Presenting Author:

Lisa Wren, B.S.

Principal Investigator:

Alfred George, Jr.

Department:

Pharmacology

Keywords:

Sudden cardiac death, arrhythmia, calmodulin, genetics, mosaicism, L-type Ca2+channels, Ca2+-dependent inactivation

Location:

Third Floor, Feinberg Pavilion, Northwestern Memorial Hospital

B151 - Basic Science

Genetic mosaicism in calmodulinopathy associated with a novel CALM3-E141K mutation

Mutations in three genes (CALM1, CALM2, CALM3) encoding the ubiquitous calcium signaling protein calmodulin (CaM) are associated with severe forms of congenital arrhythmia susceptibility referred to as calmodulinopathy. Common clinical features of calmodulinopathy are the prolongation of the corrected QT interval (QTc) on an electrocardiogram (ECG) with the onset of ventricular arrhythmias, syncope, and sudden cardiac death at an early age. Here we identified a novel mutation (E141K) in CALM3 associated with severe ventricular arrhythmia and sudden death in a proband belonging to a family with recurrent intrauterine fetal demise raising suspicion of parental mosaicism. The cellular mechanism that correlated with the clinical phenotype was also unknown. Therefore, we investigated the genetics and functional consequences of CALM3-E141K. DNA was isolated from peripheral blood and used to perform PCR-Sanger sequencing of known arrhythmia genes. Calcium binding affinity of recombinant CaM protein (WT or E141K) was measured using a fluorescence-based approach. Electrophysiology studies were performed on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transiently transfected with WT or mutant (E141K) human CaM. A newborn presented severe bradycardia, prolonged QT interval (725 ms), 2:1 AV block and T-wave alternans. She had an episode of cardiac arrest secondary to ventricular fibrillation on the 23rd day of life and was resuscitated. She was treated with placement of an ICD and propranolol. At age 3y, she had two episodes of appropriate ICD discharges while taking propranolol, but later died at age 5y of uncertain cause. Genetic testing demonstrated a novel, heterozygous missense CALM3 mutation resulting in the substitution of glutamate-141 with lysine (E141K). Ca2+ affinities for WT and E141K were 2.3 ± 0.2 μM and 75 ± 7 μM, respectively, indicating impaired Ca2+ binding caused by the mutation. hiPSC-CM transfected with CaM-E141K exhibited impaired Ca2+-dependent inactivation (CDI) of L-type Ca2+channels (LTCC), and longer action potential duration as compared to WT expressing cells. The mutation was not present in the father but the mother’s DNA exhibited the mutant allele in 25% of sequencing reads indicating genetic mosaicism. The mother had no cardiac phenotype and her QTc was 422 ms. A subsequent pregnancy was terminated at 12 weeks’ gestation because of fetal bradycardia, and another pregnancy terminated spontaneously at 20 weeks’ gestation. We report genetic mosaicism in arrhythmogenic calmodulinopathy. We also show that dysregulation of the LTCC as a result of dysfunctional CaM impairs CDI and prolongs the action potential. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or death and a child with a documented CaM mutation.