Biallelic pathogenic variants of PARS2 cause developmental and epileptic encephalopathy with spike‐and‐wave activation in sleep

Abstract Background Biallelic pathogenic variants in the mitochondrial prolyl‐tRNA synthetase 2 gene (PARS2, OMIM * 612036) have been associated with Developmental and Epileptic Encephalopathy‐75 (DEE‐75, MIM #618437). This condition is typically characterized by early‐onset refractory infantile spasms with hypsarrhythmia, intellectual disability, microcephaly, cerebral atrophy with hypomyelination, lactic acidemia, and cardiomyopathy. Most affected individuals do not survive beyond the age of 10 years. Methods We describe a patient with early‐onset DEE, consistently showing an EEG pattern of Spike‐and‐Wave Activation in Sleep (SWAS) since childhood. The patient underwent extensive clinical, metabolic and genetic investigations, including whole exome sequencing (WES). Results WES analysis identified compound heterozygous variants in PARS2 that have been already reported as pathogenic. A literature review of PARS2‐associated DEE, focusing mainly on the electroclinical phenotype, did not reveal the association of SWAS with pathogenic variants in PARS2. Notably, unlike previously reported cases with the same genotype, this patient had longer survival without cardiac involvement or lactic acidosis, suggesting potential genetic modifiers contributing to disease variability. Conclusion These findings widen the genetic heterogeneity of DEE‐SWAS, including PARS2 as a causative gene in this syndromic entity, and highlight the importance of prolonged sleep EEG recording for the recognition of SWAS as a possible electroclinical evolution of PARS2‐related DEE.


| INTRODUCTION
The PARS2 gene (OMIM * 612036) is a nuclear gene encoding for the mitochondrial prolyl-tRNA synthetase 2, an aminoacyl-tRNA synthetase essential for mitochondrial protein biosynthesis and oxidative phosphorylation (Almuqbil et al., 2020).Biallelic pathogenic variants of PARS2 are associated with Developmental and Epileptic Encephalopathy-75 (DEE-75, MIM #618437), a multisystem mitochondrial disorder, characterized by microcephaly, dysmorphic features, hypotonia, intellectual disability (ID), epileptic spasms, cerebral atrophy with delayed myelination, lactic acidemia and cardiomyopathy.Less common epileptic manifestations are focal seizures, myoclonus and generalized seizures.Most patients die within the first 10 years of life (Almuqbil et al., 2020).In these patients, the EEG usually shows multifocal spikes and hypsarrhythmia, consistent with a clinical diagnosis of Infantile Epileptic Spasms Syndrome (IESS).
Spike-and-wave activation in sleep (SWAS) is an EEG pattern typical of a spectrum of epileptic encephalopathies (EE) or DEE characterized by regression in cognitive, behavioral, or psychiatric function and motor deterioration with dyspraxia or dystonic features, recently renamed EE-SWAS or DEE-SWAS (Specchio et al., 2022).SWAS is characterized by a significant increase in slow spike-andwave complexes at 1.5-2 Hz, particularly during stage N2 of sleep.This abnormal activity is typically diffuse, but may be more localized, often in the frontal regions, or multifocal.Patients usually present with infrequent, focal, drug-responsive seizures between the ages of 2 and 5 years.As the disease progresses, multiple seizure types tend to appear.These include focal seizures with or without impaired awareness, typical and atypical absences, atonic seizures, and focal motor seizures with negative myoclonus.Some patients may present only acquired aphasia without experiencing clinical seizures (Specchio et al., 2022).
DEE-SWAS may be caused by a variety of etiologies, including brain structural abnormalities and genetic factors inherited in either monogenic or complex patterns.Up to 50% of patients have a family history of seizures (Tsai et al., 2013).GRIN2A (OMIM *138253), encoding the alpha 2 subunit of the N-methyl-D-aspartate (NMDA) glutamate receptor, is the major monogenic factor.Pathogenic variants in this gene have been associated with a range of DEE-SWAS with varying degrees of severity, included in the epilepsy-aphasia spectrum disorders (Carvill et al., 2013;Lesca et al., 2019).
We describe the case of a 25-year-old woman with DEE and an EEG pattern of SWAS carrying biallelic variants in PARS2.We also performed a literature review of all the reported cases of PARS2-associated DEE.

| MATERIALS AND METHODS
The patient was referred to the Epilepsy Center of our Institute after the transition from pediatric to adult care.She underwent an accurate clinical, neurophysiological and neuroradiological assessment.Whole exome sequencing (mean coverage: 52X; read length: 150 bp) was performed in the context of the Epi25 Project (Epi25 Collaborative, http:// epi-25.org) for which she was recruited in 2017, after obtaining specific consent.The study was approved by the local ethics committee (CE 16057).Details of variant analysis, interpretation and validation were provided elsewhere (Minardi et al., 2020).
A systematic search to map the literature with regards to PARS2-related DEE was performed between March and April 2023.The PubMed database was searched using a combination of the key words "PARS2" AND "seizures" OR "epilepsy" OR "encephalopathy", with no restrictions on publication date or language.The articles cited in the retrieved papers were also screened for additional references.The full text of the original articles was carefully reviewed for relevant content and for an in-depth characterization of the epilepsy phenotype, focusing mainly on the possible association of SWAS.

| Case presentation
This 25-year-old woman, born from unrelated parents, presented with epileptic spasms and hypsarrhythmia at 5½ months of age.Since then, regression and severe psychomotor developmental delay were evident.She was diagnosed with IESS and started on vigabatrin and valproate with seizure control and EEG normalization.The electroclinical amelioration coincided with a slow progression of motor milestones.However, there was no improvement in cognitive functioning, and structured verbal language was never acquired, except for the repetition of a few words at the age of 3. Neurologic examination (NE) showed microcephaly, profound ID, hand stereotypies, and spastic paraparesis, treated with botulinum toxin at age 2-4 years.Subsequent EEG showed multifocal epileptiform abnormalities, enhanced by sleep.Brain MRI at the age of 3 years showed mild cortical atrophy, white matter signal alteration in the bilateral subcortical retrotrigonal region suggestive of incomplete maturation, and a thinning of the corpus callosum.
From the age of 8 years, there was a progressive deterioration of the EEG, which evolved into a SWAS pattern.Neuropsychological assessment at that stage documented regression, with a complete loss of language ability.Brain MRI findings were stable.Despite the absence of obvious clinical events, levetiracetam and clonazepam were added to valproate, without improvement in the EEG.
At the age of 16, she had a seizure relapse with two convulsive seizures two hours apart, each lasting less than one minute.On that occasion, a decrease in valproate plasma levels was found.The EEG recordings consistently showed a pattern of SWAS.Brain MRI showed a greater extent of the bilateral and symmetric white matter hyperintensity in Flair, involving in addition to the retrotrigonal regions and semi-oval centers, the subcortical white matter of the pre-and post-central gyri.
Since the age of 18, she has experienced brief episodes of abrupt bilateral stretching of the oral rhyme (a kind of facial grimace) and head flexion, vocalization, facial blushing, and sometimes urinary incontinence compatible with subtle spasms.These events, occurred in clusters mainly on awakening, and lasted from 5 to 20 min, multiple times per day.A transient improvement was achieved with the optimization of valproate therapy.Over the years, she was treated with several other anti-seizure medications (ASMs) such as ethosuximide, acetazolamide, zonisamide and rufinamide.Only after steroid therapy at the age of 21 (cycles of intravenous methylprednisolone for three consecutive days/month for six months) did the seizures and EEG pattern of SWAS cease for several months.Currently, she has been experiencing multiple seizures per day despite a combination of three ASMs (valproate, lamotrigine and clobazam).The EEG still shows frequent epileptiform abnormalities during drowsiness that became continuous in sleep (Figure 1a).
Brain MRI and proton magnetic resonance spectroscopy at the age of 21 years showed diffuse hypomyelination and atrophy, stable compared with the previous one, and a modest reduction in the NAA/Cr and Cho/Cr ratios (Figure 1b).
The patient underwent extensive metabolic and cardiological exams, which were unremarkable.Genetic investigations including karyotype, PCR and fluorescence in situ hybridization for X-fragile, CGH-array, direct sequencing of MECP2, CDKL5, FOXG1, PCDH19, SLC2A1, and a multigene epilepsy panel, were all negative.At the age of 21 years, she underwent WES analysis that showed compound heterozygous variants in PARS2 (NM_152268.3):c.1091C>G (p.Pro364Arg) and c.283G>A (p.Val95Ile), inherited from the healthy father and mother, respectively, and already reported in the literature to be pathogenic (Table 1).

| DISCUSSION
We reported on a patient with DEE-SWAS carrying compound heterozygous pathogenic variants in PARS2.The literature review did not reveal other cases of PARS2related DEE-SWAS.
Our patient initially presented with IESS with hypsarrhythmia, one of the typical presentations of DEE-75.After a period of electroclinical stability, her EEG pattern changed in late childhood, showing a progressive increase in epileptiform abnormalities during sleep to continuous SWAS.It is known that DEE with hypsarrhythmia can progress to DEE-SWAS (Mir et al., 2023), an age-dependent syndrome that typically occurs between the age of 4 and 8 years, but this has never been reported in PARS2related DEE.This may be related to the short survival of the patients carrying pathogenic variants of PARS2 reported to date, most of whom died in childhood, before possibly developing a SWAS pattern.Nevertheless, no SWAS pattern was described in the older patient reported (Almuqbil et al., 2020), even though no sleep EEG recording was mentioned among the investigations performed.
Compared with the cases with identical genotypes, our patient showed a similar survival to the case reported by Almuqbil and co-authors who is the only one who died in adulthood (Almuqbil et al., 2020), at the age of 21 years due to cardiac dysfunction.Cardiac involvement is commonly observed in patients with pathological variants of PARS2, usually with initial hypertrophic cardiomyopathy followed by a dilatative evolution in a brief time before death (Almuqbil et al., 2020) usually occurring in childhood (Ciara et al., 2018;Sofou et al., 2015).No similar findings were observed in our patient, in whom the cardiac assessment repeated at 25 years of age was normal.Thus, the absence of cardiac involvement may be the reason for her long survival compared to other PARS2-mutated patients.In addition, unlike the aforementioned case (Almuqbil et al., 2020), our patient did not have recurrent infections or liver involvement.
A severe global developmental delay with ID and "absent speech" characterized most of these cases.Interestingly, our patient experienced a further cognitive regression with complete loss of language after developing SWAS.Notably, this EEG pattern may characterize conditions with subacute onset of acquired aphasia without or only with infrequent and self-limiting seizures (Specchio et al., 2022).In addition to microcephaly and axial hypotonia, which are the most common findings in NE in PARS2-DEE, our patient showed spastic paraparesis with significant gait impairment.Pyramidal signs (including limb hypertonia and Babinski's sign, possibly in combination with axial hypotonia) have been observed also in patients with the same pathogenic variants (Yin et al., 2018), as well as in patients with different genotypes (Ciara et al., 2018;Mizuguchi et al., 2017).
Although high lactate levels and myopathic changes on muscle biopsy are frequently reported in this mitochondrial DEE, our case, as well as patients with the same genotype, did not show laboratory alterations or lactate accumulation on MRI spectroscopy (Table 1).As for other cases of DEE, the application of next-generation sequencing techniques in our patient allowed genetic diagnosis decades after the onset of epilepsy, ending a long diagnostic odyssey.Genetic diagnosis also led to improved patient management through surveillance and monitoring of possible comorbidities.In terms of therapy, whereas valproate is contraindicated in other mitochondrial encephalopathies, such as Alpers syndrome due to pathogenic variants in POLG (DNA polymerase γ), as it can lead to fulminant liver failure (Pronicka et al., 2011), there are no definite data on valproate toxicity in PARS2-related DEE.In Ciara and coauthors, a patient with different compound heterozygosity of PARS2 was treated with valproic acid, which appeared to be associated with clinical worsening and liver failure (Ciara et al., 2018).In our patient, however, valproate administration coincided with a period of seizure freedom and later with a transient reduction in seizure frequency, suggesting that valproate treatment is not an absolute contraindication in mitochondrial disorders other than POLG-related ones.Steroid pulse therapy may be a treatment option for PARS2-related DEE-SWAS.In fact, several studies on the effect of steroids and ACTH at different dosage protocols in SWAS-related conditions have shown a transient positive response to steroids with normalization of the EEG and improvement of neuropsychological functions, in particular language and behavior (Veggiotti et al., 2012).Despite the temporary resolution of SWAS with corticosteroids, our patient eventually had a seizure relapse with persistence of SWAS into adulthood.The typical SWAS is a self-limited condition that usually resolves by adolescence.The longer duration of this pattern in our patient may be related to the underlying genetic etiology, responsible for mitochondrial encephalopathy with a progressive pattern of hypomyelination on brain MRI.In fact, several studies have shown that the duration of SWAS or EEG abnormalities is significantly longer in symptomatic/structural cases (Caraballo et al., 2013) or in patients with an unfavorable outcome in whom symptomatic/structural etiologies were more common (Öztoprak et al., 2021).Furthermore, in the childhood cases reported in the literature, the persistence of the SWAS pattern may have been missed due to the lack of electroclinical follow-up.
In conclusion, we first reported PARS2 as a new gene determining DEE-SWAS.The recognition of SWAS as a possible electroclinical evolution of PARS2-related DEE will help in the diagnosis and appropriate management of these patients.Moreover, it underlines the importance of a complete neurophysiological assessment including prolonged and serial sleep EEG recordings.Prolonged survival, absence of lactic acidosis and cardiac involvement, and transient electro-clinical improvement with valproate are additional atypical features in our case.Our findings widen the spectrum of genes associated with DEE-SWAS, to include PARS2.

F
I G U R E 1 (a) EEG (21 years old): during drowsiness, bilateral, synchronous, and asynchronous spike-wave (SW) over the temporalparietal regions enhanced by sleep, evolving to a pattern of continuous SW during sleep (SWAS).(b) Brain MRI (21 years old): diffuse, bilateral hyperintensity in T2-FLAIR of the subcortical white matter (retrotrigonal regions, semioval centers, pre and post-central gyrus) with reduced apparent diffusion coefficient (ADC), suggestive of diffuse hypomyelination.Posterior atrophy.The spectroscopy study was performed by placing two volumes on the right, in the altered white matter of the Rolandic region and in the morphologically apparently free over the frontal region.The results show a modest reduction in NAA/Cr and Cho/Cr ratios.
Clinical and genetic findings in PARS2-related DEE: our case and reports from literature review.