Authors :
Presenting Author: Miriam Kessi, MD – Department of Pediatrics, Xiangya Hospital, Central South University
Baiyu Chen, Second author – Department of Pediatrics, Xiangya Hospital, Central South University; Lifen Yang, Third author – Department of Pediatrics, Xiangya Hospital, Central South University; Jing Peng, Third author, head of department and co-supervisor – Department of Pediatrics, Xiangya Hospital, Central South University; Fang He, Co-supervisor and corresponding author – Department of Pediatrics, Xiangya Hospital, Central South University; Fei Yin, Corresponding author and supervisor. – Department of Pediatrics, Xiangya Hospital, Central South University
Rationale:
CACNA1A and CACNA1C variants can cause epileptic encephalopathies, status epilepticus, absence seizures, focal seizures, and sudden unexpected death in epilepsy (SUDEP). Currently, many studies have focused on the electrophysiological changes rather than exploring the molecular mechanisms, and there is no treatment for these channelopathies. Electrophysiological studies alone are not enough to explain some of the complex clinical manifestations. We aimed to explore the possible molecular mechanisms of the CACNA1A and CACNAIC related epilepsy. Our novel findings might expand the understanding of the molecular mechanisms of the CACNA1A and CACNA1C-related epilepsy resulting to the identification of the potential treatment targets.
Methods:
Clinical information and genetic results of pediatric patients with
CACNA1A and
CACNA1C pathogenic variants were collected at Xiangya Hospital, Central South University. Several electrophysiological and molecular experiments were performed on transfected HEK 293 cells to explore the effects of these variants on mitochondrial and lysosomal functions, autophagy, apoptosis and mitophagy pathways. Experiments done included electrophysiological studies, RT-qPCR, western blot, apoptosis assay, reactive oxygen species (ROS), ATP levels, mitochondrial complex I activity, mitochondrial copy numbers, mito- and lyso-tracker immunofluorescence staining, and mitochondrial calcium concentration test.
Results:
We identified six pathogenic variants in six patients: four
de novo and two inherited: p.G701R, p.R279C, p.D1644N, p.Y62C, p.L1422Sfs*8, and p. R1664Q in which two had gain-of-function (GOF) and four loss-of-function (LOF) effects. The clinical manifestations of the six cases included focal epilepsy, status epilepticus, absence seizures, intellectual disability (ID), ataxia, and cerebellar atrophy. We found that our mutants could affect mitochondrial functions by interfering with ATP production, reactive oxygen species production, mitochondrial fusion, and fission as well as mitophagy process. In addition, our mutants could affect autophagy-lysosomal system.
Three novel de novo CACNA1C variants: p.E411D, p.V622G and p.A272V were found in patients who presented with West syndrome, unclassified epilepsy, ID, and sinus arrhythmia. CACNA1C variants induced cell apoptosis, affected mitochondrial functions by interfering with mitochondrial complex 1 enzyme activity, mitochondrial calcium ions influx, ATP production, ROS production, fusion, and fission as well as mitophagy process. To the best of our knowledge, this is the first study to explore the significance of the CACNA1A and CACNA1C pathogenic variants in causing both mitochondrial and lysosomal dysfunctions.
Conclusions:
Both mitochondrial and lysosomal dysfunctions are involved in the pathogenesis of the CACNA1A- and CACNA1C-related epilepsy.
Funding:
We are grateful for the support we received from the National Natural Science Foundation of China (NO.81903221), the Natural Science Foundation of Hunan Province (2021JJ40986) and the Hunan Key Research and Development Program (NO.2019SK2081).