| Revised: 12 October 2023 | Accepted: 22 October 2023

DOI: 10.1002/jcla.24983  

CASE REPORT

Expanding phenotype heterogeneity of NARS2 by presenting
subdural hematoma and parenchymal hemorrhage
Mehrnoosh Khodaeian1 | Fatemeh Bitarafan2
| Fatemeh Garrousi1 |
Elham Amjadi Sardehie1 | Neda Pak3 | Sareh Hosseinpour4 | Marjan Shakiba5 |
Masoumeh Falah6
| Masoud Garshasbi7 | Ali Reza Tavasoli4,8
1

Department of Medical Genetics, DeNA Laboratory, Tehran, Iran

2

Department of Medical Genetics, Oslo University Hospital and University of Oslo, 0450 Oslo, Norway

3

Pediatric Radiology Division, Children's Medical Center, Pediatrics Center of Excellence, Tehran University of Medical Sciences, Tehran, Iran

4

Myelin Disorders Clinic, Pediatric Neurology Division, Children's Medical Center, Pediatrics Center of Excellence, Tehran University of Medical Sciences,
Tehran, Iran
5

Department of Pediatric Endocrinology and Metabolism, Mofid Children's Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6

ENT and Head and Neck Research Center and Department, The Five Senses Health Institute, School of Medicine, Hazrat Rasoul Akram Hospital,
Iran University of Medical Sciences, Tehran, Iran

7

Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Teheran, Iran

8

Department of Neurology, Barrow Neurological Institute, Phoenix Children's Hospital, Phoenix, Arizona, USA

Correspondence
Masoud Garshasbi, Department of
Medical Genetics, Faculty of Medical
Sciences, Tarbiat Modares University,
Tehran, Iran.
Email: [email-masked]
Ali Reza Tavasoli, Myelin Disorders Clinic,
Pediatric Neurology Division, Children's
Medical Center, Pediatrics Center of
Excellence, Tehran University of Medical
Sciences, Tehran, Iran.
Email: [email-masked]
Funding information
National Institute for Medical Research
Development, Grant/Award Number:
electronic application No: 983886; Tehran
University of Medical Scienes, Grant/
Award Number: 96-02-30-35551

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.

[Correction added on 08 December 2023, after first online publication: The affiliation of the co-authors Fatemeh Bitarafan, Masoumeh Falah, and Ali Reza Tavasolii were revised.]  

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2023 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.
J Clin Lab Anal. 2023;37:e24983.
https://doi.org/10.1002/jcla.24983

wileyonlinelibrary.com/journal/jcla

| 1 of 12

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KHODAEIAN et al.

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.
KEYWORDS

asparaginyl-tRNA synthetase 2, cerebral parenchymal hemorrhage, NARS2, subdural
hematoma, whole exome sequencing

1

|

I NTRO D U C TI O N

In this study, we investigated two unrelated Iranian male infants
from two individual families who presented with epileptic encepha-

More than 250 variants in both nuclear and mitochondrial genes

lopathy. Genetic findings revealed a reported biallelic NARS2 variant

have been reported in a heterogeneous group of disorders presented

in both cases; c.500A > G (p.His167Arg). Brain magnetic resonance

with a wide range of clinical manifestations, called mitochondrial

imaging (MRI) of one patient showed novel imaging findings includ-

1

disorders. Among the nuclear genes, those coding for the mito-

ing subdural hematoma, cerebral parenchymal hemorrhage, and cer-

chondrial aminoacyl-tRNA synthetases (mt-aaRSs) have been shown

ebellar cortical restriction in diffusion weighted sequences (DWI)

to be commonly related to human mitochondrial disorders. These

that were not reported previously.9

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. 2,3 The linkage of

2

|

M ATE R I A L S A N D M E TH O DS

2.1 | Ethical statements

amino acids to their cognate tRNAs, which is mediated by mt-aaRSs
is a crucial step in protein synthesis in mitochondria.4,5 Deficiency

Written informed consent was obtained from all participants or

of asparaginyl-tRNA synthetase (NARS2), NP_078954.4, as a mem-

their respective guardians. The study was approved by the ethical

ber of mt-aaRSs group has been reported in association with human

committee of Tehran University of Medical Sciences as well as the

disorders including both autosomal recessive nonsyndromic hearing

ethical committee of the National Institute for Medical Research

loss type 94 (MIM: 618434) and combined oxidative phosphoryla-

Development (NIMAD) of Iran under the codes of ID: IR.NIMAD.

tion deficiency 24 (COXPD24; MIM: 616239) that affects multiple

REC.1397.508 and ID: IR.NIMAD.REC.1399.066, respectively.

organs mostly the central nervous system.6–8
Up till now, 24 variants in NARS2 gene (NM_024678.6) in 35
cases including 20 patients carrying homozygous and 15 patients

2.2 | Case presentation

carrying compound heterozygous variants have been reported in the
literature and presented with a wide spectrum of clinical symptoms

Here, we report two patients from two unrelated Iranian families

even among affected individuals carrying the same variants. All of

who presented with epileptic encephalopathy and were referred to

the data related to families and variants are summarized in Table 1.

Myelin Disorders Clinic, Children's Medical Center, Tehran, Iran, and

Most of the patients who carried pathogenic variants in NARS2

DeNA Laboratory, Department of Medical Genetics, Tehran, Iran

presented with an increased level of serum lactate, refractory ep-

(Figure 1A). The written informed consent was obtained from all

ileptic seizures, sensorineural hearing impairment, intellectual dis-

participants or their legal guardians. Participants also provided writ-

ability, and global psychomotor delay or regression. Other features

ten informed consent for publication of their information included

include progressive microcephaly, renal dysfunction, optic atrophy,

in this paper.

cortical visual impairment, spastic quadriplegia, hemiparesis, flaccid
quadriplegia, urinary retention, amyotrophy, hyperCKemia, excessive fatigue, laryngomalacia, pharyngeal hypotonia, feeding difficul-

2.3 | Clinical outcome

ties, ataxia, brachymetatarsia, hallux valgus, hypotonia, upper limb
tremor, clubbed fingers, mitral valve prolapse developmental delay,

2.3.1 | Patient 1

and brisk deep tendon reflexes, and premature menopause in females was observed in some cases.6,16 The most commonly reported

The patient was a 2-year-old boy, with suspected genetic epileptic

initial phenotype based on age of disease onset is summarized in

encephalopathy, who was first visited at pediatric intensive care

Table 2.

unit (PICU) due to frequent seizures, poor general condition, and

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2 of 12

decreased level of consciousness. He was the second child of con-

range; less than 12 mmol/L). He never had hyperkalemia. In ad-

sanguine parents and was born at term pregnancy through an un-

dition, the patient had experienced episodes of sinus bradycar-

eventful normal vaginal delivery without any significant event during

dia with a heart rate of 30–35 beats per minute, occasionally for

prenatal periods. His birth weight and head circumference (HC) was

which cardiological investigations including echocardiography and

3100 g (z score = 0) and 35.5 cm (z score = 1), respectively. He had a

Holter monitoring were done with normal results. Furthermore,

healthy five-year-old brother.

the patient encountered persistent high blood pressure up to a

His parents first noted a blunted response to environmen-

maximum level of 200 mmHg and diastolic blood pressure of

tal stimuli, such as acoustic stimulus, and social events, such as

110 mmHg. On examination, hepatomegaly with a liver span of

lack of social smile, at 2 months of age. However, he was able to

3 cm below the rib edge was also detected. His admission at PICU

hold his neck. Then, he developed non-fixed transient episodes

and then general pediatric neurology ward lasted for 7 months,

of unilateral torticollis and focal motor and tonic spasm seizures

and a variety of anti-seizure medications including phenobarbital,

at 3.5 months of age. He was hospitalized and his initial labora-

levetiracetam, lamotrigin, phenytoin, Sabril, clonazepam was pre-

tory workups indicated only a low total serum calcium level (total

scribed to control his seizures. All electrolyte abnormalities were

calcium level of 6 mg/dL, normal range: 7.8–10 mg/dL), phospho-

corrected with a supplementary drug and a low dose of hydro-

rus level of 6.8 mg/dL (normal range: 4.5–6.5 mg/dL), magnesium

cortisone. Hypothyroidism, hypocalcemia, and hypoparathyroid-

level of 2 mg/dL (normal range: 1.5–5.5 mg/dL), alkaline phospha-

ism were controlled with oral levothyroxine, calcium, and vitamin

tase of 628 (normal range: 140–720 IU/L), serum lactate level of

D supplements. Hypertension was controlled with amilodipine.

18.7 mmol/L (normal range; 4.5–19.8 mmol/L), and normal level

Occupational and speech therapy was started to improve his swal-

of serum albumin and ammonia. He was prescribed calcium sup-

lowing and gross motor abilities. The patient's seizure frequency

plements in addition to phenobarbital, which resulted in seizure

was reduced to <50% and finally, after a longstanding hospitaliza-

control lasting for a few weeks just based on clinical observation.

tion, he was discharged at 16 months of age.

However, Electroencephalogram (EEG) was not performed due

Regarding his developmental milestones, the patient had never

to family dissatisfaction. On examination at discharge, motor and

achieved normal motor, speech and cognitive milestones. In addi-

cognition delays were detected.

tion, he had experienced motor and cognition regression and loos-

After a few weeks, the patient's seizures flared up with se-

ing of fix and follow ability and social smile during the long-standing

vere restlessness attacks and crying followed by apnea that was

hospitalization. He gradually lost the ability to swallow as he had

lasting up to 1 min following a mild upper respiratory tract in-

constant drooling. Therefore, feeding was established through a

fection and fever. These events resulted in a second hospitaliza-

nasogastric (NG) tube. Due to persistent abdominal distension, a

tion. His seizures evolved to mixed-type ones consisting of focal

full set of gastroenterological investigations were also performed,

motor clonic seizures with lateral gaze, head version, and focal

which confirmed gastroenterological autonomic dysfunction and it

to bilateral and multifocal motor type seizures that ultimately

was treated with Motilium syrup. Besides that, abnormal movement,

led to PICU admission at the age of 9 months. The EEG depicted

especially generalized four limbs dystonia, developed after 1 year of

generalized epileptic discharge, posterior polymorphic delta ac-

age, which was treated with trihexyphenidyl and baclofen, which re-

tivity, and diffuse fast activity due to the benzodiazepine effect

sulted in significant improvement.

(Figure 2). During hospitalization, he encountered a poor general

The first brain MRI at 16 months old revealed severe subcortical

condition and infrequent repetition of seizures. In addition, he

and deep cerebral, cerebellar and peridentate white matter involve-

experienced severe electrolyte imbalances, including low serum

ment, symmetric bilateral basal ganglia involvement, severe brain

total calcium of 6.5 mg/dL, low magnesium level of 1 mg/dL, hy-

atrophy with large cystic necrotic areas in cerebral hemispheres and

ponatremia (Na:106, range: 135–145 mEq/L), mild hypokalemia

severe thinning of the corpus callosum. Furthermore, subdural he-

(3 mmol/L, range: 3.6–5.2 mmol/L), low serum level of 25–OH–

matoma, cerebral parenchymal hemorrhage, and cerebellar cortical

Vit D3, (<8 nmol/L with a normal range of above 30 nmol/L), and

restriction in diffusion-weighted sequences of brain MRI were de-

low serum ACTH level of 3 pg/mL (normal range of 7.1–56.3 pg/

tected that were novel imaging findings in this disorder (Figure 2).

mL). Endocrinology consultation suggested panhypopituitarism.

On his last physical examination at 2 years old, he had a weight

Therefore, a low dose of hydrocortisone tablet was started which

of 8 kg (below the 3rd percentile, z score: −3) and an HC of 44 cm

was helpful to maintain serum electrolytes. Other laboratory tests

(below the 3rd percentile, z score: −3). He had poor cognition abil-

indicated severe microcytic anemia with hemoglobin level of 5 mg/

ities with no purposeful fix and follow, low-beat horizontal nys-

dL (range: 9.5–13 g/dL) and mean corpuscular volume (MCV) of

tagmus, and normal pupils' reaction to light. The gag reflex was

71 fl (range: 75–108), thrombocytopenia (13 × 10 3 , normal range

not satisfactory. He had obvious drooling and feeding was with an

of 150–450 × 10 3), hypoparathyroidism with parathyroid hor-

NG tube. Due to prolonged feeding with NG tube, Percutaneous

mone (PTH) level of 607 pg/mL (range: 15–65 pg/mL), and also

Endoscopic Gastrostomy (PEG) was placed for him. Examination

central hypothyroidism (TSH: 10.7, range: 1.7–7.5 μM). In addition,

of other cranial nerves was not feasible. He was experiencing oc-

serum lactate levels were recorded between 11 and 29 mmol/L,

casional dystonic postures. Deep tendon reflexes were exagger-

and cerebrospinal fluid (CSF) lactate level was 16 mmol/L (normal

ated (4+) with clonus and plantar reflexes were bilaterally upward.

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| 3 of 12

KHODAEIAN et al.

|

KHODAEIAN et al.

TA B L E 1 Summarized review of the phenotypic and genetic findings of all identified patients with variants in NARS2.
NARS2 variants

Pathogenicity

Important phenotype

NARS2
domain

Franklin
ACMG
classification

Seizure

Intellectual
impairment

Muscle disorders

Nucleotide

Protein

Phenotype

c.500A > G
(homozygote)

p.(His167Arg)

COXPD24
(OMM:
616239)

Catalytic

VUS

Yes

Yes

Yes

c.500A > G (1st case)
(homozygote)

p.(His167Arg)

COXPD24

Catalytic

VUS

Yes

Yes

Yes

c.500A > G (2nd case)
(homozygote)

p.(His167Arg)

COXPD24

Catalytic

VUS

Yes

Yes

Yes

c.641C > T
(homozygote)

p.(Pro214Leu)

Alpers

Catalytic

Likely
Pathogenic

Yes

Yes

Yes

c.641C > T
(homozygote)

p.(Pro214Leu)

Alpers

Catalytic

Likely
Pathogenic

Yes

Yes

Yes

c.641C > T
(homozygote)

p.(Pro214Leu)

Alpers/Leigh
syndrome

Catalytic

Likely
Pathogenic

Yes

Yes

Yes

c.822G > C
(homozygote)

p.(Gln274His)

COXPD24

Catalytic

VUS

No

No

Yes

c.822G > C
(homozygote)

p.(Gln274His)

COXPD24

Catalytic

VUS

Yes

Yes

Yes

c.822G > C
(homozygote)

p.(Gln274His)

COXPD24

Catalytic

VUS

No

Yes

Yes

c.969 T > A
c.1142A > G (compound
heterozygote)

p.(Tyr323*)
p.(Asn381Ser)

Leigh syndrome

Catalytic
Catalytic

Pathogenic
Pathogenic

Yes

No

Yes

c.969 T > A
c.1142A > G (compound
heterozygote)

p.(Tyr323*)
p.(Asn381Ser)

Leigh syndrome

Catalytic
Catalytic

Pathogenic
Pathogenic

Yes

No

Yes

c.637G > T
(4 patients)
(homozygote)

p.(Val213Phe)

Autosomal
recessive
deafness-94

Catalytic

Likely
pathogenic

Not
available

Not
available

Not available

c.641C > T
ex. 8–9 del (compound
heterozygote)

p.(Pro214Leu) -

Mitochondrial
disorder

Catalytic
Catalytic

Likely
Pathogenic

Not
available

Not
available

Not available

c.707 T > G
c.594 + 1G > A
(compound
heterozygote)

p.(Phe236Cys)
p.(Asp172_
Glu198)

COXPD24

Catalytic
Catalytic

VUS
Likely
Pathogenic

Yes

Yes

Yes

c.707 T > G
c.594 + 1G > A
(compound
heterozygote)

p.(Phe236Cys)
p.(Asp172_Glu198)

Alpers

Catalytic
Catalytic

VUS
Likely
Pathogenic

Yes

Yes

Yes

c.151C > T
c.1184 T > G
(compound
heterozygote)

p.(Arg51Cys)
p.(Leu395Arg)

COXPD24

Anti codon
binding
Catalytic

VUS
Likely
Pathogenic

Yes

Yes

Yes

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4 of 12

Paraclinical results

MRI

EEG

Lab. Tests

Other features

Survival

Ref.

Cerebral atrophy

Burst suppression pattern

Increased serum
lactate

Hearing impairment,
microcephally, cerebral atrophy

Alive (4 y)

9

Subdural hematoma,
cerebral parenchymal
hemorrhage

Generalized epileptic
discharge, posterior
polymorphic delta activity,
and diffuse fast activity

Elevated lactate in
blood

Panhypopituitarism,
hepatomegaly,
Hypothyroidism,
hypoparathyroidism, speech
problems, feeding problems,
horizontal nystagmus, Hearing
impairment

Dead (2 y)

This
Study

Normal

Not available

Increased serum
lactate

Feeding problems, hearing
impairment

Dead (2 y)

This
Study

Cerebral atrophy

Abnormal

Increased lactate

Hearing impairment, optic atrophy,
microcephaly, urinary retention

Dead (6 y)

10

Cerebral atrophy

Abnormal

Increased lactate

Hearing impairment, optic atrophy,
microcephaly, urinary retention

Dead (16 y)

10

Abnormal signal in basal
ganglia, cortical
atrophy

Abnormal

Increased lactate

Renal dysfunction

Alive (25 y)

6

Not available

Not available

Not available

Mitochondrial myopathy

Alive (34 y)

11

Not available

Not available

Not available

Epilepsy

Alive (26 y)

11

Not available

Not available

Not available

Urinary incontinence, kidney
disorder, scoliosis

Alive (29 y)

12

Hyperintensities

Not available

Increased lactate
levels

Hearing loss, laryngomalacia,
pharyngeal hypotonia

Dead (15 m)

13

Restricted diffusion left
basal ganglia

Not available

Not available

Hearing loss, feeding difficulties

Dead (6 m)

13

Cerebral atrophy,
cerebellar lesion,
basal ganglia lesion

Abnormal

Not available

Nonsyndromic hearing loss,
Premature menopause

Alive
(26-40y)

13

Abnormal

Not available

Not available

Intrauterine growth restriction,
perinatal insult, gastrointestinal
reflex, anemia, lactic acidosis,
ketosis, hyperglycemia

Not available

14

Diffuse atrophy

Abnormal

High serum lactate

Hearing impairment, microcephaly,
flaccid quadriplegia

Alive (8 y)

9

Cerebral atrophy

Abnormal

High serum lactate

Hearing impairment, microcephaly,
flaccid quadriplegia, optic
atrophy, renal dysfunction

Alive (1 y)

9

Diffuse atrophy

Abnormal

High serum lactate

Hearing loss, hemiparesis, flaccid
quadriplegia, pharyngeal
hypotonia

Alive (2 y)

9

(Continues)

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| 5 of 12

KHODAEIAN et al.

|

KHODAEIAN et al.

TA B L E 1 (Continued)
NARS2 variants

Pathogenicity

Important phenotype

NARS2
domain

Franklin
ACMG
classification

Seizure

Intellectual
impairment

Muscle disorders

Nucleotide

Protein

Phenotype

c.167A > G
c.631 T > A
(compound
heterozygote)

p.(Gln56Arg)
p.(Phe211Ile)

Alpers

Anti codon
binding
Catalytic

VUS
Likely
Pathogenic

Yes

No

No

c.167A > G
c.631 T > A
(compound
heterozygote)

p.(Gln56Arg)
p.(Phe211Ile)

Alpers

Anti codon
binding
Catalytic

VUS
Likely
Pathogenic

Yes

No

No

c.545 T > A
(homozygote)

p.(Ile182Lys)

COXPD24

Catalytic

Likely
Pathogenic

Yes

Yes

Yes

c.545 T > A
(homozygote)

p.(Ile182Lys)

COXPD24

Catalytic

Likely
Pathogenic

Yes

Yes

Yes

c.1253G > A
c.1300C > T
(compound
heterozygote)

p.(Arg418His)
p.(Leu434Phe)

COXPD24

Catalytic
Catalytic

VUS
VUS

Yes

Not
available

Not available

c.731C > G
c.1351C > T
(4 patients)
(compound
heterozygote)

p.(Ala244Gly)
p.(Arg451Cys)

Leigh syndrome

Catalytic
Catalytic

VUS
Likely
Pathogenic

Not
available

Not
available

Not available

c.951C > T
(homozygote)

p.(Asn317Asn)

Reversible COX
deficiency

Catalytic

VUS

No

Yes

Yes

c.658A > G (6 patients)
(homozygote)

p.(Met220Val)

HL
Or
NSHL

Catalytic

Likely
Pathogenic

Not
available

Not
available

Not available

c.83_84del
c.1339A > G (compound
heterozygote)

p.(Leu28Glnfs*17)
p.(Met447Val)

Fatal refractory
status
epilepticus

Catalytic

Likely
Pathogenic
Likely
Pathogenic

Yes

Not
available

Yes

c.1141A > G
c.1290G > C
(compound
heterozygote)

p.(Asn381Asp)
p.(Trp430Cys)

COXPD24

Catalytic
Catalytic

Likely
Pathogenic
VUS

Yes

Yes

Yes

Moreover, he had moderate four limbs spasticity, especially in

An auditory brain stem response test (ABR) showed moderate

lower limbs, and notable hand fisting. No scoliosis, joint deformity,

central sensory motor hearing loss in both ears. Renal ultrasonog-

or contracture was detected. His gross motor ability using Gross

raphy was normal. A repeated EEG showed a low voltage back-

Motor Function Classification System (GMFCS) score was calcu-

ground and was not remarkable for epileptiform discharge (not

lated as five out of five (5/5). On general examination, the skin was

shown). Finally, he died at the age of 2 years due to respiratory

normal without any congenital or acquired rash. Mild hepatomeg-

system failure.

aly was also detected. He had normal heart and respiratory rates
and blood pressure.
Other basic metabolic tests, including serum creatine phos-

2.3.2 | Patient 2

phokinase (CPK) and aldolase, urine organic acids and acylcarnitine profiles, and metabolic screen test (MS/MS), were all normal.

The patient was a male patient who was 6 months old at first visit and

Ophthalmologic consultation at age of 18 months was normal.

born in a family with first cousin parents without any complications

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6 of 12

Paraclinical results

MRI

EEG

Lab. Tests

Other features

Survival

Ref.

Cerebral atrophy,
subdural effusions,
cerebral restricted
diffusion

Abnormal

Increased serum
lactate

Auditory neuropathy

Dead (5.5 m)

15

Cerebral atrophy,
subdural effusions,
cerebral restricted
diffusion

Abnormal

Not available

Auditory neuropathy

Dead (9 m)

15

Not available

Bilateral synchronous spike,
polyspike waves

Not available

Auditory neuropathy, progressive
ataxia, clubbed fingers,
brachymetatarsia

Alive (17 y)

16

Not available

Bilateral synchronous spike,
polyspike waves

Not available

Auditory neuropathy

Alive (28 m)

16

Not available

Not available

Not available

Not available

Not available

17

Not available

Not available

Not available

Not available

Not available

18

Asymmetry of the
hippocampus

Abnormal

High serum lactic
acid

Hearing loss, hypotonia

Alive (22 y)

19

Not available

Not available

Not available

Hearing loss, non-syndromic
hearing loss

Not available

8

Progressive cortical and
periventricular brain
atrophy

Focal discharges, multifocal
spikes and sharp waves

Not available

Bradycardia, renal failure

Dead (14 m)

7

Normal

Slow waves, irregular spikes
in the central, parietal, and
temporal regions

Increased alanine
transaminase,
aspartate
transaminase,
alkaline
phosphatase,
lactate
dehydrogenase

Hearing impairment

Dead (6 m)

20

during pregnancy. He revealed seizures at 3 months of age for the

2.4 | Genetic findings

first time and motor delay. He suffered from bilateral sensorineural hearing loss, neurodevelopmental delay, truncal hypotonia, and

Peripheral blood samples were obtained from the affected patients

epileptic encephalopathy. Hypocalcemia, hypomagnesemia, and in-

and their parents. A High Pure PCR template preparation kit (Roche;

creased serum lactate up to 30 mmol/L were also observed. He also

Product No 11814770001) was used to extract genomic DNA. To

presented with swallowing and feeding problems resulting in using

enrich approximately 60 Mb of the Human Exome from fragmented

PEG for feeding. His first brain MRI was normal at 6 months of age,

genomic DNA, Agilent's SureSelect Human All Exon V6 kit was used.

and finally, he died as a result of respiratory system failure, at the

The generated library was sequenced on an Illumina Hiseq 4000

age of 1 year. No additional clinical information, including follow-up

platform to obtain an average coverage depth of 100. Typically, 97%

notes after the first visit at 6 months of age, was available at the time

of the targeted bases were covered >10. An end to end in-house

of study.

bioinformatics pipeline including base calling, alignment of reads

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KHODAEIAN et al.

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KHODAEIAN et al.

TA B L E 2 The most common reported clinical and symptoms in NARS2-associated disorders.
Age of Onset

Number
of cases

Most common presentations

Other reported findings (Rare)

Perinatal/
Neonatal
(Initial
phenotype)

3

Alpers phenotype, opisthotonus posturing, poor head
control, feeding problem

Spastic quadriplegia

Infantile (Initial
phenotype)

13

Alpers/Leigh phenotype, hearing and visual impairment,
psychomotor delay, seizure, microcephaly, nonsyndromic hearing loss, absent DTRs*

Urinary retention, feeding problem, limb spasticity,
nystagmus, hallux valgus, clubbed fingers, mitral
valve prolapse Flaccid or spastic quadriplegia

Childhood (Initial
phenotype)

2

Myopathy, ptosis, fatigue, epilepsy, Intellectual
disability

Dysarthria, Amyotrophy HyperCKemia

Note: *DTRs: Deep Tendon Reflexes.

to GRCh37/hg19 genome assembly, primary filtering out of low-

neurodevelopmental delay in association with significant white mat-

quality reads and probable artifacts, and subsequent annotation

ter signal changes, subdural hematoma, and cerebral parenchymal

of variants was applied. Evaluation was focused on coding exons

hemorrhage in one of these two patients with the same homozygous

along with flanking ±20 intronic bases. All disease-causing variants

c.500A > G; p.(His167Arg) variant in NARS2. The c.500A > G variant

reported in HGMD and ClinVar as well as all variants with minor al-

had been previously reported in an Israeli boy by Mizuguchi et al.9

lele frequency (MAF) of less than 1% in publicly available mutation

We also reviewed all reported patients with variants in NARS2. All

and polymorphism databases such as 1000 genome project, ExAC

data are summarized in a table to better understand the pheno-

(Exome Aggregation Consortium), ESP (Exon Sequencing Projects),

types associated with NARS2 variants and their ACMG classification

gnomAD, and Iranome database were considered.

(Table 1).

After several filtering steps, whole-exome sequencing (WES)

All 3 patients with homozygote c.500A > G variant were born at

analysis was done based on previous works16 and revealed a pre-

term without any complications in pregnancy,9 although some other

viously reported pathogenic homozygote missense variant in the

studies have reported pregnancy complications such as hyperemesis

NARS2 gene, c.500A > G (p.His167Arg), compatible with the diagno-

gravidarum,10 intrauterine growth retardation (IUGR)6 and breech

sis of autosomal recessive COXPD24 (OMIM: 616239) disorder in

position.6 Like the patient reported by Mizuguchi et al. in 2017 who

both patients. In order to check the segregation of the variant in the

manifested with generalized tonic–clonic and myoclonic seizures at

family, sanger sequencing on both patients and their parents was

4 months of age, tonic spasm seizures in case 1 at 3.5 months old,

performed (primers and conditions are available upon request). In

and frequent seizures in case 2 at age of 3 months were observed.

both families, the parents were identified as heterozygous for the

Similar to the patient reported by Mizuguchi et al., who suffered

variant (Figure 1B). Using ConSurf (http://​w ww.​consu​r f.​t au.​ac.​il)21

from intellectual disability and spastic quadriplegia, the first case

and UCSC database, 22 the evolutionary conservation of the de-

had never achieved normal motor, speech, and cognitive milestones

tected variant was analyzed, and it was shown that the variant is

and experienced motor and cognition regression and loss of fix

highly conserved and exposed (Figure 1C).

and follow ability and social smile. Besides, poor general condition,

As the c.500A > G variant was previously reported, in different

decreased level of consciousness, pyramidal, and extrapyramidal

databases such as Franklin ACMG automated Classification, it is

signs in this case were also observed. EEG in patient reported by

classified as a Variant with Unknown Significance (VUS), but accord-

Mizuguchi et al. showed a burst suppression pattern. The EEG of

ing to the recommendation of the ACMG for the classification of

our first patient depicted generalized epileptic discharge, posterior

sequence variants, 23 this variant is reclassified as a likely pathogenic

polymorphic delta activity, and diffuse fast activity.9

one (PS4, PM2, PP5, PP3, PP4).

From neurological examination standpoint, the patient re-

Protein 3D modeling was performed to better understand the

ported by Mizuguchi et al. suffered from mild axial hypotonia at

protein structure (Figure 3A). Amino acid change and its effect on

4 months of age and presented with severe hypertonia and scis-

protein 3D structure were assessed using PyMol (Figure 3B). 24

soring legs at 12 months. In this study, transient episodes of unilat-

Using I-Mutant2.0, it was revealed that the p.(His167Arg) variant

eral torticollis, dystonic postures, increased deep tendon reflexes,

could decrease the stability of the protein, which results in protein

clonus, bilaterally upward plantar reflexes, moderate four limbs

dysfunction. 25

spasticity, especially in lower limbs, and notable hand fisting in
the first patient, and truncal hypotoni and movement disorders

3

|

DISCUSSION

in the second patient were observed. Therefore, movement disorders were documented more obviously in addition to pyramidal
symptoms in our patients compared to the patient was reported

In this study, we presented the phenotypes of two unrelated male

by Mizuguchi et al. ABR showed bilateral sensorineural hearing

patients who suffered from epileptic encephalopathy and severe

loss in all three patients.9

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| 9 of 12

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.
In terms of imaging findings, brain MRI of the patient reported

of the corpus callosum. Furthermore, subdural hematoma, cerebral

by Mizuguchi et al. showed cerebral atrophy with extended vacu-

parenchymal hemorrhage, and cerebellar cortical restriction in diffu-

olization of the periventricular white matter, basal ganglia, corpus

sion-weighted sequences were novel imaging findings in this patient.

callosum, and cerebellum at 9 months. In this study, brain MRI of the

The brain MRI of the second patient was normal at age of 7 months.9

first patient at 16 months old revealed severe subcortical and deep

Progressive cerebral atrophy and subdural space volume expansion

cerebral and cerebellar white matter involvement, symmetric bilat-

could be an explanation for secondary subdural and intraparenchy-

eral basal ganglia involvement, severe cerebral atrophy with large

mal hemorrhage in the first patient. In a recently published paper

cystic necrotic areas in cerebral hemispheres, and severe thinning

describing imaging findings of a large cohort of 132 patients with

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KHODAEIAN et al.

|

KHODAEIAN et al.

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.
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.9
Comparing 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. 6,9–11,13,16,17,20 Additionally, 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.7,10,23,26,27 Developmental

delay, intellectual impairment, psychomotor regression and cognitive impairment were also observed in most of patients carryF 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.

ing 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. 6,9–12,16,19,20
Hearing impairment was another prevalent phenotype observed in

different types of mitochondrial leukodystrophies, no cerebral hem-

patients carrying homozygote c.641C > T, c.637G > T, c.545 T > A,

orrhage was reported. However, the role of mitochondria in second-

c.951C > T, c.658A > G and compound heterozygote c.969 T > A

ary brain injury and subarachnoid hemorrhage has been discussed

and c.1142A > G, c.707 T > G and c.594 + 1G > A, c.1141A > G and

in a systematic review that has been published by Zhang et al. 26–29

c.1290G > C variants. 6,8,9,13,15,16,19,20

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Other clinical outcomes reported in assessed patients include

As congenital anomalies are increasingly the most significant

muscle problems such as spastic quadriplegia, flaccid quadriplegia,

causes of infant abnormality or death, accurate assessment of

myopathy, amyotrophy, scoliosis, hypotonia, severe hypoesthesia in

causative mutations could help affected families in using advanced

lower limbs, severe hallux valgus in feet, upper limb tremor, brisk

molecular tools to prevent giving birth to affected individuals. In

deep tendon reflexes, extensor plantar responses, clubbed fingers,

recent years, various molecular testing, such as array comparative

brachymetatarsia, ataxic gait; visual impairment and optic atrophy;

genomic hybridization (CGH), single-nucleotide polymorphism

microcephaly, excessive fatigue and attention difficulties; kidney re-

(SNP) array, quantitative real-time PCR (qPCR), next-generation

lated disorders such as urinary retention, urinary incontinence and

sequencing (NGS) and non-invasive prenatal testing (NIPT), have

renal dysfunction; hemiparesis, pharyngeal hypotonia, laryngoma-

been developed to help families to have healthy children using

lacia, feeding difficulties, dysarthria and dysphagia.6,9–13,16,19 Some

preimplantation genetic diagnosis (PGD) and prenatal diagno-

least prevalent clinical outcomes observed in some patients include

sis (PND). 30–32

frequent falls, premature menopause, multisystem mitochondrial
disorder, intrauterine growth restriction, perinatal insult, progres-

AU T H O R C O N T R I B U T I O N S

sive ataxia, hyperCKemia, gastrointestinal reflex, anemia, lactic ac-

M.G and ART designed the project, M.Kh. and F.B. and E.A. did the

idosis, ketosis and hyperglycemia.11–14,16

data collection & analysis, M.Kh. and F.B. conducted the experi-

Para clinical tests, such as laboratory tests, MRI, EEG and muscle

ments, A.R.T., N.P., S.H., and M.Sh did the clinical evaluation, M.Kh.,

biopsy results, were available for some of the patients. Increased

F.B., F.G. and M.F. wrote the paper. All authors read and approved

blood lactate level was the most prevalent anomaly in laboratory

the final manuscript.

results.6,9,10,13,15,19
Although MRI results were not available for all patients, abnor-

AC K N OW L E D G M E N T S

mal results were observed in some individuals. T2-weighted hyper-

We would like to thank the families for their participation that made

intensity in different parts of the brain, cerebral atrophy, restricted

this work possible. We sincerely appreciate the staff of DeNA labo-

diffusion in the temporal lobe, left basal ganglia and cerebral cortex,

ratory for helping us in this research.

diffuse atrophy, deep gray matter, subdural effusions, asymmetry of
the hippocampus were the most brain disorders observed in available MRI results.

6,7,9,10,13–15,19

F U N D I N G I N FO R M AT I O N
Research reported in this publication was supported and funded

Bilateral synchronous spike and polyspike waves mainly in the

by the Tehran University of Medical Sciences (Grant number: 96-

posterior part of the brain, focal discharges, multifocal and spikes

02-30-35551) and the National Institute for Medical Research

and sharp waves in addition to slow waves and irregular spikes in the

Development (electronic application No: 983886).

central, parietal, and temporal regions were some of abnormal EEG
results shown in some patients.7,16,19,20
Muscle biopsy in some patients revealed slightly increased lipid/

C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.

atrophic fibers, atrophic fibers, ragged red fibers, enlarged mitochondria, increased lipid profile, mitochondrial subsarcolemmal

DATA AVA I L A B I L I T Y S TAT E M E N T

proliferation, diffuse presence of COX-negative fibers and lipid

The data that support the findings of this study are available on re-

droplets, decreased complexes II and subtle atrophy of the muscle

quest from the corresponding author. The data are not publicly avail-

fibers.6,7,10,11,19

able due to privacy or ethical restrictions.

Among all patients reviewed in this article, 8 patients died at the
ages of 5.5, 6, 9, 14 and 15 months and 6, 16 years old.6,7,10,13,15,20

ORCID

Other patients were alive at the time of the review at the ages of 2 and

Fatemeh Bitarafan

https://orcid.org/0000-0002-4489-2923

28 months and 1, 2, 8, 17, 22, 25, 26, 29 and 34 years old.6,9,11-13,16,19

Masoumeh Falah

https://orcid.org/0000-0002-2800-4199

Other patients were assessed in follow-up studies.8,13,17,18
As the previous studies reported the same variant, c.500A > G
(p.His167Arg), the novel imaging phenotype, diffuse deep cerebral
white matter signal changes, subdural hematoma, cerebral parenchymal hemorrhage, and cerebellar cortical restriction in diffusion
weighted sequences, which was revealed in one of our patients, and
well-documented movement disorders, especially dystonia, could be
considered as new spectrum of NARS2-related disorders and it shows
that more extensive investigations is needed to find out all aspects of
this rare mitochondrial disorder. Although various disease phenotypes
were reported related to NARS2 variants, as the number of known
cases is limited, genotype–phenotype correlation remains unidentified.

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How to cite this article: Khodaeian M, Bitarafan F, Garrousi F,
et al. Expanding phenotype heterogeneity of NARS2 by
presenting subdural hematoma and parenchymal hemorrhage.
J Clin Lab Anal. 2023;37:e24983. doi:10.1002/jcla.24983

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