Gene 704 (2019) 97–102

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Gene
journal homepage: www.elsevier.com/locate/gene

Short communication

Molecular diagnosis of asparagine synthetase (ASNS) deficiency in two
Indian families and literature review of 29 ASNS deficient cases

T

Akella Radha Rama Devia,b, , Shaik Mohammad Naushadb
⁎

a
b

Rainbow Children's Hospital, Banjara Hills, Road No. 2, Hyderabad, India
Sandor Speciality Diagnostics Pvt Ltd, Banjara Hills, Road No.3, Hyderabad, India

ARTICLE INFO

ABSTRACT

Keywords:
Asparagine synthetase deficiency
Microcephaly
ASNS gene
In silico characterization

In the current study, we report three cases of Asparagine Synthetase (ASNS) Deficiency from two consanguineous
families. Family 1 had two early neonatal deaths due to a novel mutation in the ASNS gene c.788C > T
(p.S263F) and both the children presented with microcephaly and one of them had severe intracranial haemorrhage. The proband from the second family was homozygous for c.146G > A (p.R49Q) and manifested
myoclonic seizures, developmental delay, coarse hair and diffuse cortical atrophy. Molecular docking studies of
both the mutations revealed alteration in the ligand binding site. Till date, 26 mutations were reported in ASNS
gene in 29 affected children indicating high degree of genetic heterogeneity and high mortality. Although asparagine depletion is not of diagnostic utility, multiple linear regression model suggested that asparagine levels
vary to the extent of 20.6% based on glutamine and aspartate levels and ASNS deficiency results in depletion of
asparagine synthesis. ASNS deficiency should be suspected in any neonate with microcephaly and epileptic
encephalopathy.

1. Introduction
Asparagine synthetase (ASNS, OMIM 615574) deficiency is an autosomal recessive neurometabolic disorder of amino acid metabolism
characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy and intractable seizures. ASNS deficiency was
first described in four families with neurological symptoms (Ruzzo
et al., 2013). Asparagine synthetase also known as aspartate-ammonia
ligase, is an enzyme involved in the biosynthesis of asparagine from
aspartate through an ATP-dependent transamination reaction in the
presence of glutamine which acts as amino group donor (Zhang et al.,
1989). Enzyme deficiency blocks asparagine synthesis resulting in low
asparagine levels in body fluids. Since ASNS is highly expressed in the
brain, the levels will be significantly low in CSF (Hongo et al., 1994).
This is a pan-ethnic disorder and has been identified within a variety
of ethnic groups with reports from Iran, Bangladesh, Arabian, French
Canadian and Indian families. Worldwide carrier frequency is reported
to be 1 in 2049, highest among Sephardic - Iranian Jewish 1:80, South
Asia 1 in 3072. (Asparagine Synthetase (ASNS) Deficiency– Sema4).
The first report of Japanese patients with ASNS deficiency is reported

recently (Yamamoto et al., 2017).
Asparagine synthetase, the human ASNS enzyme, is a 65 kDa protein that has two primary domains, termed the N- and C-terminal domains. The human ASNS gene is located at chromosome 7q21.3 and is
35 kb long with 13 exons (Heng et al., 1994). Biochemically, plasma
concentration of asparagine is variable with only a few manifesting
lower levels of asparagine. Diagnosis is difficult in suspected cases and
can only be diagnosed through DNA sequencing. Presence of microcephaly at birth suggests that brain ASNS activity is critical for the brain
development. Four patients are identified in India till date, and our
report adds three more affected infants from two families with novel
mutations.
2. Materials and methods
The patients were recruited for the study after obtaining informed
consent from the parents. The study protocol was approved by the
Institutional Ethical Committee of Rainbow Children's Hospital,
Hyderabad, India.

Abbreviations: ASNS, asparagine synthetase deficiency; SD, standard deviation; ExAC, Exome aggregation Consortium; TMS, Tandem mass spectrometry; EEG,
electroencephalogram
⁎
Corresponding author at: Rainbow Children's Hospital, Banjara Hills, Road No. 2, Sandor Speciality Diagnostics Pvt Ltd, Banjara Hills, Road No.3, Hyderabad,
India.
E-mail address: radharamadevi@sandor.co.in (A. Radha Rama Devi).
https://doi.org/10.1016/j.gene.2019.04.024
Received 5 March 2019; Received in revised form 8 April 2019; Accepted 8 April 2019
Available online 09 April 2019
0378-1119/ © 2019 Published by Elsevier B.V.

Gene 704 (2019) 97–102

A. Radha Rama Devi and S.M. Naushad

2.1. Patient 1
The first male child, to a consanguineous couple, born by caesarean
section with a birth weight of 3.0 kg and the head circumference was
30 cm and length was 50 cm. The child has been lethargic since birth
with refusal to feeds and manifested seizures on the 5th day of life.
Metabolic screening by Tandem mass spectrometry revealed normal
acyl carnitine profile and amino acids. There were no dysmorphic
features. The neonate was hemodynamically unstable and was ventilated. MRI brain revealed extensive intraventricular haemorrhage and
in cisterna magna. Diffuse cerebral atrophy, incomplete sulcal formation, no myelin signal was seen on T2 weighed images in the posterior
limb of the internal capsule. The child expired at one month of age.
2.2. Patient 2
Patient 2 was the sister of the 1st patient, was evaluated at one
month, with microcephaly at birth, excessive irritability and refusal to
feed. The baby was born by caesarean section at 40 weeks of gestation.
Birth weight was 2.9 kg, head circumference of 30 cm and length of
52 cm. At evaluation weight was 3.6 kg. Head circumference was 32 cm
(< 5th Centile) suggestive of severe microcephaly. The child was lethargic and not accepting feeds. No neurological signs were evident
except for hyperreflexia. MRI brain showed diffuse cerebral atrophy,
white matter cystic changes in the anterior frontal and temporal region
with thinned out brain stem. There were no obvious seizures but the
electroencephalogram (EEG) showed multifocal seizure disorder. The
child became comatose and expired on the 5th day of admission.
2.3. Patient 3
A 3-yr-old girl born to consanguineous parents, first in birth order,
presented with myoclonic seizures since birth. Developmental delay
and seizures were present since birth with EEG showing diffuse seizure
activity. She had coarse hair, microcephaly (head circumference < 3rd
centile). MRI brain revealed diffuse cortical atrophy. TMS was normal
with no metabolic cause. Fundus examination was normal. She was
globally delayed in development. An exome sequencing was performed
which revealed ASNS mutation along with a homozygous mutation in
ACT1 gene. In view of this, urine organic acid analysis was done which
was normal with no increased excretion of triglyl glycine. Her urine
ketones were negative. Hence the possibility of Beta ketothiolase was
excluded. Further evaluation was not possible as the child was lost to
follow up.

Fig. 1. Two dimensional representation of interactions of ASNS protein with its
ligands.
The catalytic site of ASNS protein was represented in the form of individual
amino acid residues interacting with various ligands. Aspartate, glutamate and
ATP are the reactants required for the synthesis of asparagine. Beta aspartyl
AMP intermediate is the reaction intermediate. We calculated bond length
between 256L and ATP; and 365E with that of reaction of intermediate. We
observed that R49Q mutant has lesser affinity towards ATP when compared to
wild and S263F mutant. The reaction intermediate is strongly held in S263F and
R49Q mutants than the wild protein. Hence, it is proposed that decreased
clearance of the product from the catalytic site might be resulting in feedback
inhibition of ASNS enzyme.

2.4. Molecular analysis
Exome Sequencing is performed using next generation sequencing.
The genome is sequenced to mean > 80–100× coverage and a
minimum of ~84% of bases are sequenced to at least 20× coverage.
Paired-end 151 bp reads are aligned to the NCBI reference sequence
(GRCh37.75) using the bowtie2.2.1 short read aligner, and variant calls
are made using the Annovar. Variants are subsequently filtered to
identify Variants with a minor allele frequency < 5%, Variants classified as disease causing mutations in public databases, Predicted lossof-function variants with a minor allele frequency < 5% in the patientspecific phenotype-driven gene list.

initio. The wild and mutant proteins (S180F and R49Q) were modeled
(Fig. 1). The 3DLigandSite module (Wass et al., 2010) was used for the
prediction of ligand binding site (Fig. 2). The impact of ASNS deficiency
in reducing cortical thickenning and in dilating lateral ventricles is
schematically represented (See Fig. 3.)

2.5. In silico analysis
Using the crystal structure of asparagine synthetase B from
Escherichia coli (c1ct9D) as a template, we have developed a homology
model of asparagine synthetase (Homo sapiens, NM_133436.3) using
Phyre2 web portal (Kelley et al., 2015). A total of 499 residues (89% of
target sequence) were modeled with 100% confidence using this
highest scoring template. The remaining 62 residues were modeled ab

2.6. Statistical analysis
Student t-test was used to compare the distribution of glutamine,
aspartate and asparagine in cases and controls. In order to assess the
interrelationships of asparagine with glutamine, aspartate and
98

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A. Radha Rama Devi and S.M. Naushad

Fig. 2. Multiple linear regression model of asparagine synthesis.
Using glutamine, aspartate and presence or absence of ASNS deficiency as input
variables, we have predicted the asparagine levels. Glutamine and aspartate
contribute 20.6% variability in asparagine levels. ASNS deficiency was associated with depleted asparagine synthesis. R: Spearman rank correlation coefficient.

asparagine synthase deficiency, a multiple linear regression equation
was deduced. The data of seven ASNS deficiency cases and 26 healthy
children comprising of glutamine, aspartate and asparagine levels was
the basis of this model. All these values were expressed in μmol/L. The
input variables are glutamine, aspartate and presence (1) or absence (0)
of ASNS deficiency. The output variable is asparagine level. The resultant equation is expressed as y = m1x1 + m2x2 + m3x3 + C where in
‘y’ corresponds to asparagine while x1, x2 and x3 correspond to glutamine, aspartate, ASNS deficiency (0/1). The m1, m2, m3 values signify
the impact of corresponding input variables and the ‘+’ sign denotes
positive association and ‘−’ denotes inverse association. The actual vs.
predicted values of asparagine were plotted against each other to calculate the spearman rank correlation coefficient ‘r’.
3. Results

Fig. 3. Schema depicting the clinical implications of ASNS deficiency.
In ASNS deficient subjects, as a result of reduced expression of ASNS in neuronal progenitor and post mitotic neurons, increased cell death happens in
utero contributing to reduced cortical thickness and increased dilatation of
lateral ventricles thus manifesting as microcephaly at birth.

3.1. Molecular diagnosis
In the Family 1, the dried blood spot of the 1st child was retrieved
from the new-born screening laboratory and DNA analysis was performed. Both the patients were homozygous for an unreported missense
mutation NM_133436.3 (ASNS): c.788C > T (p.S263F). This variant is
novel and not observed in 1000G database and ExAC database. This
variant is predicted to be damaging by SIFT, Polyphen2, LRT_Pred,
Mutation Taster, Mutation Assessor, RadialSVR and Phenolyzer. Parents
were carriers for the same mutation seen in the children. The mutation
is inherited as both the parents were carriers. Family 2, the affected girl
was homozygous mutant for NM_133436.3 (ASNS): c.146G > A
(p.R49Q). One carrier of this mutation was identified in ExAC database.

beta aspartyl AMP intermediate. Molecular docking studies showed that
wild protein has higher bond-length (0.31A°) compared to S180F
(0.10A°) and R49Q (0.18°) mutants between 365Leu and beta aspartyl
AMP intermediate. This indicates decreased clearance of the product in
the mutants resulting in feedback inhibition of the enzyme (Fig. 1).
The S263F mutation corresponds to 263Ser in this transcript. As
shown in Fig. 1, this mutation is altering the tertiary structure of the
protein by uncoiling of one of the helical structures probably due to
steric repulsion by 263Phe. As shown in Fig. 2, this mutation alters the
ligand binding site, which contains 256Leu, 257Leu, 258Ser, 286Phe,
287Ala, 288Ileu, 365Glu intact while 363Ser, 404Arg and 546Asp residues being the new residues forming the site. The 553Thr, 554His,
555Tyr, 556Lys and 557Ser residues are not taking part in the binding
site formation in the mutated protein.
As shown in Fig. 2, The R49Q mutation although not occurred in
residues that form ligand binding sites, the mutated protein has 256Leu,
257Leu, 258Ser, 263Ser, 286Phe, 287Ala, 288Ileu, 365Glu residues as
in the wild protein, 363Ser and 368Asp additional residues forming the
ligand binding site. Due to the mutation, 553Thr, 554His, 555Tyr,
556Lys and 557Ser residues are not part of binding site.

3.2. In silico analysis of functional implications
The wild ASNS protein was predicted to form ligand binding site
using 256Leu, 257Leu, 258Ser, 263Ser, 286Phe, 287Ala, 288Ileu,
365Glu, 553Thr, 554His, 555Tyr, 556Lys and 557Ser residues. The
256Leu, 288Ileu are the binding sites for ATP via carbonyl oxygen and
via amide nitrogen respectively. Through molecular docking studies we
calculated the H-bond length between the 256Leu residue and carbonyl
oxygen of ATP in wild vs. mutant proteins. The H-bond length was similar in wild and S180F mutant (0.02A°) while the bond length is larger
in R49Q mutant (0.09A°). The 365Glu is the site for the formation of
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Table 1
Comparison of cases from the current study with existing literature.
Variable

Value

Total number of cases
Male: female
Age group (yr)
Consanguinity
Developmental delay
Epilepsy
Microcephaly
Spasticity
Cortical blindness
Mortality
ASNS mutation

29
17:9
0.01–14
12/26 (46.15%)
28/29 (96.6%)
23/29 (79.3%)
28/29 (96.6%)
26/29 (89.7%)
8/29 (27.6%)
13/26 (50.0%)
n = 26

Patient 1

Patient 2

Patient 3

Male
0.08
Yes
Yes
Yes
Yes
No
No
Yes
c.788C > T
(S263F)

Female
0.08
Yes
Yes
No
Yes
Yes
No
Yes
c.788C > T
(S263F)

Female
3 yr
Yes
Yes
Yes
Yes
No
No
No
c.146 G > A
(R49Q)

Note: In three cases, information on gender, consanguinity and mortality could not be obtained.

3.3. Review of all the reported cases

(Gupta et al., 2017; Galada et al., 2018; Sun et al., 2016). Mutations in
Japanese were: c.434T > C, c.740T > G, c.1466T > A and
c.1623_1624del (Yamamoto et al., 2017). Arabians had c.932 A > G,
c.1121A > G, c.1193A > C, c.1219C > T mutations (Seidahmed
et al., 2016; Alfadhel et al., 2015; Ben-Salem et al., 2014). The
c.1648C > T mutation appears to be an ancestral mutation for French
population (Ruzzo et al., 2013; Gataullina et al., 2016). The
c.1439C > T in French population and c.17C > A in French-Canadian
population might have emerged after the divergence of these two population groups (Ruzzo et al., 2013; Gataullina et al., 2016). The
c.866G > C and c.1010C > T mutations were reported among Australians (Palmer et al., 2015) while c.601delA and c.1165G > C mutations were frequent in German., USA exhibited c.728 T > C and
c.1097G > A mutations (Schleinitz et al., 2018), Iranians had
c.1084 T > G mutation (Ruzzo et al., 2013). Thus high degree of genetic heterogeneity is observed in ASNS deficiency despite identical
phenotypic spectrum (Table 3).
Diffuse cerebral atrophy, incomplete sulcal formation, myelination
defects are the hallmark features of ASNS deficiency. ASNS activity in
the brain is crucial for organ development, and a tissue-specific dependence on asparagine for neural development (Ruzzo et al., 2013)
could cause the characteristic brain changes seen in these patients at
birth. Reviewing 29 individuals reported till now, a significant proportion (22/29) manifested microcephaly and abnormal gyral pattern.
Although seizures are reported to be refractory in ASNS deficiency,
both the sibs in one family did not present with clinical seizures
whereas the third child had refractory seizures. Progression of the
disease could be rapid as reported in an earlier study where in two sibs
had eventration of the diaphragm resulting in severe respiratory complications and death (Sun et al., 2016). Although it is a rare coincidental
association, it was the first abnormality reported outside the central
nervous system in this disorder indicating the possibility of multiple
organ involvements in ASNS deficiency. In the current study, extensive
intraventricular haemorrhage occurred in one infant and none of the
cases reported till now manifested cerebral haemorrhage. Asparagine is
reported to be essential for endothelial cell growth which is important
for angiogenesis. Consistently, fibroblasts proliferation was markedly
reduced under conditions of asparagine deprivation. Studies on patients
with Acute Lymphatic Leukaemia on Asparaginase treatment showed
depletion of fibrinogen (Alqasim et al., 2018) and inhibition of the
biosynthesis of hepatic coagulation factors (Rota et al., 2016). Clinical
data suggest that asparagine depletion is effective in treating hematologic malignancies. However, coagulation defects and platelet dysfunction are noted in asparaginase treatment (Jaccard et al., 2010).
This may be the possible cause of intracranial bleed in patients with
asparagine deficiency.
The c.788C > T is a novel mutation in this family and predicted to
be deleterious by different bioinformatics tools. The mutation segregating in the family with recurrence indicates the pathogenicity of the

Including the three cases reported in the current study, there are 29
cases of ASNS deficiency reported till date. The age at presentation
varied from 0.01 to 14 yr. Consanguinity was observed in 46.15% cases.
Developmental delay, epilepsy, microcephaly and spasticity are the key
features accounting for 96.6%, 79.3%, 96.6% and 89.7% of cases respectively. Cortical blindness was observed in 27.6% cases. High mortality (50.0%) is reported in the affected cases. The mean age of death
was 0.42 ± 0.42 yr while the mean age of surviving children was
4.55 ± 4.74 yr (p = 0.004) (Table 1).
Plasma and CSF levels of asparagine levels could not be performed
in the reported cases as they expired before the diagnosis. Few studies
have explored asparagine (mean ± SD: 29.4 ± 20.0 μmol/L, n = 14),
glutamine (mean ± SD: 663.6 ± 305.9 μmol/L, n = 11) and aspartate
(mean ± SD: 10.3 ± 9.9 μmol/L, n = 9) levels in ASNS deficient patients. When compared to control children, the asparagine levels were
significantly lowered in ASNS deficiency (p < 0.0001) (Table 2).
In order to assess the extent of depletion of asparagine synthesis in
ASNS patients, we have compared the data of ASNS patients with controls (n = 26). A multiple linear regression equation was deduced to
predict asparagine levels as a function of glutamine and aspartate in
cases as well as controls. Asparagine (μmol/L) = (0.021 × glutamine) +
(0.374 × aspartate) − (16.55 × ASNS deficiency) + 39.29.
This equation explains 20.6% variability in asparagine levels. There
is a significant depletion of asparagine in ASNS deficiency cases (Fig. 2).
4. Discussion
Till date, 26 different mutations were reported in the ASNS gene, 23
missense and three are non-sense mutation (two frame shift mutations
and one point mutation). Seven of these mutations occurred in exon 5.
Till date, seven cases of ASNS deficiency were reported from India. The
reported mutations in India were: c.146G > A, c.224A > G and
c.413A > C, c.788C > T of exon 1; c.1019G > A of exon 4;
c.1138G > T and c.1211G > A of exon 5; and c.1649G > A of exon 6
Table 2
Biochemical markers of ASNS deficiency.
Amino acid

Cases

Controls

P value

Asparagine (μmol/L)

29.4 ± 20.0
(n = 14)
10.3 ± 9.9
(n = 9)
663.6 ± 305.9
(n = 11)

57.9 ± 16.6
(n = 26)
10.7 ± 4.9
(n = 26)
701.3 ± 323.4
(n = 26)

< 0.0001a

Aspartate (μmol/L)
Glutamine (μmol/L)

0.87
0.74

The values were represented in mean ± standard deviation format.
a
Statistically significant.
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Table 3
Frequency of different ASNS mutations reported till date.
Exon

Nucleotide change

Protein change

No of mutated alleles

Frequency (%)

Population

1
1
1
1
1
3
3
3
4
4
4
5
5
5
5
5
5
5
6
6
6
8
8
9
10
10

c.17C > A
c.146G > A*
c.224A > G
c.413A > C
c.434T > C
c.601delA
c.728T > C
c.740T > G
c.788C > T*
c.866G > C
c.932A > G
c.1010C > T
c.1019G > A
c.1084T > G
c.1097G > A
c.1121A > G
c.1138G > T
c.1165G > C
c.1193A > C
c.1211G > A
c.1219C > T
c.1439C > T
c.1466T > A
c.1623_1624del
c.1648C > T
c.1649G > A

A6E
R49Q
N75S
D138A
L145S
M201Wfs*28
V243A
L247W
S263F
G289A
Y311C
T337I
R340H
F362V
G374E
Y374C
A380S
E389Q
Y398S
R404H
R407*
S480F
V489D
W541Cfs*5
R550C
R550H

3
2
1
1
1
2
1
1
2
1
2
1
4
6
1
4
2
2
2
2
2
2
1
1
9
2

5.17
3.45
1.72
1.72
1.72
3.45
1.72
1.72
3.45
1.72
3.45
1.72
6.90
10.34
1.72
6.90
3.45
3.45
3.45
3.45
3.45
3.85
1.72
1.72
15.52
3.45

French-Canadian
Indian
Indian
Indian
Japanese
German
USA
Japanese
Indian
Australian
Arabian
Australian
Indian
Iranian
USA
Arabian
Indian
German
Arabian
Indian
Arabian
French
Japanese
Japanese
French, French-Canadian, Bangladeshi
Indian

*: Mutations identified in the current study.

Disclosure of potential conflicts of interest

novel mutation. In silico analysis revealed that this mutation occurred
in the 263Ser residue that is crucial part of ligand binding site. The
second mutation i.e. R49Q although distant to the key binding sites,
there is conformational change leading to altered ligand binding site.
The binding affinities of both mutants are presumed to be lower than
the wild ASNS due to loss of interaction of ligand with 553Thr, 554His,
555Tyr, 556Lys and 557Ser residues. The R49Q has lesser affinity towards ATP compared to wild and S263F proteins. The beta aspartyl
AMP intermediate has higher affinity towards 365Glu in S263F than the
wild and R49Q mutant resulting in decreased clearance of the product
and feedback inhibition of ASNS.
ASNSD is one of the defects in synthesis like creatine deficiency
syndromes (Stöckler et al., 1994), glutamine synthetase deficiency
(Häberle et al., 2006), and the serine synthetic defects (Crabben et al.,
2013). Since there is a deficiency of end product in these cases, a
therapeutic benefit of supplementation has been reported in serine
biosynthetic disorders and glutamine synthetase deficiency. Worsening
of seizures after asparagine supplementation in a child with asparagine
synthetase deficiency is reported (Alrifai and Alfadhel, 2016). Value of
supplementing asparagine in ASNS deficiency is questionable as the
brain is affected at birth.

Both the authors hereby declare no conflicts of interest.
Research involving human participants and/or animals
This study complied with the ethical principles outlined in the
Declaration of Helsinki.
Informed consent
Informed consent was obtained from the parents or guardians of the
patients.
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4.1. Conclusion
ASNS deficiency is a progressive disease with no cure ultimately
leading to death. Biochemical testing is not always helpful as majority
can have normal asparagine levels in plasma. DNA testing is the ultimate diagnosis and should be done in all patients with microcephaly for
proper counselling and prenatal diagnosis in subsequent pregnancies.
The reported variants in the current study i.e. S263F and R49Q are
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Acknowledgements
We thank both the families who participated in this study. No
specific funding was obtained from any agency.
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