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Novel complex Re-Arrangement of ARG1 commonly shared by unrelated
patients with Hyperargininemia
Jafar Mohseni
a
, Chia Boon Hock
a
, Che Abdul Razak
a
, Syah Nor Iman Othman
d
, Fatemeh Hayati
a
,
Winnie Ong PeiTee
c
, Muzhirah Haniffa
c
, Bin Alwi Zilfalil
b
, Rowani Mohd. Rawi
b
,
Lock-Hock Ngu
c
, Teguh Haryo Sasongko
a,

a
Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
b
Department of Pediatrics, School of Medical Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
c
Genetic Department, Kuala Lumpur Hospital, 50586 Jalan Pahang, Kuala Lumpur, Malaysia
d
Institute of Biological Sciences, Faculty of Science, Universiti Malaya, Kuala Lumpur, Malaysia
a b s t r a c t a r t i c l e i n f o
Article history:
Accepted 23 September 2013
Available online 5 October 2013
Keywords:
Hyperargininemia
Arginase deciency
ARG1
Complex re-arrangement
Background: Hyperargininemia is a very rare progressive neurometabolic disorder caused by deciency of hepatic
cytosolic arginase I, resulting from mutations in the ARG1 gene. Until now, some mutations were reported
worldwide and none of them were of Southeast Asian origins. Furthermore, most reported mutations were
point mutations and a few others deletions or insertions.
Objective: This study aims at identifying the disease-causing mutation in the ARG1 gene of Malaysian patients
with hyperargininemia.
Methodology: We employed a series of PCR amplications and direct sequencing in order to identify the mutation.
We subsequently used quantitative real-time PCR to determine the copy number of the exons anking the muta-
tion. We blasted our sequencing data with that of the reference sequence in the NCBI in order to obtain positional
insights of the mutation.
Results: We found a novel complex re-arrangement involving insertion, inversion and gross deletion of ARG1
(designated g.insIVS1+1899GTTTTATCAT;g.invIVS1+1933_+1953;g.delIVS1+1954_IVS2+914;c.del116_188;
p.Pro20SerfsX4) commonly shared by 5 patients with hyperargininemia, each originating from different family.
None of the affected families share known relationship with each other, although four of the ve patients were
known to have rst-cousin consanguineous parents.
Conclusion: This is the rst report of complex re-arrangement in the ARG1. Further analyses showing that the
patients have shared the same geographic origin within the northeastern part of Malaysia prompted us to suggest a
simple molecular screening of hyperargininemia within related ethnicities using a long-range PCR.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Arginase I is the nal enzyme in the urea cycle that catalyzes the
hydrolysis of arginine to urea and ornithine. Arginase I is the product
of ARG1, of which mutations have been described to cause deciency of
the enzyme. ARG1 is located in the long arm of chromosome 6 (6q23)
and is made up of eight exons that code for 322 amino acids (Iyer et al.,
1998).
Hepatic Arginase I Deciency causes hyperargininemia (OMIM #
207800), a rare inborn errors of urea cycle which is inherited in an auto-
somal recessive manner withthe incidence of only 1 in2,000,000 (Scaglia
and Lee, 2006). Unlike the other inborn errors of urea cycle which usually
present with an acute hyperammonemic crisis, hyperargininemia usually
has a more chronic presentation in later childhood with neurologic
features such as delayed developmental milestones, spastic diplegia,
learning difculties, and epilepsy (Iyer et al., 1998; Lee et al., 2011). The
extreme spasticity observed in this disorder is not found in other urea
cycle defects and the frequent epileptic symptom is seldom related to
episodes of hyperammonemia (Scaglia and Lee, 2006). Some researchers
have suggested that anaccumulation of guanidino compounds that could
interfere with GABAergic transmission is the possible pathogenic
mechanism (Lee et al., 2011). Guanidino compounds have been
shown to inhibit the cerebral cortical sodiumpotassium adenosine
triphosphatase (ATPase) of rats at concentrations comparable with
those seen in affected humans (Scaglia et al., 2004). The ATPase is
Gene 533 (2014) 240245
Abbreviations: ARG1, Arginase 1 Gene; ATPase, Adenosine Triphosphatase; DNA,
DeoxyriboNucleic Acid; dNTP, deoxyribuNucleotide TriPhosphate; GABA, Gamma-
AminoButyric Acid; NCBI, National Center for Biotechnology Information; NHLBI,
National Heart, Lung and Blood Institute; OMIM, Online Mendelian Inheritance in Man;
PCR, Polymerase Chain Reaction; qPCR, quantitative real-time PCR; RefSeq, Reference
Sequence; Sdn. Bhd, Sendirian Berhad (Co. Ltd. in Malaysian).
Corresponding author. Tel.: +60 97676794/6796; fax: +60 97658914.
E-mail addresses: teguhharyosasongko@yahoo.com, teguhhs@kk.usm.my
(T.H. Sasongko).
0378-1119/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.gene.2013.09.081
Contents lists available at ScienceDirect
Gene
j our nal homepage: www. el sevi er . com/ l ocat e/ gene
Author's personal copy
essential to maintenance of the electrochemical gradient of neurons, and
its inhibition may also be involved in the pathogenesis of the seizure
disorder associated with this disease (Mori et al., 1990).
The HumanGene MutationDatabase (Stenson, 2009) reportedto date
at least 37 mutations have been identied in ARG1 gene among different
populations worldwide. Reported ARG1 mutations were mostly missense,
with only very few cases involving deletion and/or insertion without
reported complex re-arrangement (Stenson, 2009). In addition, there
has been no documented evidence of a common ARG1 mutation shared
by patients within the same or related ethnicities, thus making it difcult
for simple molecular diagnostic screening (Carvalho et al., 2012; Tsang
et al., 2012; Vockley et al., 1996). Here we reported a novel complex re-
arrangement mutation of ARG1 shared by 5 unrelated patients with
hyperargininemia andsuggesteda simple molecular diagnostic screening.
2. Methods
2.1. Patients and DNA Extraction
Six patients were referredto our laboratory inHumanGenome Centre
Universiti Sains Malaysia for molecular genetic analyses of ARG1 gene,
from Hospital Kuala Lumpur and Hospital Universiti Sains Malaysia. All
patients were of Malay ethnicity. Information on parental origins was ob-
tained through interview. Family pedigree of each patient was analysed
for three generations. Diagnosis of hyperargininemia was established
based on clinical features and laboratory analysis. Informed consent
was taken prior to blood taking. Genomic DNA was extracted from
whole blood using commercially available kit (QIAamp DNA Mini
Blood Extraction).
2.2. Polymerase Chain Reaction and Direct DNA Sequencing
Polymerase Chain Reaction was performed using GeneAmp PCR
System 9700 (Applied Biosystems, Foster City, CA, USA). The primers
for each exon of ARG1 gene were synthesized according to sequences as
described previously (Cardoso et al., 1999; Grody et al., 1992; Uchino
et al., 1992).
In order to clarify amplication failure of ARG1 exon 2, we employed
long-range PCR amplication using two of the primers described previ-
ously; forward primer of 5-TGACTGACTGGAGAGCTCAAG-3 (Uchino
et al., 1992) and reverse primer of 5-ACACAGTTATTCAACAAGACC-3
(Cardoso et al., 1999). The long-range PCR condition was 2 min at 94
C for initial denaturation, followed by 12 sec at 94 C, 30 sec at 60 C
and 15 min at 68 C for 30 cycles and a nal extension done at 68 C
for 7 min. Amplications were performed in 30 L volume containing
1g of genomic DNA, 1X Buffer S, 100M dNTPs, 1.1M of each primer
and 2 U MaxTaq polymerase (Vivantis Technology, USA) (Fig. 1).
We subsequently designed a series of primers (refer to Fig. 2(B)) in
order to identify the breakpoint deletion based on the ARG1 genomic
sequence provided in the NCBI Nucleotide Database (accession no.
GI_163954920; RefSeq NG_007086.2; Primers were synthesized by
First Base Laboratories Sdn. Bhd., Malaysia). Direct DNA Sequencing was
done to identify the mutations, using ABI Prism 3100 Genetic Analyzer
(Applied Biosystems, Foster City, CA, USA).
2.3. Real-Time qPCR
In order to provide information on homozygosity of the deletion, we
performed real-time quantitative PCR for identifying the copy number of
exons 1 and 3 which are anking the deleted region. The real-time quan-
titative PCR was performed according to previously described method
(Tran et al., 2008) using LightCycler1.5 (Roche Diagnostics, Mannheim,
Germany) and Human-actinas reference gene. Eachexperiment used 3
serial template dilutions and was done twice (duplicate experiments).
2.4. Bioinformatics Analysis
We used Basic Local Alignment Search Tool (BLAST) provided by the
NCBI Nucleotide Database, 2013 (http://blast.ncbi.nlm.nih.gov/Blast.cgi),
in order to obtain positional insights into the deleted segment.
3. Results
3.1. Patients Features
Table 1 summarizes the phenotypes, genotypes and other informa-
tion of the patients. All patients showed neurological decits as well as
striking elevation of plasma arginine level. Almost all showed delayed
developmental delay, while one of the patients showed acute infantile
hyperammonemia. All were known to have parental origin from
Kelantan, one state in the north-eastern part of Peninsular Malaysia. All
but one patient were born from rst-cousin consanguineous marriage.
Three-generation pedigree analyses of all the patients showed no rela-
tionship between any families.
3.2. Molecular Genetic Analyses
We initially tried to amplify each of the 8 exons in the ARG1of all the
6 patients. In one of the patients [patient ID 002(2)] we successfully
amplied all of the 8 exons. Upon direct DNA sequencing, we identied
a nonsense mutation in ARG1 exon 8 (Table 1). However, we experi-
enced consistent failure in amplifying exon 2 of the gene for the other
5 patients. We investigated this further by performing long-range PCR
amplication encompassing exon 1 to exon 3, where the amplication
workedwell. Our long-range PCRidentieda reductionof approximately
2.5 kilobases in the PCR product of the amplied region, which was con-
sistently found in all 5 patients and suggesting a segmental genomic loss
between exon 1 and exon 3 (Fig. 1).
We subsequently performed a series of PCRamplications anddirect
sequencing to identify the breakpoint of the deletion (Fig. 2A). Upon
Fig. 1. PCR 1 shows the amplication failure of exon 2, which was subsequently claried by PCR 2 that employed long-range primers. PCR 2 shows a notable size reduction in a region
between exon 1 and exon 3 of the patient. Amplication in PCR 2 encompassed exon 1, intron 1, exon 2, intron 2 and exon 3.
241 J. Mohseni et al. / Gene 533 (2014) 240245
Author's personal copy
identication of the breakpoint, we subjected our nding to BLAST
and nally conrmed a 10-bp insertion and a 21-bp inversion located
at nucleotides +1899 and +1933 to +1953, respectively, downstream
of exon 1 coupled by a 2357-bp deletion starting at the subsequent
nucleotides (Fig. 2C).
We submitted the novel sequence to the NCBI GenBank. The
sequence was assigned an accession number of KF539857.
In order to obtain information on whether both alleles truly carry
a homozygous mutation, we investigated the extent of genomic re-
arrangement in each allele by quantifying the copy number of exons 1
and 3 which are anking the complex re-arrangement. Our real-time
quantitative PCR found that 4 of the patients with inversiondeletion
carried 2 copies of exon 1 and 2 copies of exon 3, suggesting that both
alleles of the gene may harbour the same extent of re-arrangement
(Table 1).
4. Discussion
We postulated that both ARG1 mutations found in our cohort of
patients are diseases-causing. The nonsense mutation found in Patient
ID 002(2) will prematurely truncate the protein translation. The
inversiondeletion found in the rest of the patients spanned a total
loss of 73-bp exon 2. This will consequently lead to a frame shift muta-
tion and also truncate the protein translation.
We identied a novel complex re-arrangement of ARG1 gene in 5
unrelated patients with hyperargininemia. All of the 5 patients were
found to share the same mutation, despite coming from unrelated
families, leading us to think that this mutation could be specic to
Malay and related ethnicities of Southeast Asia. In light of the fact that
we found this novel mutation through a simple long-range PCR, it is
plausible to suggest that our method could be applicable for screening
of hyperargininemia among Malay patients in peninsular Malaysia and
other related ethnicities, such as those in the western part of Indonesia
as well as southern Thai.
The fact that all the 5 families originated fromone geographic origin
in the northeasternpart of peninsular Malaysia has also sparked suspect
of founder phenomenon, where the unrelated families may actually
share the same ancestral family. Althoughit is not possible for us to clarify
if the consanguinity resulted from multiple generations of close
marriages or from single unusually close marriages, our empirical
observations to local culture of marriage support the notion that the
patients' families belong to a relatively endogamic group of large commu-
nity. Our pedigree analyses, however, could not identify any relations
between the affected families up to 3 generations.
In order to support the suspect of founder phenomenon, we rstly
need to obtain evidence that the mutations were truly homozygous.
We initially identied the complex re-arrangement as a gross deletion
through a simple long-range PCR that reveal a genomic loss between
exon1 andexon3 of the ARG1 gene. The amplicationidentieda single
and discrete PCR product, suggesting single genomic isoformof the mu-
tation. In spite of that, one might argue that the single amplication
might rather be due to amplication of the allele with shorter deletion
extent. If one other allele carries a larger deletion beyond exon 1 and
exon 3 of the gene, our initial PCR would have not been able to identify
that. The latter situation indicated a compound heterozygous mutation,
unlikely supportive of a founder phenomenon. Here we demonstrated
A
B
C
Fig. 2. (A) Illustrative gure of ARG1gene where the mutatedgenomic segment is indicatedingrey andthe primer positions are indicatedinnumberedarrows. (B) List of primer series that
we designed, with corresponding numbers to the (A). (C) Sequence alignment between that of the mutated and that of the wildtype.
242 J. Mohseni et al. / Gene 533 (2014) 240245
Author's personal copy
Table 1
ARG1 genotype and phenotype of Malaysian Malay patients with hyperargininemia.
Patient ID
(Family ID)
Sex Age
1
Consanguinity Phenotype Genotype Parental origin
Plasma arginine
(mol/L)
2
Blood ammonia
(mol/L)
3
Clinical features Mutation Copies of exon 1
4
Copies of exon 3
4
001(1) M 11 m Yes 516 52.7 Delayed developmental milestones rst noticed
at 3 months, failure to thrive, feeding difculties,
microcephaly, limbs hypertonia and hyperreexia,
truncal hypotonia
g.insIVS1+1899GTTTTATCAT;
g.invIVS1+1933_+1953;
g.delIVS1+1954_IVS2+914;
c.del116_188;p.Pro20SerfsX4
1.9 0.11 2.1 0.12 Kelantan
002(2) M 5 y Yes 400 212 Mild delayed developmental milestones during infancy,
acute onset left hemiparesis at 5 years old and MRI
brain demonstrated right fronto-parietal infarct, followed
by cognitive regression, progressive spastic diplegia, lost
ambulation at 9 years old
c.929CNT;p.R291X Not done Kelantan
003(3) F 7 y Yes 840 194 Mild delayed developmental milestones during infancy,
an episode of sudden onset of unconsciousness at 4 years old,
recurrent episodes of leg cramp and weakness during febrile
illnesses since 7 years old,
followed by progressive spastic diplegia,
lost ambulation at 10 years old
g.insIVS1+1899GTTTTATCAT;
g.invIVS1+1933_+1953;
g.delIVS1+1954_IVS2+914;
c.del116_188;p.Pro20SerfsX4
2.1 0.12 2 0.05 Kelantan
004(3) M 6 y Yes 434 81 Normal developmental milestones,
sudden onset seizures and altered sensorium at 6 years old
g.insIVS1+1899GTTTTATCAT;
g.invIVS1+1933_+1953;
g.delIVS1 + 1954_IVS2 + 914;
c.del116_188;p.Pro20SerfsX4
2 0.05 1.95 0.1 Kelantan
005(4) F 3 m Yes 2451 530 Acute hyperammonemia at 3 months old,
subsequently has delayed developmental milestones
g.insIVS1+1899GTTTTATCAT;
g.invIVS1+1933_+1953;
g.delIVS1+1954_IVS2+914;
c.del116_188;p.Pro20SerfsX4
1.98 0.14 1.99 0.23 Kelantan
006(5) F 2 y No 475 ND Normal developmental milestones,
sudden onset abdominal pain following a high protein meal
at 2 years old, subsequently developed spastic diplegia,
lost ambulation at 6 years old
g.insIVS1+1899GTTTTATCAT;
g.invIVS1+1933_+1953;
g.delIVS1+1954_IVS2+914;
c.del116_188;p.Pro20SerfsX4
Not enough DNA sample Kelantan
1
At diagnosis.
2
Plasma arginine: normal range, 41114 mol/L,
3
Blood Ammonia: normal range, 1040 mol/L,
4
Mean SD taken from 3 serial template dilution each from duplicate experiments.
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that our patients cohort carrying the complex re-arrangement showed
normal copies of the anking exons. This may suggest that both alleles
of the gene carry the same mutation, indicating a truly homozygous
mutation. However, we admit that this nding alone is not sufcient
to demonstrate a founder phenomenon for this mutation.
Hyperargininemia is an extremely rare disorder (Scaglia and Lee,
2006). As such, there are only some mutations of ARG1 found to date.
While most disease-causing mutations were inicted by changes in one
or a few nucleotides of the gene, 2 gross deletions and 1 gross insertion
were reported (Stenson, 2009).
Our further search on other human gene variant databases showed
that there was no report of a more complex genomic re-arrangement
of this gene (Exome Variant Server, 2013; The 1000 Genomes
Project Consortium, 2012). Our report showed the rst genomic re-
arrangement of ARG1 mutation which is commonly shared by a certain
group of Malay ethnicity. In addition, this is also the rst report of ARG1
mutations among hyperargininemia patients of this ethnicity.
While the genomic re-arrangement is novel and may only be shared
within related ethnicities, another mutation [c.929CNT;p.R291X in
Patient 002(2)] was previously reported in a French-German patient
(Grody et al., 1992). This nonsense mutation affected a CGNtrinucleotide
(CGA to TGA), which also constituted a CpGdinucleotide. A C-to-T transi-
tion in this type of tri-/dinucleotide has been one of the most frequently
reported mutations in several genetic disorders, namely Hemophilia
B (factor IX gene) (Koeberl et al., 1990), Retinoblastoma (RB1 gene),
(Cowell et al., 1994) and Neurobromatosis Type 1 (NF1 gene), (Krkljus
et al., 1998). In fact, another mutation of the same nature has been
reported in ARG1 gene, which turned arginine of codon 21 into a trans-
lation stop (c.119CNT;p.R21X) (Cardoso et al., 1999).
Patients with hyperargininemia were typically presented later in life
with chronic and slowly progressing neurological problems (Scaglia and
Lee, 2006). Only very few patients were reported with neonatal or
infantile presentation of hyperammonemia (Jian-Ghai et al., 2011). One
of our patients (005(4)) was presented with acute hyperammonemia
at 3months of age. This patient carried the same mutation as the other
4 patients in which the clinical manifestation of hyperargininemia
appeared much later in life. This further conrmed the lack of
genotypephenotype correlation in hyperargininemia patients with
ARG1 mutations (Martins et al., 2011).
In conclusion, we have identied for the rst time a novel inversion
deletion that resulted from a complex re-arrangement of ARG1 gene
commonly shared by a cohort of unrelated Malay patients with
hyperargininemia. Further analyses showing that the patients have
shared the same geographic origin within the northeastern part of
Malaysia prompted us to suggest a simple molecular screening of
hyperargininemia within related ethnicities using a long-range PCR.
Funding statement
This study was supported by the Universiti Sains Malaysia Research
University Grants (1001/PPSP/812048 and 1001/PPSP/812072) for
Dr. Teguh Haryo Sasongko.
Contributorship statement
Jafar Mohseni: conception and design, acquisition of data, analysis
and interpretationof data, drafting andrevising the article for important
intellectual content and nal approval of the version to be published.
Chia Boon Hock: acquisition of data, revising the article for important
intellectual content and nal approval of the version to be published.
Norhazah Che Abd. Razak: acquisition of data, revising the article for
important intellectual content and nal approval of the version to be
published.
Syah Nor Iman Othman: acquisition of data, revising the article for
important intellectual content and nal approval of the version to be
published.
Fatemeh Hayati: acquisition of data, revising the article for important
intellectual content and nal approval of the version to be published.
Winnie Ong PeiTee: acquisition of data, revising the article for im-
portant intellectual content and nal approval of the version to be
published.
Muzhirah Haniffa: acquisition of data, revising the article for impor-
tant intellectual content and nal approval of the version to be published.
Bin Alwi Zilfalil: conception and design, revising the article for
important intellectual content and nal approval of the version to be
published.
Rowani Mohd. Rawi: conception and design, revising the article for
important intellectual content and nal approval of the version to be
published.
Ngu Lock Hock: conception and design, acquisition of data, revising
the article for important intellectual content and nal approval of the
version to be published.
Teguh Haryo Sasongko: conception and design, acquisition of data,
analysis and interpretation of data, drafting and revising the article for
important intellectual content and nal approval of the version to be
published.
What is already known on this topic
Hyperargininemia is caused by mutations in ARG1 gene and a number
of mutations have been found. Mutations found were mostly point muta-
tions with few deletions or insertions. There was no simple screening
method for this disorder as no common mutation was found so far.
What this study adds
This study reports for the rst time a complex re-arrangement of
ARG1 commonly found in un-related patients with hyperargininemia.
This suggests a simple screening method for detecting the mutation
among patients with similar ethnicity of the South East Asian.
Conict of interest
The authors declare no conict of interest.
Acknowledgment
The authors are grateful to the patients and their families for their
voluntary cooperation in this report. The authors wish to thank Asso-
ciate Professor Dr. Gan Siew Hua of the Universiti Sains Malaysia and
Dr. Gunadi of the Universitas Gadjah Mada for their inputs after critically
reading the manuscript. The authors also thank the Director General of
Health, Malaysia, for permission to publish the scientic and clinical
data from Ministry of Healths hospital.
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