1).

Figure 3.

E141K and E141V reduce Ca²⁺ binding affinity of the calmodulin C-lobe. A. Amino acid sequence alignments comparing a segment of the CaM C-domain from different species (left) and a schematic of the 4^th EF-hand motif showing the location of the E141K and E141V mutations (right). B. An overlay of WT (black), E141K (red), and E141V (blue) fluorescence change during Ca²⁺ titration for the CaM C-domain. C. Normalized fluorescence change for Ca²⁺ titration experiments. The Ca²⁺ affinities (Kd) for the CaM C-domain are provided in the inset.

Figure 4.

Effect of E141K and E141V CaM on iPSC-CM Ca²⁺ current. A. Average traces of Ca²⁺ current recorded from iPSC-CMs transfected with CaM-WT (top), CaM-E141K (middle), or CaM-E141V (bottom). B. Current density vs voltage plot comparing the peak Ca²⁺ current of WT, E141K, and E141V expressing cells (*, p < 0.05, E141K vs WT). C. Current density vs voltage plot comparing the late Ca²⁺ current recorded 100 ms after each voltage step (*, p < 0.05, both mutants vs WT; †, p < 0.05, E141K vs WT). Data symbols represent mean values of WT (n=17), E141K (n=10), and E141V (n=8) expressing cells and error bars are standard error of the mean (SEM).

Figure 5.

E141K and E141V impair Ca²⁺-dependent inactivation. A. Average traces of Ca²⁺ (black) and Ba²⁺ (red) current recorded at +10 mV from iPSC-CMs transfected with CaM-WT (top), CaM-E141K (middle), or CaM-E141V (bottom). B. Plots of I_end/I_peak for test voltages between −10 to +50 mV (*, p < 0.05, both mutants vs WT; †, p < 0.05, E141K vs WT). Data symbols represent mean values of WT (n=17), E141K (n=10), and E141V (n=8) and error bars are standard error of the mean (SEM).

Figure 6.

E141K and E141V prolong the action potential duration in iPSC-CM. A. Representative traces of action potentials recorded from human iPSC-CMs transfected with CALM3 WT or E141K (top), or with CALM1 WT or E141V (bottom). B. Box plots of action potential duration at 90% repolarization (APD₉₀) of spontaneous APs in cells expressing E141K or E141V compared to WT expressing cells. The vertical height of each box plot represents the 25^th to 75^th percentile, the solid black line within the box marks the median, and the mean value is indicated by the dashed line with the box. Whiskers (error bars) above and below the box indicate the 95^th and 5^th percentiles, respectively. All data points are plotted. C. Box plots of action potential duration at 50% repolarization (APD₅₀) of spontaneous APs in cells expressing E141K or E141V compared to WT expressing cells. D. Box plots depicting ratios of APD₉₀ to APD₅₀ determined for spontaneous AP (*, p < 0.05, comparing mutant to WT).