Clinical and Molecular Profile of Infantile-Onset Pharmaco-Resistant Epilepsy in Iraqi Children

Abstract

Background

Identifying the underlying genetic etiology of epilepsy and neurodevelopmental disorders can greatly inform clinical management from diagnosis to treatment to genetic counseling about prognosis. The aim of the study was to establish the genetic/molecular diagnosis in children presenting to two children hospitals in Baghdad, with infantile-onset epilepsy.

Methods

Twenty-nine Iraqi children with infantile-onset epilepsy, defined as epilepsy with onset in the first year of life, presenting between April 2018 and June 2021, were included in this prospective study.

Results

An overall molecular diagnostic rate of 65.5% (in 19/29 subjects) was achieved. Of the subjects with molecular diagnosis, 74% (14/19 subjects) were diagnosed by clinical exome sequencing and 26% (5/19 subjects) were diagnosed by the epileptic encephalopathy panel. Single nucleotide variants were detected in 95% (18/19 subjects) while copy number variants accounted for 5% of the cohort (1/19 subjects). Thirteen subjects (68%) had known pathogenic or likely pathogenic variants in the following genes: SCN1B, SCN1A, ALG13, STXBP1, TBC1D24, PRUNE1, ALDH7A1, PCDH19, SCN8A, and SLC13A5. Six patients (32%) had novel variants potentially explaining the phenotype in the following known genes: SYNJ1, SLC25A22, SCN2A, KCNT1, CACNA1A, and PLPBP. Half (9/19; 47%) of the molecularly diagnosed cases had therapeutic implications.

Conclusion

This is the first study to characterize the genotypic and phenotypic spectrum of infantile-onset drug-resistant epilepsy in children from Iraq. Our study demonstrates a high molecular yield with genetic heterogeneity and predominance of autosomal recessive mode of inheritance.

 

MissingLinkText

Keywords

infantile - epilepsy - pharmaco-resistant

Introduction

High prevalence of co-occurrence of epilepsy and neurodevelopmental disorders may be attributed to common pathophysiological mechanisms.[1] The International League Against Epilepsy (ILAE) defined epilepsy syndromes with onset at neonatal or infancy period as a group of well-defined electroclinical syndromes with onset in the first 2 years of life.[2] They include self-limiting epilepsies as well as developmental and epileptic encephalopathies.[2] Genetic causes underlie approximately 30 to 50% of cases with early-onset epilepsy (EOE) cases.[3] [4] Thus, a genetic etiology should be considered in cases with isolated idiopathic epilepsy even in the absence of structural brain abnormalities and inborn error of metabolism.[5] Defects in many genes, such as those encoding ion channels and proteins involved in neurotransmitter trafficking, synapsis, interorganelle communication, and cell connections, can cause epilepsy and/or neurodevelopmental disorders.[1] [6]

In children with epilepsy, developmental delay may be caused by the underlying genetic disturbances or may be a sequelae of the frequent epileptic discharges as seen in epileptic encephalopathy.[7] In such cases, early diagnosis and active management of the epileptic encephalopathy are essential to achieve better developmental outcomes.[7] Additionally, understanding the cause of epilepsy can have prognostic implications.[8] Despite the advantages of developing a precise molecular diagnosis in EOE and the significant contribution of genetic etiology to EOE in general and in the Arab region in particular, where there is a high rate of consanguinity, indeed, there are still very few studies on the prevalence, incidence, and diagnostic yield of genetic testing in EOE and epilepsy in general in Iraq and the neighboring Arab region.[9]

In this study, we aim to identify the clinical features and genetic characteristics of 29 children from Iraq presenting with epilepsy in their first year of life that is accompanied by neurodevelopmental manifestations who underwent targeted next-generation sequencing (NGS).

 

MissingLinkText

Methods

Standard Protocol Approvals, Registrations, and Patient Consents

The study protocol was approved by the Institutional Review Board of Children Welfare Teaching Hospital (IRB: 10, Dated: February 1, 2018) and was conducted according to the ethical principles of Declaration of Helsinki. Written informed consent for enrollment in the research study and the publication of relevant findings was obtained from the legal guardians of the affected individuals from all families.

Twenty-nine unrelated patients, who were presented with epilepsy in the first year of life with/without developmental delay and who were tested genetically between April 2018 and June 2021, were enrolled in the study.

 

MissingLinkText

Selection Criteria, Clinical Information, and Genetic Testing

The study involved gathering data from patients who had been examined and given their consent. Parents of all subjects who met the inclusion criteria and who were offered charity genetic testing consented, in addition to five patients who had private testing. Each child was examined and investigated by a child neurologist working at the Children Welfare Teaching Hospital and Central Children Hospital. All the children included in the study had to meet all the following inclusion criteria: (1) had seizure onset between birth and 12 months of age; (2) had abnormal epileptiform discharges that showed specific or nonspecific electrical pattern, or normal electroencephalography (EEG); (3) had drug-resistant epilepsy, with/without developmental delay or had a known epileptic encephalopathy syndrome; and (4) had no identified cause or structural brain abnormality detected on brain magnetic resonance imaging or metabolic investigations. Seizures, epilepsy, and epileptic syndromes were classified according to the 2017 classification of ILAE.[10] [11]

All clinical, neurophysiological, and imaging data were reviewed carefully to clearly define the phenotypes. All children underwent genetic testing (proband only or trio testing with their parents when affordable) by using dried blood samples on filter cards provided by the Centogene laboratory. Twenty-four cases were tested using charitable offer by the Centogene laboratory in Germany, whereas five patients had paid testing. Charity samples were sent for whole-exome sequencing; however, some of the samples were processed and analyzed by the supporting laboratory as a gene panel to reduce expenses. The tests were performed at Centogene laboratory in Germany, which is certified by the College of American Pathologists, the Clinical Laboratory Improvement Amendments, and the International Organization for Standardization basis.

In 19 patients, clinical exome sequencing (cES) was used to target coding regions of approximately 6,700 genes with known clinical significance. Whereas in 10 patients, epileptic encephalopathy panel was used and the entire coding region of 48 genes including ACY1, ADSL, ALDH7A1, AMT, ARGHEF9, ARX, CDKL5, CNTNAP2, CPT2, FLOR1, FOXG1, GABRG2, GAMT, GCSH, GLDC, GRIN2A, GRIN2B, KCNJ10, KCNQ2, MAPK10, MECP2, MTHFR, NRXN1, PCDH19, PLCB1, PNKP, PNPO, PRRT2, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, SCN1A, SCN1B, SCN2A, SCN8A, SCN9A, SLC19A3, SLC25A22, SLC2A1, SLC9A6, SPTAN1, STXBP1, TBCE, TCF4, TREX1, UBE3A, and ZEB2 genes, including 10 bp of flanking intronic sequences were targeted. All relevant variants detected by NGS were predicted damaging or likely damaging by more than one in silico prediction software (PolyPhen, Align-GVGD, SIFT [Sorting Intolerant From Tolerant], and Mutation Taster), and were confirmed by Sanger-sequencing.

 

MissingLinkText

Data Availability

All data described in this study are provided within the “may be deidentified” and made available from the corresponding authors upon request.

 

MissingLinkText

 

MissingLinkText

Results

The study included 29 patients, all of which were of Iraqi Arab ethnicity. A total of 19 subjects had cES whereas 10 subjects underwent epileptic encephalopathy panel. With both testing modalities collectively, 7 patients had negative genetic test results while 22 subjects had genetic findings; among which only 19 were clinically relevant and diagnostic molecular findings. The other three positive results were nonclinically relevant upon deep clinical characterization and thus were considered nondiagnostic results. The demographic profile of all 22 patients who had genetic findings revealed 11 girls (female-to-male ratio was 1:1), median age of seizure onset was 2 months (range 0–11 months), and median age of molecular diagnosis was 2 years (range 0.37–9.1 years).

[Table 1] demonstrates the clinical, EEG, and neuroimaging profile of the 19 children with the diagnostic molecular findings. The four most commonly reported seizure types were myoclonic and generalized tonic seizures (57.9 and 47.4%, respectively). The most frequently reported neurological (other than seizures) and systemic manifestations were intellectual disability and developmental delay (84.2 and 73.7%, respectively). No specific abnormal neuroimaging findings were identified in half of the patients.

Table 1 Demographics, types of seizures, developmental profile, and other clinical manifestations, EEG, and brain MRI findings in children presented with infantile-onset pharmaco-resistant epilepsy

Patient/sex/age/ethnicity	Age of onset (mo)	Gene	Types of seizures	Developmental and other clinical features	EEG	Neuroimaging
(1) M Died 2 year Arabic	40 days	SCN1B	Myoclonic, GTS, autonomic features, SE, fever-triggered, initial dramatic response to pyridoxine	Developmental delay, some social interaction, spasticity, scoliosis, pigeon chest, strabismus	Early infancy: normal Later: multiple epileptic discharges	Supratentorial cortical atrophic changes Ventricular dilatation
(2) M Died 6 year Arabic	3 months	SCN1B	Prolonged tonic spasms, GCS, focal with loss of consciousness, GTS, episodes of apnea/bradycardia (autonomic episodes)	Developmental delay then regression, some social interaction, impaired hearing, undescended testes, feeding difficulty, failure to thrive	Generalized spike/wave, generalized and lateralized Sharp wave discharges and phase reversal	Initially normal Later generalized brain atrophy and ventricular dilatation
(3) F 2 year 5 month Arabic	3 months	SCN1B	Myoclonus, GTS/GTCS, fever-triggered, initial dramatic response to pyridoxine	Spasticity, socially interactive, strabismus, erratic myoclonus, hypertonia	Normal	Cerebral cortical atrophy and ventricular dilatation
(4) F 8 year 7 month Arabic	7 months	SCN1A	GTS, fever-triggered, atypical absence and myoclonic (started at age of 2 years), SE	Developmental delay/regression, ambulating with assistance, autistic behavior, cognitive impairment, later: mild improvement of developmental skills	Generalized slow SW discharges (frontal predominant), multifocal spike waves, multifocal spikes	Diffused supratentorial cortical atrophy and compensated ventricular dilatation
(5) F 4 year 3 month Arabic	3 months	SCN1A	GTS, hemiclonic, myoclonic/atonic atypical absence, focal tonic seizure with impaired awareness, fever-triggered	Developmental delay, some social activity, ataxia, hypotonia, hyporeflexia, autistic behavior	Infancy: runs of spike/sharp slow waves At 3 years: runs of generalized spike wave and phase reversal or unilateral spike wave and phase reversal	Normal
(6) F 9 year Arabic	First year	ALG13	Epileptic spasms, myoclonus (dropping episodes), atypical absence	Intellectual disabilities, hypertonia, extensor plantar, hyperactive, autistic and self -mutilation behavior, visual impairment, microcephaly, facial dysmorphism “coarse facies, hypertelorism, low set ears, microcephaly, widely spaced teeth, and broad forehead”	Hypsarrhythmia Bursts generalized spike Polyspike wave discharges	Nonspecific abnormal signal intensity (high signal at T2 WI) of white matter at occipital trigon
(7) M 6 year 7 month Arabic	2 months	STXBP1	GTCS, focal clonic with impaired awareness, myoclonic	“Severe cognitive impairment, intellectual disabilities, hyperactivity, with less extensive epilepsy profile,” myoclonic / ataxic gait, autistic behavior	Generalized slowing	Normal
(8) M Died 13 month Arabic	7 days	PRUNE1	Epileptic spasms, generalized tonic (motor asymmetry), reflexive spasms	Equinavarus deformity and reduced fetal movement, global developmental delay, severe cognitive impairment, visual and swallowing impairment, hypotonia, hyporeflexia, “excessive tactile-induced startle, repeated pneumonia, transient PHT, EMG: neuronogenic markers (denervation) SMA1 like”	Generalized SW, multifocal epileptic discharges, and lateralized SW discharges	“Normal”
(9) M 1.1 year Arabic	10 days	ALDH7A1	GCS, multifocal tonic with facial flushing, tonic spasms	Normal examination, normal development	“Burst – suppression”	Normal CT
(10) M 9 month Arabic	10 days	SCN8A	Focal clonic with, impaired awareness, generalized clonic seizures with asymmetry	Developmental regression, severe cognitive impairment, visual impairment, axial and appendicular hypotonia	Slow background, multifocal epileptiform discharges	Normal MRI
(11) M Died at 2 year Arabic	3 days	SYNJ1	GCS with asymmetry, focal clonic with impaired awareness and secondary generalization, spasms, myoclonic seizures “50% response to pyridoxine and KD”	GDD, severe cognitive impairment, visual impairment/squint,, scoliosis, inverted feet deformity, axial hypotonia, appendicular hypertonia, NG feeding, “choreoathetosis/dystonia”	2 months: Lateralized sharp wave discharges, periodic-like pattern (PLED), generalized and localized SW discharges, abnormal sleep background 17 months: multifocal epileptic discharges	Normal
(12) F 2 year 7 month Arabic	15 days	TBC1D24	GTS (focal semiology), GCS, clonic EPC, “reflexive myoclonus aborted by manual closure of the eyes”	LBW, GDD, constipation, hand-foot chilblains, respiratory and urinary tract infections, abnormal visual behavior, fat pads, spasticity, hyperreflexia, aortic incompetence	Not available	Widening of subarachnoid space (frontal and temporal)
(13) F 6 year 7 month Arabic	11 months	PCDH19	Brief GTC, clusters of fever-triggered focal clonic/focal tonic with impaired awareness, GTS with asymmetry, SE	Hyperactivity, psychomotor delay, normal cognitive status, cafe au-lait spots, stereotypes of hands	Normal	Normal
(14) F 2 year Arabic	3 days	SLC13A5	Generalized clonic seizures with focal semiology They discontinued on the 2nd year by multiple ASD	Initially mild developmental delay, improved and plateaued at age of 18 months, unambulated, moderate cognitive impairment, socially interactive, apparently abnormal teeth, central hypotonia, appendicular hypertonia, hyporeflexia, weak muscle strength	Normal	Normal
(15) M 6 year Arabic	10 days	SLC25A22	Erratic myoclonus (early myoclonic epilepsy), asymmetrical startle, epileptic spasms, screaming, atypical absence	Severe cognitive decline and motor impairment, hypotonia, hyporeflexia, bad dentition, dysmorphology, recurrent chest infection, microcephaly, feeding difficulty, growth retardation	Multifocal spike wave and generalized spike wave / polyspike wave discharges	Mild frontal atrophy and enlarged subarachnoid space
(16) M 26 month Arabic	Birth	SCN2A	Clusters, GTS (with focal semiology), spasms and myoclonic seizures, status epilepticus	Developmental delay, severe cognitive impairment, “auditory-induced startle, spasticity, swallowing difficulty,” visual impairment, constipation, dental problem,, plantar extensor, weakness of limbs, microcephaly, recurrent chest infection during infancy	Burst suppression (Ohtahara syndrome) at 2 months	Normal
(17) F 20 month Arabic	2 months	KCNT1 De novo	Generalized tonic (asymmetric) / alternating asymmetry from side to side, automatism, seizures stopped or reduced (> 80%) after placing VNS	Delayed milestones, severe cognitive impairment, visual impairment, feeding difficulty, truncal hypotonia, appendicular hypertonia, cafe au-lait patch, hyperreflexia, clonus	Burst suppression pattern (Ohtahara syndrome), multifocal epileptic discharges intermixed with BS pattern	Mild atrophy and ventricular dilatation
(18) M 16 month Turkmen	6 months	CACNA1A	Myoclonic seizures	Developmental delay, severe cognitive and visual impairment, small palpebral fissure, fat pads of the dorsum of hands/feet, “perioral/peripalpebral and tongue myokymia,” hypertonia of lower limbs, absent DTR, weakness of limbs	Asymmetrical burst suppression pattern (age of 8 months)	Normal
(19) M 9 year Arabic	4 days	PLPBP	Fever-triggered GTS, GTCS Pharmacoresistant Vitamin B6- dependent	Developmental delay, intellectual difficulty, hyperactivity/autistic behavior, subtle dysmorphology, normal physical and neurological examination	Focal epileptic discharges	Normal

Abbreviations: ASD, antiseizure drug; CT, computed tomography; DTR, deep tendon reflex; EEG, electroencephalography; EMG, electromyography; EPC, epilepsia partialis continua; F, female; GCS, generalized clonic seizure; GDD, global developmental delay; GTS, generalized tonic seizure; GTCS, generalized tonic clonic seizure; KD, ketogenic diet; LBW, low birth weight; MRI, magnetic resonance imaging; NG, nasogastric; NA, not available; M, male; PHT, pulmonary hypertension; PLED, periodic lateralized epileptic discharge; SMA1, spinal muscular atrophy type 1; SE, status epilepticus; SW, spike wave; WI, weighted image; VNS, vagal nerve stimulation.

Note: “” information inside the quotation marks represent novel features (clinical, EEG or MRI) or rare features of some variants reported in few previous studies or cohorts.

Of the 19 patients, 18 had single nucleotide variant while one subject had a copy number variant (CNV), as seen in [Table 2]. Thirteen patients had known pathogenic/likely pathogenic variants. Two out of the three unrelated subjects with the SCN1B variant had the same exact variant in homozygous state and were unrelated, raising the possibility of an Iraqi founder variant, while the two subjects with the SCN1A variant had different variants.

Table 2 Molecular characterization in the 19 children with infantile-onset pharmaco-resistant epilepsy and diagnostic findings

Gene (transcript no.)	Variant	Zygosity	Known versus novel variant	Variant classification	Variant type	Mode of inheritance	# of affected children in the family
SCN1B (NM_199037.4) cES	c.449–2A > G; p.?	Homozygous	Known	P	SNV	AR	2 (the brother died)
SCN1B (NM_199037.4) EE panel	c.254G > A; p.(Arg85His)	Homozygous	Known	P	SNV	AR	3 (the 2 siblings died)
SCN1B (NM_199037.4)	c.254G > A; p.(Arg85His)	Homozygous	Known	P	SNV	AR	3 (the 2 siblings died)
SCN1A (NM_001165963.4) cES	c.777C > A; p.(Ser259Arg)	Heterozygous	Known	P	SNV	AD	1 (proband only)
SCN1A (NM_001165963.4) cES	c.1663–2A > C; p.?	Heterozygous	Known	LP	SNV	AD	1 (proband only)
ALG13 (NM_001040142.1) EE panel	c.320A > G; p.(Asn107Ser)	Heterozygous	Known	P	SNV	XLD	1 (proband only)
STXBP1 (NM_003165.3) EE panel	c.875G > T; p.(Arg292Leu))	Heterozygous (de novo)	Known	P	SNV	AD	1 (proband only)
PRUNE1 (NM_021222.2) cES	c.316G > A; p.(Asp106Asn)	Homozygous	Known	P	SNV	AR	1 (proband only)
ALDH7A1 (NM_001182.3) cES trio	c.1597del; p.(Ala533Profs*109)	Homozygous (parents carriers)	Known	P	SNV	AR	1 (proband only)
SCN8A (NM_014191.3) cES	c.4398C > G; p.(Asn1466Lys)	Heterozygous	Known	P	SNV	AD	1 (proband only)
SYNJ1 (NM_003895.3) cES	c.865C > T; p.(Arg289*)	Homozygous	Novel	LP	SNV	AR	1 (proband only)
TBC1D24 (NM_001199107.1) EE panel	c.866C > T; p.(Ala289Val)	Homozygous	Known	LP	SNV	AR	1 (proband only)
PCDH19 (NM_001184880.1) cES	c.838_839del; p.(Tyr280Argfs*39)	Heterozygous	Known	LP	SNV	XLD	1 (proband only)
SLC13A5 (NM_177550.3) cES	deletion chromosome 17 region: (6589531–6659473)	Homozygous	Known	LP	CNV	AR	2 (the brother died)
SLC25A22 (NM_001191060.1) cES	c.835G > A; p.(Glu279Lys)	Homozygous	Novel	VUS	SNV	AR	1 (proband only)
SCN2A )NM_001040142.1( EE panel	c.4913_4914delins AA; p.(Arg1638Gln)	Heterozygous	Novel	VUS	SNV	AD	1 (proband only)
KCNT1 (NM_020822.2) cES	c.2278A > C; p.(Ile760Leu)	Heterozygous (de novo)	Novel	VUS	SNV	AD	1 (proband only)
CACNA1A cES	c.7529_7532dup; p.(Trp2511fs)	Heterozygous	Novel	VUS	SNV	AD	1 (proband only)
PLPBP )NM_007198.3( cES	c.668T > C; p.(Met223Thr)	Homozygous Parents carriers	Novel	VUS	SNV	AR	1 (proband only)

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; cES, clinical exome sequencing; CNV, copy number variant; EE panel, epileptic encephalopathy panel; LP, likely pathogenic; P, pathogenic; SNV, single nucleotide variant; VUS, variant of unknown significance; XLD, X-liked dominant.

Six patients had novel variants, one of which was classified as likely pathogenic in SYNJ1 (n = 1). The other five patients had novel variants classified as variants of unknown significance (VUS) that most probably explain the clinical symptoms of the index patient and were identified in: SLC25A22 (n = 1), SCN2A (n = 1), KCNT1 (n = 1), CACNA1A (n = 1), and PLPBP (n = 1). The functional study of each variant has not been performed; yet, deep clinical characterization and phenotyping suggest these variants are responsible for the observed clinical features in the patients. [Table 2] shows detailed molecular findings (gene, variant, and classification of the variant and protein changes) in each of the 19 patients with a diagnostic molecular result. The three patients with nondiagnostic positive results had a VUS in a gene associated with a disease, including MLYCD (n = 1), CLN3 (n = 1), and PNKP (n = 1), that could not fully and tightly explain the clinical findings observed in the patient.

Autosomal recessive mode of inheritance was the most commonly reported type, 52.6% (10/19 children including 8 with pathogenic/likely pathogenic variants and 2 with VUS compatible with clinical phenotype), as shown in [Table 2].

[Table 2] reveals the type of genetic test performed in each patient. cES was used to test 19/29 patients, and an epileptic encephalopathy panel was used to test the remaining 10. Collectively, the diagnostic yield of the cohort was 65.5% combining both tests. The diagnostic rate of the epileptic encephalopathy panel was 50% (5/10) including 4/10 patients with pathogenic/likely pathogenic variants and 1/10 patients (10%) with clinically relevant VUS. The diagnostic rate of cES was higher at 74% (14/19) including 10/19 patients (53%) with pathogenic/likely pathogenic variants and 4/19 (21.1%) with clinically significant VUS.

Therapeutic changes were considered in 47% (9/19) of patients. Among those 15.7% (3/19) cases were potentially treatable inherited metabolic disorders due to biallelic homozygous deleterious variants in ALDH7A1, PLPBP, and SLC13A5. Treatment changes and impact are summarized in [Table 3].

Table 3 Treatment implications in 9 children with infantile-onset pharmaco-resistant epilepsy with diagnostic results

Gene	Variant classification	Treatment implication	Outcome
SCN1A	Likely pathogenic	Sodium valproate + clonazepam ± stiripentol[a]	• Marked reduction of seizures (Sodium valproate and clonazepam) with very infrequent breakthrough fever-triggered episodes  • > 50% reduction of seizure frequency with marked reduction of severity
PCDH19	Likely pathogenic	Ganaxolone	• Seizure freedom (ASMs: carbamazepine, topiramate, levetiracetam, and clobazam)  • Mild intellectual disabilities
ALDH7A1	Pathogenic	Pyridoxine + Lysine restricted diet[a]	• Seizure freedom  • Normal cognition and development
PLPBP	VUS (clinically relevant)	Pyridoxine[a]	• Seizure freedom  • Mild intellectual disabilities
SLC13A5	likely pathogenic	Ketogenic diet	• Her seizure was intractable to ketogenic diet, however as the child aged, seizure frequency decreases
SCN2A	VUS (clinically relevant)	Sodium channel blockers (carbamazepine, phenytoin)[a]	• Reduced seizure frequency
KCNT1	VUS (clinically relevant)	Quinidine	• Quinidine is not available. The child had dramatic reduction of seizure frequency by VNS
SCN8A	Pathogenic	Sodium channel blockers (carbamazepine, phenytoin)[a]	• > 50% reduction of seizure frequency
CACNA1A	VUS (clinically relevant)	Acetazolamide[a]	• Follow up was lost since the addition of acetazolamide

Abbreviations: ASM, antiseizure medication; VNS, vagal nerve stimulation; VUS, variant of unknown significance.

^a Treatment was available and applied to the patient.

 

MissingLinkText

Discussion

In this study, the overall diagnostic yield was 65.5% (19/29 case), among which 73.7% (14/19) were pathogenic/likely pathogenic variants and 26.3% (5/19) were clinically compatible VUS/novel variants.

The diagnostic yield was found to be higher than previous studies. Rim et al assessed 74 patients with epilepsy with seizure onset before 3 years of age by performing a customized NGS panel that included 172 genes and reported a diagnostic yield of 37.8%.[12] Møller et al used 46-gene NGS panel that was performed on 216 patients who had a range of different epilepsies and the study revealed a diagnostic yield of 23%.[13] Helbig et al conducted a study on large cohort of epilepsy patients (1,131 children and adults) using clinical exome trio that showed diagnostic yield of 33.4%.[14] Mercimek-Mahmutoglu et al found out that targeted NGS panels increased the genetic diagnostic yield from < 10 to > 25% in patients with epileptic encephalopathy.[15] Similar to our study, the previous studies included novel variants/VUS in their diagnostic rate calculation. The higher yield in the current study might be attributed to the cumulative impact of using different molecular tests, the population background, and the more homogenous and well-defined phenotypes of our cohort.

Myoclonic seizures were evident in children with epilepsies related to the following genes: SCN1B, SCN1A, SCN2A, STXBP1, ALG13, CACNA1A, TBC1D24, SLC25A22, and SYNJ1. While epileptic spasms were associated with SCN1B, ALG13, PRUNE1, SYNJ1, ALDH7A1, SCN2A, and SLC25A22. A study investigating a cohort of Chinese children with unexplained early infantile epileptic encephalopathy (EIEE) demonstrated higher prevalence of epileptic spasms (70.6%).[5] Another study that investigated a cohort of patients with intractable epilepsy, global developmental delay, and cognitive dysfunction showed that generalized tonic and tonic–clonic seizures were the two most common seizure types occurring in 50% of the patients, while myoclonic seizures were present in 25% of patients.[15]

Fever was found to be the main seizure-triggering factor in epilepsy related to SCN1A, SCN1B, PCDH19, and PLPBP genes. Fever-associated seizure is commonly reported in children harboring mutations in the cholinergic receptor nicotinic α 4 subunit (CHRNA4), in the voltage-gated sodium channel subunit genes (SCN1A, SCN2A, and SCN1B), and in the GABA(A) receptor subunit genes (GABRG2 and GABRD).[16]

Based on functional classification of the genes with the implicated variants, nearly half (47.4%) of the patients with positive diagnostic results had variants in genes encoding ion channels (SCN1B, SCN1A, SCN2A, SCN8A, KCNT1, and CACNA1A). This finding is comparable with previous study that reported by Blazekovic et al who retrospectively collected data from 277 idiopathic epilepsy cases aged 6 months to 17 years in which around a third of their molecularly diagnosed cases (34.38%) had genetic changes in genes encoding ion channels (CACNA1A, GABRA1, SCN1B, SCN5A, SCN1A, HCN4, KCNQ2, SCN9A, SCN2A, and SCN8A).[17] Nashabat et al identified 28/72 (38.9%) patients with EIEE and had molecular changes in genes responsible for ion channels.[8]

An evaluation and literature review of some of the molecular findings in the current cohort were provided.

A homozygous CNV (88.5 kb deletion) in SLC13A5 on Chr17p13.1 was found, which was previously reported in three siblings from an Iraqi family with developmental and epileptic encephalopathy.[18] They shared similar clinical manifestations to that exhibited in our patient. It is possible that this CNV is a founder variant in our Iraqi population, which results in loss of function, and therefore, was considered likely pathogenic.

A variant in STXBP1 (c.875G > T; p.(Arg292Leu)) was previously described in a child from a Chinese cohort, who exhibited a more severe form of epilepsy, in contrast to the less extensive epilepsy profile reported in our patient.[19] Additional genetic and environmental factors might contribute to the less severe epileptic profile in the present patient.

The variant (c.866C > T) in TBC1D24 was previously found in a patient with myoclonic seizures in a compound heterozygous state in trans with another nonsense variant (p.Gln207*; p.Ala289Val).[20] [21]

SYNJ1 patient exhibited dystonia/choreoathetosis, which was reported to be a key feature in the majority of patients with missense mutations in SYNJ1.[22] [23]

Genetic studies are highly recommended in children with Ohtahara syndrome.[24] An argument that was evidenced in our SCN2A patient, in whom the cooccurrence of symptoms like spasticity, microcephaly, and auditory-induced startle supports the variant's pathogenicity and clinical significance. Even though they are also present in other sodium channelopathies, such as SCN8A.[25] [26]

Clinical compatibility has been demonstrated for a VUS variant found in the KCNT1 gene. Previous studies have demonstrated that codon 760 mutations have deleterious effects and to be disease-causing.[27] [28] [29] [30] [31]

The current study reports myoclonic seizures and myokymia in a patient with VUS in CACNA1A, highlighting the clinical findings in the little existing literature.[32] [33]

The clinicoelectric symptoms of early myoclonic encephalopathy seen in SLC25A22 patient resembled those previously identified in small patient groups.[34] [35] [36] [37] [38] [39]

Identifying the genetic etiology of epilepsy is the first step toward practicing precision medicine and offering targeted treatment.[40] Therapeutic implications depend on the underlying pathophysiology and type of mutation identified. Common targeted therapies include supplementation of pyridoxine in pyridoxine-dependent epilepsy, channel-modifying treatment, that is, use or avoidance of sodium channel blockers in sodium channel mutations and use potassium channel opener in potassium channel mutations, diet modifications (such as use of ketogenic diet in GLUT1 deficiency), and mammalian target of rapamycin (mTOR) pathway regulations in mTOR pathway mutations.[40] Similar to our study, genetic testing in epilepsy and EOE in several previous studies has led to various therapeutic implications.[13] [15] Mercimek-Mahmutoglu et al found that 4.5% of the entire 110 patient cohort (25% of those with molecular diagnosis) had treatable inherited metabolic disorders.[15]

While 10 cases had either nondiagnostic genetic result (3/10 cases) or negative genetic testing (7/10 cases), genetic etiology is still suspected. In the seven cases with negative genetic testing, four underwent the epileptic encephalopathy panel while three had cES.

In this study, we found that cases with autosomal recessive inheritance (52.6%) outnumbered those with autosomal dominant inheritance (36.8%), a result that is expected in populations with high consanguinity rate. Similar to our study, Nashabat et al studied a cohort of EIEE from Saudi Arabia and found that autosomal recessive mode of inheritance accounted for more cases than the autosomal dominant inheritance (50 and 45.8%, respectively).[8] This is to the contrary of western countries with low rate of consanguinity in which studies of EOE/epileptic encephalopathies show higher prevalence of variants associated in autosomal dominant inheritance.[41]

The limitations in our present study include the small sample size, the inability to perform family-based (proband-parents trio) cES for all patients, the utilization of nonhomogeneous NGS testing modalities, and limited ability to deduce ancestry information from the exome data. Yet, despite these challenges listed above, a high molecular diagnostic yield was achieved, which guided diagnosis, management, and counseling in most of the cohort.

This study is the first report to portray the genotype–phenotype profile of Iraqi children with infantile-onset epilepsy that is pharmaco-resistant and with/without developmental delay. Our patient cohort exhibited genetic heterogeneity with predominance of autosomal recessive mode of inheritance. Precise diagnosis guided patient care toward appropriate treatment and accurate prognosis. We highlighted phenotypic expansion and phenotypic variability and reported many novel variants in genes known to cause EOEs. It is hoped that this study will fuel the conductance of future larger, multicenter studies in Iraq and the region on the genetics of epilepsy in children.

 

MissingLinkText

 

MissingLinkText

Conflict of Interest

None declared.

Acknowledgments

We would like to express our special thanks and gratitude to the parents of the patients who on one way or another shared their support and helped us in doing the research by providing their consent to publish the information and photos of the patients. We would like to show our gratitude to Professor Arndt Rolfs, Professor Peter Bauer, and the workers in the Centogene laboratory, Germany, for providing charitable facility for genetic analysis (cES and panel testing).

Authors' Contributions

N.W.S. and N.M. contributed to the conceptualization of the manuscript. N.W.S. and A.K.A. contributed to data collection. N.W.S. wrote the original draft. D.M. critically reviewed the manuscript.

• References

• 1 Zhang Y, Kong W, Gao Y. et al. Gene mutation analysis in 253 Chinese children with unexplained epilepsy and intellectual/developmental disabilities. PLoS One 2015; 10 (11) e0141782
• 2 Zuberi SM, Wirrell E, Yozawitz E. et al. ILAE classification and definition of epilepsy syndromes with onset in neonates and infants: position statement by the ILAE Task Force on Nosology and Definitions. Epilepsia 2022; 63 (06) 1349-1397
• 3 Berg AT, Coryell J, Saneto RP. et al. Early-life epilepsies and the emerging role of genetic testing. JAMA Pediatr 2017; 171 (09) 863-871
• 4 Calhoun JD, Carvill GL. Unravelling the genetic architecture of autosomal recessive epilepsy in the genomic era. J Neurogenet 2018; 32 (04) 295-312
• 5 Arafat A, Jing P, Ma Y. et al. Unexplained early infantile epileptic encephalopathy in Han Chinese children: next-generation sequencing and phenotype enriching. Sci Rep 2017; 7: 46227
• 6 Wang J, Wen Y, Zhang Q. et al. Gene mutational analysis in a cohort of Chinese children with unexplained epilepsy: Identification of a new KCND3 phenotype and novel genes causing Dravet syndrome. Seizure 2019; 66: 26-30
• 7 Scheffer IE, Liao J. Deciphering the concepts behind “Epileptic encephalopathy” and “Developmental and epileptic encephalopathy”. Eur J Paediatr Neurol 2020; 24: 11-14
• 8 Nashabat M, Al Qahtani XS, Almakdob S. et al. The landscape of early infantile epileptic encephalopathy in a consanguineous population. Seizure 2019; 69: 154-172
• 9 Idris A, Alabdaljabar MS, Almiro A. et al. Prevalence, incidence, and risk factors of epilepsy in Arab countries: a systematic review. Seizure 2021; 92: 40-50
• 10 Scheffer IE, Berkovic S, Capovilla G. et al. ILAE classification of the epilepsies: position paper of the ILAE Commission for Classification and Terminology. Epilepsia 2017; 58 (04) 512-521
• 11 Fisher RS, Cross JH, D'Souza C. et al. Instruction manual for the ILAE 2017 operational classification of seizure types. Epilepsia 2017; 58 (04) 531-542
• 12 Rim JH, Kim SH, Hwang IS. et al. Efficient strategy for the molecular diagnosis of intractable early-onset epilepsy using targeted gene sequencing. BMC Med Genomics 2018; 11 (01) 6
• 13 Møller RS, Larsen LH, Johannesen KM. et al. Gene panel testing in epileptic encephalopathies and familial epilepsies. Mol Syndromol 2016; 7 (04) 210-219
• 14 Helbig KL, Farwell Hagman KD, Shinde DN. et al. Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy. Genet Med 2016; 18 (09) 898-905
• 15 Mercimek-Mahmutoglu S, Patel J, Cordeiro D. et al. Diagnostic yield of genetic testing in epileptic encephalopathy in childhood. Epilepsia 2015; 56 (05) 707-716
• 16 Nakayama J. Progress in searching for the febrile seizure susceptibility genes. Brain Dev 2009; 31 (05) 359-365
• 17 Blazekovic A, Gotovac Jercic K, Meglaj S. et al. Genetics of pediatric epilepsy: next-generation sequencing in clinical practice. Genes (Basel) 2022; 13 (08) 1466
• 18 Duan R, Saadi NW, Grochowski CM. et al. A novel homozygous SLC13A5 whole-gene deletion generated by Alu/Alu-mediated rearrangement in an Iraqi family with epileptic encephalopathy. Am J Med Genet A 2021; 185 (07) 1972-1980
• 19 Li T, Cheng M, Wang J. et al. De novo mutations of STXBP1 in Chinese children with early onset epileptic encephalopathy. Genes Brain Behav 2018; 17 (08) e12492
• 20 Shang KW, Zhang YH, Yang XL. et al. Clinical features and gene mutations in epilepsy of infancy with migrating focal seizures [in Chinese]. Zhonghua Er Ke Za Zhi 2016; 54 (10) 735-739
• 21 Poulat AL, Ville D, de Bellescize J. et al. Homozygous TBC1D24 mutation in two siblings with familial infantile myoclonic epilepsy (FIME) and moderate intellectual disability. Epilepsy Res 2015; 111: 72-77
• 22 Samanta D, Arya K. Electroclinical findings of SYNJ1 epileptic encephalopathy. J Pediatr Neurosci 2020; 15 (01) 29-33
• 23 Al Zaabi N, Al Menhali N, Al-Jasmi F. SYNJ1 gene associated with neonatal onset of neurodegenerative disorder and intractable seizure. Mol Genet Genomic Med 2018; 6 (01) 109-113
• 24 Pisani F, Percesepe A, Spagnoli C. Genetic diagnosis in neonatal-onset epilepsies: back to the future. Eur J Paediatr Neurol 2018; 22 (03) 354-357
• 25 Wolff M, Brunklaus A, Zuberi SM. Phenotypic spectrum and genetics of SCN2A-related disorders, treatment options, and outcomes in epilepsy and beyond. Epilepsia 2019; 60 (Suppl 03): S59-S67
• 26 Gardella E, Marini C, Trivisano M. et al. The phenotype of SCN8A developmental and epileptic encephalopathy. Neurology 2018; 91 (12) e1112-e1124
• 27 Barcia G, Fleming MR, Deligniere A. et al. De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy. Nat Genet 2012; 44 (11) 1255-1259
• 28 Heron SE, Smith KR, Bahlo M. et al. Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 2012; 44 (11) 1188-1190
• 29 McTague A, Appleton R, Avula S. et al. Migrating partial seizures of infancy: expansion of the electroclinical, radiological and pathological disease spectrum. Brain 2013; 136 (Pt 5): 1578-1591
• 30 McTague A, Nair U, Malhotra S. et al. Clinical and molecular characterization of KCNT1-related severe early-onset epilepsy. Neurology 2018; 90 (01) e55-e66
• 31 Martin HC, Kim GE, Pagnamenta AT. et al; WGS500 Consortium. Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis. Hum Mol Genet 2014; 23 (12) 3200-3211
• 32 Epi4K Consortium. De novo mutations in SLC1A2 and CACNA1A are important causes of epileptic encephalopathies. Am J Hum Genet 2016; 99 (02) 287-298
• 33 Hu Y, Jiang H, Wang Q, Xie Z, Pan S. Identification of a novel nonsense mutation p.Tyr1957Ter of CACNA1A in a Chinese family with episodic ataxia 2. PLoS One 2013; 8 (02) e56362
• 34 Molinari F, Kaminska A, Fiermonte G. et al. Mutations in the mitochondrial glutamate carrier SLC25A22 in neonatal epileptic encephalopathy with suppression bursts. Clin Genet 2009; 76 (02) 188-194
• 35 Lemattre C, Imbert-Bouteille M, Gatinois V. et al. Report on three additional patients and genotype-phenotype correlation in SLC25A22-related disorders group. Eur J Hum Genet 2019; 27 (11) 1692-1700
• 36 Molinari F, Raas-Rothschild A, Rio M. et al. Impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy. Am J Hum Genet 2005; 76 (02) 334-339
• 37 Cohen R, Basel-Vanagaite L, Goldberg-Stern H. et al. Two siblings with early infantile myoclonic encephalopathy due to mutation in the gene encoding mitochondrial glutamate/H+ symporter SLC25A22. Eur J Paediatr Neurol 2014; 18 (06) 801-805
• 38 Poduri A, Heinzen EL, Chitsazzadeh V. et al. SLC25A22 is a novel gene for migrating partial seizures in infancy. Ann Neurol 2013; 74 (06) 873-882
• 39 Giacomini T, Pisciotta L, Prato G. et al. Severe early-onset developmental and epileptic encephalopathy (DEE) associated with novel compound heterozygous pathogenic variants in SLC25A22: Case report and literature review. Seizure 2019; 70: 56-58
• 40 Striano P, Minassian BA. From genetic testing to precision medicine in epilepsy. Neurotherapeutics 2020; 17 (02) 609-615
• 41 D'Gama AM, Mulhern S, Sheidley BR. et al; Gene-STEPS Study Group, IPCHiP Executive Committee. Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study. Lancet Neurol 2023; 22 (09) 812-825

Publication History

Received: 26 December 2024

Accepted: 06 June 2025

Article published online:
11 August 2025