Genetic Epilepsies and Developmental Epileptic Encephalopathies with Early Onset: A Multicenter Study

The genetic causes of epilepsies and developmental and epileptic encephalopathies (DEE) with onset in early childhood are increasingly recognized. Their outcomes vary from benign to severe disability. In this paper, we wished to retrospectively review the clinical, genetic, EEG, neuroimaging, and outcome data of patients experiencing the onset of epilepsy in the first three years of life, diagnosed and followed up in four Italian epilepsy centres (Epilepsy Centre of San Paolo University Hospital in Milan, Child Neurology and Psychiatry Unit of AUSL-IRCCS di Reggio Emilia, Pediatric Neurology Unit of Vittore Buzzi Children’s Hospital, Milan, and Child Neurology and Psychiatry Unit, IRCCS Mondino Foundation, Pavia). We included 168 patients (104 with monogenic conditions, 45 with copy number variations (CNVs) or chromosomal abnormalities, and 19 with variants of unknown significance), who had been followed up for a mean of 14.75 years. We found a high occurrence of generalized seizures at onset, drug resistance, abnormal neurological examination, global developmental delay and intellectual disability, and behavioural and psychiatric comorbidities. We also documented differing presentations between monogenic issues versus CNVs and chromosomal conditions, as well as atypical/rare phenotypes. Genetic early-childhood-onset epilepsies and DEE show a very wide phenotypic and genotypic spectrum, with a high risk of complex neurological and neuropsychiatric phenotypes.


Introduction
Epilepsy is the most frequently occurring neurological disease in the pediatric age range.Onset in early childhood is common.In 2017, the International League Against Epilepsy (ILAE) proposed a revision of previous classifications for seizures and epilepsy.This latest classification incorporates six possible aetiologic categories into a taxonomy: genetic, structural (which may be congenital or acquired), metabolic, immune, infectious, and unknown [1].
On a clinical basis, genetic epilepsies can be distinguished into generalized genetic epilepsies, focal genetic epilepsies, and developmental and epileptic encephalopathies (DEE) [1].
Technological developments, in particular with the availability of next-generation sequencing (NGS) techniques, have brought exome (ES) and genome (GS) sequencing into clinics, allowing the identification of a growing number of genes linked to epilepsy.Nowadays, more than 500 epilepsy-linked genes have been identified [6].
The aim of this multicentre study was to analyze a cohort of patients with genetic epilepsies or DEE with onset in the first three years of life.In particular, the primary objective of this study was to report on a detailed phenotypic description, starting from genotypes, including clinical, EEG, neuroimaging, and seizure outcome data.

Results
In total, we enrolled 168 patients (97 females and 71 males) with a range of genetic epilepsies and or developmental and epileptic encephalopathies with onset within the first 3 years of age.
Mean age at epilepsy onset was 11 months (range 0-36 months) and mean age at last follow-up was 177 months (range 0-672 months).
Several variants were reported in additional genes encoding proteins with different cell functions (Table 1), of which 28 were identified in single patients (Figure 1).

Clinical Findings Family History
In most patients (63/104, 60%), family history was negative for neurological diseases.The remaining patients (41/104, 40%) had a positive family history for neurological or neurodevelopmental disorders.

Clinical Findings Family History
In most patients (63/104, 60%), family history was negative for neurological diseases.The remaining patients (41/104, 40%) had a positive family history for neurological or neurodevelopmental disorders.

Seizure Outcome at the Latest Follow-Up Visit
At the latest follow-up visit, approximately half (54/104, 52%) of the patients had drug-resistant seizures.For two patients, data on outcomes were not available, while other patients (48/104, 46%) were seizure-free.Among seizure-free patients, 8/48 (16.7%) were not on regular antiseizure medications.
EEG Pattern at the Latest Follow-Up Visit EEG at latest follow-up visit revealed the poor organization of background activities, with frequent multifocal or generalized discharges in 32 patients (31%), focal epileptiform discharges in 12 patients (12%), multifocal discharges in 7 patients (7%), and diffuse discharges in 7 patients (8%).Excess slow activity with or without interictal epileptiform discharges was seen in 11 cases (11%) and there was a DEE-SWAS pattern in three patients (3%).One patient (1%) presented a Lennox-Gastaut pattern.In 25 patients (24%), EEG was normal.For the remaining five patients (5%), data on EEG at the latest follow-up visit were not available for review.
By comparing EEG features at onset and at the latest follow-up, we documented an improvement in 14 patients (13.5%) and a worsening in 10 (9.6%), while EEG findings were stable in 61 patients (58.6%).We were unable to comment on the evolution of EEG patterns in 19 cases (18.3%) because either the first or the last EEG (or both) were unavailable for review.

Clinical Findings Family History
In the majority of patients (32/45, 71%), family history was negative for neurological diseases.A small number of patients (12/45, 26%) had positive family history for neurological diseases (i.e., epilepsy, febrile seizures) or neurodevelopmental disorders (autism spectrum disorder, intellectual disability, and speech delay).Family history was not available for one adopted child.

Clinical Findings Family History
In the majority of patients (32/45, 71%), family history was negative for neurological diseases.A small number of patients (12/45, 26%) had positive family history for neurological diseases (i.e., epilepsy, febrile seizures) or neurodevelopmental disorders (autism spectrum disorder, intellectual disability, and speech delay).Family history was not available for one adopted child.

Neurological Examination
In most patients within this group (39/45, 87%), neurological examination results were abnormal.Gait abnormalities and abnormal muscle tone were the most common neurological signs.Detailed characteristics (when available) are depicted in Table 5.  4).

Neuroimaging Findings
More than half of the patients in this group had brain MRI abnormalities (25/45, 55% with 21 showing brain malformation and four progressive changes).In 13/45 (29%) patients, brain MRI was unremarkable, while in 7 (16%) patients' neuroimaging data were not available.A detailed description of neuroimaging findings is reported in Table 6.Neuroimaging Findings More than half of the patients in this group had brain MRI abnormalities (25/45, 55%, with 21 showing brain malformation and four progressive changes).In 13/45 (29%) patients, brain MRI was unremarkable, while in 7 (16%) patients' neuroimaging data were not available.A detailed description of neuroimaging findings is reported in Table 6.
NGS gene panel led to a diagnosis for three patients [one patient 46,XX,del(4p16.3),and two patients with Angelman syndrome, the first carrying the c. 1347_1348delGA (p.Asn450Glnfs*23) variation on the UBE3A gene and the second a 46,XX,del(15)(q11q13)], while direct gene sequencing (UBE3A gene) was diagnostic for two patients.

Seizure Outcome
At the latest follow-up visit, more than half of the patients (26/45, 58%) were seizure-free and among these only two [one with Down's syndrome and one with 46,XY,del(16p11.2)]were not on medication.The remaining patients (19/45, 42%) were drug-resistant.
EEG Pattern at the Latest Follow-Up Visit EEG at the end of follow-up revealed the poor organization of background activity, with frequent multifocal or generalized discharges in 20 patients (44%), diffuse interictal epileptiform discharges in seven patients (16%), focal or multifocal interictal epileptiform discharges in five patients (11%), and an excess of slow activities with or without interictal discharges in three cases (7%).In seven patients (15%), EEG was normal.
For the remaining three patients (7%), data on EEG at latest follow-up visit were not available.
By comparing EEG features at onset and at the latest follow-up, we documented an improvement in five patients (11.1%) and a worsening in four (8.9%), while EEG findings were stable in 18 patients (40%).We are unable to comment on the evolution of EEG patterns in 18 cases (40%) because either the first or the last EEG (or both) were unavailable for review.

Clinical Findings Family History
The majority of patients (11/19, 57.9%) had no family history of neurological diseases.However, more than one-third (7/19, 36.8%) had a positive family history.Family history was not available in one.

Epilepsy
The mean age at epilepsy onset was 17.1 months (range: 0-36 months).The main seizure type at onset is generalized (8/19, 42.1%).Among patients with generalized seizures at onset, four (4/8, 50%) had tonic-clonic seizures, two had absence seizures (typical in one case and atypical in one), one had myoclonic seizures, and one myoclonic-atonic seizures.Six ( Eight (8/19, 42.1%) patients had behavioural and/or psychiatric comorbidities, including aggressive behavior, ASD or autistic traits, and there was inattention in two patients.Obsessive traits, hyperactivity, ideomotor slowing, and irritability were present in one patient each.These impairments were combined in two cases.

Seizure Outcome
Nine (9/19, 47.4%) patients were drug-resistant.Six were seizure-free on therapy, one was seizure-free without therapy, while for three seizure-free patients the information regarding whether they were or not on medications was not available.Drug-resistant patients mainly experienced focal motor seizures (5/19, 31.6%), while generalized seizures (absences in one, tonic-clonic seizures in one) were less represented (2/19, 10.5%).One patient with Lennox-Gastaut syndrome experienced multiple seizure types (atypical absences, tonic and focal motor seizures).The seizure type at the latest follow-up visit was not reported in one patient.

Discussion
We are reporting on a retrospective multicenter Italian cohort of patients that had their onset of genetic epilepsies or developmental and epileptic encephalopathies within the first three years of life.We aimed to better define their electroclinical, neuroimaging, and genetic profiles, their epilepsy outcomes at the end of the follow-up, and the occurrence of neurodevelopmental and psychiatric comorbidities.
Although our study design included patients with epilepsy onset within 36 months of age, the mean age of onset is significantly lower (11 months), reflecting previous literature data [24][25][26] and the decline of diagnostic yield of genetic testing with increasing age at epilepsy onset [27][28][29].
In our cohort, the most represented seizure type at onset is generalized, especially in infants and children with CNVs and chromosomopathies (62% versus 37.5% in children with monogenic conditions).Previous studies [30,31] also reported on the prevalence of generalized seizures, the most common within the group being either tonic-clonic seizures [30] or epileptic spasms [31].Interestingly, we found that the distribution of seizure types at onset is different between patients with monogenic conditions and patients harbouring a CNV or chromosomal abnormality, with focal seizures being more than three times more common in monogenic conditions, while the frequency of febrile seizures, focal-to-bilateral seizures, and status epilepticus at onset is similar in the two groups.
We also documented very high figures of abnormal neurological examination in both subgroups.We believe that this strongly confirms that, differently from older age groups, early-onset genetic epilepsies often occur in the context of complex neurological phenotypes, in which epilepsy is just one of many dynamic clinical targets needing to be addressed with a holistic approach.In particular, the association of epilepsies and DEE with movement disorders is gaining increasing attention in the literature.In a recent paper analyzing a single centre's experience in the follow-up of persons with monogenic conditions and clinically affected by epilepsy and movement disorder, the investigators found that, in their sample, the semiology of movement disorders (especially the presence of hypokinetic versus hyperkinetic movement disorders) tended to identify two aetiologically different groups: the first mainly involving neurodegenerative conditions and the second mainly involving defects of neurotransmission, neuronal excitability, or neural development [32].However, this finding should not be interpreted in absolute terms, as it must be noted that hyperkinetic movement disorders (such as ataxia or spasticity) are well-described features of various neurodegenerative disorders [33,34].Additional relevant phenotypic clusters in our cohort include hereditary spastic paraplegias (HSP).Within complex HSP cases, epilepsy is found in a relevant subset of pediatric-onset cases [35].Thus neurological features, together with the epilepsy phenotype, can represent useful handles to formulate the correct diagnostic hypotheses.
In line with these observations, we documented heterogeneous neuroimaging features, which can be divided into three main groups: normal, aspecific and abnormal.According to a study performed on an unselected cohort of children with new-onset epilepsy starting before 3 years of age, aetiologically relevant findings were present in 40% and incidental findings in an additional 15% of patients [24].In our series, normal neuroimaging findings prevailed in children with monogenic conditions, while the majority of patients with CNVs or chromosomal aberrations had abnormal neuroimaging.Patients with monogenic conditions had malformations in 32 cases and progressive MRI changes in 16, while in the group with CNVs and chromosomal aberrations the ratio was 21/4.We found typical brain MRI findings (i.e., cortical malformations in TUBB-related disorder or lissencephaly with a pathogenic LIS1 variant), but also aspecific findings.Importantly, the presence of progressive MRI changes, such as cerebral or cerebellar atrophy, identified a subgroup of children for whom receiving neuroimaging is even more critical for correct management and diagnosis.In some cases, neuroimaging features pointed out overt neurodegenerative conditions (i.e., large subcortical cysts in megalencephalic leukoencephalopathy with subcortical cysts), thus informing further investigations into neurogenetic and neurometabolic disorders.
Unsurprisingly [36][37][38], comorbidities with neurodevelopmental and psychiatric disorders were common.The vast majority of patients had DD or ID (80.7% of individuals with monogenic conditions and 91% of those with CNVs or chromosomal abnormalities), and autism spectrum disorder was diagnosed in 11.5% of individuals with monogenic conditions and in one person with a CNV.Behavioural issues/psychiatric disorders were more common in those harbouring CNVs or chromosomal abnormalities than in monogenic conditions (33.3% versus 14.4%).The associations we documented have been well established in the literature: 1p36 deletion syndrome and abusive/aggressive behaviour [39], PCDH19 pathogenic variants and hyperactive, autistic, and obsessive-compulsive features [40], CHD2 pathogenic variants and hyperactivity [41], MBD5 and limited social interactions, aggressive and self-injurious behavior, short attention span, and autistic features [42].Although the prevalence of behavioural and psychiatric comorbidities generally increases in persons with intellectual disability [43], growing evidence supports the view that the link between epilepsy and neurobehavioral impairments is based on specific neurobiological mechanisms [44], including changes in neurotransmitters/neuromodulators, hypothalamic-pituitary adrenal axis dysfunction, network dysfunction, altered neurogenesis, neurotrophic factors, and neuroinflammation [45].The complex relationship between epilepsy and its neurobehavioural comorbidities is further suggested by one retrospective observational study in Norway, highlighting how the prevalence of these comorbidities is similar in focal and generalized epilepsies, but significantly higher in focal epilepsy of unknown cause compared to lesional epilepsy, and independent of seizure control [46].This might suggest a more critical role for intrinsic (genetically based) susceptibility factors.This hypothesis is also corroborated by the lower age of seizure onset in persons with focal epilepsy with comorbidities compared to those without [46].
At the end of the follow-up period, drug resistance occurred slightly more frequently in individuals with monogenic epilepsies than in those harbouring CNVs or chromosomal abnormalities, and 42% versus 58% of patients were seizure-free at their latest evaluation.In a population-based study on patients with early-childhood-onset epilepsy, 28% were drug-resistant, of whom 47% had monogenic epilepsy [30].
DEE-and epilepsy-related genes can be grouped into five categories: ion transport; cell growth and differentiation; regulation of synaptic processes; transport and metabolism of small molecules; and regulation of gene transcription and translation [47,48].Among monogenic disorders, the largest group in our cohort includes children harbouring P/LP variants in genes encoding ion channels (30%), in line with previous research [47,49,50].However, 28 genes were involved on only one occasion, strikingly highlighting the vast genetic heterogeneity underlying early-onset epilepsies and DEE [51].
Even if it is well known that the association between genomic disorders and epilepsy varies in terms of prevalence and semiology, and that in syndromic epilepsies seizures are part of a multisystem abnormality, with different types of potentially associated seizures [38], we decided to include patients presenting with CNVs and chromosomal abnormalities.We based our choice on the presence of epilepsy-related genes inside the deleted/duplicated regions, of documented enrichment in epilepsy, or on their relationship to genetic OMIM syndromes featuring neurological symptoms, including epilepsy [52].Our results are in line with the literature in that the most common CNVs include 1p36 deletion syndrome [53] and rearrangements involving chromosome 16 [54].
In our cohort, we documented a higher percentage of DEE in those with monogenic conditions (54% versus 38%).This is in agreement with a previous observation that, when epilepsy manifests as DEE, it is more likely to be caused by pathogenic variants in single genes rather than by CNVs [52].
After careful diagnostic work-up and re-evaluation of clinical and genetic reports and variants classification, the detected genetic variations had an uncertain clinical significance in 11% of cases.This was lower than in a recently published pediatric cohort in which 16.4% of tested patients had at least one VUS detected with chromosomal microarray and 41.9% via NGS sequencing of a panel of epilepsy-related genes [55].However, study design was different from ours.
For each and every involved gene, we confirmed that the phenotypic spectrum was very wide.We documented some clinical features partially, rarely, or never described in the literature (Supplementary Table S1).Two sisters carrying a compound heterozygous variation in the ALDH18A1 gene presented with the so-far-unreported phenotype of DEE-SWAS in the context of spastic paraplegia.In fact, to the best of our knowledge, only one patient with complex spastic paraplegia featuring epilepsy has been described, but he experienced temporal lobe seizures [56].Among patients harbouring SCN1A LP/P variants, although the largest group was composed of patients with Dravet syndrome followed by those with a GEFS plus phenotype [57][58][59], we also reported on one patient [12] with the recently defined phenotype of neonatal developmental and epileptic encephalopathy with movement disorders and arthrogryposis, associated with gain-of-function SCN1A variants [60].One female patient carried a pathogenic ARHGEF9 single-gene variant, which was an atypical finding because females are usually healthy carriers and few descriptions of affected subjects are available [18].Furthermore, in one patient with atypical Rett syndrome, we found a pathogenic mosaic variant in the GABRG2 gene, which is usually associated with different epilepsy phenotypes but has not been reported elsewhere in association with Rett syndrome [17].A female patient harbouring an LP variant in the PIGW gene with early-onset epilepsy and a complex neurological phenotype achieved seizure control in late childhood.She is currently the oldest known patient out of a total of 7 published worldwide [15,61].A final patient with Snyder-Robinson syndrome (secondary to a pathogenic hemizygous SMS gene variant) had myoclonic seizures, which have been reported in only one additional patient [62].We think that such cases are good examples of the role of NGS technologies (and especially ES) in solving atypical, unusual, or complex phenotypes.Reaching a precise and timely genetic diagnosis is important in order to correctly define the recurrence risk, and (when applicable) to aim for a targeted therapy [63][64][65][66].
Our study had several limitations.Due to the retrospective design, in some cases we were unable to retrieve all the relevant information for each patient.Furthermore, diagnostic tests were selected at the discretion of the treating physician and not as part of a trial, although evidence-based international recommendations were followed.Finally, we did not perform a statistical analysis of our data, but rather qualitatively described our findings.
However, we think that there are also some strengths to this work, such as the collection of detailed clinical, EEG, neuroimaging, and genetic data over a mean follow-up period of 14.75 years.Data analysis involved both clinical geneticists and pediatric neurologists at each of the four collaborating centres.

Materials and Methods
This retrospective observational cohort study was carried out at four Italian epilepsy centers (Epilepsy Center of San Paolo University Hospital in Milan, Child Neurology and Psychiatry Unit of AUSL-IRCCS di Reggio Emilia, Pediatric Neurology Unit of Vittore Buzzi Children's Hospital, Milan, and Child Neurology and Psychiatry Unit, IRCCS Mondino Foundation, Pavia).

Inclusion Criteria
Inclusion criteria were as follows: (a) genetic epilepsies with pathogenic or likely pathogenic variants and VUS; (b) age of epilepsy onset in the first three years of life.
The choice to include children with epilepsy onset within 36 months of age was made because the risk of cognitive impairment, behavioral comorbidities, and drug resistance was higher in this age group [67].

Exclusion Criteria
Exclusion criteria were as follow: (a) epilepsies related to other aetiological causes (such as inborn metabolic diseases and acquired structural aetiologies); (b) patients with a tuberous sclerosis complex and a typical Rett syndrome harbouring pathogenic variants on the methyl-CpG binding protein 2 (MECP2) gene.This choice was made to ensure better homogeneity of the sample because the Epilepsy Centre of San Paolo University Hospital in Milan has been a reference centre for these two diseases since 2006-2007.

Data Collection
Detailed clinical features were retrospectively collected by reviewing medical charts, consultations reports, and discharge letters.Apart from reading and annotating the reports, neuroimaging and electroencephalogram (EEG) data were directly reviewed.All data were gathered in a database.
Informed consent for genetic testing was obtained from all children's parents.For this paper, a formal approval from the local ethics committee was waived because we retrospectively reported on observational data.
For each patient, information about the following variables were collected: gender, family history for epilepsy and/or febrile seizures, epileptic features, neurologic examination, cognitive impairments and behavioral issues, neuroimaging features, metabolic and genetic findings.
Regarding the epileptic phenotype, we evaluated age at epilepsy onset, type of seizures at onset and at the last follow-up, EEG pattern at onset and at the latest follow-up, drug therapy, and drug resistance.
We classified seizure types according to the 2017 ILAE Classification [1] and epilepsy syndromes according to the 2022 ILAE Classification and definition of epilepsy syndromes with onset in childhood [68].Moreover, we categorized genetic variants according to the guidelines and recommendations of the American College of Medical Genetics and Genomics (ACMG) [69].
Brain magnetic resonance imaging or computed tomography were performed according to clinical presentation at the discretion of the treating physician.The presence of any acquired structural abnormalities was an exclusion criterion.
Furthermore, we evaluated genetic consultations and assessed which genetic test led to diagnosis for each patient.Performed genetic tests include karyotype, CGH-array, singlegene Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA), and NGS (targeted gene panels, ES or GS).NGS results were all confirmed by Sanger sequencing [70].The specific genetic test result was considered as diagnostic based on a thorough evaluation by the multidisciplinary team of pediatric neurologists and clinical geneticists at each participating center, and according to well-established guidelines and recommendations [71].The selection of the genetic test (s) to administer to each patient was made by the treating physician, on a clinical basis, according to current evidence and best clinical practice [71].The functional role of detected genes was categorized based on [48,72,73].
Psychomotor and cognitive development was evaluated by formal neuropsychological testing (such as Griffiths Mental Development Revised Scales [74], Wechsler Preschool and Primary Scale of Intelligence-WPPSI [75], Wechsler Intelligence Scale for Children-WISC) [76,77] or, if unavailable, best clinical assessment (based on developmental milestones and academic achievements) [78,79].
We divided our patients' cohort into two groups: patients with pathogenic and likely pathogenic variants and patients with VUS.Within the group of patients with pathogenic and likely pathogenic genetic variations, we further distinguished between monogenic conditions and chromosomal abnormalities (copy number variations-CNV-and structural defects).In the subgroup with monogenic conditions, we also included microdeletions containing genes known to be associated with diseases, which act with a loss of function mechanism (i.e., 16p11.2microdeletion syndrome and proline-rich transmembrane protein 2-PRRT2-gene) [80,81].

Conclusions
In conclusion, the main findings in our retrospective multicentre study of genetically caused epilepsies and DEE with onset within the first three years of life are: a high occurrence of generalized seizures at onset, drug resistance, abnormal neurological examination, global developmental delay and intellectual disability, and comorbidities with behavioural and psychiatric issues.We also documented different presentations between monogenic versus CNVs and chromosomal conditions, and atypical or rare phenotypes.A subgroup of patients with progressive neuroimaging changes highlighted how the diagnostic work-up and clinical management of early-childhood epilepsies can significantly diverge from that of older age groups and be more complex.

Cyclin-dependent kinase-like 5 (Figure 1 .
Figure 1.(A) Distribution of aetiological diagnoses within genes encoding ion channels in our cohort.(B) Distribution of aetiological diagnoses involving other cell functions.

Figure 1 .
Figure 1.(A) Distribution of aetiological diagnoses within genes encoding ion channels in our cohort.(B) Distribution of aetiological diagnoses involving other cell functions.

Figure 2 .
Figure 2. Behavioural and neuropsychaitric comorbidities in patients with monogenic disorders.

Figure 3 .
Figure 3. Distribution of chromosomal abnormalities and CNVs in our cohort.

Figure 3 .
Figure 3. Distribution of chromosomal abnormalities and CNVs in our cohort.

Figure 4 .
Figure 4. Behavioural and neuropsychiatric comorbidities in patients with chromosomal abnormal ities and CNVs.

Figure 4 .
Figure 4. Behavioural and neuropsychiatric comorbidities in patients with chromosomal abnormalities and CNVs.

Table 1 .
Cell functions of causative genes.

Table 2 .
Neurological examination findings in patients with monogenic disorders.

ASD ADHD Irritability and Psychomotor Agitation Attachment Disorder OCD Psychotic Disorders
Figure 2. Behavioural and neuropsychaitric comorbidities in patients with monogenic disorders.

Table 4 .
Brain MRI findings in monogenic disorders.

Table 5 .
Neurological examination findings in patients with chromosomal abnormalities and CNVs.

Table 6 .
Brain MRI findings in chromosomal abnormalities and CNVs.

Table 6 .
Brain MRI findings in chromosomal abnormalities and CNVs.

Table 7 .
Characteristics of patients carrying a VUS.