Expanding phenotype heterogeneity of NARS2 by presenting subdural hematoma and parenchymal hemorrhage

Abstract Background NARS2 encodes mitochondrial Asparaginyl‐tRNA Synthetase 2, which catalyzes the aminoacylation of tRNA‐Asn in the mitochondria. To date, 24 variants have been reported in NARS2 gene in 35 patients. The phenotypic variability of NARS2‐associated disorder is broad, ranging from neurodevelopmental disorders to hearing loss. In this study, we report some novel imaging findings in an Iranian patient suffering from epileptic encephalopathy, caused by a previously reported variant, c.500A > G; p.(His167Arg), in NARS2. Methods The spectrum of clinical manifestations of two Iranian patients was investigated and genetic analysis was performed by Whole‐exome sequencing (WES). Additionally, we also reviewed the literature and summarized the phenotypes of previously reported patients with variants in the NARS2 gene. Results Here, we present the phenotypic and genetic features of 2 unrelated Iranian infants presented with neurodevelopmental delay, seizures, hearing impairment, feeding problems, elevated serum lactate levels in addition to subdural hematoma and cerebral parenchymal hemorrhage in the brain magnetic resonance imaging (MRI) of one of the patients. Genetic analysis revealed a biallelic missense variant in NARS2: c.500A > G; p.(His167Arg). We described the subdural hematoma and cerebral parenchymal hemorrhage of the brain for the first time. Conclusions Our study provides new clinical findings, subdural hematoma, and parenchymal hemorrhage, in NARS2‐related disorders. Our findings along with previous studies provide more evidence of the clinical presentation of the disease caused by pathogenic variants in NARS2. Expanding the clinical spectrum increases the diagnostic rate of molecular testing and improves the quality of counseling for at‐risk couples.


| INTRODUC TI ON
More than 250 variants in both nuclear and mitochondrial genes have been reported in a heterogeneous group of disorders presented with a wide range of clinical manifestations, called mitochondrial disorders. 1Among the nuclear genes, those coding for the mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) have been shown to be commonly related to human mitochondrial disorders.These enzymes charge mitochondrial tRNA molecules with their cognate amino acids.Although most mitochondrial proteins are encoded by the nuclear genome in the cytosol, a mitochondrial translation system is necessary to translate the 13 mitochondrial polypeptide subunits that are vital for oxidative phosphorylation. 2,3The linkage of amino acids to their cognate tRNAs, which is mediated by mt-aaRSs is a crucial step in protein synthesis in mitochondria. 4,5Deficiency of asparaginyl-tRNA synthetase (NARS2), NP_078954.4,[8] Up till now, 24 variants in NARS2 gene (NM_024678.6) in 35 cases including 20 patients carrying homozygous and 15 patients carrying compound heterozygous variants have been reported in the literature and presented with a wide spectrum of clinical symptoms even among affected individuals carrying the same variants.All of the data related to families and variants are summarized in Table 1.

Most of the patients who carried pathogenic variants in NARS2
presented with an increased level of serum lactate, refractory epileptic seizures, sensorineural hearing impairment, intellectual disability, and global psychomotor delay or regression.Other features include progressive microcephaly, renal dysfunction, optic atrophy, cortical visual impairment, spastic quadriplegia, hemiparesis, flaccid quadriplegia, urinary retention, amyotrophy, hyperCKemia, excessive fatigue, laryngomalacia, pharyngeal hypotonia, feeding difficulties, ataxia, brachymetatarsia, hallux valgus, hypotonia, upper limb tremor, clubbed fingers, mitral valve prolapse developmental delay, and brisk deep tendon reflexes, and premature menopause in females was observed in some cases. 6,16The most commonly reported initial phenotype based on age of disease onset is summarized in Table 2.
In this study, we investigated two unrelated Iranian male infants from two individual families who presented with epileptic encephalopathy.Genetic findings revealed a reported biallelic NARS2 variant in both cases; c.500A > G (p.His167Arg).Brain magnetic resonance imaging (MRI) of one patient showed novel imaging findings including subdural hematoma, cerebral parenchymal hemorrhage, and cerebellar cortical restriction in diffusion weighted sequences (DWI) that were not reported previously. 9

| Case presentation
Here, we report two patients from two unrelated Iranian families who presented with epileptic encephalopathy and were referred to Myelin Disorders Clinic, Children's Medical Center, Tehran, Iran, and DeNA Laboratory, Department of Medical Genetics, Tehran, Iran (Figure 1A).The written informed consent was obtained from all participants or their legal guardians.Participants also provided written informed consent for publication of their information included in this paper.

| Patient 1
The patient was a 2-year-old boy, with suspected genetic epileptic encephalopathy, who was first visited at pediatric intensive care unit (PICU) due to frequent seizures, poor general condition, and Conclusions: Our study provides new clinical findings, subdural hematoma, and parenchymal hemorrhage, in NARS2-related disorders.Our findings along with previous studies provide more evidence of the clinical presentation of the disease caused by pathogenic variants in NARS2.Expanding the clinical spectrum increases the diagnostic rate of molecular testing and improves the quality of counseling for at-risk couples.

K E Y W O R D S
asparaginyl-tRNA synthetase 2, cerebral parenchymal hemorrhage, NARS2, subdural hematoma, whole exome sequencing decreased level of consciousness.He was the second child of consanguine parents and was born at term pregnancy through an uneventful normal vaginal delivery without any significant event during prenatal periods.His birth weight and head circumference (HC) was 3100 g (z score = 0) and 35.5 cm (z score = 1), respectively.He had a healthy five-year-old brother.
His parents first noted a blunted response to environmental stimuli, such as acoustic stimulus, and social events, such as lack of social smile, at 2 months of age.However, he was able to hold his neck.Then, he developed non-fixed transient episodes of unilateral torticollis and focal motor and tonic spasm seizures at 3.5 months of age.He was hospitalized and his initial laboratory workups indicated only a low total serum calcium level (total calcium level of 6 mg/dL, normal range: 7.8-10 mg/dL), phosphorus level of 6.8 mg/dL (normal range: 4.5-6.5 mg/dL), magnesium level of 2 mg/dL (normal range: 1.5-5.5 mg/dL), alkaline phosphatase of 628 (normal range: 140-720 IU/L), serum lactate level of 18.7 mmol/L (normal range; 4.5-19.8mmol/L), and normal level of serum albumin and ammonia.He was prescribed calcium supplements in addition to phenobarbital, which resulted in seizure control lasting for a few weeks just based on clinical observation.However, Electroencephalogram (EEG) was not performed due to family dissatisfaction.On examination at discharge, motor and cognition delays were detected.
After a few weeks, the patient's seizures flared up with severe restlessness attacks and crying followed by apnea that was lasting up to 1 min following a mild upper respiratory tract infection and fever.These events resulted in a second hospitalization.His seizures evolved to mixed-type ones consisting of focal motor clonic seizures with lateral gaze, head version, and focal to bilateral and multifocal motor type seizures that ultimately led to PICU admission at the age of 9 months.The EEG depicted generalized epileptic discharge, posterior polymorphic delta activity, and diffuse fast activity due to the benzodiazepine effect (Figure 2).During hospitalization, he encountered a poor general condition and infrequent repetition of seizures.In addition, he experienced severe electrolyte imbalances, including low serum total calcium of 6.5 mg/dL, low magnesium level of 1 mg/dL, hyponatremia (Na:106, range: 135-145 mEq/L), mild hypokalemia Therefore, a low dose of hydrocortisone tablet was started which was helpful to maintain serum electrolytes.Other laboratory tests indicated severe microcytic anemia with hemoglobin level of 5 mg/ dL (range: 9.5-13 g/dL) and mean corpuscular volume (MCV) of 71 fl (range: 75-108), thrombocytopenia (13 × 10 3 , normal range of 150-450 × 10 3 ), hypoparathyroidism with parathyroid hormone (PTH) level of 607 pg/mL (range: 15-65 pg/mL), and also central hypothyroidism (TSH: 10.7, range: 1.7-7.5 μM).In addition, serum lactate levels were recorded between 11 and 29 mmol/L, and cerebrospinal fluid (CSF) lactate level was 16 mmol/L (normal range; less than 12 mmol/L).He never had hyperkalemia.In addition, the patient had experienced episodes of sinus bradycardia with a heart rate of 30-35 beats per minute, occasionally for which cardiological investigations including echocardiography and Holter monitoring were done with normal results.Furthermore, the patient encountered persistent high blood pressure up to a maximum level of 200 mmHg and diastolic blood pressure of 110 mmHg.On examination, hepatomegaly with a liver span of 3 cm below the rib edge was also detected.His admission at PICU and then general pediatric neurology ward lasted for 7 months, and a variety of anti-seizure medications including phenobarbital, levetiracetam, lamotrigin, phenytoin, Sabril, clonazepam was prescribed to control his seizures.All electrolyte abnormalities were corrected with a supplementary drug and a low dose of hydrocortisone.Hypothyroidism, hypocalcemia, and hypoparathyroidism were controlled with oral levothyroxine, calcium, and vitamin D supplements.Hypertension was controlled with amilodipine.
Occupational and speech therapy was started to improve his swallowing and gross motor abilities.The patient's seizure frequency was reduced to <50% and finally, after a longstanding hospitalization, he was discharged at 16 months of age.
Regarding his developmental milestones, the patient had never achieved normal motor, speech and cognitive milestones.In addition, he had experienced motor and cognition regression and loosing of fix and follow ability and social smile during the long-standing hospitalization.He gradually lost the ability to swallow as he had constant drooling.Therefore, feeding was established through a nasogastric (NG) tube.Due to persistent abdominal distension, a full set of gastroenterological investigations were also performed, which confirmed gastroenterological autonomic dysfunction and it was treated with Motilium syrup.Besides that, abnormal movement, especially generalized four limbs dystonia, developed after 1 year of age, which was treated with trihexyphenidyl and baclofen, which resulted in significant improvement.
The first brain MRI at 16 months old revealed severe subcortical and deep cerebral, cerebellar and peridentate white matter involvement, symmetric bilateral basal ganglia involvement, severe brain atrophy with large cystic necrotic areas in cerebral hemispheres and severe thinning of the corpus callosum.Furthermore, subdural hematoma, cerebral parenchymal hemorrhage, and cerebellar cortical restriction in diffusion-weighted sequences of brain MRI were detected that were novel imaging findings in this disorder (Figure 2).
On his last physical examination at 2 years old, he had a weight of 8 kg (below the 3rd percentile, z score: −3) and an HC of 44 cm (below the 3rd percentile, z score: −3).He had poor cognition abilities with no purposeful fix and follow, low-beat horizontal nystagmus, and normal pupils' reaction to light.The gag reflex was not satisfactory.He had obvious drooling and feeding was with an NG tube.Due to prolonged feeding with NG tube, Percutaneous Endoscopic Gastrostomy (PEG) was placed for him.Examination of other cranial nerves was not feasible.He was experiencing occasional dystonic postures.Deep tendon reflexes were exaggerated (4+) with clonus and plantar reflexes were bilaterally upward.
TA B L E 1 Summarized review of the phenotypic and genetic findings of all identified patients with variants in NARS2.Motor Function Classification System (GMFCS) score was calculated as five out of five (5/5).On general examination, the skin was normal without any congenital or acquired rash.Mild hepatomegaly was also detected.He had normal heart and respiratory rates and blood pressure.

Phenotype
Other basic metabolic tests, including serum creatine phosphokinase (CPK) and aldolase, urine organic acids and acylcarnitine profiles, and metabolic screen test (MS/MS), were all normal.
Ophthalmologic consultation at age of 18 months was normal.
An auditory brain stem response test (ABR) showed moderate central sensory motor hearing loss in both ears.Renal ultrasonography was normal.A repeated EEG showed a low voltage background and was not remarkable for epileptiform discharge (not shown).Finally, he died at the age of 2 years due to respiratory system failure.

| Patient 2
The patient was a male patient who was 6 months old at first visit and born in a family with first cousin parents without any complications

| Genetic findings
Peripheral blood samples were obtained from the affected patients to GRCh37/hg19 genome assembly, primary filtering out of lowquality reads and probable artifacts, and subsequent annotation of variants was applied.Evaluation was focused on coding exons along with flanking ±20 intronic bases.All disease-causing variants reported in HGMD and ClinVar as well as all variants with minor allele frequency (MAF) of less than 1% in publicly available mutation and polymorphism databases such as 1000 genome project, ExAC (Exome Aggregation Consortium), ESP (Exon Sequencing Projects), gnomAD, and Iranome database were considered.
After several filtering steps, whole-exome sequencing (WES) analysis was done based on previous works 16 and revealed a previously reported pathogenic homozygote missense variant in the NARS2 gene, c.500A > G (p.His167Arg), compatible with the diagnosis of autosomal recessive COXPD24 (OMIM: 616239) disorder in both patients.In order to check the segregation of the variant in the family, sanger sequencing on both patients and their parents was performed (primers and conditions are available upon request).In both families, the parents were identified as heterozygous for the variant (Figure 1B).Using ConSurf (http:// www.consu rf.tau.ac.il) 21 and UCSC database, 22 the evolutionary conservation of the detected variant was analyzed, and it was shown that the variant is highly conserved and exposed (Figure 1C).
As the c.500A > G variant was previously reported, in different databases such as Franklin ACMG automated Classification, it is classified as a Variant with Unknown Significance (VUS), but according to the recommendation of the ACMG for the classification of sequence variants, 23 this variant is reclassified as a likely pathogenic one (PS4, PM2, PP5, PP3, PP4).
Protein 3D modeling was performed to better understand the protein structure (Figure 3A).Amino acid change and its effect on protein 3D structure were assessed using PyMol (Figure 3B). 24ing I-Mutant2.0, it was revealed that the p.(His167Arg) variant could decrease the stability of the protein, which results in protein dysfunction. 25

| DISCUSS ION
In this study, we presented the phenotypes of two unrelated male patients who suffered from epileptic encephalopathy and severe neurodevelopmental delay in association with significant white matter signal changes, subdural hematoma, and cerebral parenchymal hemorrhage in one of these two patients with the same homozygous c.500A > G; p.(His167Arg) variant in NARS2.The c.500A > G variant had been previously reported in an Israeli boy by Mizuguchi et al. 9 We also reviewed all reported patients with variants in NARS2.All data are summarized in a table to better understand the phenotypes associated with NARS2 variants and their ACMG classification (Table 1).
All 3 patients with homozygote c.500A > G variant were born at term without any complications in pregnancy, 9 although some other studies have reported pregnancy complications such as hyperemesis gravidarum, 10 intrauterine growth retardation (IUGR) 6 and breech position. 6Like the patient reported by Mizuguchi et  The brain MRI of the second patient was normal at age of 7 months. 9ogressive cerebral atrophy and subdural space volume expansion could be an explanation for secondary subdural and intraparenchy- Written informed consent was obtained from all participants or their respective guardians.The study was approved by the ethical committee of Tehran University of Medical Sciences as well as the ethical committee of the National Institute for Medical Research Development (NIMAD) of Iran under the codes of ID: IR.NIMAD.REC.1397.508and ID: IR.NIMAD.REC.1399.066,respectively.
mal hemorrhage in the first patient.In a recently published paper describing imaging findings of a large cohort of 132 patients with F I G U R E 1 The Pedigree of the investigated families, Sanger sequencing chromatograms, and multiple sequence alignment.(A) The Pedigree of the investigated families and in these pedigrees, an asterisk (*) indicates the patient that was selected for performing WES.Red symbol: affected and homozygous for the variant; dotted symbols: carriers for the variant; squares: males; circles: females; parallel lines: consanguineous marriage.(B) Sanger sequencing chromatograms showing nucleotide sequences of NARS2 in the regions of c.500A > G which is found in the families.(C) UCSC database used to show the multiple sequence alignment displaying evolutionary conservation of c.500A nucleotide and His167 (H) in the NARS2 gene among different species.Meanwhile, the amino acid sequence of NARS2 protein colored according to the conservation scores provided by the ConSurf server.differenttypes of mitochondrial leukodystrophies, no cerebral hemorrhage was reported.However, the role of mitochondria in secondary brain injury and subarachnoid hemorrhage has been discussed in a systematic review that has been published by Zhang et al.[26][27][28][29] Electrolyte abnormalities and elevated blood lactate levels were detected in both patients.In the patient reported by Mizuguchi et al.9 only elevated serum lactate was reported.Our patients died at the age of 2 years and 1 year, respectively, due to respiratory failure, while in the Mizuguchi et al., the patient was alive at 4 years of age. 9Comparing the phenotype of patients, especially who carried same homozygous or compound heterozygous mutations could show the pleiotropic nature of NARS2-related disorder, which may play an important role in disease diagnosis.The most prevalent phenotype in the reported patients carrying homozygote c.641C > T, c.822G > C, c.1184 T > G, c.545 T > A, and compound heterozygote c.969 T > A and c.1142A > G, c.707 T > G and c.594 + 1G > A, c.1141A > G and c.1290G > C, c.1253G > A and c.1300C > T variants was different types of seizures including epileptic seizures, tonic-clonic seizures, myoclonic seizures. 6,9-11,13,16,17,20Additionally, epilepsy phenotype was reported in patients carrying homozygote c.822G > C, c.631 T > A and compound heterozygote c.83_84del and c.1339A > G, c.707 T > G and c.594 + 1G > A variants. 7,10,23,26,27Developmental delay, intellectual impairment, psychomotor regression and cognitive impairment were also observed in most of patients carrying homozygote c.641C > T, c.822G > C, c.1184 T > G, c.545 T > A, c.951C > T and compound heterozygote c.707 T > G and c.594 + 1G > A, c.1141A > G and c.1290G > C variants. 6,9-12,16,19,20Hearing impairment was another prevalent phenotype observed in patients carrying homozygote c.641C > T, c.637G > T, c.545 T > A, c.951C > T, c.658A > G and compound heterozygote c.969 T > A and c.1142A > G, c.707 T > G and c.594 + 1G > A, c.1141A > G and c.1290G > C variants. 6,8,9,13,15,16,19,20

F I G U R E 3
Protein 3D modeling of NARS2.(A) Schematic structure of NARS2.(B) Amino acid change and its effect on protein 3D structure.F I G U R E 2 Brain MRI of a 16-month old boy (case 1) with mutation in NARS2.Axial T2-Weighted, T1-Weighted and FLAIR sequences, respectively (A-C), and coronal T2-Weighted image (D) show significant supra and infratentorial parenchymal destruction as white matter cystic change and vacuolization with involvement of U fibers, involvement of cerebral and cerebellar cortex, abnormal T2 hyperintensity and T1 hypointensity, cystic change and atrophy of basal ganglia and thalami, involvement of dentate nuclei and ventriculomegaly.The cerebral atrophy and secondary subdural hematoma in different ages are shown by white stars-B.In sagittal T1-Weighted image (E) note the cystic change and abnormal signal intensity of vermis.Axial diffusion weighted images (DWI) images (F, H) and ADC map sequence (G, I) demonstrate parenchymal hemorrhage (white arrowheads) as blooming artifact and restriction of vermis and cerebellar cortex (white arrows) representing acute phase in restricted areas.Axial T2-Weighted views (J-L) show mid brain, pons and medulla oblongata involvement, respectively.
The most common reported clinical and symptoms in NARS2-associated disorders.
olization of the periventricular white matter, basal ganglia, corpus callosum, and cerebellum at 9 months.In this study, brain MRI of the first patient at 16 months old revealed severe subcortical and deep cerebral and cerebellar white matter involvement, symmetric bilateral basal ganglia involvement, severe cerebral atrophy with large cystic necrotic areas in cerebral hemispheres, and severe thinning sion-weighted sequences were novel imaging findings in this patient.