Front. Pediatr.Frontiers in PediatricsFront. Pediatr.2296-2360Frontiers Media S.A.10.3389/fped.2025.1471965PediatricsOriginal ResearchEvaluation of the etiology of epilepsy and/or developmental delay in children via next-generation sequencing: a single-center experienceKavaHandan1†Akgun-DoganOzlem234†YesilyurtAhmet5AlanayYasemin236*IsikUgur7Department of Pediatrics, School of Medicine, Acibadem Mehmet Ali Aydinlar University, Istanbul, TürkiyeDivision of Pediatric Genetics, Department of Pediatrics, School of Medicine, Acibadem Mehmet Ali Aydinlar University, Istanbul, TürkiyeAcibadem Mehmet Ali Aydinlar University Rare Diseases and Orphan Drugs Application and Research Center (ACURARE), Acibadem Mehmet Ali Aydinlar University, Istanbul, TürkiyeDepartment of Transitional Medicine, Health Sciences Institute, Acibadem Mehmet Ali Aydinlar University, Istanbul, TürkiyeAcibadem Labgen Genetic Diagnosis Center, Istanbul, TürkiyeDepartment of Genome Studies, Health Sciences Institute, Acibadem Mehmet Ali Aydinlar University, Istanbul, TürkiyeDivision of Pediatric Neurology, Department of Pediatrics, School of Medicine, Acibadem Mehmet Ali Aydinlar University, Istanbul, Türkiye
Edited by: Piero Pavone, University of Catania, Italy
Reviewed by: Raffaele Falsaperla, Policlinico San Marco, Italy
Eulàlia Turon-Viñas, Universitat Autònoma de Barcelona, Spain
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Background
We aimed to understand the genetic etiology in children presenting with epilepsy and/or developmental delay by using next-generation sequencing (NGS).
Materials and methods
We included children presenting to our pediatric neurology clinic with a diagnosis of epilepsy and/or developmental delay between January 2019 and December 2021. We evaluated the patients using the NGS equipment in our genetic laboratory.
Results
In total, 90 patients were included in the study. Twenty (34.4%) out of 58 patients who had undergone whole-exome sequencing (WES) had pathogenic or likely pathogenic (P/LP) variants and 11 (18.9%) had variants of unknown significance (VUS). Five (41.6%) out of 12 patients who had undergone whole-genome sequencing had P/LP variants and 5 (41.6%) had VUS. Eleven (55%) out of 20 patients who had undergone WES and chromosomal microarray had P/LP variants and 2 (10%) had VUS. Twenty-six novel variants were described. Twelve patients (13.3%) were diagnosed using a known specific treatment.
Conclusion
NGS aids in precisely diagnosing children with epilepsy and/or developmental delay. Furthermore, it provides a correct prognosis, specific treatment methods, and a multidisciplinary approach.
The incidence of epilepsy in children ranges from 0.5 to 8 per 1,000 individuals (1–4). Its estimated lifetime prevalence is 1%, and the highest incidence is observed during the first year of life (5). Various developmental disorders, such as developmental delay (DD), intellectual disability (ID), autism spectrum disorder (ASD), and attention deficit and hyperactivity disorder (ADHD) are more common in children with epilepsy (6).
In developed countries, the absence of expected psychomotor development is observed in approximately 1% of children (7). DD and ID may occur alone or in conjunction with congenital malformations or neurological findings such as epilepsy, hypotonia, ataxia, electroencephalogram (EEG) abnormalities, and behavioral problems such as ASD and ADHD. DD and ID are phenotypically and genetically heterogeneous, and a specific diagnosis cannot be reached in most cases. Therefore, genetic tests play a crucial role in determining the cause of abnormality in this patient group (8).
Next-generation sequencing (NGS) technologies enable the reliable identification of candidate genes responsible for epilepsy and DD in a single, cost-effective, and time-efficient process (9). There are compelling reasons to make a specific diagnosis in a child presenting with DD. Diagnosis permits families to understand the long-term prognosis, access special education services, and learn about potential treatment options. Moreover, it is crucial for counseling parents regarding recurrence risk, prenatal diagnosis, and preimplantation genetic diagnosis. Furthermore, a diagnosis can help parents access research studies and connect with other families facing similar challenges (10).
Chromosomal microarray (CMA) has long been utilized as a first-line genetic test in children with DD and ID. With the advancement of NGS techniques, whole-exome sequencing (WES) and whole-genome sequencing (WGS) have become pivotal diagnostic tools for children with DD and ID. The American College of Medical Genetics and Genomics (ACMG) guidelines strongly recommend considering WES/WGS as a first- or second-line test for patients with DD and ID (10).
NGS has unveiled new candidate genes implicated in the pathogenesis of epilepsy. Ion channels play a crucial role in maintaining proper neuronal excitability; these include voltage-gated sodium, potassium, calcium, and chloride ion channels, and ligand-gated acetylcholine receptor-mediated ion channels. Mutations in genes encoding components of these ion channels lead to ion channel dysfunction, known as channelopathy, and form the basis for the development of epilepsy syndromes. Genetic changes causing channelopathies may contribute to the pathogenesis of epilepsy via the gain or loss of ion channel function (11).
Early genetic testing and diagnosis are foundational for accurate treatment of epilepsy. However, they should be complemented with functional tests to explore the characteristics of the pathophysiological mechanisms and the functional impact underlying the detected variant. Specifically in ion channel disorders, understanding gain-of-function mutations and loss-of-function mutations is critical when making therapeutic decisions (12, 13).
Experience with most of the above mentioned treatments is limited to a relatively small number of patients. With a better understanding of the pathogenesis and cellular electrophysiology of genetic epilepsies, more treatment options are likely to emerge (14).
Materials and methods
This retrospective cohort study included 90 patients aged 0–18 years, who were observed at the Acibadem Altunizade Hospital Pediatric Neurology Outpatient Clinic between January 2019 and December 2021 and had been diagnosed with epilepsy and/or DD. These patients had undetermined etiology despite undergoing biochemical and metabolic tests and magnetic resonance imaging (MRI). The genetic analyses were performed as part of the diagnostic process when patients first presented for evaluation. Informed consent for genetic testing was obtained by the accredited diagnostic laboratory prior to the tests. The WES and CMA results were processed at the Acibadem Labgen Genetic Diseases Evaluation Center, and the WGS results were obtained from the Centogene Laboratory (Germany).
Retrospective evaluation of the WES, CMA, and WGS results was conducted in collaboration with the Pediatric Genetics Department of Acibadem University. Patient medical records were retrospectively reviewed; these encompassed their medical history; background; family history; neurological symptoms; motor and mental developmental history; neurological examination findings; liver and kidney function test results; metabolic screening test, EEG, and MRI results; antiepileptic treatments; and genetic diagnoses.
Ethical approval for the retrospective study was obtained from the Scientific Research Ethics Committee of Acibadem University School of Medicine (Decision No: 2021/25/26).
Whole-exome sequencing
Genomic DNA isolation was performed using peripheral blood leukocytes. Enrichment was performed using the Twist® Human Core Exome kits. Prepared libraries were sequenced using the Novaseq 6000 (Illumina) instrument. Using the Burrows–Wheeler Aligner (BWA-MEM; Sentieon), sequenced data were aligned with genome reference to GRCh37 (hg19) (15). Duplicate marking, indel realignment, baseline quality recalibration, and variant calling were performed using the Genome Analysis Toolkit (GATK) algorithms (Sentieon) (16). Variant annotation, filtering, and interpretation were performed with the VarSome Clinical platform (Spatter). Variants in the coding and splicing regions and known pathogenic variants in the noncoding regions were included in the analysis. Variants with minor allele frequencies (below 1%) in the gnomAD database were evaluated. Filtering and prioritization of potentially disease-causing variants were performed based on the inheritance pattern, information in the ClinVar database, Human Phenotype Ontology (HPO) terms associated with the patient's findings, and in silico pathogenicity estimates. Variants were classified according to the ACMG guidelines (17) and ClinGen recommendations (18), pathogenic (P), and likely pathogenic (LP), and clinically relevant variants of uncertain significance (VUS) associated with the patient's clinical findings were reported.
WGS
Genomic DNA was fragmented enzymatically, and libraries were generated using polymerase chain reaction-mediated addition of Illumina-compatible adapters. Libraries were sequenced with paired ends on an Illumina platform to achieve an average depth of coverage of ∼30×. Alignment of the GRCh37/hg19 genome and human mitochondrial DNA (NC_012920) to the revised Cambridge Reference Sequence (rCRS), variant calling, annotation, and extensive variant filtering were applied through an inhouse bioinformatics pipeline. The copy number variant (CNV) recall was based on Illumina's DRAGEN pipeline. All variants in the GnomAD database with minor allele frequency of <1% and disease-causing variants reported in the Human Gene Mutation Database, ClinVar, and CentoMD were evaluated. During the evaluation, the entire gene region was examined for candidate variants associated with the phenotype in exons and in ±20 intronic regions. All potential patterns were considered for the inheritance pattern. Furthermore, family history and clinical information were used to assess the pathogenicity and probability of the disease-causing effect of the detected variants. Variants were divided into five classes (pathogenic, possibly pathogenic, of uncertain clinical significance, possibly benign, and benign) according to the ACMG guidelines. All potential variants associated with the patient's phenotype were reported. CNVs of uncertain clinical significance were not reported. Variants with heteroplasmy levels of up to 15% were reported as mitochondrial variants. Centogene has established specific quality criteria and validation processes for variants detected by NGS. Variants with poor sequence quality and/or indeterminate zygosity were confirmed by orthogonal methods. As a result, a specificity of >99.9% was guaranteed for all reported variants.
Chromosomal microarray
Technical work was conducted using the Affymetrix Cytoscan 750 K Microarray Platform. Findings that fell within the resolution thresholds (at least 25 markers for decreases of 100 kb and greater and increases of 200 kb and greater in known genomic areas) were analyzed using filters in the “Chromosome Analysis Suite (CHAS)” analysis program. Findings outside the reporting standards; absence of heterozygosity regions; additional mosaic findings; findings below the resolution threshold overlapping with the Online Mendelian Inheritance in Man (OMIM) genes, above-threshold findings without the OMIM genes, the OMIM genes not detected but findings in loci associated with the phenotype; findings that cannot be directly associated with the reason for referral; and results other than the OMIM gene point mutations, small deletions or duplications, low rate mosaicism, and balanced chromosomal changes such as reciprocal and the Robertsonian translocations were not evaluated. Whether the findings were familial or not was determined only through the studies requested by the parents. When required, the segregation analysis of the CNVs detected in the mother and father via array analysis was investigated.
GRCh37/hg19 was used as the reference genome in the analysis. The ClinGen, Decipher, UNIQUE, OrphaNet, DGV, ClinVar, OMIM®, dbSNP, and HPO databases associated with the patient's clinical findings were used to assess the variants. Detected variants were assessed using the patient's clinical findings.
Detected variants were classified according to the ACMG-ClinGen Variant Classification Guidelines. Variants classified as pathogenic, possibly pathogenic, and of uncertain clinical significance associated with the patient's clinical findings were reported. Reported variants were named according to the ISCN 2020 nomenclature (19).
Statistical analysis
The demographic and clinical characteristics of the patients evaluated in the study were analyzed via descriptive statistical analyses (number, percentage, mean, standard deviation, etc.). The comparisons of age, clinical findings, perinatal history, demographic characteristics, MRI, and EEG findings according to the genetic analysis results of the patients evaluated in the study were conducted using chi-square analysis. The significance level for all analyses was set at p < 0.05. The conformity of the data to normal distribution was checked with the values of kurtosis and skewness (±1.5). IBM SPSS 22.0 was used to conduct the analyses.
Results
In total, 90 patients were included in the study. Of the 90 patients, 53 (58.9%) were male, and 37 (41.1%) were female. The age of the patients at the time they were seen at our clinic was 0.04–16 years. The mean age of the patients was 3.99 ± 2.71 years, and the median age was 2.71 years. Forty-seven patients were from Turkey; the remaining 43 patients were international (Table 1). EEG abnormalities were detected in a total of 58 (64.4%) patients. EEG was not available from four patients (P68, P77, P79, P81) (Table 2).
Cranial MRI was abnormal in 44 patients (48.8%). The MRI results were abnormal in 27 (50%) of 54 patients with P/LP variants and clinically relevant VUS variants. MRI was not available in two patients (P68, P82) (Table 3).
Magnetic resonance imaging findings of all patients.
MRI findings
N*
%
Corpus callosum atrophy
15
16.6
Ventriculomegaly
13
14.4
White matter changes
12
13.3
Cerebral atrophy
10
11.1
Cerebellar atrophy
9
10
Basal ganglia and/or thalamus lesions
6
6.6
Hypoxic ischemic brain injury
5
5.5
Hydrocephalus
3
3.3
Cortical dysplasia
2
2.2
Cerebral venous sinus thrombosis
1
1.1
Intracranial hemorrhage
1
1.1
Hypothalamic hamartoma
1
1.1
Holoprosencephaly
1
1.1
Optic nerve atrophy
1
1.1
MRI, magnetic resonance imaging.
*Some patients had more than one finding.
In 15 (16.6%) patients, parental consanguinity/origin from same village was reported. There was first-degree cousin marriage between the parents in seven patients (7.8%), and second-degree cousin marriage between the parents in five patients (5.6%). In total, 24 patients (26.7%) had a family history of similar diseases. Thirty-one patients (34.4%) had abnormal perinatal history, such as asphyxia, infection, and hospitalization in the neonatal intensive care unit (Supplementary Table S1).
The patients were classified into three main groups; epilepsy only (n = 9; 10%), DD and epilepsy (n = 53; 58.9%), and only DD (n = 28; 31.1%) (Figure 1).
Phenotype of patients.
Molecular findings
Among all patients (n = 90), WES was studied in 58 patients, WGS was studied in 12 patients, and WES and CMA were studied in 20 patients. Trio-WES was performed in four, and trio WGS was performed in two families. Among the 58 patients who had undergone WES, P/LP variants were detected in 20 (34.4%), and clinically relevant VUS was detected in 11 (18.9%). Among the 12 patients who had undergone WGS, P/LP variants were detected in 5 (41.6%), and VUS was detected in 5 (41.6%). Among the 20 patients who had undergone WES and CMA, P/LP variants were detected in 11 (55%), and VUS was detected in 2 (10%) (Figure 2). A molecular diagnosis enabling a potential treatment option was established 12 patients (13.3%), (Table 4).
Molecular analysis results of patients.
Molecular diagnosis enabling potential treatment in epilepsy only and epilepsy+DD groups.
In our cohort, several genes were frequently implicated in cases of genetic epilepsy and developmental delay, including PRRT2 (n:2), KCNQ2 (n:2), SCN1A (n:2), PLA2G6 (n:2), and KDM6A (n:2). Among molecularly diagnosed cases SCN1A, SCN2A, KCNQ2, SCN8A, and ATP1A2 were identified as channelopathy-related genes, representing 7 patients. While channelopathies were less frequent compared to other conditions in the cohort, their distinct clinical characteristics warrant further attention.
Developmental delay was present in 71.4% of patients with channelopathies, slightly lower than the 93.6% observed in patients with other conditions. This lower prevalence may reflect the primary impact of channelopathies on seizure activity and excitability rather than broader neurodevelopmental processes. Similarly, dysmorphic features were observed in 14.2% of patients with channelopathies, significantly less frequent compared to 36.1% in other genetic conditions, highlighting the subtler phenotypic manifestations of channelopathies.
Abnormal MRI findings were detected in 28.5% of patients with channelopathies, which was markedly lower than the 53.1% observed in patients with other genetic conditions. This suggests that structural brain abnormalities are less commonly associated with channelopathies, reinforcing their functional nature. In contrast, EEG abnormalities were present in 71.4% of patients with channelopathies, slightly higher than the 61.7% seen in other genetic conditions. This reflects the prominent role of electrical disturbances in the pathophysiology of channelopathies.
Table 5 provides a detailed summary of the molecular findings in the diagnosed cohort, further illustrating the differences in clinical and genetic characteristics between channelopathies and other neurodevelopmental conditions.
Molecular findings in the diagnosed cohort.
P
G
Molecular test
Gene (Transcript)
Nucleotide
Protein
Zygosity
Inheritance
Variant
ACMG
Novel/Previously reported variant
Diagnosis (#OMIM number)
Epilepsy only
P2
M
WES CMA
PRRT2 (NM_001256442.1.)
c.629dup
p.Ala211Serfs*14
Het
AD
fs
P
Clinvar 31171
BFIS, 2 (#605751)
P3
M
WES
PRRT2 (NM_145239.3)
c.694dup
p.Arg217Profs*8
Het
AD
fs
P
Clinvar 65758
BFIS, 2 (#605751)
P4
M
Trio-WGS
KCNQ2, EEF1A2
20q13.33 (62074225_62129854)x1
Het
AD, de novo
CNV
LP
Novel
DEE 7 (#613720), BFNS1 (#121200), DEE 33(#616409)
P5
M
WES
SCN2A (NM_001040143.2)
c.379G>C
p.Val127Leu
Het
AD, de novo
mis
LP
Novel
DEE, 11(#613721), BFIS, 3(#607745)
P6
F
WES
NBEA (NM_015678.5)
c.1370G>T
p.Cys457Phe
Het
AD
mis
VUS
Novel
NEDEGE (#619157)
Epilepsy and developmental delay
P10
M
WES
FOLR1 (NM_016729.3)
c.322G>A
p.Glu108Lys
Hom
AR
mis
VUS
Clinvar 450195
NCFTD (#613068)
P11
M
Trio-WES
SCN1A (NM_001353958.2)
c.5341T>A
p.Tyr1781Asn
Het
AD, de novo
mis
LP
C Depienne et al. (20)
Dravet syndrome (#607208)
P12
M
WES
KNL1 (NM_170589.4)
c.6700C>T
p.His2234Tyr
Hom
AR
mis
VUS
Novel
MCPH 4 (#604321)
P13
F
WES
ATP1A2 (NM_000702.4)
c.2429G>A
p.Gly810Asp
Het
AD
mis
VUS
Novel
DEE 98 (#619605)
P14
F
WGS
22q11 del.
22q11 (18877539-21681527)x1
Het
AD
CNV
P
Decipher 472470 Akgun-Dogan et al. (21)
22q11 deletion syndrome (#611867)
P15
M
WES
ASH1l (NM_018489.3)
c.7189C>T
p.Arg2397*
Het
AD
ns
LP
HGMD CM1513932
IDD, AD 52 (#617796)
P16
M
Trio-WES
PCK1 (NM_002591.4)
c.524G>A
p.Arg175Gln
Hom
AR
mis
VUS
Clinvar 1368065
PCKDC (#261680)
P17
M
WES CMA
ABCC8 (NM_000352.6)
c.4432G>A
p.Gly1478Arg
Het
AD, paternal
mis
LP
Clinvar 434045
HHF 1 (#256450)
P18
M
WES CMA
KAT6A (NM_006766.4)
c.5828T>C
p.Met1943Thr
Het
AD
mis
VUS
Novel
MRD 32 (#616268)
MT-ATP8 (NC_012920.1)
c.98A>G*
p.Tyr33Cys
Homoplasmic
mt
mis
VUS
Novel
MC5DM2 (#516070)
P19
F
WGS
2q37del.
2q37.3 (237531981_242782258)x1
Het
AD
CNV
P
Decipher 324169 Akgun-Dogan et al. (21)
2q37 microdeletion syndrome (#600430)
3q29 dup.
3q29.32q29 (176456169_197851444)x3
Het
AD
CNV
P
S. Goobie et al. (22) 2008 Akgun-Dogan et al. (21)
3q29 microduplication syndrome (#611936)
P20
F
WES
GNB1 (NM_002074.5)
c.233A>G
p.Lys78Arg
Het
AD
mis
P
Clinvar 224714
IDD, AD 42 (#616973)
P21
M
WES
UBA5 (NM_024818.6)
c.863T>C
p.Met288Thr
Hom
AR
mis
VUS
Clinvar 1429996
DEE 44(#617132)
P22
F
WES
PACS2 (NM_001100913.3)
c.625G>A
p.Glu209Lys
Het
AD
mis
P
Clinvar 495141
DEE 66 (#618067)
P23
M
WES
PPT1 (NM_000310.4)
c.538dupC
p.Leu180Profs*9
Hom
AR
fs
P
Clinvar 56201
NCLs 1(#256730)
P24
M
WES
ADAM22 (NM_021723.5)
c.1312C>A
p.Pro438Thr
Hom
AR
mis
VUS
Marieke M. van der Knoop et al. (23)
DEE 61 (#617933)
P25
M
WES CMA
PNPO (NM_018129.3)
c.674G>A
p.Arg225His
Hom
AR
mis
P
Clinvar 223153
PNPOD (#610090)
P27
F
WES
SLC2A1 (NM_6516.4)
c.275+1del
p.?
Het
AD, de novo
S
VUS
Clinvar 1048756
GLUT-1 deficiency syndrome (#606777)
P28
M
WES
PLA2G6 (NM_003560.4)
22q13.1 (38484755_38511688)x0
Hom
AR
CNV
P
Neil V Morgan et al. (24)
INAD 1 (#26600)
P29
F
WES CMA
KDM6A (NM_001291415.2)
c.4180C>T
p.Leu1394Phe
Het
XLD
mis
VUS
Novel
Kabuki syndrome (#300867)
P30
M
WGS
SPTBN4 (NM_020971.2)
c.455T>G
p.Leu152Arg
C/H
AR
mis
VUS
Akgun-Dogan et al. (21)
NEDHND (#617519)
SPTBN4 (NM_020971.2)
c.7149del
p.Pro2384Argfs*60
C/H
AR
fs
VUS
Clinvar 1709124 Akgun-Doganet al. (21)
NEDHND (#617519)
P31
F
WES CMA
WWOX (NM_016373.4)
c.716T>G
p.Leu239Arg
Hom
AR
mis
LP
Clinvar 871669
DEE 28 (#616211)
P32
F
WGS
MACF1 (NM_012090.5)
c.3512T>C
p.Leu1171Pro
Het
AD
mis
VUS
Novel
LIS 9 (#618325)
P33
F
WES
KCNQ2 (NM_172107.4)
c.793G>A
p.Ala265Thr
Het
AD
mis
P
Milh et al. (25)
DEE 7 (#613720), BFNS1 (#121200)
P34
F
Trio-WES
GFAP (NM_002055.5)
c.1217G>T
p.Arg406Met
Het
AD, de novo
mis
LP
Novel
Alexander Disease(#203450)
P35
M
WES
ALDH7A1 (NM_001201377.2)
c.988C>T
p.Arg330*
Hom
AR
ns
P
Clinvar 1457545
EPD (#266100)
SLC22A5 (NM_003060.4)
c.1088T>C
p.Leu363Pro
Hom
AR
mis
LP
Clinvar 25408
CDSP (#212140)
P36
F
WES
PLA2G6 (NM_003560.4)
c.753dup
p.Asn252Glnfs*130
Hom
AR
fs
LP
Sallevelt et al. (26)
INAD 1 (#256600)
P37
F
WGS
NAGA (NM_000262.2)
c.973G>A
p.Glu325Lys
C/H
AR, paternal
mis
P
Clinvar 18162 Akgun-Dogan et al. (21)
NAGA deficiency type 3 (#609241)
NAGA (NM_000262.2)
c.103C>T
p.Arg35Cys
C/H
AR, de novo
mis
VUS
Clinvar 429497 Akgun-Dogan et al. (21)
NAGA deficiency type 3 (#609241)
P39
M
Trio-WGS
LSS (NM_001001438.2)
c.1970T>G
p.Met657Arg
C/H
AR
mis
VUS
Novel
APMR 4 (#618840)
LSS (NM_001001438.2)
c.2123G>T
p.Arg708Met
C/H
AR
mis
VUS
Novel
APMR 4 (#618840)
TUBB (NM_001293212.1)
c.334T>G
p.Phe112Val
Het
AD,de novo
mis
VUS
Novel
CDCBM 6 (#615771)
P40
M
WES
CUX1 (NM_001913.5)
c.1733_1734dupGC
p.Leu579Alafs*18
Het
AD
fs
LP
Novel
GDDI (#618330)
P41
M
WES CMA
SCN1A (NM_001202435.1)
c.1070 del
p. Asn357llefs*22
Het
AD
fs
LP
Novel
Dravet syndrome (#607208)
P44
F
WES
SETD1A (NM_014712.3)
c.2642G>T
p.Gly881Val
Het
AD,maternal
mis
VUS
Novel
EPEDD (#618832)
Developmental delay
P63
M
WES
BCL11A (NM_022893.4)
c.1486G>T
p.Glu496Ter
Het
AD,de novo
ns
P
Novel
Dias-Logan Syndrome (#617101)
SCN8A (NM_014191.4)
c.1743_1744delTG
p.Asn581LysfsTer5
Het
AD,de novo
fs
P
Novel
BFIS 5(#617080), CIAT (#614306), DEE 13(#614558)
P64
M
WGS
THOC2 (NM_001081550.1)
c.521A>G
p.Lys174Arg
Hemizygous
XLR, maternal
mis
VUS
Novel
XLID 12 (#300957)
P65
M
WES CMA
3q13.31 mikrodel.
3q12.1q13.32 (98920626_118556822)x1
Het
AD
CNV
P
Molin et al. (27)
3q13.31 deletion syndrome (#615433)
P66
M
WES
BCL11B (NM_138576.4)
c.2438_2459del
p.Val813Alafs*24
Het
AD,de novo
fs
LP
Goos et al. (28)
IDDSFTA (#606558)
P67
F
WGS
ERCC6 (NM_000124.3)
c.543+1G>T
p.?
Hom
AR
S
LP
Clinvar 974894 Akgun-Dogan et al. (21)
Cockayne syndrome (#133540)
P68
M
Trio-WES
ECEL1 (NM_004826.4)
c.1507-9G>A
p.?
C/H
ARl
S
LP
Clinvar 210907
Arthrogryposis, distal type (#615065)
ECEL1 (NM_004826.4)
c.1630C>T
p.Arg544Cys
C/H
AR
mis
VUS
Clinvar 816801
Arthrogryposis, distal type (#615065)
P69
F
WES CMA
KDM6A
Xp11.3 (44733823_44837306)x1
Het
XLD
CNV
P
Clinvar 564836
Kabuki syndrome (#300867)
P70
M
WES CMA
TCF 20 (NM_005650.4)
c.5452G>T
p.Glu1818*
Het
AD, de novo
ns
P
Novel
DDVIBA (#618430)
P71
F
WES
DHCR7 (NM_001360.3)
c.452G>A
p.Trp151*
C/H
AR
ns
P
Clinvar 21273
Smith-Lemli-Opitz Sendromu (#270400)
DHCR7 (NM_001360.3)
c.1295A>G
p.Tyr432Cys
C/H
AR
mis
P
Clinvar 1076651
Smith-Lemli-Opitz Syndrome (#270400)
P72
F
WES
GCDH (NM_000159.4)
c.383G>A
p.Arg128Gln
Het
AR
mis
LP
Clinvar 189063
Glutaric acidemia type 1(#231670)
GCDH (NM_000159.4)
c.1282A>G
p.lle428Val
Het
AR
mis
VUS
Novel
Glutaric acidemia type 1(#231670)
P73
M
WES
DARS1 (NM_001349.4)
c.606C>G
p. lle202Met
Hom
AR
mis
VUS
Novel
HBSL (#615281)
ERLIN2 (NM_007171.2)
c.78A>G
p.Ile26Met
Het
AD
mis
VUS
Novel
SPG 18 (#611225)
P74
F
WES CMA
ZEB2 (NM_014795.4)
c.1091C>G
p.Ser364*
Het
AD
ns
LP
Novel
Mowat-Wilson Syndrome (#235730)
P75
M
WES CMA
SOX5
12p12.1 (23805762_23967495)x1 del
Het
AD
CNV
P
Lamb et al. (29)
Lamb-Shaffer Syndrome (#616803)
P76
M
WES
PANK2 (NM_153638.3)
c.676G>A
p.Val226Ile
Hom
AR
mis
LP
Akcakaya et al. (30)
NBIA 1 (#234200)
P77
F
WES
PIK3R2 (NM_005027.4)
c.1117G>A
p.Gly373Arg
Mozaic
AD,de novo
mis
P
Clinvar 39808
MPPH 1 (#603387)
P78
M
WES
SLC1A4 (NM_003038.5)
c.911G>A
p.Gly304Asp
Hom
AR
mis
VUS
Novel
SPATCCM (#616657)
P79
M
WGS
LAMB1 (NM_002291.2)
c.4756A>T,
p.Thr1586Ser
C/H
AR
mis
VUS
Novel
LIS 5 (#615191)
LAMB1 (NM_002291.2)
c.4859T>C
p.Ile1620Thr
C/H
AR
mis
VUS
Clinvar 999947 Akgun-Dogan et al. (21)
LIS 5 (#615191)
P80
F
WES CMA
KIF1A
2q37.3 (240058543_242782258)x1 del
Het
AD
CNV
P
Decipher 411683, 277875
Nescav Syndrome (#614255)
Significant variants detected in the patients in our study are listed along with their characteristics. Variants reported in the literature are given with Decipher, HGMD, and ClinVar numbers. Variants that had not been reported previously in the literature are called new variants. These genetic variants’ classification detected according to by June 2024.
ACMG, American College of Medical Genetics and Genomics; APMR4, alopecia-intellectual disability syndrome 4; BFIS, benign familial infantile seizures; BFNS, benign familial neonatal seizures; CDCBM 6, cortical dysplasia, complex, with other brain malformations 6; CDSP, carnitine deficiency, systemic primary; CIAT, cognitive impairment with or without cerebellar ataxia; CNV, copy number variant; DDVIBA, developmental delay with variable intellectual impairment and behavioral abnormalities; C/H, compound heterozygous; DEE, developmental and epileptic encephalopathy; EPD, epilepsy, pyridoxine-dependent; EPEDD, epilepsy, early-onset, with or without developmental delay; F, female; fs, frameshift variant, G, gender; GDDI, global developmental delay with or without impaired intellectual development; GLUT1DS1, GLUT-1 deficiency syndrome; HBSL, hypomyelination with brainstem and spinal cord involvement and leg spasticity; Het, heterozygous; Hom, homozygous; HGMD, human gene mutation database; HHF, hyperinsulinemic hypoglycemia; IDD, AD, intellectual developmental disorder, autosomal dominant; IDDSELD: epilepsi ve konuşma geriliği ile birlikte zihinsel yetersizlik; IDDSFTA, intellectual developmental disorder with dysmorphic facies, speech delay, and T-cell abnormalities; INAD, infantile neuroaxonal dystrophy; LIS 9, lissencephaly 9 with complex brainstem malformation; M, male; MCPH 4, primary microcephaly 4; MC5DM2, mitochondrial complex 5 (ATP synthase) deficiency, mitochondrial type 2; mis, missense variant; MPPH1, megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 1; MRD 32, mental retardation, autosomal dominant; mt, mitochondrial; NBIA1, neurodegeneration with brain iron accumulation 1; NCFTD, neurodegeneration due to cerebral folate transport deficiency; NCLs, neuronal ceroid lipofuscinosis; NEDEGE, neurodevelopmental disorder with or without early-onset generalized epilepsy; NEDHND, neurodevelopmental disorder with hypotonia, neuropathy, and deafness; ns, nonsense variant; OMIM, online Mendelian inheritance in man; PCKDC, phosphoenolpyruvate carboxykinase deficiency, cytosolic; PNPOD, pyridoxamine 5′-phosphate oxidase deficiency; SPATCCM, spastic tetraplegia, thin corpus callosum, and progressive microcephaly; SPG 18, spastic paraplegia 18; S, splice-site variant; XLID 12, intellectual developmental disorder, X-linked 12.
Dysmorphic findings were significantly more frequent in the molecularly diagnosed group (P/LP/VUS variants), whereas other features, such as MRI abnormalities, neurobehavioral findings, and ocular findings, did not show a statistically significant difference (Table 6).
Comparison of clinical characteristics according to patients' genetic results.
Clinical findings
Genetic results
P
P/LP/VUS variants
–
n
%
n
%
MRI findings
27
49.1
17
51.4
0.828
Spasticity
16
29.6
11
30.6
0.925
Hypotonia
20
37.0
7
19.4
0.074
Dystonia
4
7.4
1
2.8
0.348
Ataxia
0
0.0
2
5.6
0.080
ADHD
8
14.8
3
8.3
0.358
Autism
1
1.9
1
2.8
0.720
Microcephaly
8
14.8
5
13.9
0.903
Macrocephaly
4
7.4
0
0.0
0.095
Dysmorphism
18
33.3
2
5.6
0.002
Strabismus
4
7.4
2
5.6
0.730
Nystagmus
0
0.0
1
2.8
0.218
Optic atrophy
2
3.7
1
2.8
0.811
Coloboma
1
1.9
0
0.0
0.412
Cataract
1
1.9
1
2.8
0.770
Visual impairment
1
1.9
0
0.0
0.412
Microphthalmia
0
0.0
1
2.8
0.218
Swallowing difficulty
7
13.0
4
11.1
0.793
Hearing loss
2
3.7
1
2.8
0.811
Status epilepticus
2
3.7
1
2.8
0.811
Cleft palate lip
1
1.9
1
2.8
0.770
Hypothyroidism
3
5.6
2
5.6
0.999
Hydrocephalus
2
3.7
1
2.8
0.811
Sialorrhea
4
7.4
1
2.8
0.348
ADHD, Attention deficit and hyperactivity disorder; MRI, Magnetic resonance imaging.
Discussion
Several epilepsy phenotypes exhibit chromosomal imbalances or alterations in genes encoding ion channels and transcription factors. Genetic testing is crucial for elucidating the underlying cause in patients with epilepsy and DD (31). Despite advanced diagnostic technologies nearly half of the affected individuals remain undiagnosed.
In the literature, the rate of diagnosis of DD and epilepsy by utilizing WES is reported to be between 25% and 55% (32–36). Hiraide et al., have reported that WES, including CNV analysis, resulted in a diagnostic rate of 53.5% (37). In our study, the percentage of LP/P variants with WES was 34.4%. Clinically relevant VUS were observed in an additional 18.9%, providing a similar diagnostic rate (53.3%) despite absence of CNV analysis. Similarly, the diagnostic rate of WGS in DD and epilepsy cohorts is reported to be 21%–50% (38–42). Among the 12 patients who had undergone WGS, 5 (41.6%) had P/LP variants, and 5 (41.6%) had clinically relevant VUS. WGS was performed in patients without prior testing. Two out 5 P/LP WGS results showed CNV as the underlying cause which would have been missed by WES alone. WGS shortened the diagnostic duration in these patients as first-tier genetic test.
The findings of our study indicate that the most frequently observed genes are consistent with those reported in the literature (11–14). Furthermore, although a direct comparison is not feasible due to the significant differences in the numbers, channelopathy-related genes stood out prominently in our study. Similarly, in the literature, channelopathy-related genes have also been highlighted as a significant group, in line with our data. Compared to other genetic etiologies underlying epileptic encephalopathies, channelopathies are less frequently associated with dysmorphic features and abnormal MRI findings, while EEG abnormalities are more prominently observed. This highlights the critical importance of genetic analysis in establishing an accurate diagnosis. Furthermore, genetic findings play a pivotal role in informing and tailoring treatment strategies (11, 13).
In this study, a molecular genetic diagnosis with available treatment was established in 13.3% of the patients. Only one patient with an actionable variant was left untreated due to absence of clinical seizures. Mahler et al. reported a treatment change rate of 6%. The higher rate of specific treatment changes in our study may be attributed to our patients having a more heterogeneous phenotype, as well as the emergence of new treatment options involving genes related to ion channels (31). By re-evaluating the previously negative WES data of patient P27, a de novo VUS variant in the SLC2A1 gene was identified; this was followed by segregation analysis, which led to a diagnosis of GLUT-1 deficiency (43). Seizures were controlled using a ketogenic diet, demonstrating the importance of data reanalysis in treatment modifications.
Trio WES analysis was conducted in 4 families due to economic burden. Segregation analysis was performed following initial WES analysis. 20.7% of the diagnosed individuals showed de-novo monoallelic variants. Brunet et al. reported a much higher frequency of de- novo variants (40.3%) following trio WES in their study. The lower percentage of de-novo monoallelic variants in our cohort reflects the higher rate of consanguinity leading to a higher incidence of biallelic disorders (22.2%) in comparison to 16% reported by Brunet et al. (44).
Samati et al. reported abnormal MRI findings in one-third of individuals with epilepsy in a recent review. The most frequently observed abnormalities were encephalomalacia related to chronic infarcts, cerebral atrophy, disorders of neuronal migration, periventricular leukomalacia. Accompanying EEG finding was mainly focal discharges (78%) (45). In our study, corpus callosum atrophy (16.6%), ventriculomegaly (14.4%), and white matter changes (13.3%) were present in 48.8% of our patients and focal discharges were most common (40%) as well. Our cohort mainly consists of unexplained patients without evident focal lesions. The relatively lower percentage of focal discharges may be related to the lower number of focal lesions on MRI in our patient group who underwent genetic testing.
The main limitation of our study is the retrospective nature and heterogeneity of the genetic tests. Some patients had only WES, some had WES and CMA, and some had WGS. Therefore, the number of patients having each test was limited. However, our study adds novel genetic variants that have not been described previously in the literature. All patients received pre-test and post-test genetic counseling from two pediatric geneticists who also contributed to deep-phenotyping and reverse phenotyping of the diagnosed cohort. Patients with negative genetic results will be recalled for reanalysis yearly. Genetic counseling is provided by clinical geneticists in Turkey. Families with a diagnosis were counseled regarding recurrence risks and future reproductive options. This is the largest single center study with a multidisciplinary approach from our country.
In conclusión, NGS helps to determine the underlying molecular etiology of neurogenetic conditions. Moreover, an increasing percentage of patients receive personalized treatment after molecular diagnosis. WES and WGS increase the diagnosis rate and shorten the diagnostic odyssey for families when applied as first-tier genetic tests. Determining the genetic diagnosis of patients with epilepsy and/or DD using NGS is crucial in terms of an accurate prognosis, potential treatment methods, and a multidisciplinary approach involving molecular and clinical genetic expertise.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Ethics statement
The studies involving humans were approved by Scientific Research Ethics Committee of Acibadem University School of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin.
Author contributions
HK: Data curation, Formal Analysis, Investigation, Writing – original draft, Methodology, Project administration. OA-D: Conceptualization, Data curation, Writing – review & editing, Supervision, Validation. AY: Methodology, Writing – review & editing, Data curation. YA: Data curation, Writing – review & editing, Supervision, Conceptualization, Validation. UI: Data curation, Investigation, Project administration, Supervision, Writing – review & editing, Conceptualization.
Funding
The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2025.1471965/full#supplementary-material
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