Epilepsy in Legius syndrome: Coincidence or causation?

Legius syndrome is a rare genetic disorder, caused by heterozygous SPRED1 pathogenic variants, which shares phenotypic features with neurofibromatosis type 1 (NF1). Both conditions typically involve café‐au‐lait macules, axillary freckling, and macrocephaly; however, patients with NF1 are also at risk for tumors, such as optic nerve gliomas and neurofibromas. Seizure risk is known to be elevated in NF1, but there has been little study of this aspect of Legius syndrome. The reported epilepsy incidence is 3.3%–5%, well above the general population incidence of ~0.5%–1%, but the few reports in the literature have very little data regarding epilepsy phenotype. We identified two unrelated individuals, both with Legius syndrome and epilepsy, and performed thorough phenotyping. One individual's mother also had Legius syndrome and now‐resolved childhood epilepsy, as well as reports of more distant relatives who also had multiple café‐au‐lait macules and seizures. Both probands had experienced childhood‐onset focal seizures, with normal brain MRI. In one patient, EEG later showed apparently generalized epileptiform abnormalities. Based on the data from this small case series and literature review, seizure risk is increased in people with Legius syndrome, but the epilepsy prognosis appears to be generally good, with patients having either self‐limited or pharmacoresponsive courses.


| INTRODUCTION
Legius syndrome, also known as neurofibromatosis 1-like syndrome, is a rare genetic disorder caused by heterozygous pathogenic variants in SPRED1 (OMIM 609291), located at 15q14, which encodes Sproutyrelated EVH1 domain-containing protein 1 (SPRED1).Legius syndrome shares some phenotypic features with neurofibromatosis type 1 (NF1, OMIM 162200), namely hyperpigmented macules, also known as café-au-lait macules (Brems et al., 2007;Legius et al., 2021).While the overrepresentation of seizures in people with NF1 is welldocumented (Pecoraro et al., 2017), epilepsy risk has not generally been considered increased in Legius syndrome; however, this is based on relatively few data.In this study, we report two probands with epilepsy in the context of Legius syndrome and review the evidence for a causal link between the two entities.

| METHODS
We reviewed our clinical and research databases for patients with epilepsy and pathogenic or likely pathogenic variants in SPRED1 and performed thorough phenotyping.The study was approved by the local research ethics boards.Patients or caregivers provided written informed consent.

| RESULTS
Two patients were identified.

| Patient #1
An 18-year-old female had seizures from the age of 8 years.The events primarily occurred from sleep, and involved nausea, unresponsiveness, confusion, and headache.Seizures were wellcontrolled on levetiracetam, with breakthrough focal or focal to bilateral tonic-clonic seizures only occurring in times of poor medication compliance.Past medical history was unremarkable.
Early developmental milestones were normal, and she was an overall average student in school but sometimes struggled with motivation.
On physical examination, she had macrocephaly and nine café-aulait macules >1.5 cm diameter, with several others that were smaller, as well as axillary freckling.Neurologic and ophthalmologic examinations were normal.
EEG at age 12 years showed occasional to frequent bursts of generalized medium to high voltage spike-wave and polyspike-wave discharges (Figure 1a).On family history, both parents were of Cree descent, with no known consanguinity.There were multiple individuals with café-aulait macules and/or epilepsy.The proband's mother carried the same SPRED1 pathogenic variant and had clinical features of Legius syndrome, with 17 café-au-lait macules and bilateral axillary freckling.
The mother reported that she believed she had seizures in childhood, but did not recall being on anti-seizure medication.The mother had also been diagnosed with breast cancer at age 43 years and unfortunately died at age 52 years of complications of liver cancer.There was also a maternal uncle with multiple café-au-lait macules and epilepsy.The patient's mother also reported that she had a cousin with epilepsy who was still on anti-seizure medications, as well as a niece who had childhood-onset epilepsy that spontaneously resolved.Unfortunately, the proband was lost to follow-up, preventing construction of a more extensive pedigree and collection of more detailed clinical information from other affected individuals in the family.

| Patient #2
A 10-year-old female had a febrile seizure at 9 months of age and then subsequently developed afebrile seizures at age 4 years.Her events were all in keeping with focal impaired awareness seizures, though semiology was variable.Her initial afebrile seizure presentation involved two consecutive events 25 and 15 min in duration, occurring from sleep, and included gurgling noises, left hemiclonic jerking, and pallor with perioral cyanosis.After the seizures, she had transient hemiparesis.She later had events involving impaired awareness and head and eye version to the right, with subsequent post-ictal dyspnea and facial dysesthesia; duration was 3-8 min.There were also events from sleep involving teeth-grinding and being difficult to rouse, which were suspicious for seizures.She was started on clobazam which completely controlled seizures until she was weaned off just before her ninth birthday.She has remained seizure-free off medication since then.
She was born at term following an uncomplicated pregnancy and delivery.Her medical history was significant for hereditary spherocytosis with associated mild splenomegaly and biliary colic.
She has severe ADHD on methylphenidate and a learning disorder.
Her family history was significant only for an 8-year brother with three café-au-lait macules, ADHD, speech delay and autism spectrum disorder and a mother with multiple café-au-lait macules.The mother was of First Nations (Cree/Iroquois) and northwestern European background, and the father First Nations (Squamish/ Haida) and Greek ancestry; there was no known consanguinity.The paternal grandmother had cancer affecting multiple organs including breast, brain, and lung.The proband's physical examination was significant for multiple hyperpigmented macules, but no neurologic deficits were noted.Initial EEG at age 4 years showed normal background activity with occasional focal spikes over the left frontal region in drowsiness and sleep, as well as occasional sharply contoured paroxysmal theta activity over the mid-frontal area in sleep.A follow-up overnight ambulatory EEG at age 6 years showed frequent right occipital spikewave discharges limited to non-REM sleep (Figure 1b).Brain MRI was normal.Chromosomal microarray was also normal, but whole exome sequencing revealed a heterozygous variant in SPRED1 (c.1246T>C, p.Cys416Arg) classified as likely pathogenic.She was also found to carry two variants in SPTA1 (OMIM 182860), c.2898+2T>C (likely pathogenic) and c.6531-12C>T (disease modifier), which were thought to explain her hereditary spherocytosis.The SPRED1 variant was suspected to be maternally inherited based on the mother's caféau-lait macules; the family was referred to medical genetics for parental testing but the family declined.

| DISCUSSION
Legius syndrome is an autosomal dominant disorder involving dysfunction of SPRED1, a protein which negatively regulates mitogen-activated protein kinase (MAPK) activity (Wakioka et al., 2001).Disruption of the Ras/MAPK pathways is the defining common feature of "RASopathies," a group of genetic conditions that also includes NF1, Noonan syndrome, capillary malformationarteriovenous malformation syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, and LEOPARD syndrome (Rauen, 2013).
Seizures have been reported in 3.3%-5% of individuals with Legius syndrome, which is higher than the general population prevalence of epilepsy, of 0.5%-1% (Denayer et al., 2011;Messiaen et al., 2009;Picot et al., 2008).It seems epilepsy risk should be considered elevated in patients with SPRED1 pathogenic variants; however, little is known regarding the typical phenotypic spectrum as clinical details in published reports have been limited.It is notable that the café-au-lait macule endophenotype in both families was maternally inherited with Cree descent.From our literature review, we identified five patients with SPRED1 pathogenic variants and a history of seizures, but almost no specifics were available (Table 1) (Denayer et al., 2011;Laycock-van Spyk et al., 2011;Messiaen et al., 2009;Romanisio et al., 2021).However, from the information from the patients we describe in this report, we can now say that epilepsy may have onset in childhood, initially presenting with focal semiologic features and EEG findings, perhaps like self-limited epilepsy with autonomic seizures (SeLEAS), though EEG may later be more suggestive of a genetic generalized epilepsy.The course of epilepsy may be self-limited, but some patients require anti-seizure medication into adulthood.These conclusions must of course be considered with caution, given the small number of patients involved.Additionally, it should be noted that patient #1 did not have an epilepsy gene panel or other broader testing performed, thus it is possible there is a second genetic factor contributing to the incidence of epilepsy in her family.
Recently published data have reported somatic pathogenic variants in Ras/MAPK pathway genes in patients with low-grade epilepsy-associated tumors and focal cortical dysplasias, raising the possibility that development of subtle focal brain malformations, possibly related to overactivity of the mammalian target of rapamycin (mTOR) pathway, could be an epileptogenic mechanism (Bedrosian et al., 2022;Fujita et al., 2023;L opez-Rivera et al., 2023;Zhao et al., 2019).However, enrichment of somatic genetic variants predicted to activate the Ras/MAPK pathway was also recently reported in the hippocampi of patients with drug-resistant mesial temporal lobe epilepsy, a form of focal epilepsy in which the pathology of resected specimens is quite different from that of focal cortical dysplasia and low-grade epilepsy-associated tumors (Khoshkhoo et al., 2023).While these findings are interesting, further research is needed to more clearly elucidate mechanisms that lead to epilepsy in patients with Legius syndrome and other RASopathies.
Brain MRI was normal.A 21-gene neurofibromatosis gene panel (MNG Laboratories) revealed a SPRED1 pathogenic variant (c.271delC, p.His91ThrfsX30).F I G U R E 1 EEG samples: (a) EEG for patient #1 at age 12 years in wakefulness.On longitudinal bipolar montage, a burst of generalized high voltage spike-wave and polyspike-wave discharges is seen (arrow).(b) EEG for patient #2 at age 6 years in non-REM sleep.On longitudinal bipolar montage, focal spike-wave discharges are seen, maximal over the right occipital region (O2) (arrows illustrate several examples).