Genetic and phenotypic landscape of pediatric-onset epilepsy in 142 Indian families: Counseling and therapeutic implications

The application of genomic technologies has led to unraveling of the complex genetic landscape of disorders of epilepsy, gaining insights into their underlying disease mechanisms, aiding precision medicine, and providing informed genetic counseling. We herein present the phenotypic and genotypic insights from 142 Indian families with epilepsy with or without comorbidities. Based on the electroclinical findings, epilepsy syndrome diagnosis could be made in 44% (63/142) of the families adopting the latest proposal for the classification by the ILAE task force (2022). Of these, 95% (60/63) of the families exhibited syndromes with developmental epileptic encephalopathy or progressive neurological deterioration. A definitive molecular diagnosis was achieved in 74 of 142 (52%) families. Infantile-onset epilepsy was noted in 81% of these families (61/74). Fifty-five monogenic, four chromosomal, and one imprinting disorder were identified in 74 families. The genetic variants included 65 (96%) single-nucleotide variants/small insertion-deletions, 1 (2%) copy-number variant, and 1 (2%) triplet-repeat expansion in 53 epilepsy-associated genes causing monogenic disorders. Of these, 35 (52%) variants were novel. Therapeutic implications were noted in 51% of families (38/74) with definitive diagnosis. Forty-one out of 66 families with monogenic disorders exhibited autosomal recessive and inherited autosomal dominant disorders with high risk of recurrence.


| INTRODUCTION
Epilepsy is one of the most common neurological disorders which can be present in isolation or can be associated with other comorbidities such as global developmental delay (GDD), intellectual disability (ID), autism spectrum disorder (ASD), and/or behavioral abnormalities.[4] With the advent of next-generation sequencing techniques, a plethora of epilepsy-associated genes and disease-causing variants have been identified in association with epileptic disorders.High genotypic and phenotypic heterogeneity observed among these epileptic disorders substantiates the use of exome or genome sequencing (ES/GS) as a first-tier genetic test in establishing a definitive molecular diagnosis. 5,69][10][11] Identification of precise genetic etiology further facilitates tailored precision medicine, and targeted genetic counseling including prognosis and recurrence risk.
Numerous studies have highlighted the significance of elucidating the genetic basis of epilepsy across diverse populations.Large-scale cohort studies play a pivotal role in understanding the diverse phenotypic and genotypic landscape of epilepsies, offering crucial insights into the effectiveness of genomic tests, especially in resource-limited settings.The distinct genetic makeup, environmental factors, possible genetic variations unique to the population, and resource constraints in India emphasize the need for a comprehensive study to assess the genetic burden of epilepsy.We hereby present the phenotypic and genotypic spectrum of epilepsies from 142 Indian families with an emphasis on the utility of sequential genomic testing for achieving genetic diagnosis, selecting optimal treatment, and assisting genetic counseling.

| Subject recruitment
We ascertained and recruited individuals presenting with epilepsy with/without comorbidities from October 2019 till June 2023 as a part of an ongoing study.The affected individuals recruited for the study were inpatients as well as outpatients from either genetic or pediatric or pediatric neurology clinics.The diagnosis of epilepsy was based on the current definition of International League Against Epilepsy (ILAE). 1 Individuals with epilepsy due to acquired causes (stroke, trauma, tumors, neonatal hypoxia, infections) were excluded from the study.3][14][15][16] Informed consents for genetic testing and publication of data were obtained from the families.The informed consents were approved by the institutional ethics committee, Kasturba Medical College, and Kasturba Hospital, India as per the declaration of Helsinki.

| Genetic testing
Genomic DNA was extracted from the peripheral blood sample of the proband, parents and siblings (as required) using the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA; cat # 51106).The testing strategy included either an exome first or a sequential testing approach in which targeted tests such as fragile-X screening, TP-PCR, Methylation-specific MLPA (MS-MLPA), targeted gene Sanger sequencing or chromosomal microarray (CMA) was followed by ES for the affected individuals based on the clinical phenotype (Figure 1).
The disease-causing variants identified in the affected individuals were further validated and segregated in the families using Sanger sequencing.Copy-number variant (CNV) analysis from exome data was performed for individuals in whom no clinically relevant singlenucleotide variants (SNVs) or insertion/deletions (indels) were detected on ES.The CNVs identified using ES data were further validated using CMA, or MS-MLPA.The detailed description of the genetic tests employed along with description of CNV and ES analysis is provided in Data S1.

| Therapeutic implications and impact on genetic counseling
After achieving molecular diagnosis, literature review was done to ascertain if any anti-epileptic drugs (AED) and/or any specific therapy is recommended or contraindicated.The following search terms were used in PubMed to find the publications: "seizure + gene name + treatment," "epilepsy + gene name + treatment," and "specific gene name + treatment."The evidence was classified as "strong" when treatment guidelines recommended the use of a particular AED or treatment, as "emerging" when multiple publications supported its use and as "sparse" when benefit was shown only in a single report.
The impact of definitive diagnosis on genetic counseling was assessed by analyzing its influence on recurrence risk and identifying the number of families that could benefit from targeted prenatal diagnosis or preimplantation genetic diagnosis and thus prevention of untreatable disorders.

| RESULTS
We ascertained a total of 161 affected individuals from 152 unrelated families with epilepsy with or without any comorbidities.[19][20][21][22][23][24][25][26] The current cohort consists of 149 individuals from 142 families with epilepsy and their demographics, clinical, and molecular details have been shown in Table 1.Of these, 88 (59%) are males and 61 (41%) are females.Consanguinity was noted in 46 (32%) families.isolated/familial epilepsy was observed in only five individuals (4%) while epilepsy with additional comorbidities was observed in most (144 individuals, 96%).These comorbidities included GDD, ID, tone abnormalities, movement abnormalities, behavioral abnormalities, ASD, dysmorphism, and eye abnormalities (Figure 2A).Sixty-three of the 142 families (44%) could be classified into one of the epilepsy syndromes.Forty-eight families (76%, n = 63) had a diagnosis of neonatal and infantile-onset syndromes.Of these, three were defined as genetic epilepsy with febrile seizure plus (GEFS+), 24 as infantile epileptic spasm syndrome (IESS), 11 as early-infantile developmental and/or epileptic encephalopathy (EI-DEE), four as epilepsy of infancy with migrating focal seizures (EIMFS), three as Dravet syndrome, one as progressive myoclonic epilepsy (PME), and two with etiology-specific DEE.Among the 15 families with childhood onset syndromes (24%, n = 63), one was diagnosed with epilepsy with myoclonic-atonic seizures (EMAS), three with Lennox-Gastaut syndrome (LGS), five with EE-SWAS, and six with PME.The spectrum of epilepsy syndromes by age of onset with its corresponding molecular diagnosis is described in detail in Table 2.
A definitive molecular diagnosis was achieved in 74 of 142 families (52%) using either a single or sequential testing involving a targeted test, CMA, Mendeliome and/or ES.The details of these testing strategies and diagnostic yield are depicted in Figure 1.A total of 60 genetic disorders were identified in 74 families with epilepsy.
These included 55 monogenic disorders in 66 families, four chromosomal disorders in four families, and one imprinting disorder in four families.Out of the 55 monogenic disorders, 33 were autosomal recessive (AR), 19 were autosomal dominant, and 3 were X-linked dominant disorders.
A total of 67 causative variants were identified in 66 families with monogenic disorders, of which 65 (96%) were SNVs/indels, 1 (2%) was CNV, and one (2%) was triplet-repeat expansion.The type of variants observed in the cohort have been illustrated in Figure 2B.Thirtyfive of the 67 causative variants (52%) were noted to be novel.All these variants have been submitted to the ClinVar database.According to the standards and guidelines for the interpretation of sequence LGS, Lennox-Gastaut syndrome; PME, progressive myoclonic epilepsy.
Among the 66 families with monogenic epilepsy, 37 families har- The landscape of epilepsy genetics is broadening due to advancements in cutting-edge genomic methodologies which has led to the identification of over 800 genes associated with epilepsy within the last 20 years.This substantial discovery has significantly contributed to the extensive genetic diversity observed in epileptic disorders. 28,29In this study, we present a systematic analysis of 142 Indian families, revealing the phenotypic and genotypic spectrum of epilepsy and its implications for counseling and therapy.
We applied the latest ILAE 2022 diagnostic criteria for the electroclinical syndromic classification of our patient cohort.Epilepsy syndromes exhibit a wide electroclinical variability, spanning from focal epilepsy syndrome (FES) to syndromes involving DEE or with progressive neurological deterioration. 1,14In our study, majority (93%) of the families with the epilepsy syndrome classification fell within the DEE or neurological deterioration spectrum.Of these, IESS was most observed in 38% (24/63) of the families which is aligning with previously published cohorts with 22% and 21% of families with IESS. 8,30is was followed by EI-DEE in 18% ( 11 Genetic disorders with epilepsy encompass a spectrum of disorders ranging from monogenic disorders, chromosomal disorders, triplet-repeat disorders, and other multifactorial disorders.The most frequently occurring monogenic disorders in pediatric onsetepilepsy cohorts include channelopathies (e.g., SCN1A/SCN2Arelated disorder, KCNQ2-related disorder), followed by metabolic conditions (e.g., SLC2A1-related GLUT1 deficiency syndrome; 2][33] A study by Boonsimma et al. (2022) showed that channelopathies comprised of the majority (53%) of the monogenic causes in infantile-onset epilepsies followed by neurometabolic causes (15%), and other rare genetic disorders with epilepsy (32%). 6In contrast, the findings of the current study show that channelopathies and metabolic disorders represent only 16% and 15% of the 55 monogenic disorders, and the majority (69%) being other rare genetic disorders with epilepsy (Table 1).The broader range of epi- T A B L E 3 Gene-specific therapeutic implications observed in the cohort.

Proband ID Gene
Anti Though disorders with all inheritance patterns are associated with epilepsy, de novo variants causing dominant disorders constitute the most common group of genetic conditions identified in these individuals.Previous studies have shown that 50%-70% of the disease-causing variants occur de novo causing autosomal or X-linked dominant disorders. 6,11,33However, in the current study, it is observed that only 33% of the causative variants occurred de novo and 53% of the variants were identified in families with autosomal recessive disorders.This high rate of autosomal recessive disorders could be attributed to the prevalence of consanguinity and inbreeding in specific communities and geographic regions of India. 34,35e diagnostic yield of genetic testing relies significantly on factors such as age of seizure onset, presence of additional comorbidities, and the type of testing employed in individuals with epilepsy. 5,8,32,36e high diagnostic yield observed in our cohort can be attributed primarily to the 80% of affected individuals with seizure onset <2 years of age, and the presence of comorbidities in 94% of the individuals.
Recent studies have reported cohorts of early-onset epilepsy with increased diagnostic yield ranging from 50% to 60% within the first year of life. 6,8,11A study by Zou et al. 11 reported 117 of 320 affected individuals with definitive diagnosis.Of these, 74% of individuals with genetic etiology had seizure onset within the first year of life. 11In the current study, we found that 94% of individuals (70/74) with genetic etiology had infantile-onset epilepsy (<2 years), while 4% (4/74) had childhood-onset epilepsy (>2 years).These findings align with previously reported findings, reinforcing the association between earlyonset epilepsy and a high diagnostic yield. 8,11 have used targeted or genomic testing first or sequential testing approach in the current study which led to an overall diagnosis of 52% in the cohort.The choice of the genetic testing employed for an individual was based on the clinical diagnosis, targeted region of interest, and the potential variant characteristics.This strategy of testing was limited to the choice of tests in order to make optimal use of the resources.We, therefore, emphasize that the clinical diagnosis and the appropriate tests employed after deep phenotyping are a rational approach for both a high yield of molecular diagnosis as well as optimal use of resources.
Advanced NGS techniques, such as exome or genome sequencing as well as epilepsy-focused gene panels have become an invaluable tool for elucidating the genetic underpinnings of epilepsies.Familybased or a trio-ES is often the preferred choice of testing for the ease of identification of de novo variants in disorders with epilepsy.Previous studies have demonstrated varied diagnostic yields for various testing modalities, that is, epilepsy-focused gene panels yielding between 15% and 47%, 32,36,37 WES between 30% and 64%, 6,8,38,39 and GS achieving a diagnostic rate ranging from 36% to 43%. 11,33In the current study, we observed a diagnostic yield of 68% and 45% Identification of precise genetic etiology has significantly advanced precision medicine in epileptic disorders.Till date, the management of epilepsies heavily relies on use of conventional AEDs.
However, several recent studies have highlighted the benefits of using a targeted treatment approach based on the underlying etiology to reduce seizure frequency and improve developmental outcomes. 11,36 the current study, treatment implications have been noted in 51% of individuals with a definitive diagnosis, which is comparable to the previously reported studies 30%-55%. 6,33,36Based on this, immediate actionable treatment for biochemical disorders was available for eight families with variants in ALDH7A1, GCDH, TPP1, GCH1, and PDHA1, and AED modifications for five families with variants in SCN1A, KCNQ2, and PRRT2.We have also listed the usage or avoidance of certain AEDs for which the evidence is emerging, or sparse, and further studies or additional reports are needed to prove its efficacy (Table 2).Consequently, treatment intervention early in childhood has a great potential to improve the prognosis and developmental outcomes of the epilepsy-affected individuals.In conclusion, the spectrum of epileptic disorders is expanding with a rapid rate driven by technological advances and gene discovery.Therefore, the integration of early genetic testing and deep phenotyping of individuals with epilepsy in healthcare settings will help establish accurate diagnosis and has potential benefits in terms of reducing the economic burden by preventing additional investigations.
We further demonstrate the feasibility of using singleton ES as a firsttier test in a resource-limited setting to facilitate early genetic diagnosis, genetic counseling, and precision medicine.Comprehensive and systematic studies across diverse populations are likely to contribute to the increasing pool of disease-causing variants and epileptic disorders globally which will further help us understand the complete genetic landscape of epilepsy.
The age at examination ranged from the newborn period to 21 years with a median age of 3 years.The age of seizure onset ranged from day 1 to 15 years with a median age of 7 months.It was observed that 79% (118/149; 115 families) of the affected individuals had infantile-onset epilepsy (<2 years), and 21% (31/149; 27 families) had childhood-onset epilepsy (>2 years).Of these 149 individuals, F I G U R E 1 Flowchart depicting the genetic testing strategy employed to achieve molecular diagnosis in 142 families with epilepsies.CMA, chromosomal microarray; ES, exome sequencing; MLPA, multiplex ligation-dependent probe amplification; MS-MLPA, methylation-specific MLPA; TP-PCR, triplet repeat primed PCR.[Colour figure can be viewed at wileyonlinelibrary.com] mutated genes were PRRT2 (four families), TPP1 (three families), STXBP1 (three families), SCN1A (two families), ALG11 (two families), MECP2 (two families), CLN6 (two families), CACNA1A (two families), and KCNT1 (two families).Variants in remaining 44 epilepsy-associated genes were observed in one family each.These genes were further classified into 10 gene ontology categories and are represented in Figure 2C.Additional details pertaining to the clinical features, EEG findings, genetic testing performed, disease-causing variants, and ClinVar submission IDs have been provided in Tables bored variants causing autosomal recessive disorders carrying 25% risk of recurrence.Additionally, there were five families with inherited variants causing autosomal dominant disorders posing a 50% risk of recurrence in other family members.Another 22 families with de novo variants causing autosomal/X-linked dominant disorders have a negligible (<1%) risk of recurrence.F I G U R E 2 (A) Bar graph of the clinical features observed in addition to epilepsy in 144 affected individuals.ASD, autism spectrum disorder; GDD, global developmental delay; ID, intellectual disability; (B) Types of 65 single-nucleotide variants/insertion-deletions observed in the cohort (C) Schematic representation of the functional category of 53 epilepsy-associated genes observed in the cohort.[Colour figure can be viewed at wileyonlinelibrary.com]T A B L E 2 Epilepsy syndrome classification and molecular diagnosis observed in the cohort (n = 142 families).
/63), and PME in 13%(8/63)   of the families in the current cohort.The ILAE 2022 classification has introduced a new category of etiology-specific syndromes which currently encompasses only six genetic conditions.However, this category is likely to expand with increasing genetic testing and consequent knowledge expansion.Hence, many of the genetic etiologies identified in the cohort which currently could not be classified into electroclinical syndromes, such as affected individuals with KCNT1, CACNA1A, PUM1, FGF12, AP3B2, PRRT2, GRIN2A, and Angelman syndrome may be classified as etiology-specific syndromes in the future.
Abbreviations: AD, autosomal dominant; AR, autosomal recessive; DEE, developmental and/or epileptic encephalopathy; F, Female; ID, identifier; M, Male; NA, not applicable; VUS, variant of uncertain significance; XLD, X-linked dominant; Y, years.a Novel variants not reported in the literature.b Paternal sample is not available for segregation.
It is noteworthy that, majority of the individuals in the current cohort present with additional comorbidities which makes the treatment more challenging.Further insights into the underlying genetic mechanisms and cellular pathways in future are likely to pay the way for development of novel therapeutic strategies for management of epilepsy.There are a few limitations of the study.The current cohort consists of majority of individuals who have epilepsy with additional comorbidities.This may have resulted in higher diagnostic yield of genetic testing when compared to the cohorts of individuals with isolated epilepsy.Despite the application of state-of-the-art genetic techniques, 50% of the families in the current cohort remained undiagnosed.Application of trio ES, genome sequencing, long-read sequencing, and additional-omics approach can be further employed to identify other novel causative genes, nonexonic/structural variants, somatic alterations, epimutations, and digenic or oligogenic etiologies.Reanalysis of exome data too may increase the diagnostic yield based on the updated new information in the literature.In addition, though the study highlights the implications of genetic testing on treatment, it lacks longitudinal clinical outcomes, hindering insights into precision medicine's efficacy for epilepsy.
AUTHOR CONTRIBUTIONS PM contributed to data collection, analysis of the exome sequencing data, Sanger validation, reporting the variant and drafting the manuscript, preparing figures and tables.LPR, SM, NK, SP has contributed to analyzing the exome sequencing data and validation of the results.DN, VB has contributed to clinical assessment, analysis, interpretation of genomic data and genetic counseling of the recruited families.SN contributed to the analysis and reporting of chromosomal microarray data.KN, AP, AC, BH, NJ contributed to operating and modulating the in-house exome sequencing and copy number variant analysis pipeline and analysis of the data.SF, MY, VK, contributed to the collection of clinical history of the affected patients.RN and VB contributed to the Sanger validation of the identified genomic variants.SA, LL, JP, RBY, PBK, YBL, SJP, S Nampoothiri, NK, have referred families and contributed to patient evaluation and genetic counseling.S Siddiqui has contributed to radiodiagnosis.KMG has contributed to funding acquisition, patient evaluation, study recruitment, analysis, interpretation of the genomic testing, and genetic counseling to the families.S Bielas has contributed to funding acquisition and planning of additional wet lab experiments.AS and SS have contributed to funding acquisition, conceptualization, patient evaluation, study recruitment, and conceptualizing the manuscript and overall supervision.All authors have read and approved the final version of the manuscript.
T A B L E 1 Cohort characteristics of families with epilepsy.
(Continues)T A B L E 1 (Continued)