Application of induced pluripotent stem cells in epilepsy.

Epilepsy is among the most common neurological disorders, affecting approximately 50 million people worldwide. Importantly, epilepsy is genetically and etiologically heterogenous, but several epilepsy types exhibit similar clinical presentations. Epilepsy-associated genes are being identified. However, the molecular pathomechanisms remain largely unknown. Approximately one-third of epilepsy is refractory to multiple conventional anti-epileptic drugs (AEDs). Induced pluripotent stem cells (iPSCs) provide an excellent tool to study the pathomechanisms underlying epilepsy and to develop novel treatments. Indeed, disease-specific iPSCs have been established for several genetic epilepsies. In particular, the molecular mechanisms underlying certain developmental and epileptic encephalopathies, such as Dravet syndrome, have been revealed. Modeling epilepsy with iPSCs enables new drug development based on the elucidated pathomechanisms. This can also be used to evaluate conventional AEDs and drug repurposing. Furthermore, transplanting neuronal cells derived from iPSCs into the brain has great potential to treat refractory epilepsies. Recent advances in iPSC technology have enabled the generation of neuronal organoids, or "mini brains." These organoids demonstrate electrophysiological activities similar to those of the brain and have the potential for extensive epilepsy research opportunities. Thus, the application of iPSCs in epilepsy provides insight into novel treatments based on the molecular pathomechanisms of epilepsy. In this review, we comprehensively discuss the studies conducted on iPSCs established for genetic epilepsy or epilepsies without major structural dysmorphic features.

Clinical and genetic analysis of benign familial infantile epilepsy caused by PRRT2 gene variant.

Objective: This study presents the clinical phenotypes and genetic analysis of seven patients with benign familial infantile epilepsy (BFIE) diagnosed by whole-exome sequencing. Methods: The clinical data of seven children with BFIE diagnosed at the Department of Neurology, Children's Hospital Affiliated to Zhengzhou University between December 2017 and April 2022 were retrospectively analyzed. Whole-exome sequencing was used to identify the genetic causes, and the variants were verified by Sanger sequencing in other family members. Results: The seven patients with BFIE included two males and five females ranging in age between 3 and 7 months old. The main clinical phenotype of the seven affected children was the presence of focal or generalized tonic-clonic seizures, which was well controlled by anti-seizure medication. Cases 1 and 5 exhibited predominantly generalized tonic-clonic seizures accompanied by focal seizures while cases 2, 3, and 7 displayed generalized tonic-clonic seizures, and cases 4 and 6 had focal seizures. The grandmother and father of cases 2, 6, and 7 had histories of seizures. However, there was no family history of seizures in the remaining cases. Case 1 carried a de novo frameshift variant c.397delG (p.E133Nfs*43) in the proline-rich transmembrane protein 2 (PRRT2) gene while case 2 had a nonsense variant c.46G > T (p.Glu16*) inherited from the father, and cases 3-7 carried a heterozygous frameshift variant c.649dup (p.R217Pfs*8) in the same gene. In cases 3 and 4, the frameshift variant was de novo, while in cases 5-7, the variant was paternally inherited. The c.397delG (p.E133Nfs*43) variant is previously unreported. Conclusion: This study demonstrated the effectiveness of whole-exome sequencing in the diagnosis of BFIE. Moreover, our findings revealed a novel pathogenic variant c.397delG (p.E133Nfs*43) in the PRRT2 gene that causes BFIE, expanding the mutation spectrum of PRRT2.

Proline-rich transmembrane protein 2 knock-in mice present dopamine-dependent motor deficits.

Mutations of proline-rich transmembrane protein 2 (PRRT2) lead to dyskinetic disorders such as paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements precipitated by suddenly initiated motion, and some convulsive disorders. Although previous studies have shown that PKD might be caused by cerebellar dysfunction, PRRT2 has not been sufficiently analyzed in some motor-related regions, including the basal ganglia, where dopaminergic neurons are most abundant in the brain. Here, we generated several types of Prrt2 knock-in (KI) mice harboring mutations, such as c.672dupG, that mimics the human pathological mutation c.649dupC and investigated the contribution of Prrt2 to dopaminergic regulation. Regardless of differences in the frameshift sites, all truncating mutations abolished Prrt2 expression within the striatum and cerebral cortex, consistent with previous reports of similar Prrt2 mutant rodents, confirming the loss-of-function nature of these mutations. Importantly, administration of l-dopa, a precursor of dopamine, exacerbated rotarod performance, especially in Prrt2-KI mice. These findings suggest that dopaminergic dysfunction in the brain by the PRRT2 mutation might be implicated in a part of motor symptoms of PKD and related disorders.

Benign familial infantile epilepsy associated with KCNQ3 mutation: a rare occurrence or an underestimated event?

Benign familial infantile epilepsy (BFIE) is the most genetically heterogeneous phenotype among early-onset familial infantile epilepsies. It has an autosomal dominant inheritance pattern with incomplete penetrance. Although PRRT2 is the most mutated gene detected in families with BFIE, other mutations in KCNQ2, SCN2A, and GABRA6 genes have also been described. To date, KCNQ3 mutations have been detected in only four patients with BFIE. Here, we describe the clinical pattern and course of an additional individual with BFIE associated with a novel missense heterozygous KCNQ3 c.1850G>C variant inherited by his unaffected father. The incidence of KCNQ3 mutations among BFIE patients is reported to be low in the literature, however, whether this is underestimated is unclear as not all current epilepsy gene panels include KCNQ3.

Paroxysmal and cognitive phenotypes in Prrt2 mutant mice.

Mutations in proline-rich transmembrane protein 2 (PRRT2) cause a range of episodic disorders that include paroxysmal kinesigenic dyskinesia and benign familial infantile epilepsy. Mutations are generally loss of function and include the c649dupC frameshifting mutation that is present in around 80% of affected individuals. To investigate how Prrt2 loss of function mutations causes disease, we performed a phenotypic investigation of a transgenic Prrt2 knockout (Prrt2 KO) mouse. We observed spontaneous paroxysmal episodes with behavioural features of both seizure and movement disorders, as well as unexplained deaths in KO and HET animals. KO mice showed spatial learning deficits in the Morris water maze, as well as gait abnormalities in the quantitative Digigait analysis; both of which may be representative of the more severe phenotypes experienced by homozygous patients. These findings extend the described phenotypes of Prrt2 mutant mice, further confirming their utility for in vivo investigation of the role of Prrt2 mutations in episodic diseases.

Association of a synonymous SCN1B variant affecting splicing efficiency with Benign Familial Infantile Epilepsy (BFIE).

Benign Familial Infantile Epilepsy (BFIE) is clinically characterized by clusters of brief partial seizures progressing to secondarily generalized seizures with onset at the age of 3-7 months and with favorable outcome. PRRT2 mutations are the most common cause of BFIE, and found in about 80% of BFIE families. In this study, we analyzed a large multiplex BFIE family by linkage and whole exome sequencing (WES) analyses. Genome-wide linkage analysis revealed significant evidence for linkage in the chromosomal region 19p12-q13 (LOD score 3.48). Mutation screening of positional candidate genes identified a synonymous SCN1B variant (c.492T>C, p.Tyr164Tyr) affecting splicing by the removal of a splicing silencer sequence, shown by in silico analysis, as the most likely causative mutation. In addition, the PRRT2 frameshift mutation (c.649dupC/p.Arg217Profs*8) was observed, showing incomplete, but high segregation with the phenotype. In vitro splicing assay of SCN1B expression confirmed the in silico findings showing a splicing imbalance between wild type and mutant exons. Herein, the involvement of the SCN1B gene in the etiology of BFIE, contributing to the disease phenotype as a modifier or part of an oligogenic predisposition, is shown for the first time.

Characteristics of patients with benign partial epilepsy in infancy without PRRT2 mutations.

Mutations in the proline-rich transmembrane protein 2 gene (PRRT2) are known to cause clinical symptoms of paroxysmal kinesigenic dyskinesia (PKD), benign partial epilepsy in infancy (BPEI), and infantile convulsions with choreoathetosis (ICCA) syndrome; however, not all patients with BPEI have PRRT2 mutations, and the genetic backgrounds for such patients are still unknown. To characterize BPEI patients without PRRT2 mutations, we analyzed unrelated 63 patients with BPEI. Sanger sequencing identified PRRT2 mutations in 33 probands (52%). The most common insertion, c.649dup, was identified in 28 probands. Two novel truncation mutations, c.232dup and c.503_504del were identified independently. 16p11.2 microdeletion was not detected in patients without PRRT2 mutations. PRRT2 mutation detection rates were 21/31 (68%) and 12/32 (38%) in probands who were positive or negative for family history, respectively, indicating a significant difference between the two groups. In this study, 20 probands with BPEI were negative for family history of BPEI and negative for PRRT2 mutation. BPEI in these probands may be due to complex genetic predispositions. Because the possibility remains that a second gene contributes to BPEI, further studies are necessary in patients with BPEI but no PRRT2 mutation, especially in Asian people.