CELSR3 variants are associated with febrile seizures and epilepsy with antecedent febrile seizures

Abstract Aims To identify novel pathogenic gene of febrile seizures (FS)/epilepsy with antecedent FS (EFS+). Methods The trio‐based whole‐exome sequencing was performed in a cohort of 462 cases with FS/EFS+. Silico programs, sequence alignment, and protein modeling were used to predict the damaging of variants. Statistical testing was performed to analyze gene‐based burden of variants. Results Five heterozygous missense variants in CELSR3 were detected in five cases (families) with eight individuals (five females, three males) affected. Two variants were de novo, and three were identified in families with more than one individual affected. All the variants were predicted to be damaging in silico tools. Protein modeling showed that the variants resulted in disappearance of multiple hydrogen bonds and one disulfide bond, which potentially caused functional impairments of protein. The frequency of CELSR3 variants identified in this study was significantly higher than that in controls. All affected individuals were diagnosed with FS/EFS+, including six patients with FS and two patients with EFS+. All cases presented favorable outcomes without neurodevelopmental disorders. Conclusions CELSR3 variants are potentially associated with FS/EFS+.


| INTRODUC TI ON
Febrile seizures (FS) are the most common convulsive event in humans. Generally, about 4%-5% of population suffer at least one FS during lifetime, whereas the incidence of FS in the Asian population is up to 8%-10%. 1,2 FS alters susceptibility of nervous system under exposure to fever. 3 The biological basis of FS remains unknown.
Increasing evidence supports that genetic factor is a predominantly pathogenic element of FS. 2 CELSR3 (OMIM* 604264) encodes cadherin EGF LAG sevenpass G-type receptor 3 (CELSR3), which is a special subgroup of adhesion G protein-coupled receptors that is expressed mainly in the brain across whole lifespan. 6 Although function of CELSR3 is uncertain, previous studies have shown that CELSR3 plays a crucial role in dendrite development, axon guidance, and brain wiring. [6][7][8][9] Mice of homozygous CELSR3 null exhibit neonatal lethality and abnormal nervous system development, such as abnormal morphology of embryonic or fetal subventricular zone, globus pallidus malformation, anomalous innervation, and neuronal migration disorder. 10,11 These findings suggest an essential role of CELSR3 in neurodevelopment.
In this study, we performed trios-based whole-exome sequencing (WES) in a cohort of patients with FS/EFS+ to screen novel genetic variants. Five heterozygous missense variants in CELSR3 were detected in five unrelated cases. This study suggests that CELSR3 is potentially a candidate causative gene of FS/EFS+.

| Subjects
We recruited the patients with FS/EFS+ from five hospitals in Epileptic seizures and epilepsy syndromes were diagnosed according to the criteria of the Commission on Classification and Terminology of the International League Against Epilepsy (1981, 1989, 2001, 2010, and 2017

| WES and bioinformatic analysis
The blood samples were collected from the probands, their parents, and other available family members to determine the origin of the identified genetic variants. The genomic DNAs from the blood samples were extracted by using the Qiagen Flexi Gene DNA kit (Qiagen). Trio-based WES was performed with NextSeq500 sequencing instruments (Illumina) according to the standard procedures as previously described. [24][25][26] We adopted a case-by-case analytical approach to identify candidate causative variants in each trio. Firstly, we prioritized the rare variants with a minor allele frequency <0.005 in the 1000 Genomes Projects, Exome Aggregation Consortium, and Genome Aggregation Database (gnomAD, gnomad.broadinstitute.org). We retained potentially pathogenic variants, including frameshift, nonsense, canonical splice site, initiation codon, and missense variants predicted as being damaging in silico tools (VarCards, http://varca rds.biols.ac.cn/). Finally, we analyzed the potential disease-causing variants in each case under the follow-

| Identification and analysis of CELSR3 variants
All the variants were predicted to be damaging in more than one of the commonly used silico prediction tools (VarCards, http://varca rds.biols.ac.cn/).
Variant p.Cys1970Tyr was not present in gnomAD database.
The frequencies of variants p.Arg1075Trp, p.Gln2713Arg, p.Ar-g3141Gln, and p.Arg3189Trp were far below 0.001 in gnomAD (  Three-dimensional structural model of CELSR3 showed that the affected residues were all located on the outer side of the domains ( Figure 2B-E). Originally, residue Arg1075 formed two hydrogen bonds with Glu1073 at distances of 3.0 Å and 3.3 Å, respectively.

| Structural alteration of CELSR3 protein
When arginine was replaced by tryptophan at residue 1075, one of the hydrogen bonds with residue Glu1073 was destroyed and the other hydrogen bond was shortened to a distance of 1.9 Å ( Figure 2B). Residue Cys1970 linked Cys1955 with a disulfide bond, which was destroyed as cysteine at residue 1970 being replaced by tyrosine ( Figure 2C). Residue Gln2713 formed two hydrogen bonds with Gly2712 and Thr2711. When glutamine was replaced by arginine at residue 2713, the hydrogen bond with Gly2712 was destroyed ( Figure 2D). Residue Arg3141 formed five hydrogen bonds with Ser3140, Leu3144, and Asp3145. When arginine was replaced by glutamine, three hydrogen bonds were destroyed while the two hydrogen bonds with Leu3144 and Asp3145 were kept. Residue Arg3189 interacted with Gln3004, Gln3006, Lys3008, and Arg3014 by forming five hydrogen bonds. When arginine at residue 3189 was replaced by tryptophane, all hydrogen bonds above were destroyed ( Figure 2E).

| Clinical features of the cases with CELSR3 variants
In this study, the CELSR3 variants were identified in five cases (families) with eight individuals (five females, three males) affected ( Figure 1A, Table 1). All affected individuals were diagnosed with FS/EFS+, including six individuals with FS, one patient with generalized tonic-clonic seizures with antecedent FS, and one patient with secondarily generalized tonic-clonic seizures with antecedent

Allele count/number in the controls of gnomAD-East Asian population
Identified

| DISCUSS ION
CELSR3 is a cell adhesion protein and largely expressed in the brain, functioning as a signaling receptor. CELSR3 converts cell-  In conclusion, we identified five heterozygous CELSR3 variants in five unrelated cases (families) with eight individuals affected by FS/EFS+. All patients presented favorable outcomes without neurodevelopmental disorders. The gene-based burden of variants showed a significant association between CELSR3 variants and FS/EFS+. Protein modeling suggested that the variants led to structure alterations of CELSR3 but were located on the outer side of the domains, potentially explaining the mild phenotype. These findings suggest that CELSR3 variants are potentially associated with FS/EFS+.

ACK N OWLED G M ENTS
We thank the patients and their families for participating in the study. We thank the staffs of Cipher Gene LLC (Beijing) and Chigene (Beijing) Translational Medical Research Center Co. Ltd. for providing next-generation sequencing, analyzing genetic diseases, and discussion.

CO N FLI C T O F I NTE R E S T
The authors have stated that they had no interests that might be perceived as posing a conflict or bias.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.