Soft cerebellar signs unveil RARS2‐related epilepsy

Pathogenic RARS2 variants, by critically reducing mitochondrial arginyl-tRNA aminoacylation activity, cause a rare autosomal recessive disease, generally presenting as a severe encephalopathy with onset at birth, premature death, microcephaly, drug-resistant epilepsy, and hypotonia.1-9 Epilepsy was reported in ~90% of the cases, often with myoclonic and clonic seizures.2 As in other primary mitochondrial diseases, epilepsy may be attributed to ATP depletion, resulting in loss of neuron hyperpolarization (Na+/K+ ATPase activity impairment) and increased excitation (loss of GABA-mediated inhibition).10 The most frequent neuroradiological finding is cerebral atrophy, followed by pontocerebellar hypoplasia.1-9 We report the clinical and genetic findings of a 16-year-old boy with RARS2-related encephalopathy distinguished for later onset, longer survival, and milder phenotype compared to previously reported cases. A 13-year-old Italian boy without family history of neurological diseases was brought to the emergency room for a sleep-related focal-to-bilateral seizure. His mother reported the following ictal semiology: paroxysmal arousal from sleep, sitting up on the bed, head deviation, drooling, grunting, and asymmetric limb tonic posturing with right upper limb extension and left upper limb flexion (figure 4 sign), without awareness nor response to stimuli, with full recovery in ~10 min. EEG showed a 7 Hz background rhythm with brief trains of synchronous and asynchronous frontal 3 Hz sharp waves, prevalent on the right side (Figure 1A). After a similar second seizure on the following day, levetiracetam 500 mg twice a day was started achieving a seizure-free three-year follow-up and a consistent EEG improvement. The patient's medical history revealed normal pregnancy, birth, and early motor development with autonomous walking at 15 months. A single episode of febrile seizure at 1 year of age was reported. At 3 years, he was referred to a pediatric neurologist for language delay with increased nonverbal communication; square wave jerks, interrupted pursuit, mild dysmetria, and tandem gait inability were observed at that time. Psychomotor and speech therapy alongside with professional school support were provided with benefit. A brain MRI at the age of 13 years revealed isolated cerebellar vermis atrophy (Figure 1B). At the last neurological examination, at 16 years, the patient displayed mild intellectual disability and cerebellar features including dysarthria, gaze difficulties, postural and kinetic tremor of upper limbs, limbs dysmetria, dysdiadochokinesia, and gait ataxia. The combination of focal epilepsy with additional clues including cerebellar ataxia and language delay induced us to perform genetic tests: array-comparative genomic hybridization (array-CGH) resulted negative, whole-exome sequencing revealed two compound heterozygous variants in RARS2 (NM_020320.5): c.685C>T (p.Arg229*) and c.972C>T (p.Thr324=) (Figure S1A). The p.Arg229* is a known pathogenic variant.11 The c.972C>T, affecting the third to last nucleotide of exon 11, is extremely rare (gnomAD AF = .000004) and predicted in silico to likely impact splicing (SpliceAI = Donor Loss .49/1, dbscSNV ADA score = .919/1, VarSeak = Exon Skipping, class 4/5). Transcript analyses on patient's cDNA demonstrated that the c.972C>T disrupts splicing generating a defective transcript missing exon 11, although allowing the synthesis of some wild-type transcript (Figure 1C), likely able to ensure a significant residual Rars2 enzymatic activity. The presence of this rare splice-disrupting variant in combination with a known RARS2 pathogenic variant in a patient with epilepsy and ataxia prompted us to classify it as likely pathogenic (ACMG criteria: PM2, PM3, PS3). Functional analyses on patient's cultured lymphocytes demonstrated a moderate reduction (~39%) of normal-length Rars2 protein compared to two healthy controls (Figure 1D) and a normal mitochondrial respiratory chain activity, consistently with his relatively mild phenotype. In conclusion, RARS2-related disease, commonly known as a severe syndromic encephalopathy, may present with more shaded phenotypes representing a diagnostic challenge.3, 5, 6, 9 Our patient, distinguished for a mild phenotype, characterized by longer survival, drug-responsive epilepsy, cerebellar ataxia, and mild intellectual disability, highlights that RARS2-encephalopathy should be considered also in older patients evaluated for epilepsy, when subtle additional features like cerebellar ataxia and mild intellectual disability are recorded in medical history. His milder phenotype is likely attributable to a relevant residual wild-type RARS2 transcript, supporting the hypothesis that the phenotypic severity is limited by the presence of significant residual functional enzyme. Further research is necessary to delineate a reliable genotype–phenotype correlation for the RARS2 gene, to provide patients and their families with improved genetic counseling and clinical assistance. We thank Associazione Amici del Centro Dino Ferrari for their support. None of the authors has any conflict of interest to disclose. Data S1: Data S2: Figure S1: Figure S2: Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Which of the following clinical features tends to appear in patients with RARS2-related disease displaying longer survival? Which of the following affirmations about the RARS2 c.972C>T variant is true? Which among the following is probably a protective factor in RARS2-related encephalopathy? Answers may be found in the supporting information.