Novel NARS2 variants in a patient with early-onset status epilepticus: case study and literature review

Asparaginyl-tRNA synthetase 2 (Asn-RS) encoded by NARS2 is a member of the class II family of aminoacyl-tRNA synthetases (aaRSs) which play a crucial role in biosynthesis by catalyzing the ligation of asparagine to tRNA molecules [1]. This protein was first identified in 2005 [2] and contains 477 amino acids. It is expressed ubiquitously throughout the body both in humans and mice [1]. Moreover, it is expected to function as a dimer [2].

Pathogenic variants in NARS2 have been subsequently identified [3, 4] and were correlated to the combined oxidative phosphorylation deficiency 24 (COXPD24) (OMIM: 616,239) which is an autosomal recessive mitochondrial disorder that exhibits pleiotropic phenotypes. It is associated with visual and hearing abnormalities, myopathy, neurodevelopmental disorder, and mitochondrial dysfunction [5‐9].

The aaRSs are a group of enzymes that facilitate the ligation of 20 amino acids to their molecular cognate tRNA [29]. Variations in aaRSs were reported leading to central nervous system (CNS) pathologies with epileptic encephalopathy, developmental delay, and intellectual disability [30]. NARS2 is a member of the class II family of aaRSs to catalyze the ligation of asparagine to tRNA molecules in the mitochondrion. The variant of NARS2 was first reported in two siblings with myopathy and combined complex I and IV deficiency in skeletal muscle [4]. NARS2 deficiency may cause a decrease in oxygen consumption rates and electron transport chain activities in patient fibroblasts [1]. The specific cardiac dysfunction and neonatal diabetes phenotypes are supplied in NARS2 variant individuals. On the whole, they mainly present status seizures, visual hearing disorder, and severe myopathy that was identified as the pathogenic gene of COXPD24 (OMIM:616,239).

A comprehensive review of NARS2 mutations was performed. Up to now, only 28 variants in NARS2 gene have been reported, and the exact genotype-phenotype correlation is not clear. The number of reported cases related to NARS2 deficiency has been gradually increasing [3, 5‐7, 9, 26‐28]. Recently, more individuals of NARS2 variants have been reported [9, 22‐25]. Data from this study was compared with 28 variants in NARS2 gene published studies. Their diagnosis, phenotype, variant type, zygote type, survival outcome, and clinical finding are summarized in Table 2. Domain structure and modeling of the known NARS2 variations in previous studies in Fig. 2D.

Epileptogenesis is commonly associated with neurodegeneration and bioenergetic defects and mitochondrial dysfunction decline of energy by dysfunction of the electron transport chain leading to apoptotic neuronal death [31]. As previous studies, neurodevelopmental disorders were the main features of NARS2 deficiency. Most of the patients with NARS2 variants had focal, generalized, or myoclonic seizures and mitochondrial abnormalities such as combined complexes decreased and structurally abnormal [3]. In this study, a female infant with intermittent fever, status seizures, and GDD was described. GDD presented as difficulty in head control and roll over at her four-month-old. Status frequent focal subclinical or clinical seizures in the left frontotemporal were observed by long-term EEG monitor. Moreover, the brain structure abnormal was also detected in our patient with abnormal single and bilateral white matter atrophy in MRI. These clinical features were commonly in diseases with aaRSs gene mutations, including leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) cases with NARS2 variations, leukoencephalopathy with brainstem and spinal cord involvement, lactate elevation (LBSL) with Aspartyl-tRNA Synthetase 2 (DARS2) variations, and Alanyl-tRNA Synthetase 2 (AARS2)-related leukoencephalopathy [29]. It seems that there may be a shared mechanism of mitochondrial dysfunction in these disorders.

Severe myopathy was another characteristic clinical feature for cases with NARS2 variant. It is well known that mitochondrial dysfunction will affect tissues request high-energy such as brain, muscle, and heart. Patients with NARS2 deficiency usually develop muscle weakness of limbs and face muscles. Myocardial dysfunction in this case was represented with CK-MB and PRO-LPBN evaluated. Heart phenotype in patients with NARS2 deficiency was rare with mitral valve prolapse [9] and cardiac dysfunction [22], while myocardial dysfunction has been reported in other aaRSs, including AARS2 [32] and Lysyl-tRNA synthetase (KARS) [7, 33]. The reported patient with cardiac dysfunction has same phenotype with our patient and persistent elevation of serum hepatic and myocardial enzymes, but further investigation is necessary to verify whether NARS2 variants lead to cardiomyopathy.

Individuals with the same variant could exhibit different phenotypes in identical [4] or unrelated [7] families. While some clinical features with vision impairment were specifically present in some cases but not found in our patient. This may be explained by tissue specificity that consistent with other mitochondrial diseases [34]. The broad phenotypic variability of NARS2 related disease present from an infantile lethal phenotype to mild non-progressive disease. Therefore, there may be no strong association between the genetic variants and disease severity [7].

All variants observed in NARS2 were located in functional domains of NARS2 (Fig. 1D). Most of them were missense and may lead to protein dysfunction by changing the stability or interactions with other biological molecules [35]. The compound heterozygous variations of our patient in NARS2 [c.1352G > A (p.Arg451His); c.707T > C (p.Phe236Ser)] are located in the aminoacyl-tRNA synthetase domain. This domain contains three conservative motifs which are also found in other classII aaRSs. Among them, motif 3 contains strictly conserved arginine (Arg) residue that plays a crucial role in adenosine triphosphate (ATP) binding function [36]. Based on protein modeling analyses, the variant c.1352G > A; p.Arg451His (Fig. 3) changes Arg to His that is from a conserved non-aromatic to an aromatic, differently shaped, and this changing conserved Arg in motif 3 may affect ATP binding and the NARS2 function. Furthermore, another pathogenic mutation for the change of the Arg residue (from Arg to Cys) was also shown in another patient with Leigh syndrome [20]. Another variant in our patient in the 236th amino acid changes one amino acid to another that is more polar, smaller, and more flexible. It was found to have intermolecular hydrogen bonds with the 176th and 178th residues that were contained in conserved motif 1 (Fig. 3D). The crucial role of motif 1 [37] in dimerization may be affected by Phe236Ser. Meanwhile, another changed residue in 236th (from Phe to Cys) was found in patients with the infantile-onset neurodegenerative disorder [5] which explains the pathogenicity of this variation. The two NARS2 variants in our patient were predicted by mCSM and DUET software to have a stability change in the structure of the protein (Fig. 3). Unfortunately, our study was lacking in the validation of in vivo or vitro experiments. Given the patient’s ultimate demise, we will address this shortcoming in our future research.

In conclusion, we identified the novel compound heterozygous variants in an infantile-onset patient with status epilepticus and neurodegenerative disorder with final diagnosis as mitochondrial encephalomyopathy. Our study expands the genotype spectrum of COXPD24 and highlights the critical role of NARS2 in epilepsy and neurodevelopment.