A Novel X-Linked Variant of IQSEC2 is Associated with Lennox-Gastaut Syndrome and Mild Intellectual Disability in Three Generations of a Korean Family

Abstract

Aim:

Lennox-Gastaut syndrome (LGS) is a severe type of childhood-onset epilepsy with multiple types of seizures, specific discharges on electroencephalography, and intellectual disability. However, LGS-related genes are largely unknown. To identify causative genes related to LGS, we collected and analyzed data from a three-generation Korean family in which one member had LGS and two had intellectual disability.

Methods:

Genomic DNAs were extracted from blood samples of all participants and used in whole-exome sequencing (WES). Genetic variants were detected by the Genome Analysis Toolkit and confirmed by Sanger sequencing. Variant pathogenicity was evaluated by prediction programs and the American College of Medical Genetics criteria. The LGS patient had generalized slow spike-and-wave discharges, multiple types of seizures, and developmental delay.

Results:

Analyses of the WES data from the family revealed a novel variant (c.1048G>A, p.Ala350Thr) in the IQ motif and Sec7 domain 2 (IQSEC2). This variant is within a highly evolutionarily conserved IQ-like motif, indicating a decrease in the calmodulin-binding capacity or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid transmission. The hemizygous variant in the male with LGS was a maternally inherited X-linked variant from the heterozygous maternal grandmother and mother, both of whom had intellectual disability.

Conclusion:

These findings indicate that the variant of IQSEC2 triggered both LGS and intellectual disability dependent on sex in this family. We report a novel X-linked inherited IQSEC2 variant for LGS and intellectual disability, which enhances the spectrum of variants in the IQ-like motif of IQSEC2.

Introduction

Lennox-Gastaut syndrome (LGS) is a type of childhood epilepsy with an onset age of 3-5 years (Al-Banji et al., 2015). The main features of LGS are multiple types of seizures (generalized tonic, atonic, and atypical absence) and generalized slow spike-and-wave (GSSW) or generalized paroxysmal fast activity (GPFA) discharges on electroencephalography (EEG) (Camfield, 2011). Brain magnetic resonance imaging (MRI) scans of LGS patients can appear abnormal, but this is not always the case (Asadi-Pooya and Sharifzade, 2012). For 65% to 75% of patients, LGS occurs due to a central nervous system infection, brain malformation, or brain injury. However, in ∼30% of patients, LGS occurs due to genetic variants (Camfield, 2011). More than 80% of LGS patients also have intellectual disabilities that are associated with impaired cognitive and adaptive behaviors (Camfield, 2011; Bourgeois et al., 2014; Al-Banji et al., 2015). Intellectual disabilities can also be caused by genetic abnormalities (Vissers et al., 2016). Therefore, the identification of causative genes and variants related to LGS and intellectual disability is important for treatment and therapeutic development.

Recently, genetic variants in the IQ motif and Sec7 domain 2 (IQSEC2) gene have been recognized as being associated with disorders such as autism, intellectual disability, epilepsy, and LGS (Shoubridge et al., 2010; Allen et al., 2013; Olson et al., 2015; Zerem et al., 2016; Mignot et al., 2019). The IQSEC2 gene is on the X chromosome and encodes a guanine-nucleotide exchange factor for the ADP-ribosylation factor family of small GTPases. IQSEC2 has multiple domain structures, including calmodulin binding IQ-like, catalytic ADP-ribosylation factor guanine-nucleotide exchange factor (ARF-GEF, Sec7), pleckstrin homology (PH), and PDZ binding (PSD-95/Dlg1/zo-1) motifs (Myers et al., 2012; Zerem et al., 2016). The IQSEC2 variants that have been detected in association with intellectual disability are X-linked inherited forms that are more severe and affect males (Shoubridge et al., 2010). In LGS and LGS-like syndrome, only eight de novo variants of IQSEC2 have been detected, and two of them were stop-gain variants (p.Gln1108X and p.Gln1033X) (Redin et al., 2014; Zerem et al., 2016; Mignot et al., 2019). Many of the variants were located in the IQ-like motif that binds with calmodulin or the Sec7 domain, which has ARF-GEF catalytic activity (Shoubridge et al., 2010; Zerem et al., 2016; Mignot et al., 2019).

In this study, we collected samples from a Korean family with a clinical history of LGS and used whole-exome sequencing (WES) data analysis to identify causative genetic variants related to LGS. In the process of this analysis, we identified a novel X-linked inherited variant in the IQ-like motif of IQSEC2 that was a causative variant associated with LGS and mild intellectual disability in a three-generation family. This study may contribute to the identification of genetic variants and to an understanding of the effects of these variants that are associated with LGS and intellectual disability.

Materials and Methods

Clinical description

Data were collected from a three-generation Korean family in which one member was diagnosed with LGS and two with intellectual disability by the Department of Pediatrics, Chungnam National University Hospital. The male patient had an early global developmental delay in infancy but no infantile spasms. The patient experienced febrile convulsions for ∼20 min at 7 months of age. He developed generalized tonic seizures at 3.5 years of age, and sudden atonic seizures occurred at 5 years of age. At the age of 5 years, his social quotient based on the Social Maturity Scale was 39, and his intellectual quotient based on the Korean Wechsler Intelligence Scale for Children-Profiles IV was 47. He was diagnosed with intellectual disability, and EEG showed GSSW discharges and GPFAs (Fig. 1B, C). Brain MRIs at 3 years of age revealed no abnormal signal intensity lesions in the brain parenchyma (Fig. 1D-F). LGS is defined by the clinical triad of (1) multiple types of seizures, including generalized tonic, atonic, and myoclonic seizures, atypical absences, and spasms; (2) GSSW and/or GPFA on EEG; and (3) progressive developmental delay (Camfield, 2011). Based on these criteria and the clinical data, we diagnosed the patient with LGS. Thus, we treated him with various antiepileptic drugs, such as levetiracetam, valproic acid, zonisamide, rufinamide, and clobazam, but they were ineffective, and the patient continued to have monthly seizures. His maternal grandmother (I:1) and mother (II:2) had mild intellectual disability, and his father (II:1) and older sister (III:1) had normal cognitive abilities (Fig. 1A).

FIG. 1.

Pedigree, EEG, and MRI of the LGS patient. (A) Pedigree of the family. Males and females are represented as squares and circles, respectively. The filled symbol represents the LGS patient (III:2). The half-filled symbols indicate mild intellectual disability (I:1 and II:2). The dotted box indicates no sample. (B) Generalized slow spike-and-wave discharge on EEG. (C) Generalized paroxysmal fast activities on EEG. (D) T1 axial MRI scan. (E) T1 coronal MRI scan. (F) T1 sagittal MRI scan. EEG, electroencephalography; LGS, Lennox-Gastaut syndrome; MRI, magnetic resonance imaging. Color images are available online.

This study was approved by the institutional review board (IRB) and ethics committee at the Chungnam National University Hospital and the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Written informed consent was obtained from the grandmother and the parents of the patient.

WES and genetic variant analysis

Genomic DNAs were extracted from blood samples of participants by using the DNeasy Blood and Tissue Kit (69506; Qiagen, Hilden, Germany). We prepared sequencing libraries from the gDNA of five individuals from Korean families using the SureSelectXT Library Prep kit. WES was performed on a HiSeq 2500 platform (Illumina, Santa Clara, CA) with a paired-end 101 bp read length. The obtained sequencing reads were mapped to the human reference genome (hg19) using Burrows-Wheeler software (v0.7.12) (Li and Durbin, 2009). The calling of single nucleotide variants was performed by the Genome Analysis Toolkit (McKenna et al., 2010) and the functional annotation of variants was performed by ANNOVAR (Wang et al., 2010). The uncommon genetic variants with minor allele frequencies <5% were selected based on 1000 Genomes Project data and Korean ethnic sequencing data (KOVA, v1). Variants with >0.909 PolyPhen2 scores (probably damaging) were selected as disease-causing variants, and we examined genes related to LGS or epilepsy. The selected variant was confirmed by Sanger sequencing through the ABI3730XL DNA sequencer, and IQSEC2 primers were designed by NCBI Primer-Blast: 5'-GAAGCAGGAGGAGGAGGAGA-3′ (forward) and 5′-AAAGGAGAACTGCATCCGCA-3′ (reverse).

Pathogenicity prediction

We estimated the pathogenicity of the IQSEC2 variant using ANNOVAR, PANTHER, PROVEAN, PredictSNP, and PhD-SNP (Bendl et al., 2014; Choi and Chan, 2015; Schiemann and Stowell, 2016; Tang and Thomas, 2016). The pathogenicity of the variant was also evaluated by the pathogenicity prediction guideline from the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) (Richards et al., 2015).

Results

Genetic findings

We obtained WES data for the family members with LGS and intellectual disability and analyzed the data using the Genome Analysis Toolkit. The results showed a novel variant (c.1048G>A) in IQSEC2 associated with LGS and intellectual disability, which was also confirmed by Sanger sequencing (Fig. 2A). The variant converts alanine to threonine on amino acid residue 350 (p.Ala350Thr), which was not reported in previous studies and not found in databases such as ClinVar, dbSNP, the 1000 Genomes Project, ESP6500, and the Exome Aggregation Consortium (ExAC). The logarithm of odds score of the variant was 3.97, meaning that the variant occurred by linkage (Greenberg et al., 1998). The genetic variant is X-linked; it was inherited from the maternal grandmother and mother. The father (II:1) and female sibling (III:1) did not have the variant. The variant was heterozygous in the mother and grandmother and hemizygous in the male patient. It is located in the evolutionarily highly conserved IQ-like motif (Fig. 2B). These results indicate that the heterozygous variant in females, with two X chromosomes, results in mild intellectual disability but that the hemizygous variant in males, with one X chromosome, results in LGS in this Korean family.

FIG. 2.

IQSEC2 variant detected in a three-generation family with LGS and mild intellectual disability. (A) Representative Sanger sequencing results of normal and affected individuals. Arrows indicate the position of IQSEC2 variants (c.1048G>A). (B) Conservation of the IQ-like motif of IQSEC2 in various species. The position of the missense variant is shown by the arrow. Conserved amino acids are indicated in blue. (C) Variants summary of IQSEC2. The variant detected in the patient in this study (p.Ala350Thr) is shown in red. Color images are available online.

Pathogenicity validation for the IQSEC2 variant

We estimated the pathogenicity of the IQSEC2 variant using various prediction programs. The analysis revealed that our IQSEC2 variant is a damaging, deleterious, and disease-causing variant. In addition, using the ACMG/AMP criteria, the variant was classified as a pathogenic variant with the criteria of PS3, PS4, PM2, and PM5 (Table 1).

Table 1.

Pathogenicity Prediction Result of IQSEC2 Variant (c.1048G>A, p.Ala350Thr)

Prediction programs								ACMG/AMP
PolyPhen2	SIFT	MutationTaster	LRT	PANTHER	PROVEAN	PredictSNP	PhD-SNP	Criteria	Classification
Damaging (0.998)	Damaging (0)	Disease causing (1)	Deleterious	Probably damaging	Deleterious	Deleterious	Disease	PS3, PS4, PM2, PM5	Pathogenic

PolyPhen2, polymorphism phenotyping v2 (damaging: 0.909-1, probably damaging: 0.447-0.908, benign: 0-0.446); SIFT, scale-invariant feature transform (tolerated: >0.05, damaging: ≤0.05); MutationTaster, online prediction software (disease causing: >0.5, polymorphism: ≤0.5); LRT, likelihood ratio test.

ACMG/AMP, American College of Medical Genetics/Association for Molecular Pathology; IQSEC2, IQ motif and Sec7 domain 2.

Conclusions

IQSEC2 is expressed in the central nervous system, including the brain and spinal cord, and plays a key role in cytoskeletal and synaptic organization and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic transmission (Murphy et al., 2006; Sakagami et al., 2008; Myers et al., 2012). Variants in IQSEC2 have been found in many types of disorders, such as epilepsy, intellectual disability, Rett syndrome, and LGS (Redin et al., 2014; Kalscheuer et al., 2015; Olson et al., 2015; Zerem et al., 2016). In previous studies, both de novo and X-linked variants of IQSEC2 were detected, but only de novo variants were found in LGS and LGS-like disorders, although the patient in our study inherited the X-linked IQSEC2 variant (Supplementary Table S1) (Allen et al., 2013; Redin et al., 2014; Zerem et al., 2016; Mignot et al., 2019). These findings indicate that our study may be the first case in which an inherited X-linked IQSEC2 variant was discovered in a male LGS patient and in maternal family members with mild intellectual disability.

We deeply explored the pathogenicity of this variant of IQSEC2 (c.1048G>A) using prediction programs and ACMG/AMP criteria, and it was predicted to be a pathogenic variant. The variant in the IQ-like motif might affect the calmodulin-binding capacity or AMPA receptor-mediated synaptic transmission. Furthermore, the analyses of our WES data implied that the effect of the X-linked inherited IQSEC2 variant depended on sex and caused LGS in males and mild intellectual disability in females in this Korean family.

Among the previously reported IQSEC2 variants, one variant (c.1049C>T) that was observed in epilepsy converted an alanine to valine at amino acid 350 of IQSEC2 (Fig. 2C) (Zerem et al., 2016; Zipper et al., 2017). Both alanine and valine have hydrophobic side chains; however, the A350V IQSEC2 mutation results in decreased surface AMPA receptors (Rogers et al., 2019). Since our c.1048G>A variant converted the alanine to threonine, which has polar and uncharged side chains (p.Ala350Thr), our variant might have the strong potential to affect AMPA receptors. These findings indicate that the different amino acid polarities of these variants may alter the structure and function of IQSEC2 and might result in differences in the symptoms.

In summary, we report a novel X-linked c.1048G>A variant of IQSEC2 that is a causative variant associated with LGS and mild intellectual disability in a three-generation Korean family. This variant enhances the spectrum of variants in the IQ-like motif of IQSEC2 and needs to be included in the targeted or broader sequencing analysis pipeline of both LGS and intellectual disability.

Footnotes

Acknowledgments

We thank the patient and his family for participating in our study.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2014M3A9A5034157 and 2014M3C9A2064619) and the KRIBB Research Initiative Program.

Supplementary Material

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Supplementary Material

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