Biochem Genet

DOI 10.1007/s10528-017-9825-6

REVIEW

Aminoacidopathies: Prevalence, Etiology, Screening,

and Treatment Options

Muhammad Wasim1 • Fazli Rabbi Awan1 • Haq Nawaz Khan1 •

Abdul Tawab1 • Mazhar Iqbal1 • Hina Ayesha2

Received: 12 January 2017 / Accepted: 18 September 2017

Ó Springer Science+Business Media, LLC 2017

Abstract Inborn errors of metabolism (IEMs) are a group of inherited metabolic

disorders which are caused by mutations in the specific genes that lead to impaired
proteins or enzymes production. Different metabolic pathways are perturbed due
to the deficiency or lack of enzymes. To date, more than 500 IEMs have been
reported with most of them being untreatable. However, fortunately 91 such dis-
orders are potentially treatable, if diagnosed at an earlier stage of life. IEMs have
been classified into different categories and one class of IEMs, characterized by
the physiological disturbances of amino acids is called as aminoacidopathies. Out
of 91 treatable IEM, thirteen disorders are amino acid related. Aminoacidopathies
can be detected by chromatography and mass spectrometry based analytical
techniques (e.g., HPLC, GC–MS, LC–MS/MS) for amino acid level changes, and
through genetic assays (e.g., PCR, TaqMan Genotyping, DNA sequencing) at the
mutation level in the corresponding genes. Hence, this review is focused to
describe thirteen common aminoacidopathies namely: Phenylketonuria (PKU),
Maple Syrup Urine Disease (MSUD), Homocystinuria/Methylene Tetrahydrofo-
late Reductase (MTHFR) deficiency, Tyrosinemia type II, Citrullinemia type I and
type II, Argininosuccinic aciduria, Carbamoyl Phosphate Synthetase I (CPS)
deficiency, Argininemia (arginase deficiency), Hyperornithinemia–Hyperam-
monemia–Homocitrullinuria (HHH) syndrome, N-Acetylglutamate Synthase
(NAGS) deficiency, Ornithine Transcarbamylase (OTC) deficiency, and Pyruvate
Dehydrogenase (PDH) complex deficiency. Furthermore, the etiology, prevalence
and commonly used analytical techniques for screening of aminoacidopathies are

& Fazli Rabbi Awan

awan.fr@gmail.com
1
Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering
(NIBGE) / [Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad], Jhang
Road, P.O. Box. 577, Faisalabad 38000, Pakistan
2
DHQ Hospital, Faisalabad Medical University, Faisalabad, Pakistan

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briefly described. This information would be helpful to researchers and clinicians

especially from developing countries to initiate newborn screening programs for
aminoacidopathies.

Keywords Aminoacidopathies
Metabolic disorders
HPLC
LC–MS/MS

GC–MS
Newborn screening

Introduction

Inborn errors of metabolism (IEMs) are inherited biochemical disorders which are
caused by mutations in the genes leading to impaired proteins or enzymes
production which affect physiologically important metabolic pathways resulting
in impaired changes in the vital metabolites. IEMs are rare diseases when taken
individually, but collectively these disorders are quite frequent in different
populations (Scaturro et al. 2013). Overall, IEMs affect around 2–3% of the
global population and if the diagnosis are missed or delayed, then the
management of such patients become a challenge that results in an enormous
psychological and financial burden on the family and society (van Karnebeek
2014; Genereaux et al. 2015). As IEMs are comprised of a variety of diseases,
these are grouped based on their underlying pathology, location of cellular defect,
or the type of biological molecules being involved. One class of IEMs that is
caused by the abnormal changes of physiological amino acids is called
Aminoacidopathies. Aminoacidopathies are metabolic disorders which are caused
by defective metabolic pathways due to the deficiencies of functional enzymes
(van Vliet et al. 2014). All the enzymes are proteins in nature and are made up of
different types and composition from twenty physiologically important amino
acids. Every amino acid has a specific structure and function, so after joining
different amino acids, these make proteins in a specific three dimensional
configuration which is the functional form of all the proteins (Yan et al. 2014).
When functional protein is formed, then interactions with other proteins are also
important to perform their different biological functions (Ashenberg and Laub
2013). If any change in the structure of protein or enzyme takes place, then its
function may alter or abolish, ultimately affecting its bioactivity (Schaefer and
Rost 2012). Due to mutations in the respective genes, the expression levels or
conformation of the corresponding proteins can change, which is associated with
certain diseases (Anoosha et al. 2015).
Owing to complex etiology, the screening and diagnosis of such disorders is a
bottleneck, hence collective efforts of scientists and clinicians are required for their
precise and timely diagnosis (van Karnebeek et al. 2014). If such collective efforts
are missing; it results in undiagnosed diseases which cause distress for the patients
and their families throughout their lives (Gramer et al. 2014).

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Newborn Screening

Different diseases are screened through NBS program including aminoacidopathies

but the list of diseases vary from country to country according to the prevalence data
(Ozben 2013; Scaturro et al. 2013). Early detection and treatment of IEMs through
NBS program can reduce mortality and minimize morbidity (Golbahar et al. 2013).
NBS program plays a vital role for the diagnosis of IEMs in the developed countries
and thus reduce the burden of such diseases over there. Therefore, to minimize the
burden of IEMs, developing countries need to set up such NBS programs; however,
it requires prior prevalence data of such diseases in those countries.

Types of Aminoacidopathies

As mentioned earlier that more than 500 IEMs have been reported so far; however,
fortunately 91 such disorders are potentially treatable, if diagnosed at an earlier
stage of life. Out of these 91 conditions, 13 disorders are caused by the amino acid
disturbance which are: Phenylketonuria (PKU), Maple Syrup Urine Disease
(MSUD), Homocystinuria/Methylene Tetrahydrofolate Reductase (MTHFR) defi-
ciency, Tyrosinemia type II, Citrullinemia type I and type II, Argininosuccinic
aciduria, Carbamoyl Phosphate Synthetase I (CPS) deficiency, Argininemia
(arginase deficiency), Hyperornithinemia–Hyperammonemia–Homocitrullinuria
(HHH) syndrome, N-Acetylglutamate Synthase (NAGS) deficiency, Ornithine
Transcarbamylase (OTC) deficiency, and Pyruvate Dehydrogenase (PDH) complex
deficiency collectively called as aminoacidopathies. Different symptoms appear in
these disorders such as: hyperactivity, intellectual disability, developmental delay,
vomiting, and seizures (van Karnebeek and Stockler 2012).

Prevalence of Aminoacidopathies

In most of the countries, prevalence of aminoacidopathies is low and all the

developed countries have established NBS programs and collected prevalence data
for such disorders. Aminoacidopathies are caused due to the genetic mutations in
the specific genes and most of them are monogenic disorders. In the Table 1,
thirteen potentially treatable aminoacidopathies are summarized along with their
respective genes, OMIM number, and worldwide prevalence data.
Majority of these disorders arise due to consanguineous marriages. In the Middle
East, consanguinity rate is about 40% among first cousins and up to 60% in
intermarriages between relatives, this is the main reason for increased incidence of
genetic disorders over there due to narrow genetic pool (Golbahar et al. 2013;
Karam et al. 2013). In developed countries, the rate of phenylketonuria (PKU) is
higher as compare to other aminoacidopathies (Pimentel et al. 2014; Sherry et al.
2015), like those in the Chinese and the Korean population (Hardelid et al. 2008);
but this ratio is very high and alarming in Turkey with a range of 1 in 2600 to 1 in

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6500 births (Williams et al. 2008; Dobrowolski et al. 2011; Khemir et al. 2011; Al
Hafid and Christodoulou 2015).

Etiology of Aminoacidopathies

Etiological factors at the level of genes and enzymes are given below for the
common aminoacidopathies.

Phenylketonuria

It is caused by the deficiency of phenylalanine hydroxylase enzyme (EC number:

1.14.16.1) coded by PAH gene as listed in Table 1. Due to mutations in PAH gene
(NC_000012.12), its respective enzyme is not functional which leads to elevated
level of phenylalanine that causes classical PKU. If it remains untreated, this disease
can lead to intellectual disability, developmental delay, and death (Williams et al.
2008).

Maple Syrup Urine Disease

It is a condition in which body cannot break down leucine, isoleucine, and valine. It
is mainly caused by a defect in these three genes BCKDHA (NC_000019.10),
BCKDHB (NC_000006.12), and DBT (NC_000001.11) listed in Table 1. These
amino acids then get stored in the body, affect the brain and other organs. If
untreated, it can lead to developmental delay, intellectual disability, seizures, coma,
and death (Li et al. 2016; Su et al. 2017).

Tyrosinemia Type II

This disorder is caused by the elevated blood levels of tyrosine, by the deficiency of
an enzyme tyrosine aminotransferase and the shortage of this enzyme is due to the
mutations in the TAT gene (NC_000016.10) as listed in Table 1. If untreated, it can
lead to serious health problems like developmental delay and intellectual disability
(Carvalho-Silva et al. 2017; Pena-Quintana et al. 2017).

Citrullinemia Type I

It is caused by the accumulation of citrulline and ammonia in the blood, due to

mutations in the ASS1 gene (NC_000009.12) as shown in Table 1. Affected infants
typically appear normal at birth, but if remain untreated, they experience poor
feeding, vomiting, seizures, loss of consciousness, and intellectual disability (Kose
et al. 2017).

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Table 1 Causative genes, OMIM number, and prevalence data of common aminoacidopathies
Sr. Aminoacidopathies Respective genes Worldwide References
no prevalence

1 Phenylketonuria PAH (OMIM: 261600) 1:10,000–25,000 Williams et al. (2008),

(PKU) Ozben (2013) and
Dehghanian et al.
(2017)
2 Maple syrup urine BCKDHA, BCKDHB 1:185,000 Herber et al. (2015) and
disease (MSUD) and DBT (OMIM: Imtiaz et al. (2017)
248600)
3 Homocystinuria MTHFR (OMIM: 1:150,000 Ozben (2013) and
236250) and CBS Froese et al. (2016)
(OMIM: 236200)
4 Tyrosinemia type II TAT (OMIM: 276600) 1:250,000 Ozben (2013),
Carvalho-Silva et al.
(2017) and Pena-
Quintana et al. (2017)
5 Citrullinemia type I ASS1 (OMIM: 1:44,300–200,000 Ozben (2013), Woo
215700) et al. (2014) and Kose
(2017)
6 Citrullinemia type II SLC25A13 (OMIM: 1:17,000–230,000 Ozben (2013), Wen
605814) et al. (2014) and Woo
et al. (2014)
7 Argininosuccinic ASL (OMIM: 207900) 1:70,000 Ozben (2013) and Wen
aciduria et al. (2016)
8 Carbamoyl CPS1 (OMIM: 1:62,000–800,000 Martinez et al. (2010)
phosphate 237300) and Choi et al. (2017)
synthetase I (CPS)
deficiency
9 Argininemia ARG1 (OMIM: 1:3,50,000–2,000,000 Lee et al. (2011),
(arginase 207800) Ozben (2013) and
deficiency) Zhang et al. (2017)
10 Hyperornithinemia– SLC25A15 (OMIM: More than 100 Ersoy Tunali et al.
Hyperammonemia– 238970) affected individuals (2014) and Martinelli
Homocitrullinuria have been reported et al. (2015)
(HHH) syndrome worldwide
11 N-Acetylglutamate NAGS (OMIM: Very rare. Incidence Caldovic et al. (2010
Synthase (NAGS) 237310) is unknown and Al Kaabi and El-
deficiency Hattab (2016)
12 Ornithine OTC (OMIM: 311250) 1:62,000–77,000 Caldovic et al. (2015)
Transcarbamylase and Shao et al. (2017)
(OTC) deficiency
13 Pyruvate PDHA1 (OMIM: Very rare. Incidence Okajima et al. (2008),
dehydrogenase 312170), PDHB is unknown Patel et al. (2012),
(PDH) complex (OMIM: 614111), Tajir et al. (2012),
deficiency DLAT (OMIM: Steller et al. (2014),
245348), DLD Castiglioni et al.
(OMIM: 246900) 2015, Ciara et al.
PDHX (OMIM: (2016) and Pliss et al.
245349) and PDP1 (2016)
(OMIM: 608782)

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Citrullinemia Type II

It is a condition in which the body is unable to make citrin, a protein that helps to
move different substances within the cells. Mutations in the SLC25A13 gene
(NC_000007.14) as shown in Table 1 typically prevent cells from making any
functional citrin. If untreated, it leads to developmental delay, vomiting, seizures,
and intellectual disability (Woo et al. 2014).

Homocystinuria

It is a disorder of methionine metabolism, leading to an abnormal accumulation of

homocysteine in blood and urine. Mutations in MTHFR (NC_000001.11) and CBS
(NC_000021.9) genes listed in Table 1 lead to this disease (Froese et al. 2016).
Signs and symptoms of this disorder are dislocation of the lens, abnormal blood
clotting, osteoporosis, or other skeletal abnormalities. If untreated, it leads to serious
problems such as developmental delay and learning problems.

Argininosuccinic Aciduria

It is a heterogeneous urea cycle disorder mainly caused due to mutations in the ASL
gene (NC_000007.14), which leads to the accumulation of argininosuccinic acid in
the blood and urine (Wen et al. 2016). Signs and symptoms of this disease are
lethargy, loss of appetite, coma, and seizures; if untreated, it can lead to
developmental delay, intellectual disability, liver damage, and skin lesions.

Hyperornithinemia–Hyperammonemia–Homocitrullinuria (HHH)
Syndrome

It is a condition in which the body is unable to process and remove the waste
ammonia. It is caused by the mutations in the SLC25A15 gene (NC_000013.11)
which result in the deficiency of an enzyme ornithine translocase as listed in
Table 1. If left untreated, this can result in developmental delay, learning
disabilities, and other health related problems (Ersoy Tunali et al. 2014, Martinelli
et al. 2015).

Ornithine Transcarbamylase (OTC) Deficiency

It is an inherited disorder that causes ammonia to accumulate in the blood as in

HHH syndrome but in this disorder mutations in the OTC gene (NC_000023.11)
cause this disease as listed in Table 1. This gene provides instructions for making
the ornithine transcarbamylase enzyme, but due to deficiency of this enzyme,
ammonia accumulate in the blood that damage nervous system, liver, and causes
other neurological problems (Caldovic et al. 2015; Shao et al. 2017; Wang et al.
2017).

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N-Acetylglutamate Synthase (NAGS) Deficiency

It is another disorder in which ammonia accumulates in the blood as in OTC

deficiency and in HHH syndrome; but in this disorder, mutations in the NAGS gene
(NC_000017.11) are responsible for this disorder as listed in Table 1. If left
untreated, it can lead to brain damage or even death (Caldovic et al. 2010).

Carbamoyl Phosphate Synthetase I (CPS) Deficiency

It is an inherited disorder that causes ammonia to accumulate in the blood as in the

case of OTC deficiency, HHH syndrome, and in NAGS deficiency but mutations in
the CPS1 gene (NC_000002.12) causes this disease as listed in Table 1. Sign and
symptoms of this condition are unusual sleepiness, poorly regulated breathing rate
or body temperature, unwillingness to feed, vomiting after feeding, unusual body
movements, seizures, or coma (Choi et al. 2017; Diez-Fernandez and Haberle
2017).

Pyruvate Dehydrogenase (PDH) Complex Deficiency

This disorder is caused by the buildup of lactic acid in the body. Children having
this disorder have delayed developmental milestones of mental abilities and motor
skills such as sitting and walking, intellectual disability, seizures, weak muscle
(Pliss et al. 2016). Mutations in PDHA1 (NC_000023.11), PDHB (NC_000003.12),
DLAT (NC_000011.10), DLD (NC_000007.14), PDHX (NC_000011.10), and PDP1
(NC_000008.11) genes are involved in this disorder (Ciara et al. 2016).

Argininemia

It is an inherited disorder that is caused by the accumulation of arginine in the

blood. Arginase function is to utilize arginine in the body but due to mutations in the
ARG1 gene (NC_000006.12), this enzyme does not work properly as listed in
Table 1. Sign and symptoms are stiffness (especially in the legs), developmental
delay, intellectual disability, seizures, tremor, and difficulty with balance and
coordination (Zhang et al. 2017).

Genetics of Aminoacidopathies

Genetic factors play a major part in IEMs, e.g., aminoacidopathies causing ID, but it
is challenging to study the genetic basis of these diseases because of the variable
clinical information and genetic heterogeneity of such patients. Recently, progress
has been made by using next-generation sequencing approach (Vissers et al. 2016)
for understanding and rapidly elucidating the genetic basis of such diseases. IEMs
are inherited group of disorders and generally inherited in autosomal recessive
manner (Al Riyami et al. 2012). The underlying genetic defects of these 13 amino
acid disorders are given in Table 1. Most of the infants with metabolic diseases

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generally look healthy at the time of birth but at later stage of life, these infants
show different abnormal symptoms.

Screening and Diagnosis of Aminoacidopathies

Initial screening of amino acid disorders could be done through simple assays like
ferric chloride, dinitrophenylhydrazine, and cyanide-nitroprusside tests. These tests
have been helpful in the early detection of various rare metabolic disorders, and can
easily be performed without specialized training or advanced analytical equipment.
Ferric chloride and dinitrophenylhydrazine tests have been used for the early
diagnosis of different aminoacidopathies with specific color; PKU (green color),
Tyrosinuria (blue green color), MSUD (grayish color), and for homocystinuria,
Cyanide-nitroprusside test gives (Pink to beet-red color). Since, these colorimetric
assays are not very specific or sensitive, so other advance analytical techniques like
HPLC, GC–MS, LC–MS, and genetic assays are employed for the screening and
diagnosis of IEM including aminoacidopathies.
Screening of aminoacidopathies is a challenging task for many developing
countries like Pakistan. For the screening and diagnosis of these rare disorders,
every country needs to have NBS program. Such set ups should be equipped with
the latest analytical technologies like HPLC, GC–MS, LC–MS, and PCR with new
sensitive methods of detection. Progress related to these analytical methods and
equipment is briefly summarized for applications to aminoacidopathies.
Aminoacidopathies can easily be detected by advance chromatographic tech-
niques and in most of the cases by reverse-phase chromatography (RP-HPLC). Ion-
exchange chromatography is also used but it has also been reported that ion-
exchange chromatography fails to determine low levels of free amino acids form
biofluids (Wu et al. 2016). Although, ion-exchange chromatography has been used
for the detection of amino acids in the urine samples (Ferreira and Cusmano-Ozog
2016), but this is time consuming, laborious, and is not convenient to perform in
clinical laboratories (Yi et al. 2011). However, in case of RP-HPLC, it is easy to
perform and require small quantity of sample for the detection of free amino acids
(Perucho et al. 2015; Wu et al. 2016). It has been reported that hydrophilic
interaction liquid chromatography (HILIC) column is an ideal separation mode for
the hydrophilic compounds like amino acids. Using HILIC, detection of 36 un-
derivatized amino acids from plasma has been reported for the early detection of
aminoacidopathies (Prinsen et al. 2016).
Quantification of amino acids from different biological fluid such as blood
plasma and urine can be done using HPLC within 45 min (Babu et al. 2002), then
the suspected disorder can be confirmed by LC–MS. It has also been reported that
the detection of 26 amino acids through HPLC by pre-column derivatization
(Sharma et al. 2014). Derivatization of amino acids with O-Phthalaldehyde is
considered as best method, with high signal response and chromatographic
resolution in HPLC (Babu et al. 2002; Perucho et al. 2015); but recently, it has
been reported that O-Phthalaldehyde with MercaptoPropionic Acid (MPA) as a
derivatizing agent gives high signal response in RP-HPLC-FLD (Wu et al. 2016).

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The introduction of mass spectrometry (MS/MS) has revolutionized the analysis

of amino acids, even when present in trace amounts, eliminating the need for
derivatization. Now, the analysis of un-derivatized amino acids, present in very
complex samples or matrix can be done, using these sensitive techniques like
HPLC–ESI–MS/MS. Around 40 amino acids and their isomers have been separated,
screened, and around 20 amino acids have been quantified from dried blood spots
within 10 min by using HPLC–ESI–MS/MS. In such assays, use of shorter C18
column has reduced the run time substantially (Zoppa et al. 2006). On the other
hand, owing to high accuracy, sensitivity, and capacity to analyze multiple
compounds simultaneously, gas chromatography- mass spectrometry (GC–MS) has
been used worldwide in the diagnosis of IEM. Using, GC–MS many metabolic
conditions were simultaneously screened by urinary analysis (Jiang et al. 2015).
Advance analytical methods like GC–MS and LC–MS are being used in
developed countries for the screening of aminoacidopathies in addition to RP-
HPLC, which is a cost effective method. So, in our opinion, it is reasonable to use
RP-HPLC in countries with limited resources like Pakistan for initial screening of
amino acid and other related IEM disorders, which could be later confirmed with
GC–MS or LC–MS based assays.
For genetic diagnosis of IEM, PCR based strategies commonly used are PCR–
RFLP, ARMS–PCR, and RT–PCR. Moreover, after the availability of human
genome data, whole-exome sequencing is also applied for the diagnosis of such
disorders (Monroe et al. 2016).

Treatment Options for Aminoacidopathies

As an increasing number of IEMs are present in different populations, there should

be a treatment option for such disorders. Dietary treatments have been used in many
of the developed countries for the most prevalent diseases. Diet options are
important to attain metabolic control and for the prevention of damage to organs. In
case of aminoacidopathies, specific metabolic nutrition therapy and medications can
reduce the morbidity and improve patient outcomes. For example, leucine and
phenylalanine restricted diet is useful in the cases of MSUD and PKU diseases,
respectively (van Vliet et al. 2014; Ho et al. 2016). It is also reported that dietary
treatments have a positive impact on kids having neurodevelopmental problems
(Coughlin et al. 2015). Recently, a child is reported with liver failure having
Citrullinemia type 1 disorder, referred to a liver transplantation center, (Bindi and
Eiroa 2017). Hence, there is a need for early treatment for such disorders to avoid
transplant because most of the IEM damage the liver. Furthermore, for all
aminoacidopathies, diet treatments are the main options to reduce the burden of
these disorders; there are also other treatment options available as mentioned in
Table 2. However, there is a need for more genetic-based and pharmacological
therapies with only beneficial effects, e.g., in the case of PKU the PAH gene therapy
is required for the proper treatment of classical PKU patients. If treatment of such
the diseases is not started promptly then their abnormal symptoms may cause

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Table 2 List of aminoacidopathies and treatment options

Sr. Disorders Treatment options References
no

1 PKU Phenylalanine and protein restricted Al Hafid and Christodoulou (2015),

diet, BH4 therapy, duration of Banta-Wright et al. (2015a, b),
breastfeeding, fish free diet Htun et al. (2015) and Pena et al.
(2015)
2 MSUD Leucine restricted diet van Vliet et al. (2014)
3 HHH syndrome Protein restricted diet Qadri et al. (2016)
4 NAGS deficiency N-Carbamylglutamate drug Kim et al. (2015)
5 Homocystinuria Methionine restricted diet, vitamin Kumar et al. (2016)
B6 and cystathionine beta-
synthase (CBS) gene therapy
6 Tyrosinemia type II Omega-3 fatty acid supplementation Carvalho-Silva et al. (2017
7 Citrullinemia type I Liver transplant, development of Bindi and Eiroa (2017) and
induced pluripotent stem cells Yoshitoshi-Uebayashi et al. (2017)
(iPSCs)
8 Citrullinemia type Supplemented formula of medium- Hayasaka et al. (2015)
II chain triglyceride (MCT)
9 PDH complex High-fat diet (ketogenic diet) given Stenlid et al. (2014) and Pliss et al.
deficiency to mothers during pregnancy (2016)
10 OTC deficiency Adeno-associated virus (AAV) gene Wang et al. (2017)
therapy
11 Argininosuccinic Arginine therapy and dietary Nagamani et al. (2012a, b)
aciduria modifications
12 Argininemia Arginine therapy by using adeno- Cantero et al. (2016)
(arginase associated virus
deficiency)
13 Carbamoyl Protein-restriction, supplementation Diez-Fernandez and Haberle (2017)
phosphate with citrulline and/or arginine, N-
synthetase I carbamoyl-L-glutamate,
(CPS) deficiency pharmacological chaperones and
gene therapy

serious health problems, disability, and death. Therefore, with timely treatment, a
patient may live a healthy life.

Summary

Rare disorders are neglected in most of the developing countries due to burden of
other common infectious, non-communicable, and genetic disorders. However, rare
disorders need be addressed as well because 2–3% of global population suffers from
inborn errors of metabolism (IEM). Due to huge burden of these disorders, NBS
program is an important urgency for all the developing nations to screen and
diagnose the prevalent IEMs including aminoacidopathies. By now, treatment
options are available in most of the developed countries because they have

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prevalence of data for such rare disorders. Conversely, developing countries like
Pakistan have a very limited data for such diseases which is not sufficient to initiate
such diet management plans. Hence, prevalence data of IEMs in the developing
nations should be collected and special diet(s) and other treatment/management
plans specific for respective diseases should be initiated to reduce the burden of such
disorders in developing countries. Different analytical techniques are routinely used
for screening in advanced countries but countries with limited resources like
Pakistan, these methods and techniques are not easily available owing to lack of
expertise or financial sources. Hence, this review will act as primer to help
researchers, clinicians, and scientists from developing countries while planning to
set up NBS program for screening and diagnosing aminoacidopathies and related
disorders.

Acknowledgement This review originated as a result of our research project, ‘‘Diagnosis of

treatable inborn metabolic disorders of intellectual disability’’ (Project No. CRP/PAK14-02; Contract
No. CRP/14/012) funded by the International Centre for Genetic Engineering and Biotechnology
(ICGEB), Italy.

Author Contributions FRA conceived the idea and revised/approved the manuscript. MW wrote the
draft and revised it several times, HNK provided information on genetics of the diseases, AT and MI
wrote and revised the analytical/diagnostic section, and HA provided advice on clinical aspects of the
described diseases.

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