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Review

Diseases caused by defects of mitochondrial carriers: A review

Ferdinando Palmieri ⁎
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy

a r t i c l e i n f o a b s t r a c t

Article history:
Received 31 January 2008
Accepted 18 March 2008
Available online 25 March 2008

A strikingly large number of mitochondrial DNA (mtDNA) mutations have been found to be the cause of
respiratory chain and oxidative phosphorylation defects. These mitochondrial disorders were the first to be
investigated after the small mtDNA had been sequenced in the 80s. Only recently numerous diseases resulting
from mutations in nuclear genes encoding mitochondrial proteins have been characterized. Among these,
nine are caused by defects of mitochondrial carriers, a family of nuclear-coded proteins that shuttle a variety of
metabolites across the mitochondrial membrane. Mutations of mitochondrial carrier genes involved in
mitochondrial functions other than oxidative phosphorylation are responsible for carnitine/acylcarnitine
carrier deficiency, HHH syndrome, aspartate/glutamate isoform 2 deficiency, Amish microcephaly, and
neonatal myoclonic epilepsy; these disorders are characterized by specific metabolic dysfunctions, depending
on the physiological role of the affected carrier in intermediary metabolism. Defects of mitochondrial carriers
that supply mitochondria with the substrates of oxidative phosphorylation, inorganic phosphate and ADP, are
responsible for diseases characterized by defective energy production. Herein, all the mitochondrial carrier-
associated diseases known to date are reviewed for the first time. Particular emphasis is given to the molecular
basis and pathogenetic mechanism of these inherited disorders.

© 2008 Elsevier B.V. All rights reserved.

Keywords:
Carrier deficiencies
Mitochondria
Mitochondrial carriers
Mitochondrial diseases
Mitochondrial carrier-related diseases
Transporters

1. Introduction

Mitochondrial diseases are hundreds of clinical phenotypes caused
by defects in one or more components of the mitochondrial proteome.
This proteome consists of more than 1000 proteins that are encoded
by either nuclear DNA or mitochondrial DNA (mtDNA). The mitochon-
drial genome encodes only 13 proteins which are subunits of the four
respiratory chain enzyme complexes (I–IV) and ATP synthase complex
(V). Both the subunits encoded by mtDNA or nuclear DNA are essential
for the activity of these complexes and therefore for the accomplish-
ment of oxidative phosphorylation.

Since 1988, when the first mutations in mtDNA were described
[1,2], more than 400 mutations in mtDNA have been identified as
being responsible for respiratory chain and oxidative phosphorylation
diseases (http://www.mitomap.org). These mutations concern not
only mtDNA genes encoding the above-mentioned 13 proteins but
also mtDNA genes encoding 2 ribosomal RNAs and 22 transfer RNAs
which are part of the protein synthesis machinery present in the
mitochondrial matrix. Respiratory chain and oxidative phosphoryla-
tion diseases can also be caused by mutations of nuclear DNA genes
that encode proteins required for a) the replication, transcription,
translation, repair and maintenance of mtDNA; b) the functioning of
complexes I–V (the nuclear-coded subunits) and their assembly; c) the
import of the substrates of oxidative phosphorylation (see section 4 of
this review); and d) the import, modification and insertion of respi-

ratory chain cofactors such as heme, metals and the iron–sulphur
clusters. mtDNA-dependent oxidative phosphorylation diseases are
due either to large-scale rearrangements including deletions and
duplications, which are usually sporadic and invariably heteroplasmic,
or to point mutations which may be heteroplasmic or homoplasmic
and are transmitted maternally. Nuclear-dependent oxidative phos-
phorylation diseases can induce secondary multiple mtDNA deletions
or loss of mtDNA in affected tissues and follow the Mendelian pattern
of inheritance.

Although the primary function of mitochondria is the synthesis of
ATP by oxidative phosphorylation, these organelles play many other
important roles. They contain several metabolic pathways, such as the
citric acid cycle, or parts thereof, are the main site where reactive
oxygen species (ROS) are generated, and are fundamental for certain
biological processes such as Ca2+ cell signaling, cell proliferation and
necrotic or apoptotic cell death. Some enzymes involved in ROS
management, in amino acid catabolism and in the biosynthesis of fatty
acids, cholesterol, steroid hormones, farnesyl and geranyl side chains,
cardiolipin, ubiquinol, heme and urea are located inside mitochondria.
Furthermore, mitochondria communicate with other cell compart-
ments by means of transport proteins (carriers) present in the mito-
chondrial membrane that allow the selective passage of solutes in and
out of the mitochondrial matrix. All these functions are accomplished
by a large number of proteins which are encoded by the nuclear
genome, translated in the cytosol and imported into the mitochondria.

In recent years, the completion of human nuclear genome se-
quencing and the ongoing identification of nuclear gene function have
accelerated the disclosure of several mitochondrial disorders (apart

Biochimica et Biophysica Acta 1777 (2008) 564–578

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from oxidative phosphorylation diseases) resulting from mutations in
nuclear genes encoding mitochondrial proteins. These include
disorders caused by mutations of proteins involved in the citric acid
cycle [3], fatty acid β-oxidation [4], cardiolipin metabolism (Barth's
syndrome) [5,6], the utilization of iron (Friedreich's ataxia, X-linked
ataxia and sideroblastic anemia) [7], antioxidant defense [8], protein
import (X-linked deafness distonia) [9], network dynamics (fission/
fusion) [10] and mitochondrial metabolite carriers (this review). In
addition, some cancers are caused by mutations in nuclear genes en-
coding mitochondrial proteins [11,12]. Several reviews on mitochon-
drial diseases resulting from defects in the respiratory chain and
oxidative phosphorylation have been published [13–19]. The con-
tribution of mtDNA mutations, ROS generation and, in general, mito-
chondrial dysfunction to cardiovascular disease, diabetes, cancer,
aging and aging-related neurodegenerative disorders, such as Parkin-
son's and Huntington's disease, has also been reviewed [12,20–27].

This review highlights for the first time the current molecular,
biochemical, clinical and pathophysiological information about all the
diseases known thus far to be associated with defects of mitochondrial
carriers. As a premise, some features of mitochondrial carriers are
summarized.

2. Characteristics and physiological role of mitochondrial carriers

Because mitochondria and cytosol cooperate in a large number of
metabolic cellular processes, a continuous and highly diversified flux
of metabolites, nucleotides, and cofactors into and out of the

mitochondria is needed. The transport of solutes across the inner
mitochondrial membrane is catalyzed by a family of nuclear-coded,
membrane-embedded proteins called mitochondrial carriers. The
carriers are involved in the Krebs cycle, oxidative phosphorylation,
modulation of the nucleotide and deoxynucleotide pools in the mito-
chondrial matrix, the synthesis and breakdown of mtDNA, mtRNA and
mitochondrial proteins, fatty acid oxidation, gluconeogenesis, lipo-
genesis, transfer of reducing equivalents, urea synthesis, amino acid
degradation, and other functions shared by cytosol and mitochondria.
The central role in cell metabolism of most mitochondrial carriers
identified so far (except the pyruvate carrier, PyC) is illustrated in Fig. 1.

In man, mitochondrial carriers are encoded by the SLC25 gene
family ([28] for a review). Several carriers, such as the ADP/ATP, ATP-
Mg/Pi, glutamate and ornithine carriers, have isoforms encoded by
different genes, and only the phosphate carrier (PiC) has two
alternatively spliced isoforms. The main carriers importing substrates
for oxidative phosphorylation are widely distributed in human tissues.
In contrast, other carriers are tissue specific and have a limited dis-
tribution reflecting their importance in special functions. The majority
of mitochondrial carriers catalyze obligatory solute exchange reac-
tions. Those mediating H+-compensated undirectional substrate flux
may also fall into the above category, given that at least PiC has been
shown to function in a phosphate/OH− antiport mode. The carnitine–
acylcarnitine carrier catalyzes both unidirectional transport of carni-
tine and the carnitine/acylcarnitine exchange, whereas the uncoupling
protein catalyzes uniport as the exclusive transport mode. As their
driving force, mitochondrial carriers utilize the concentration gradient

Fig. 1. Metabolic roles of mitochondrial carriers. The scheme shows 15 carriers catalyzing metabolite transport through the inner mitochondrial membrane. These carriers are
involved in: oxidative phosphorylation (AAC, PiC, UCP); oxidation/reduction pathways (AGC, OGC, DIC, CIC, CAC, PyC); homeostasis of the intramitochondrial adenine nucleotide pool
(APC); methylation of mtDNA, mtRNA and some intramitochondrial proteins (SAMC); import of the essential coenzyme thiamine pyrophosphate (ThPP) required for pyruvate– and
oxoglutarate–dehydrogenase complex activities (TPC); and, with partial overlap, amino acid metabolism (AGC, ORC, GC, ODC). The scheme does not show all the metabolic pathways
in which the already identified carriers are involved.

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of the solutes and/or the H+ electrochemical potential gradient (ΔμH+)
generated across the inner mitochondrial membrane by the function of
the respiratory chain. Mitochondrial carriers can be divided into
electrogenic (or electrophoretic) and electroneutral transporters
depending on whether their transport reactions result in charge
imbalance. The intra-/extra-mitochondrial distribution of solutes
transported by electophoretic carriers is regulated by the electrical
component of ΔμH+, whereas the distribution of solutes carried by the
proteins transporting anions together with an equivalent amount of H+

(anion/H+ symport) or in exchange for OH− (anion/OH− antiport) is
regulated by the transmembrane pH gradient [29].

All primary structures of the mitochondrial carrier family mem-
bers consist of three tandemly repeated homologous domains of about
100 amino acids in length. Each repeat contains two hydrophobic
stretches, that span the membrane as α-helices, and a characteristic
signature motif P-X-[DE]-X-X-[RK]-(20–30 residues)-[DE]-G-X-X-X-X-
[YWLF]-[RK]-G. Based on the sequence features described above and
on the accessibility of carriers to peptide-specific antibodies, proteo-
lytic enzymes and impermeable reagents, each mitochondrial carrier
monomer has six helices traversing the membrane connected by
hydrophilic loops with both the N- and C-termini on the cytosolic side
of the inner mitochondrial membrane. In 2003, the 3D structure of a
member of this protein family, the ADP/ATP carrier in complex with its
inhibitor, carboxyatractyloside, was determined [30]. This structure,
whose pseudo-three-fold symmetry [30,31] is in agreement with the
three-fold sequence repeats, consists of six transmembrane α-helices
(H1–H6) and three short α-helices (h12, h34 and h56) parallel to the
membrane plane on the matrix side [30]. In each repeated domain, the
first part of the signature motif P-X-[DE]-X-X-[RK] is located at the end
of the odd-numbered transmembrane α-helices, whereas the second
part [DE]-G-X-X-X-X-[YWLF]-[RK]-G is located immediately before
the even-numbered transmembrane α-helices. The prolines of the
signature motif P-X-[DE]-X-X-[RK] sharply kink the odd-numbered
transmembrane α-helices, and the charged residues of the three
signature motifs form a salt bridge network that closes the water-
accessible cavity that is exposed towards the cytosolic side of the
mitochondrial membrane and is occupied by the inhibitor.

By using comparative models of carriers with known substrate
specificity, a common substrate-binding site was proposed [32]. This
site is formed by residues which belong to the three even-numbered
transmembrane α-helices and protrude into the cavity at the midpoint
of the membrane, one-and-a-half helix turns above the salt bridge
network. The significant sequence conservation in the mitochondrial
carrier family suggests that the structural fold and transport mechan-
ism are similar for all carriers. Although the conformational changes
involved in substrate translocation are not yet known, it is currently
thought that translocation might be triggered by substrate-induced
rearrangement of the salt bridge network from inter- to intra-domain
interactions [[33] and references therein].

The human genome encodes about 50 different mitochondrial
carriers. In the last decade, a significant advance in the field of
mitochondrial transport has been the identification of many mito-
chondrial carrier family members by over-expression in Escherichia
coli and/or Saccharomyces cerevisiae, purification and reconstitution
into liposomes for functional characterization by transport assays.
Until now, almost 30 human carriers have been characterized in terms
of substrate specificity, inhibitor sensitivity, transport mechanism,
tissue distribution and kinetic characteristics [[28] for a review, [34–
37]]. Naturally, this extension of the mitochondrial carrier family
members has greatly favoured the discovery of their involvement in
several diseases.

3. Diseases associated with mitochondrial carriers

Mitochondrial carrier-related diseases are rare errors of metabo-
lism caused by alterations of nuclear genes encoding mitochondrial

carriers. The nine known disorders are listed in Table 1. Carrier
acronyms throughout the text are those used in ref. [28]; aliases are
mentioned in the same reference. These disorders are all transmitted
by Mendelian inheritance; with the exception of autosomal dominant
progressive external ophthalmoplegia (adPEO), the others are inher-
ited in an autosomal recessive manner.

Mitochondrial carrier-related diseases can be divided into two
groups. The first concerns defects of mitochondrial carriers directly
linked to oxidative phosphorylation, such as the ADP/ATP carrierand the
PiC. The symptomatology of these diseases is characterized by
insufficient energy production in tissues where these carriers are
expressed and play an important role in oxidative phosphorylation.
These diseases should also include adPEO, which is caused by hete-
rozygous mutations of the ADP/ATP carrier isoform 1 gene (as well as of
other nuclear genes), because these mutations cause mtDNA instability.

The second group of diseases is due to alterations of the genes
encoding mitochondrial carriers that are required for mitochondrial
functions other than oxidative phosphorylation and, in particular,
intermediary metabolism. Their symptomatology depends on the
metabolism affected and its significance in specific tissues.

4. Diseases due to defects of mitochondrial carriers directly linked
to oxidative phosphorylation

Given that the catalytic site of the ATP synthase complex is
exposed towards the mitochondrial matrix, the uptake of inorganic
phosphate (Pi) and ADP into the mitochondria is essential for the
oxidative phosphorylation of ADP to ATP (see Fig.1). These uptakes are
catalyzed by two distinct carriers, the PiC and the ADP/ATP carrier,
respectively; very recently, deficiencies of one isoform of both carriers
have been described.

4.1. AAC1 deficiency

In 2005, a homozygous mutation (c.368C→A) was found in the
SLC25A4 gene of a 25-year-old male patient of Slovenian origin [38].
SLC25A4 encodes the heart-/muscle-specific ADP/ATP carrier (AAC1,
also termed ANT1). The mutation involves a highly conserved alanine
at position 123 that is replaced with an aspartic acid (A123D). In the
crystallographic structure of bovine AAC1, A123 protrudes into the
central cavity two helix turns above the salt bridge network (slightly
above the level of the proposed binding site) and is localized in the
third transmembrane segment within the consensus sequence
GXXXG, which is thought to be involved in high-affinity associations
between transmembrane α-helices [39]. The substitution of A123D
results in a complete loss of the protein's ability to transport ADP and
ATP in reconstituted liposomes [38]. Interestingly, in contrast with the
other AAC1 mutations found so far in humans (see 4.4), A123D is the
first recessive mutation found in the AAC1 gene.

The patient affected with AAC1 deficiency (OMIM 103220) mani-
fested exercise intolerance, lactic acidosis, hypertrophic cardiomyo-
pathy and a mild myopathy with no PEO. Exercise intolerance with

Table 1
Diseases associated with mitochondrial carriers

Disorder Gene Carrier Substrates

AAC1 deficiency SLC25A4 AAC1 ADP/ATP
Sengers' syndrome ? AAC1 ADP/ATP
PiC deficiency SLC25A3 PiC Phosphate
adPEO SLC25A4 AAC1 ADP/ATP
CAC deficiency SLC25A20 CAC Carnitine/acylcarnitines
HHH syndrome SLC25A15 ORC1 Ornithine/citrulline
NICCD/CTLN2 SLC25A13 AGC2 Aspartate/glutamate
Amish microcephaly SLC25A19 TPC Thiamine pyrophosphate
Neonatal myoclonic epilepsy SLC25A22 GC1 Glutamate

Carrier acronyms are taken from ref. [28]; aliases are mentioned in the same reference.

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easy fatigability and muscle pain was present since early childhood
and ventricular hypertrophy was diagnosed during puberty, indicating
that the disease is slowly progressive. Serum lactate levels in fasting
conditions and at rest were higher than normal. In the skeletal muscle
of the patient, numerous ragged-red fibers and multiple mtDNA large-
scale deletions were present (without a reduction of the wild-type
mtDNA copy number). The activities of the three mtDNA-dependent
respiratory chain complexes (I, III and IV) were lower than normal and
citrate synthase activity was much higher than normal, probably due
to mitochondrial proliferation. Interestingly, this symptomatology
closely resembles that of the AAC1 knock-out mice [40]. As in the
mouse model, loss of AAC1 function is compatible with adult life,
presumably due to compensation by glycolysis and/or transport
activities of AAC isoforms or other adenine nucleotide mitochondrial
carriers, such as the ATP-Mg/Pi carrier.

4.2. Sengers' syndrome

AAC1 deficiency was previously demonstrated in two unrelated
patients with Sengers' syndrome (OMIM 212350), an inherited
autosomal recessive disease characterized by congenital cataracts,
hypertrophic cardiomyopathy, mitochondrial myopathy and lactic
acidosis but not PEO [41]. In the muscle tissues of both patients, the
protein content of AAC1 was drastically reduced. Furthermore, ade-
nine nucleotide transport was strongly impaired in liposomes
reconstituted with mitochondrial extracts of the patients' skeletal or
heart muscle. No mutation was found in the SLC25A4 gene and
involvement of the AAC1 locus was excluded by linkage analysis. The
genetic defect underlying Sengers' syndrome is still unknown.
Nevertheless, AAC1 deficiency appears to be causally related to the
clinical symptoms and tissue specificity of the disease. It may be that
defective transcription, translation or targeting of AAC1 is responsible
for this disorder.

4.3. PiC deficiency

In 2007, a homozygous mutation in the gene encoding the PiC
(SLC25A3) (c.215G→A) was found in two ill siblings of non-
consanguineous Turkish parents (the parents were heterozygous for
the same mutation) [42]. The human gene, which is spread over 8.3 kb
of DNA, maps to 12q23.1 and contains 9 coding exons; of these, exons
3A and 3B are closely related and spliced alternatively [43]. The
resulting isoforms, termed PiC-A and PiC-B, differ in 13 amino acids
and kinetic parameters but have the same substrate specificity and
inhibitor sensitivity [44]. Both catalyze the uptake of phosphate, either
by H+ co-transport or in exchange for OH−, into the mitochondria at
the expense of the ΔpH component of the protonmotive force
generated by electron transport. This uptake of Pi into mitochondria
is essential for oxidative phosphorylation of ADP to ATP. Like isoforms
of other mitochondrial proteins involved in oxidative phosphoryla-
tion, PiC-A is abundantly expressed only in heart and muscle, whereas
PiC-B is expressed in all tissues [44,45]. The differences between the
two PiC isoforms in kinetic properties (Km of PiC-B is 3-fold lower than
that of PiC-A, and Vmax of PiC-B is 3-fold higher than that of PiC-A)
and abundance in tissue (69 and 0 pmol PiC-A per mg protein, and 10
and 8 pmol PiC-B per mg protein in heart and liver bovine mito-
chondria, respectively1) may account for the variation in reliance of
the tissues on oxidative phosphorylation. The ubiquitous PiC isoform B
might match the basic energy requirement of all tissues, and isoform
PiC-A might become operative to accommodate the higher energy
demands associated with contraction of striated muscle fibers.

Interestingly, the c.215G→A mutation found in the two patients is
localized in exon 3A and therefore affects the heart-/muscle-specific
isoform, PiC-A. In the PiC-A of both patients glycine 72, that is
conserved in all known PiC sequences and in most other mitochon-
drial carriers and is located in the first transmembrane helix three
helix turns from the membrane's cytosolic side, is substituted by a
glutamic acid residue; this substitution is deleterious for protein
function [42].

PiC deficiency (OMIM 610773) is characterized by muscular
hypotonia, progressive hypertrophic cardiomyopathy, elevated
plasma lactate levels, and lactic acidosis. In one of the two affected
siblings, cyanosis and poor weight gain were also observed. The
patients died of heart failure at 4 and 9 months.

In accordance with the tissue specificity of disease symptomatol-
ogy, the synthesis of ATP by oxidative phosphorylation was defective
only in muscle. Indeed, it was found that in heart mitochondria, but
not in digitonin-permeabilized fibroblasts, ADP-stimulated respira-
tion of pyruvate was drastically reduced, whereas uncoupler-stimu-
lated respiration was normal [42]. Furthermore, the activities of
respiratory chain enzymes, pyruvate dehydrogenase and oligomycin-
sensitive ATP-ase were normal. No mutation was detected in the genes
of ATP synthase subunits encoded by mtDNA or in SLC25A4 encoding
the heart-/muscle-specific isoform of the ADP/ATP carrier. In view of
the above-reported results, the disease is due to a defect in the PiC-A-
mediated import of Pi into mitochondria, which prevents ATP
synthesis by oxidative phosphorylation.

4.4. Autosomal dominant progressive external ophthalmoplegia (adPEO)

adPEO is a clinically and genetically heterogeneous disorder that is
usually inherited as a dominant trait. It is caused by defects in certain
nuclear genes and is characterized by multiple deletions of mtDNA in
post-mitotic tissues [46–48]. Therefore, adPEO constitutes an example
of nuclear gene defects affecting mtDNA. The mtDNA deletions result
in a lack of respiratory chain proteins that lead to defective energy
production. adPEO manifests an adult-onset phenotype (typically at
20–40 years of age); however, early onset has also been reported.
Typical symptoms of the disorder are PEO due to weakness of the
external eye muscles, ptosis, and mild descending myopathy. Addi-
tional variable symptoms include bilateral cataract, sensorineural
hypocusia, tremor, ataxia, sensorimotor peripheral neuropathy, gen-
eralized muscle weakness, exercise intolerance, depression, parkin-
sonism and endocrine dysfunction. Serum lactate levels are normal or
slightly increased. Muscle biopsies of affected individuals exhibit
ragged-red fibers, cytochrome c oxidase-negative fibers, and dimin-
ished activity of respiratory chain complexes. Southern blot analysis of
muscle DNA reveals the presence of heterogeneous mtDNA species
due to multiple large-scale deletions ranging from 3.0 to 8.5 kb in
length. mtDNA lesions are present and accumulate in post-mitotic
tissues, mainly brain, muscle and heart. This is consistent with the
tissue distribution of AAC1 in man, since AAC1 is the predominant
isoform of the ADP/ATP carrier in skeletal and heart muscle but is also
present in brain.

PEO is one of the most common diseases caused by primary mtDNA
deletions. However, more recently, mutations of three nuclear genes
have also been shown through linkage analysis to be responsible for
adPEO by causing secondary multiple deletions of mtDNA. One of these
nuclear genes is SLC25A4 located on chromosome 4q34 and encoding
the heart-/muscle-specific mitochondrial AAC1 (OMIM 609283). The
other two genes are Twinkle, on chromosome 10q24 encoding an
mtDNA helicase (OMIM 606075), and POLG1 on chromosome 15q25
encoding the catalytic subunit of mtDNA-specific polymerase γ (OMIM
157640). Furthermore, at least a fourth locus must be implicated in
view of cases that could not be linked to any of the three genes reported
above. Nuclear-dependent PEO is usually inherited as an autosomal
dominant disorder, but cases with autosomal recessive inheritance

1 It should be mentioned that according to Mayr et al. [42], “the small proportion of
isoform B” detectable in all muscle tissues “may originate from either myoblasts or
contaminating connective tissue or blood”.

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have been described in families carrying heterozygous mutations in
the POLG1 gene. Mutations in the POLG1 and Twinkle genes are the
most frequent cause of PEO, whereas those in the SLC25A4 gene are
rarer. Four heterozygous missense mutations of AAC1 (A90D, A114P,
L98P, D104G) have been found in adPEO families and one (V289M) in a
sporadic case. In the crystallographic structure of bovine AAC1, these
mutations interface with the membrane except for D104G which is
located in the cytosolic loop between H2 and H3. All the mutations are
near the cytosolic side of the carrier protein, three to four helix turns
from the common binding site. AAC1-related adPEO is relatively
benign because the symptoms are usually limited to skeletal and facial
muscles. As for all diseases caused by mutations of mtDNA, treatment
of adPEO is symptomatic and includes eye props and ptosis surgery.

The Twinkle and POLG1 genes are directly involved in the repair
and replication of mtDNA, whereas AAC1 catalyzes the exchange of
cytosolic ADP for intramitochondrial ATP. Therefore, the mechanism
by which AAC1 mutations cause mtDNA instability is unclear. Several
hypotheses have been set forth; the mutations may: modify the
transport properties of AAC1; alter the intramitochondrial pool of
adenine nucleotides causing a shortage of ADP, which may be insuf-
ficient for dATP synthesis through the action of ribonucleotide
reductase and nucleoside diphosphokinase and, consequently, yield
a high error rate of mtDNA polymerase; and cause a defective import
of the mutated proteins into mitochondria, leading to misfolded
proteins that are more susceptible to ROS or to an increased pro-
duction of ROS within the mitochondria and damage of mtDNA. To
investigate the functional consequences of the human AAC1 muta-
tions found in adPEO patients, the mutations were introduced at
equivalent positions in AAC2, the yeast ortholog of human AAC1 [49].
Interestingly, expression of some of these mutants in aac2-defective
haploid strains of S. cerevisiae caused a growth defect on nonfermen-
table substrates, a decrease of cytochrome content and reduced
respiratory activity, but variable inhibition of ATP and ADP transport
as deduced from measurements in liposomes reconstituted with
mitochondrial extracts obtained from cells transformed with either
each mutant or wild-type AAC2. In addition, heteroallelic strains,
expressing both the aac2 mutant allele and wild-type aac2, behaved
as a recessive trait for oxidative growth and as a dominant trait for
mtDNA instability. Therefore, the dominant feature of mtDNA
alteration induced by pathogenic aac2 alleles is similar in yeast and
adPEO patients.

5. Diseases due to defects of mitochondrial carriers involved in
intermediary metabolism

The diseases of this group known so far are CAC deficiency, HHH
syndrome, AGC2 deficiency, Amish microcephaly and neonatal
myoclonic epilepsy. They will be reviewed in the following sections
in the order in which they have been associated for the first time to
alterations of a specific mitochondrial carrier gene.

5.1. CAC deficiency

Carnitine–acylcarnitine carrier (CAC) deficiency2 (OMIM 212138) is
the first disorder to have been associated with a member of the SLC25
gene family, SLC25A20 [50,51]. This gene, which spans about 42 kb,
contains 9 coding exons, maps to chromosome 3p21.31 and encodes the
CAC, a protein of 301 amino acids. The rat CAC was purified in 1990 and
7 years later its cDNA was cloned in rat and man [[50,52] and references
therein]. CAC catalyzes a 1:1 electroneutral exchange between a mo-
lecule of acylcarnitine, that enters mitochondria, and a molecule of free
carnitine, that exits the organelles (see Fig. 3K of ref. [28]). The CAC
function is conserved in all eukaryotes, although mammalian CAC has a

higher affinity for long-chain rather than short-chain acylcarnitines and
the lowest affinity for free carnitine, in contrast with the fungal carriers
[[53] and references therein]. In particular, CAC is the key component of
thecarnitine cyclewhichconsists of three steps: i)transferof acyl groups
from acyl-CoA to carnitine through carnitine palmitoyltransferase
(CPT I), an outer mitochondrial membrane enzyme; ii) CAC-mediated
transport of acylcarnitines across the inner mitochondrial membrane in
exchange for free carnitine; and iii) transfer of acyl groups from
acylcarnitines to CoA inside the mitochondria through CPT II, an inner
mitochondrial membrane enzyme (see Fig. 3K of ref. [28]). By catalyzing
the carnitine/acylcarnitine exchange, CAC allows the import of fatty acyl
moieties into the mitochondria where they are oxidized by the β-
oxidation pathway. This pathway is the major source of energy for heart
and skeletal muscles during fasting and physical exercise. Besides the
exchange, CAC is also able to perform unidirectional transport of
substrates across the inner mitochondrial membrane but at a much
lower rate (about one tenth that of the exchange); nevertheless, uniport
of carnitine into carnitine-depleted mitochondria is physiologically
important to balance the matrix level of carnitine with that present in
the cytosol, a prerequisite for optimal carnitine/acylcarnitine activity.
Given that CAC-mediated transport is an essential step in the long-chain
fattyacid β-oxidationpathway, CAC deficiency is a fatty acid β-oxidation
disorder that prevents the body from converting long-chain fatty acids
into energy.

CAC deficiency was first described in 1992 by Stanley et al. [54]. It is
a severe autosomal recessive, nonpopulation-specific disorder pre-
senting an equal male-to-female ratio. The most affected organs are
heart, liver, brain, and skeletal muscles. The disorder manifests with
life-threatening episodes of coma upon fasting (due to hypoglycemia,
since the liver is unable to produce ketone bodies from fat during
fasting and muscles use glucose), cardiomyopathy, cardiac arrhythmia,
muscle weakness and abnormal liver function (all likely due to
the accumulation of long-chain fatty acids and acylcarnitines that
cannot be oxidized). Other symptoms include vomiting, lethargy, hy-
potonia, weakness, hepatomegaly, cardiac insufficiency, respiratory
distress, and seizures. In addition to hypoglycemia, metabolic alte-
rations in blood include hypoketosis (caused by lack of hepatic fatty
acid β-oxidation), acidosis, hyperammonemia (possibly due to a
compensatory increase in amino acid oxidation), dicarboxylic acid-
uria, increased long-chain acylcarnitines, decreased free carnitine,
mildly elevated creatine kinase and liver enzyme levels and occa-
sional hypocalcemia. When examined in cultured cells, CAC and fatty
acid β-oxidation activities are markedly reduced or nearly abo-
lished, although the other carnitine cycle enzymes, CPT I and CPT II,
and β-oxidation enzymes exhibit normal activity.

CAC deficiency presents two infantile phenotypes: the first, and
most common, with early onset in the neonatal period, and the second
milder form with onset in infancy or, less frequently, in childhood.
Neonates with the early-onset variety deteriorate rapidly after birth;
their mortality rate is high, generally displaying a good correlation
between the severe phenotype and null genotype. Infants whose CAC
retains some residual activity usually have no cardiac involvement and
can respond to treatment with medium-chain triglycerides that do not
require carnitine to enter the mitochondria. When both phenotypes
are untreated, patients with CAC deficiency experience hypoglycemic
crises that may lead to brain damage, coma and death, or may die from
cardiac arrest.

Since the first reported mutation in the SLC25A20 gene [50], 26
others have been identified (Fig. 2). Interestingly, some missense
mutations (D32N, R133W, P230R and D231H; Fig. 2A) regard residues
of the signature motifs present in all mitochondrial carriers, and two
others (R178Q and G81R; Fig. 2B) concern residues whose equivalents
in yeast CAC have been proposed to bind the substrate [32],
underscoring the importance of the motifs and of the proposed
“common substrate-binding site” [32] for the structure and function of
these proteins.2 Also known as carnitine/acylcarnitine translocase (CACT) deficiency.

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Early recognition and treatment is crucial in CAC-deficient patients.
CAC deficiency can be identified by newborn screening programs
using tandem mass spectrometry, although in severe infantile cases
the results of testing might return after the child is already symp-
tomatic. Management and long-term treatment of the CAC patient
consist of fasting prevention with frequent meals, a diet rich in
carbohydrates, low in lipids (of which about 80% should be provided as
medium-chain triglycerides), and supplemented with essential poly-
unsaturated fatty acids. Patients should have an emergency protocol in
place to assure administration of intravenous glucose in case of fever,
vomiting or other illness preventing oral intake. Intake of L-carnitine
may be helpful, particularly in individuals with severely deficient car-
nitine levels. CAC deficiency can be differentiated from other disorders
of the carnitine cycle and fatty acid β-oxidation in cultured fibroblasts
(e.g., medium-chain acyl-CoA dehydrogenase deficiency, OMIM
201450) by measurement of urine organic acid and plasma acylcarni-
tine profiles. Diagnosis should be confirmed in vitro by measurements
of fatty acid β-oxidation activity, CAC activity (in cultured fibroblasts
[55], in reconstituted liposomes [56] or by complementation in S.
cerevisiae [57] or Aspergillus nidulans [58]) and DNA testing. Determi-
nation of carrier activity is also important to establish that a newly
found mutation is disease-causing.

5.2. HHH syndrome

Another error of metabolism is HHH syndrome (OMIM 238970)
that is caused by mutations in the SLC25A15 gene [59]. This gene,
which maps to band 13q14.1, spans about 23 kb and contains 6 coding
exons, encodes isoform 1 of the ornithine carrier, named ORC1. The
ornithine carrier was purified from rat liver mitochondria in 1992 and
termed ornithine/citrulline carrier for its ability to catalyze a highly
active ornithine/citrulline exchange and for its importance in the urea

cycle [60]. It was cloned for the first time in S. cerevisiae in 1997 [61].
Later, starting from the yeast ortholog, two human isoforms, ORC1 and
ORC2, were identified [59,62]. The two human isoforms were over-
expressed and characterized in reconstituted liposomes. Both trans-
port L-isoforms of ornithine, lysine, arginine, and citrulline by a 1:1
substrate exchange or, to a lesser extent, by an exchange of basic
amino acids for H+; both are inactivated by spermine and spermi-
dine and stimulated by malate and phosphate. However, these iso-
forms differ in some respects. Firstly, ORC2 has a broader specificity
than ORC1, since it transports L- and D-histidine, L-homoarginine and
D-isoforms of ornithine, lysine and arginine with the same efficiency
as L-isoforms. Secondly, ORC2 has a higher affinity for lysine and
arginine and a lower affinity for ornithine and citrulline than does
ORC1. Thirdly, ORC1 is expressed at higher levels than ORC2 in all the
tissues investigated, particularly in liver, lung, pancreas and testis [62].

ORC plays a number of important roles in cell metabolism. For
example, under conditions of low arginine content in the diet and/or in
tissues where argininase activity is negligible, ornithine produced
intramitochondrially from glutamate must be exported to the cytosol
to accomplish polyamine biosynthesis. Conversely, when dietary
content of arginine is high, the ornithine formed by arginine hydro-
lysis in the cytosol must be imported into the mitochondria where it is
converted to glutamate and proline by ornithine aminotransferase, an
enzyme that is located in the mitochondrial matrix and abundant in
liver and kidney. Hepatic ornithine aminotransferase is localized in the
pericentral hepatocytes that contain glutamine synthetase and not in
the periportal hepatocytes containing the urea cycle enzymes [63]. In
periportal hepatocytes, ORC carries out the important function of
exchanging cytosolic ornithine for mitochondrial citrulline. By provid-
ing this function, ORC links the enzyme activities of urea synthesis in
the cytosol to those in the mitochondria and is therefore an essential
component of the urea cycle, as previously proposed (see Fig. 3E of ref.

Fig. 2. Mutations in patients with CAC deficiency. (A) Lateral view of the structural-homology model of human CAC (in cartoon representation) showing the positions of the 9 amino
acid substitutions found to date in CAC deficiency. (B) A view of the proposed substrate-binding site from the cytoplasmic side. The carnitine substrate is shown in stick
representation. (C) List of type and number of mutations found to date in patients with CAC deficiency. All mutations are from the HGMD database (http://www.hgmd.cf.ac.uk/ac/
index.php). The colour code for the transmembrane α-helices is as follows: H1, red; H2, orange-green; H3, light green; H4, dark green; H5, light blue; and H6, blue. Purple surfaces
highlight the salt bridge network between residues K35 and D231, K234 and E132, and K135 and D32.

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[28]) [[64] and references therein]. Under physiological conditions, it is
highly probable that the key step of the urea cycle consisting in the
ornithine/citrulline exchange is catalyzed, mainly or exclusively, by
ORC1, since ORC2 has a lower affinity for ornithine and citrulline and is
expressed in liver at a much lower level. Deficiency of ORC1 in patients
with HHH syndrome supports this interpretation.

As indicated by its name, HHH syndrome is characterized by
hyperammonemia, hyperornithinemia and homocitrullinuria. These
metabolic alterations result from a defect in ORC1 as do many clinical
symptoms of the disease. Hyperammonemia is due to impairment of
the urea cycle at the level of ORC1. Defective ORC1 does not allow
import of ornithine from the cytosol to the mitochondria and, con-
sequently, impedes the function of intramitochondrial ornithine
transcarbamoylase that condenses carbamoyl phosphate and orni-
thine to form citrulline. Accumulation of ornithine in the cytosol
explains hyperornithinemia and leads to an increased production of
polyamines. In the absence of ornithine inside the mitochondria, car-
bamoyl phosphate accumulates and either condenses with lysine to
form homocitrulline, leading to homocitrullinuria, or enters the
cytosolic pyrimidine biosynthetic pathway, leading to increased
excretion of orotic acid and uracil. Other laboratory findings include
increased levels of glutamine, alanine, liver transaminases and alka-
line phosphatase.

HHH syndrome may present at any age from the neonatal phase to
adulthood. It usually manifests in early childhood, with 10% of patients
presenting in the neonatal period. The most common symptoms of the
disorder are episodes of confusion, lethargy, and coma due to hyper-
ammonemia, and neurologic features such as mental retardation,
learning difficulties, spastic paraplegia and seizures. Interestingly,
most patients present with pyramidal dysfunction, often resulting in
early adulthood in frank spastic paraplegia, whose course appears to
be unrelated to treatment and whose pathogenetic mechanism(s) is

still poorly understood [65]. Patients also often present abnormal liver
function leading to hepatitis-like attacks and coagulopathy with
factor-specific defects (factors VII, IX and X). Some symptoms present
in an acute way (e.g., intermittent episodes of vomiting, ataxia,
lethargy, confusion, coma, myoclonic jerks, and hepatitis-like attacks),
whereas others follow a more chronic course (e.g., aversion for
protein-rich foods, coagulation abnormalities, hypotonia, develop-
mental delay, progressive encephalopathy with mental regression,
pyramidal dysfunction). Similarly to other urea cycle defects, neonates
may manifest symptoms soon after feeding. Children and adults have
a tendency to refuse high-protein foods. Although variable, the
phenotype of HHH patients is somewhat milder than that associated
with defects of urea cycle enzymes. It may be that ORC2 partially
compensates for ORC1. Gain of function polymorphism of ORC2 and a
haplogroup (mtDNA lineage) have been suggested [66] as possible
factors that may influence the ORC1 deficiency phenotype. Because in
mouse isoform 2 of ORC is a pseudogene (given the presence of a
single nucleotide deletion near the 5′-end causing a frame shift), it
would be interesting to delete the murine ORC1 and compare the
resulting phenotype with that of HHH patients and the phenotype of
urea cycle enzyme knock-out mice.

HHH syndrome is an autosomal recessive disease and has a pan-
ethnic distribution with a higher proportion of cases reported in
Canada (a founder mutation located in Quebec), Italy, and Japan. The
male-to-female ratio is approximately 2:1. Since its first description in
1969 by Shih et al. [67], 71 additional cases have been reported. In
Fig. 3, all the 33 naturally occurring mutations in ORC1 known to date
are listed, including 16 from Dr. Dionisi-Vici that are yet unpublished.
Many of the mutations have been functionally analyzed in recon-
stituted liposomes and found to inactivate the carrier [62,68]. Inter-
estingly, two of the disease-causing mutations, R275Q and E180K,
concern residues involved in the proposed substrate-binding site,

Fig. 3. Mutations in patients with HHH syndrome. (A) Lateral view of the structural-homology model of human ORC1 (in cartoon representation) showing the positions of the 14
amino acid substitutions found to date in HHH syndrome. The transmembrane α-helices are coloured as reported in Fig. 2. Purple surfaces highlight the salt bridge network between
residues K34 and D231, K234 and E128, and K131 and D31. (B) A view of the proposed substrate-binding site from the cytoplasmic side. The ornithine substrate is shown in stick
representation. (C) List of type and number of mutations found thus far in patients with HHH syndrome. Seventeen mutations were taken from the HGMD database (http://www.
hgmd.cf.ac.uk/ac/index.php); the remainder was obtained through the courtesy of Dr. Dionisi-Vici.

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whereas T32R and P126R residues belonging to the signature motifs
of the mitochondrial carrier family (Fig. 3). Furthermore, 6 of the
14 missense mutations affect residues protruding toward the cavity
occupied by the entering substrate in the cytosolic conformation of
the carrier (Fig. 3).

Early diagnosis and treatment of HHH syndrome are important to
prevent neurologic and cognitive deterioration. However, diagnosis is
often delayed as a result of non-specific symptoms. Differential diag-
nosis is based mainly on biochemical characteristics: persistently
elevated plasma ornithine levels 5 to 10 times higher than normal,
which distinguishes the disorder from other urea cycle defects; the
presence of homocitrulline and orotic acid in urine; and episodic or
postprandial hyperammonemia, differentiating HHH syndrome from
gyrate atrophy (OMIM 258870) which is caused by ornithine trans-
aminase deficiency. Measurement of carrier activity in cultured fibro-
blasts [59]3 and in reconstituted liposomes followed by genetic testing
constitutes evidence of the disease.

Treatment of HHH syndrome is similar to that of other urea cycle
defects and is based mainly on a low-protein diet. It is usually
accompanied by supplementation of citrulline and ornithine (to
reduce ammonemia), although the long-term side effects of hyper-
ornithinemia are not known. Arginine, sodium benzoate and sodium
phenylbutyrate have also been administered to decrease ammonia
levels. Treated patients exhibit good metabolic control and very
infrequent relapses of hyperammonemia. The mortality rate for the
disease is low.

5.3. AGC2 deficiency

Aspartate/glutamate carrier isoform 2 (AGC2) deficiency is a highly
widespread autosomal recessive disease in the Far East and Southeast
Asia with a carrier prevalence of about 1 in 70 individuals and a male-
to-female ratio of 2.4:1 [69]. AGC2 deficiency was also detected in the
Middle East, including Israel. More recently, some cases have been
found in the US and the United Kingdom, pointing towards a pan-
ethnic distribution. AGC2 deficiency has been extensively studied by
Dr. Saheki in Japan who identified SLC25A13 as the gene responsible
for the disease by means of homozygosity mapping and positional
cloning [70]. The SLC25A13 gene, spanning about 201 kb, maps to
chromosome 7q21.3 and consists of 18 exons. Subsequently, it was
found that the gene product of SLC25A13, previously termed citrin,
and its closely related human homolog, aralar, function as Ca2+-
regulated aspartate/glutamate carriers, abbreviated as AGC2 and
AGC1, respectively [71]. AGC1 (encoded by SLC25A12 on chromosome
2q24) is highly expressed in brain, heart, and skeletal muscle, whereas
AGC2 is found in several tissues but most abundantly in liver where
AGC1 is absent [72,73]. These proteins are localized in the inner
mitochondrial membrane, bind Ca2+, and consist of two domains: the
C-terminal domain, containing all the sequence features of the mito-
chondrial carrier family, and the N-terminal domain, a long extension
containing four EF-hand Ca2+-binding motifs and protruding outside
the inner mitochondrial membrane. In reconstituted liposomes, AGC1
and AGC2 transport aspartate and glutamate by an obligatory 1:1
exchange. The exchange is electrophoretic because aspartate is tran-
sported as an anion, whereas glutamate is transported with a proton.
Consequently, in energized mitochondria with a positive membrane
potential outside, exit of aspartate and entry of glutamate are strongly
favoured and therefore the AGC-catalyzed reaction is essentially
unidirectional and irreversible. The Km values of both isoforms for
external aspartate and glutamate are about 0.05 and 0.2 mM, re-
spectively, do not change with membrane potential in reconstituted
liposomes, and are virtually identical to those determined with native

AGC. The C-terminal domains of AGC1 and AGC2 account for the
activities and all transport properties of the entire two proteins and
constitute their catalytic portion, whereas the N-terminal domains,
containing the Ca2+-binding sites, make up their regulatory portion. In
fact, the activity of AGC1 and AGC2 is stimulated by Ca2+ on the
external side of the inner mitochondrial membrane, since this
stimulation is not affected by ruthenium red, a specific inhibitor of
Ca2+ entry into mitochondria [71]. Furthermore, by using mitochond-
rially targeted luciferase to monitor ATP formation, a surplus
production of ATP was observed in the mitochondria of agonist-
stimulated cells overexpressing AGC1 or AGC2 but not in stimulated
cells overexpressing the C-terminal domains lacking the Ca2+-binding
sites. Notably, the intramitochondrial Ca2+ level was the same in cells
overexpressing either the entire protein (AGC1 or AGC2) or each of the
two C-terminal domains alone [74]. Therefore, cytosolic Ca2+ activates
mitochondrial oxidative metabolism through its binding to the N-
terminal domain of AGC.

The major function of AGC is to supply aspartate to the cytosol. This
is particularly important in hepatocytes that have a negligible capacity
of taking up aspartate from the blood. In hepatocytes, AGC plays a
fundamental role in urea synthesis by supplying aspartate to argi-
ninosuccinate synthetase (ASS), a urea cycle enzyme that is localized
in the cytosol and condenses citrulline and aspartate to produce
argininosuccinic acid. The reactions of the urea cycle, their subcellular
localization and site of aspartate involvement are shown in Fig. 3E of
ref. [28]. Besides urea synthesis, cytosolic aspartate is necessary for
gluconeogenesis from reduced substrates and for protein, purine and
pyrimidine synthesis. Cytosolic aspartate is also very important for the
oxidation of cytosolic NADH+H+, since AGC is a key component of the
malate–aspartate shuttle that transfers the reducing equivalents of
NADH+H+ from cytosol into mitochondria (see Fig. 3H of ref. [28]). In
this shuttle, AGC again provides the cytosol with aspartate; in the
cytosol, aspartate is transaminated to oxaloacetate which is reduced
by cytosolic malate dehydrogenase to malate, leading to the oxidation
of cytosolic NADH+H+. Malate enters the mitochondria via the
oxoglutarate carrier and in the matrix is reduced to oxaloacetate by
mitochondrial malate dehydrogenase, resulting in the reduction of
intramitochondrial NAD+. Subsequently, oxaloacetate is transami-
nated to aspartate by mitochondrial aspartate aminotransferase, com-
pleting the cycle. It should be emphasized that the malate–aspartate
shuttle operates in the direction of transferring reducing equivalents
from cytosol to mitochondria because the reaction catalyzed by AGC is
unidirectional and irreversible. By oxidizing cytosolic NADH, the
malate–aspartate cycle allows aerobic glycolysis and alcohol metabo-
lism to occur. It is important to note that the glycerol-3-phosphate
shuttle, which is the main physiological alternative mechanism for the
oxidation of cytosolic NADH, exerts low activity in human liver
(whereas it is very active in murine liver).

Because AGC2 is the only isoform, or at least the most prevalent
isoform, of this carrier expressed in liver, lack of its function in this
tissue explains many symptoms of the disease. AGC2 deficiency causes
two age-dependent phenotypes: neonatal intrahepatic cholestasis
caused by citrin (AGC2) deficiency (NICCD, OMIM 605814) and adult-
onset type II citrullinemia (CTLN2, OMIM 603471). Type II citrulline-
mia usually manifests in adults between the ages of 20 and 50 years. A
shortage of aspartate in the cytosol of hepatocytes causes citrulline-
mia, hypoproteinemia, and recurrent episodes of hyperammonemia
responsible for the observed encephalopathy and neuropsychiatric
symptoms (e.g., sudden aberrant behavior, disturbance of conscious-
ness, disorientation, delirium, tremor, convulsive seizures and coma).
The inhibition of cytosolic NADH oxidation, with the consequent
increase in the NADH/NAD+ ratio, causes a distaste for carbohydrates,
a preference for fat and especially for proteins as well as an incapacity
to consume alcohol. In fact, in patient dietary surveys carbohydrates
average only ~34% of total caloric intake, whereas proteins average
~23%. In particular, a strong desire for beans and nuts, which are

3 The activity, which is based on measurement of the incorporation of [14C]ornithine,
as compared to that of [3H]leucine, into cellular protein, is also markedly decreased or
null in patients with gyrate atrophy.

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particularly rich in aspartate and asparagine, has been described.
Citrullinemia and hyperammonemia are also due to a unique feature
of the disease, i.e., a variable liver-specific reduction in ASS protein and
activity with no alterations in either its gene or hepatic ASS mRNA
levels [75]. Conversely, in type I citrullinemia (CTLN1, OMIM 215700)
the gene for ASS is mutated, leading to ASS deficiency in all tissues
[76]. It is not yet clear why the level of hepatic ASS is low in CTLN2. It
may be that critical aspartate levels are required to make ASS active or
to stabilize the enzyme. Patients also manifest fatty liver most likely
because they oxidize cytosolic NADH+H+ by yet another pathway, the
citrate–malate shuttle (see Fig. 3D of ref. [28]), whose normal function
is to synthesize fatty acids by transferring acetyl-coenzyme A from the
mitochondrial matrix to the cytosol. In fact, the key enzyme of the
citrate–malate shuttle, the citrate carrier, exports citrate from
mitochondria to the cytosol where it is cleaved to acetyl-coenzyme
A and oxaloacetate by ATP-citrate lyase. Furthermore, some patients
develop hyperlipidemia which likely results from the functioning of
the citrate–malate shuttle and from the accumulation of glycerol-3-
phosphate due to poor activity of the glycerol-3-phosphate cycle in
human liver. Other laboratory findings are increased arginine and
pancreatic secretory trypsin inhibitor levels, an elevated threonine-to-
serine ratio, and a decreased ratio between branched-chain amino
acids (BCAAs) to aromatic amino acids (AAAs) in patient sera. Finally,
pancreatitis and hepatocellular carcinoma occur with a higher
incidence in CTLN2 patients. Adult-onset type II citrullinemia is pro-
gressive and patients usually die from hyperammonemic encephalo-
pathy and complications of brain edema. Given the poor prognosis of
the adult AGC2-deficiency phenotype, patients should consider
undergoing liver transplantation.

The neonatal phenotype, NICCD, was recognized more recently by
Saheki and co-workers [77]. Affected children commonly present
transient intrahepatic cholestasis, fatty liver, hepatomegaly, growth
retardation, citrullinemia, aminoacidemias (elevated methionine,
threonine, tyrosine, phenylalanine, lysine and arginine), an increased
threonine-to-serine ratio and galactose concentration, ketotic hypo-
glycemia, and hypoproteinemia. Some patients present with hepatitis,
jaundice, decreased coagulation factors, hemolytic anemia and bleed-
ing diathesis as a result of liver dysfunction and hypoproteinemia. High
levels of plasma α-fetoprotein have been detected in NICCD but not in
CTLN2. The neonatal phenotype of AGC2 deficiency is usually benign.
Symptoms disappear by the age of 12 months; afterwards, these
patients become seemingly healthy (or manifest non-specific symp-
toms) but display a preference for protein- and lipid-rich foods and an
aversion for foods high in carbohydrate content. One or several
decades later, some AGC2-deficient individuals go on to develop
CTLN2. The clinical spectrum of AGC2 deficiency has recently been
expanded with the report of infantile presentation characterized by
failure to thrive and bleeding diathesis and no evidence of neonatal
intrahepatic cholestasis [78].

In the compensatory period (which corresponds to the transition
from NICCD to the onset of CTLN2), affected individuals live with
metabolic defects that mainly involve liver, i.e., a high cytosolic NADH/
NAD+ ratio and impairment of urea synthesis. It is likely that in the
AGC2-deficient liver, partial oxidation of cytosolic NADH is accom-
plished by the low activity of the glycerol-3-phosphate NADH shuttle
and by the citrate–malate cycle, as mentioned above. Given that in
patient liver the export of aspartate from the mitochondria is nearly
null and aspartate is virtually not taken up by the blood, ureagenesis is
dependent on aspartate production in the cytosol. Aspartate can be
produced in the cytosol in two ways: from asparagine by the action of
asparaginase and from oxaloacetate by cytosolic aspartate amino-
transferase. Asparagine can be synthesized by asparagine synthetase
or can be supplied from the diet via the portal venous blood. Oxa-
loacetate can be formed from fumarate, a product of the urea cycle
enzyme argininosuccinate lyase, by the successive actions of cytosolic
fumarase and malate dehydrogenase. In both cases serious draw-

backs emerge, i.e., the production of 1 mol of ammonia per each mol
of aspartate provided by asparagine and the generation of 1 mol of
NADH+H+ per each mol of aspartate provided by oxaloacetate origi-
nating from malate (see Fig. 6B of ref. [79]). Therefore, in the AGC2-
deficient patient, the deficit of ureagenesis is due not only to a
decreased availability of aspartate to ASS but also to an increased
cytosolic NADH/NAD+ ratio as a consequence of the defective malate–
aspartate NADH shuttle and of aspartate production from fumarate, as
mentioned above.

SLC25A13 is the only gene known to be associated with AGC2
deficiency. The first mutations observed in SLC25A13 led to either
truncation of the protein or extensive deletions causing AGC2 de-
ficiency [80]. More recently, missense mutations were also detected.
Until now, no significant correlation has been observed between
SLC25A13 mutation types and the age of CTLN2 onset.

Forty-four mutations have been found thus far in patients with AGC2
deficiency, 24 of which have not yet been published (T. Saheki, personal
communication). All together, 14 missense mutations have been found.
The newly identified mutation R588Q that inhibits transport of
glutamate and aspartate in reconstituted liposomes by about 90% [81]
regards a residue belonging to the proposed common substrate-binding
site formed by R588, R492, E404 and K405 — residues equivalent to
those proposed to bind the substrate in yeast AGC1 (R792, R692, E600
and K601) [32]. R588 corresponds to R288 of the bovine oxoglutarate
carrier, to R294 of theyeast AAC2, and to R276 of the murine UCP1 which
also do not tolerate replacement with other amino acids [[82] and
references therein]. R588 also corresponds to R275 of the human ORC1;
its substitution with Q causes HHH syndrome (see 5.2).

To examine the consequences of the R588Q mutation, docking of
glutamate was performed using the residues of the common substrate-
bindingsite of the human AGC2 [83]. Docking with thehumanwild-type
AGC2 shows the substrate directly on top of the salt bridge network
interacting with R588 and the charged residues K453 and K353 of the
network (Fig. 4A). In the case of the R588Q mutant, the substrate is
positioned far from the salt bridge network at the level of the binding
site (Fig. 4B). Therefore, lack of R in position 588 impedes the interaction
of the substrate with the salt bridges causing loss of activity. It is
tempting to speculate that residue R588 functions as a mobile side-chain
that transports the substrate onto the salt bridges, determining their
aperture and the substrate's progression through the protein toward the
matrix.

The pathophysiology of AGC2 deficiency was recently investigated
by elegant studies conducted on knock-out mice [84]. SLC21A13−/−

mice exhibited a marked decrease in mitochondrial aspartate tran-
sport and malate–aspartate shuttle activities in vitro. In liver perfused
with ammonia, the rate of urea production was drastically reduced
and the lactate-to-pyruvate ratio, which reflects the cytosolic NADH/
NAD+ redox state, was increased. Under these conditions, both the
deficit in ureagenesis and increase in the NADH/NAD+ ratio were
partially corrected by the administration of asparagine that enters
hepatocytes and is hydrolyzed to aspartate. Likewise, pyruvate (but
not aspartate or citrate) reduces the deficit in ureagenesis and
decreases the NADH/NAD+ ratio in the liver-perfused system. Further-
more, in liver perfused with lactate (but not pyruvate) gluconeogen-
esis was diminished. These results demonstrate that hepatic cytosolic
aspartate is rate-limiting in SLC25A13−/− mice. However, AGC2 knock-
out mice did not manifest the key symptoms of AGC2 deficiency
present in man. The mice exhibited neither hyperammonemia and
changes in amino acid levels even following administration of protein-
rich diets nor hypoglycemia upon fasting.

The most likely explanation for the lack of CTLN2-like phenotype in
the AGC2 knock-out mice is the high activity of the glycerol-3-
phosphate cycle in their liver. Consequently, Saheki et al. generated
mice with a combined disruption of the AGC2 gene and the gene for
mitochondrial glycerol-3-phosphate dehydrogenase, a key compo-
nent of the glycerol phosphate cycle [85]. Double knock-out mice

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investigated after 40 days of age manifested: (i) a reduction in growth
rate, which is consistent with the observation that usually CTLN2
patients are thin and NICCD patients exhibit growth retardation; (ii)
hyperammonemia and increased lactate-to-pyruvate ratio under fed
conditions, which were further increased after sucrose administra-
tion; (iii) elevated plasma citrulline levels and threonine/serine ratio
under fed and fasted conditions (not altered by sucrose administra-
tion); (iv) decreased plasma BCAA/AAA ratio and alanine level after
sucrose administration (both were normal under fed or fasted
conditions); (v) hypoglycemia upon fasting due to the increased
cytosolic NADH/NAD+ ratio leading to decreased gluconeogenesis
from reduced substrates and to the lowered oxaloacetate level re-
quired by phosphoenolpyruvate carboxykinase; and (vi) fatty liver,
elevated levels of plasma free fatty acids and glycerol; the latter being
due to loss of the glycerol-3-phosphate shuttle activity in all the
tissues and to loss of the malate–aspartate shuttle in liver. Finally, the
hepatic aspartate content was lower in the double knock-out mice
compared to wild-type, glycerol-3-phosphate shuttle and AGC2 single
knock-out mice [85]. Therefore, the double knock-out mice exhibited
most of the features of the human AGC2-deficient phenotype.

AGC2 deficiency should be suspected in children with hypoglyce-
mia, hepatomegaly, growth retardation or failure to thrive. In addition,
AGC2 deficiency should also be considered in children presenting with
elevated blood galactose levels. It is important to confirm the diagnosis
of AGC2 deficiency, as treatment of these patients highly contrasts that
of urea cycle disorders. Individuals with urea cycle enzyme defects are
given protein-restricted, high-caloric diets to prevent episodes of
hyperammonemia. In contrast, CTLN2-affected patients manifest
severe episodes of hyperammonemia upon consumption of high-
carbohydrate diets. Likewise, severe episodes of hyperammonemia
have been observed after administration of glycerol or fructose for the
treatment of hyperammonemia and brain edema [85]. Alcohol intake
and the use of anti-inflammatory and analgesic drugs and/or surgery
may often provoke CTLN2 symptoms. At present, liver transplantation

is the only effective treatment for type II citrullinemia. To prevent
hyperammonemia (and resolve growth retardation in children), a diet
high in protein and lipid content and low in carbohydrates is highly
recommended. This recommendation is further supported by the
observed food preference of these patients. Treatment with pyruvate
has also been suggested to decrease the cytosolic NADH/NAD+ ratio
[79]. Moreover, in children with NICCD, supplementation with fat-
soluble vitamins and use of lactose-free formula or high protein and
low carbohydrate diet have also been applied [78]. In summary,
treatment of NICCD requires continued dietary control and growth
monitoring. Continuous monitoring of these patients is necessary to
prevent a severe outcome later in life.

5.4. Amish microcephaly (MCPHA)

Amish microcephaly (MCPHA, OMIM 607196) is an inborn error of
metabolism presenting severe microcephaly and 2-oxoglutaric acid-
uria [86]. The disorder is caused bya defect in the SLC25A19 gene which
encodes a protein of 320 residues [87]. The protein was first identified
as a deoxynucleotide carrier (DNC) [88] and later as a thiamine pyro-
phosphate carrier (TPC) [89]. In reconstituted liposomes, TPC trans-
ports the important coenzyme thiamine pyrophosphate (ThPP),
thiamine monophosphate (ThMP) and deoxynucleotides in descend-
ing order of potency, dNDPNdNTPNdNMP, by an obligatory exchange
mechanism. Nucleotides are also transported, though less efficiently
than the corresponding deoxynucleotides. Furthermore, the dideox-
ynucleoside triphosphates that are produced in the cytosol from
dideoxynucleosides, including antiviral and anticancer nucleoside
analogs, are equally efficient substrates as dNDPs [88].

To date, Amish microcephaly has only been observed in the Old
Order Amish community in Pennsylvania, U.S.A. with a high pre-
valence of about 1 in 500 individuals. In this community, 23 nuclear
families affected with MCPHA are connected to a single ancestral
couple that lived in the 1700s. The disease is characterized by severe

Fig. 4. Structural-homology model of human AGC2 illustrating the role of R588 in substrate translocation through the carrier. Docking of glutamate in the R588 wild-type AGC2 (A)
and the Q588 mutated AGC2 (B) viewed from the lateral side. Glutamate is shown in the van der Waals representation and coloured in yellow. Stick representations highlight some
residues that are located within 4 Å from the substrate. Rectangular boxes contain residues R588 (A) and Q588 (B). Portions of helices IV, V and VI (in A and B) and of the salt bridge
network (in A) were rendered transparent to facilitate viewing of the substrate and side-chains of some amino acids. The transmembrane α-helices are coloured as reported in Fig. 2.
Purple surfaces highlight the salt bridge network between residues K353 and D542, K545 and E450, and K453 and D350.

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congenital microcephaly, elevated levels of α-ketoglutarate in urine,
premature death (observed maximum survival, 14 months) and, as a
result of microcephaly, nearly absent cranial convexity, frontal sloping
and distorted facial features. The only non-CNS physical anomaly is
moderate micrognathia. Patients manifest no orientation to sight or
sound and no fine or gross motor development, have metabolic
acidosis enhanced by episodic viral illnesses, in some cases mild
hepatomegaly, difficulty maintaining normal body temperature and
develop increasing irritability.

It was first considered that the increased levels of α-ketoglutarate
might be the result of a defect in the α-ketoglutarate dehydrogenase
complex which comprises three enzymes: 2-oxoglutarate dehydro-
genase (OMIM 203740), dihydrolipoamide succinyltransferase (OMIM
126063) and dihydrolipoamide dehydrogenase (OMIM 246900). The
genes encoding these enzymes were excluded on the basis of genetic
linkage and haplotype analysis, indicating that this disorder must be
associated with another genetic locus.

Through a whole-genome scan, fine mapping and haplotype ana-
lysis, the gene affected in MCPHA was localized to a region of 2 Mb, on
chromosome 17q25 [87,90]. In this region, the genes encoding proteins
with known mitochondrial functions were analyzed in view of the
oxoglutarate abnormality in MCPHA, which suggested mitochondrial
dysfunction. A homozygous nucleotide change in the coding region of
TPC, c.530G→C (NM_021734), was identified in affected children. The
parents of individuals affected by MCPHA are obligate heterozygotes.

The mutation, which produces a glycine-to-alanine substitution at
position 177, alters a highly conserved residue in the mitochondrial
carrier family that is located in the second part of the signature motif
between helices h34 and H4, in proximity to one of the important salt
bridges that close the substrate translocation path in the cytosolic
conformation of the carrier (Fig. 5). The substitution co-segregates
with the disease in the 16 families investigated [87] and is deleterious
for protein function. Indeed, in reconstituted liposomes the transport
activity of ThPP, ThMP or dATP mediated by the G177A mutant TPC
was 25% to 30% that mediated by the wild-type TPC [89]. In addition,
substitutions of G177 with bulkier residues such as valine, cysteine and
leucine are not tolerated at all (F. Palmieri, unpublished data), as was
also found for the equivalent glycine in other members of the mito-
chondrial carrier family [[82] and references therein]. It may be hy-
pothesized that G177 functions as a hinge between helices h34 and H4
during the conformational changes that occur in the catalytic cycle of
the carrier.

SLC25A19 is the only gene known to be associated with Amish
microcephaly. To elucidate the pathogenic mechanism of this disease,
a knock-out mouse for the SLC25A19 gene was generated, and the
phenotype of both the knock-out mice and patients was studied at the
cellular level [89].

All SLC25A19−/− mice died prior to 12.5 days' gestation. The homo-
zygous embryos at E 10.5 exhibited an abnormally developed CNS
with a neural-tube closure defect and exencephaly that commonly

Fig. 5. Structural-homology model of human TPC showing the position of the mutation G177A responsible for Amish microcephaly. Cartoon representations of the G177 wild-type
TPC (A) and the A177 mutated TPC (B) viewed from the cytoplasmic side. The residues G177 and A177 are in surf representation and coloured in yellow. The transmembrane α-helices
are coloured as reported in Fig. 2. Purple surfaces highlight the salt bridge network between residues K241 and D140, R143 and D37, and K40 and D238. Cartoon and stick
representations showing the proximity of G177 (C) and A177 (D) to the salt bridge between K241 and D140. G177 and A177 are in surf representation and coloured in yellow. In C and
D, the signature motifs (P-X-D/E-X-X-K/R) of H1, H3 and H5 are coloured blue, green and orange, respectively, with the side-chains in stick representation. The salt bridges are
indicated by black dotted lines.

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involves the entire brain. In addition, they manifested erythropoietic
failure, being nearly devoid of erythrocytes at E 9.5, and elevated
oxoglutarate levels in the amniotic fluid.

In the mitochondria of knock-out mice fibroblasts and patient
lymphoblasts, no differences in ribonucleoside- and deoxynucleoside-
triphosphate levels, aswell as nodepletion or deletions in mitochondrial
DNA, were found in comparison to controls. These findings demonstrate
that although SLC29A19 may be able to transport dNDPs and dNTPs in
vivo, this function is not critical, owing perhaps to a redundancy of
deoxynucleotide carriers in mammalian mitochondria.

In agreement with the other function of TPC (i.e., to import ThPP
produced in the cytosol into the mitochondria in exchange for intra-
mitochondrially generated ThMP), it was found that ThPP and ThMP
were not detectable in the mitochondria of knock-out mice fibroblasts
and were markedly decreased in the mitochondria of patient lym-
phoblasts as compared to controls. (Of note, ThPP and ThMP were
increased in the cytosol.) Secondly, in the media of both cell lines
lactate was markedly increased, indicating mitochondrial dysfunction.

Lastly, the mitochondrial ThPP-dependent ketoglutarate dehydrogen-
ase (KGDH) and pyruvate dehydrogenase (PDH) complex activities
were nearly absent in knock-out mice mitochondrial extracts and
strongly diminished in patient mitochondrial extracts as compared to
controls. Interestingly, the addition of ThPP to the KGDH and PDH assay
mixtures restored full activity, showing that the lower activities of
KGDH and PDH in knock-out mice and patients is caused by lack of
ThPP in mitochondria [89]. Taken together, these data suggest that
transport of ThPP is the primary physiological function of TPC.

Table 2 summarizes and compares the phenotypes of the SLC25A19
knock-out mice and patients with Amish microcephaly. It can be
observed that all the metabolic abnormalities of the human disease,
starting from the decrease in ThPP level in the mitochondria, were also
present in the knock-out mice. However, these abnormalities, which
are caused by lack of ThPP in the mitochondria, were more severe in
the knock-out mice. The only exception concerns increased oxoglu-
tarate levels in amniotic fluid which were found to be less dramatic
than those in the urine of patients. This is due to the fact that mouse
embryos died prior to kidney formation and therefore did not
concentrate the metabolite. The increased severity of the mouse
model is also illustrated by the embryonic lethality of the SLC25A19
knock-out animals, by the arrest of brain formation at the level of the
neural tube closure, probably caused by decreased fluxes through
KGDH and PDH reactions, and by erythropoietic failure perhaps
caused by lack of succinyl-CoA, a product of KGDH. The most likely
explanation for the differences in the mouse and human phenotypes is
that human point mutation retains some ThPP transport activity
whereas the murine null mutation does not. In conclusion, Amish
microcephaly is due to a defect of ThPP transport into mitochondria,
which primarily affects brain development because of the brain's great
need for oxidative metabolism.

Metabolic screening and eventual imaging studies are warranted
for all children with congenital microcephaly. No intervention or
vitamin therapy has proven to be effective for treating and/or im-
proving the disorder. Only supportive therapy is available for MCPHA-

Table 2
Phenotypes of the knock-out mice and patients with Amish microcephaly

Knock-out mice Patients

TPC activity Absent Reduced to 25–30%
Lethality Embryonic 1–14 months
CNS defects Arrest of brain formation

(open neural tube)
Microcephaly

Mitochondrial ThPP and ThMPa Not detectable Decreased
Cytosolic ThPP and ThMPa Highly increased Increased
KGDH and PDH activitiesa Highly decreased Decreased
KGa Increasedb Highly increased
Lactatea Highly increased Increased
mtDNA abnormalitiesa Absent Absent
Mitochondrial (d)NTP levelsa Normal Normal

a Data obtained from SLC25A19−/− mouse fibroblasts and patient lymphoblasts.
b In amniotic fluid.

Fig. 6. Partial sequence alignment of the glutamate and aspartate/glutamate carriers in man and other species displaying high conservation of P206 and G236 of the human glutamate
carrier isoform 1. Abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Rn, Rattus norvegicus; Xt, Xenopus tropicalis; Dr, Danio rerio; Gg, Gallus gallus; Dm, Drosophila
melanogaster; Ag, Anopheles gambiae; Nv, Nasonia vitripennis; Sc, Saccharomyces cerevisiae; Nf, Neosartorya fischeri; Af, Aspergillus fumigatus; Nc, Neurospora crassa; Dd, Dictyostelium
discoideum.

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affected patients. Phenobarbital has been administered for the treat-
ment of seizures, and physical therapy is encouraged for alleviating
contractures or other secondary neurologic manifestations. Genetic
risk of MCPHA is best determined in the pre-gestational phase.

5.5. Neonatal myoclonic epilepsy

Mutations in the SLC25A22 gene encoding isoform 1 of the gluta-
mate carrier (GC1) cause a form of early myoclonic epilepsy (EME),
named neonatal myoclonic epilepsy (OMIM 609304) [91]. The
SLC25A22 gene maps to chromosome 11p15.5 and contains 8 coding
exons. GC1 and its isoform GC2, which is encoded by SLC25A18, were
identified upon gene expression in E. coli as glutamate carriers by
their transport properties in reconstituted liposomes [92]. Both iso-
forms are located in the mitochondrial membrane (M. Lasorsa and F.
Palmieri, unpublished data) and catalyze a glutamate/H+ symport with
high specificity. However, GC1 has a high Km for glutamate (∼5 mM),
whereas the Km value of GC2 is low (∼0.22 mM) and the Vmax value of
GC1 is higher than that of GC2. Furthermore, isoform 1 is expressed at
higher levels than isoform 2 in all the tissues investigated, particularly
in liver, pancreas and brain. The differences in expression levels and
kinetic parameters of the two isoforms suggest that isoform 2 matches
the basic requirement of all tissues, especially with respect to amino
acid degradation, and that isoform 1 becomes operative to accom-
modate higher demands associated with specific metabolic functions.
Because glutamate is co-transported with an H+ by the GC, and
therefore its distribution across the mitochondrial membrane is
dependent on ΔpH, entry of glutamate is favoured in energized mi-
tochondria. However, when glutamate is generated intramitochond-
rially (e.g., by proline oxidation), the GC may operate in the reverse
direction to limit intramitochondrial accumulation of glutamate.

The first GC1 mutation, a substitution of proline 206 with leucine,
was found in a consanguineous Arab Muslim family with 4 affected
children among 8 [91]. A second mutation (a change from glycine 236
to tryptophan) was identified in a consanguineous family from Algeria
with 1 affected child among 3 (L. Colleaux and F. Palmieri, unpublished
data). All affected individuals were homozygous for the above-
mentioned mutations, whereas the parents were heterozygous.

The symptoms of the affected children in both families were
similar: very-early-onset intractable myoclonic seizures, hypotonia,
progressive microcephaly, abnormal visual nerve conduction, and
rapid evolution into encephalopathy and spasticity. Concerning labo-
ratory features, neonatal myoclonic epilepsy is characterized by a
typical EEG pattern with suppression burst and by an abnormal visual-
evoked potential with low amplitude signal and slow response.

Both the P206L and G236W substitutions, which are located in H4
and H5 two and three helix turns from the cytosolic membrane side,
respectively, alter highly conserved residues in the glutamate and
aspartate/glutamate carriers found in man and other species (Fig. 6)
and abolish glutamate transport in reconstituted liposomes. In
digitonin-permeabilized fibroblasts from patients, glutamate oxida-
tion was also strongly defective [[91] and unpublished data]. These
results have provided the first evidence that, despite normal oxidative
phosphorylation, impaired mitochondrial glutamate import/metabo-
lism leads to an alteration of neuronal excitability.

Although the pathogenesis of neonatal myoclonic epilepsy remains
an open question, the following information should be stressed:
i) during fetal development, GC1 is expressed first in brain, specifically
within territories that contribute to the genesis and control of myo-
clonic seizures [91]; ii) GC1 expression is much greater in astrocytes
than in neurons [93]; iii) astrocyte mitochondria do not contain the
aspartate/glutamate carrier [94] and thus take up glutamate only via
the glutamate carrier; iv) glutamate is a major excitatory neurotrans-
mitter; and v) after its release into the synapse glutamate is taken up
by astrocytes. It may be that GC1-dependent epilepsy is due to a defect
of mitochondrial glutamate transport in astrocytes, which would

consequently lead to an increase in intrasynaptic glutamate
concentration.

There is no effective treatment for neonatal myoclonic epilepsy;
affected children die within 1 to 2 years after birth or survive in a
vegetative state. Some severe cases of the disorder may be confused
with febrile seizures, which are of shortened duration and recur less
frequently. It is also important to distinguish neonatal myoclonic
epilepsy from benign neonatal sleep myoclonus. The latter condition
is seen in healthy infants, occurs only in sleep, and EEG is normal.
Aside from metabolic errors, EME syndromes may be caused by
perinatal insults and brain malformations. A lumbar puncture may
help to rule out neurodegenerative diseases.

6. Conclusions

Since 1997, when carnitine–acylcarnitine carrier deficiency was
associated to the nuclear SLC25A20 gene, the number of nuclear gene
defects impairing the function of mitochondrial metabolite transpor-
ters has been rapidly increasing. To date, eight mitochondrial carrier-
related diseases have been well characterized biochemically and
genetically. This new group of diseases contributes to the current
expansion of the field of mitochondrial disorders which were first
thought to be limited to those related to respiratory chain dysfunction.
In the last decade, the number of mitochondrial diseases caused by
defects of the nuclear-coded components of the mitochondrial
proteome has been growing at a booming rate to justify what is
now known as “mitochondrial medicine”.

Despite the substantial progress that has been made in our
understanding of the molecular bases of mitochondrial carrier-asso-
ciated diseases, etiologic therapy is not yet available. Current therapy is
symptomatic and also addresses disease complications. However, some
mitochondrial carrier-associated diseases, like other metabolic disor-
ders, benefit enormously from appropriate dietary measures. Effective
disease management should include wider diffusion of newborn
screening and close collaboration among physicians, geneticists and
metabolic disease experts in specialized centers. Preventive therapy
through genetic counseling and prenatal diagnosis is essential and
already available. As we wait for gene therapy to become available for
practical application, further understanding of pathogenetic mechan-
isms through a multidisciplinary approach combining in vitro assays
with in vivo studies on patients, cellular and animal models will help to
develop new therapeutic strategies that may prove useful to this group
of metabolic disorders. Further studies are likely to reveal additional
mitochondrial carrier-related diseases (particularly in view of the fact
that the function of about 20 human genes of the SLC25 family has yet to
be identified) and provide new insight into this exciting field.

Acknowledgments

This work was supported by grants from Ministero dell'Università
e della Ricerca (FIRB and PRIN), Ministero della Salute, Apulia Region,
Center of Excellence in Genomics (CEGBA) and European Community
contract LSHM-CT-2004-503116.

References

[1] I.J. Holt, A.E. Harding, J.A. Morgan-Hughes, Deletions of muscle mitochondrial DNA
in patients with mitochondrial myopathies, Nature 331 (1988) 717–719.

[2] D.C. Wallace, G. Singh, M.T. Lott, J.A. Hodge, T.G. Schurr, A.M. Lezza, L.J. Elsas II, E.K.
Nikoskelainen, Mitochondrial DNA mutation associated with Leber's hereditary
optic neuropathy, Science 242 (1988) 1427–1430.

[3] P. Rustin, T. Bourgeron, B. Parfait, D. Chretien, A. Munnich, A. Rötig, Inborn errors of
the Krebs cycle: a group of unusual mitochondrial diseases in human, Biochim.
Biophys. Acta 1361 (1997) 185–197.

[4] P. Rinaldo, D. Matern, M.J. Bennett, Fatty acid oxidation disorders, Annu. Rev.
Physiol. 64 (2002) 477–502.

[5] M. Schlame, J.A. Towbin, P.M. Heerdt, R. Jehle, S. DiMauro, T.J. Blanck, Defi-
ciency of tetralinoleoyl-cardiolipin in Barth syndrome, Ann. Neurol. 51 (2002)
634–637.

576 F. Palmieri / Biochimica et Biophysica Acta 1777 (2008) 564–578



Author's personal copy

[6] F. Valianpour, R.J. Wanders, H. Overmars, P. Vreken, A.H. Van Gennip, F. Baas, B.
Plecko, R. Santer, K. Becker, P.G. Barth, Cardiolipin deficiency in X-linked cardio-
skeletal myopathy and neutropenia (Barth syndrome, MIM 302060): a study in
cultured skin fibroblasts, J. Pediatr. 141 (2002) 729–733.

[7] I. Napier, P. Ponka, D.R. Richardson, Iron trafficking in the mitochondrion: novel
pathways revealed by disease, Blood 105 (2005) 1867–1874.

[8] A.G. Estévez, J.P. Crow, J.B. Sampson, C. Reiter, Y. Zhuang, G.J. Richardson, M.M.
Tarpey, L. Barbeito, J.S. Beckman, Induction of nitric oxide-dependent apoptosis in
motor neurons by zinc-deficient superoxide dismutase, Science 286 (1999)
2498–2500.

[9] J.A. MacKenzie, R.M. Payne, Mitochondrial protein import and human health and
disease, Biochim. Biophys. Acta 1772 (2007) 509–523.

[10] S.A. Detmer, D.C. Chan, Functions and dysfunctions of mitochondrial dynamics,
Nat. Rev. Mol. Cell Biol. 8 (2007) 870–879.

[11] P. Rustin, Mitochondria, from cell death to proliferation, Nat. Genet. 30 (2002)
352–353.

[12] M. Brandon, P. Baldi, D.C. Wallace, Mitochondrial mutations in cancer, Oncogene
25 (2006) 4647–4662.

[13] S. DiMauro, E.A. Schon, Mitochondrial respiratory-chain diseases, New Engl. J.
Med. 348 (2003) 2656–2668.

[14] M. Zeviani, A. Spinazzola, Mitochondrial disorders, Curr. Neurol. Neurosci. Rep. 3
(2003) 423–432.

[15] J. Schmiedel, S. Jackson, J. Schäfer, H. Reichmann, Mitochondrial cytopathies,
J. Neurol. 250 (2003) 267–277.

[16] A. Rötig, A. Munnich, Genetic features of mitochondrial respiratory chain
disorders, J. Am. Soc. Nephrol. 14 (2003) 2995–3007.

[17] S. DiMauro, G. Davidzon, Mitochondrial DNA and disease, Ann. Med. 37 (2005)
222–232.

[18] A.H. Shapira, Mitochondrial disease, Lancet 368 (2006) 70–82.
[19] J.A. Smeitink, M. Zeviani, D.M. Turnbull, H.T. Jacobs, Mitochondrial medicine: a

metabolic perspective on the pathology of oxidative phosphorylation disorders,
Cell Metab. 3 (2006) 9–13.

[20] S. Ohta, A multi-functional organelle mitochondrion is involved in cell death,
proliferation and disease, Curr. Med. Chem. 10 (2003) 2485–2494.

[21] M.R. Duchen, Roles of mitochondria in health and disease, Diabetes 53 (2004)
96–102.

[22] D.C. Wallace, A mitochondrial paradigm of metabolic and degenerative diseases,
aging, and cancer: a dawn for evolutionary medicine, Annu. Rev. Genet. 39 (2005)
359–407.

[23] E. Mariani, M.C. Polidori, A. Cherubini, P. Mecocci, Oxidative stress in brain aging,
neurodegenerative and vascular diseases: an overview, J. Chromatogr. B 827
(2005) 65–75.

[24] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurode-
generative diseases, Nature 443 (2006) 787–795.

[25] D.C. Chan, Mitochondria: dynamic organelles in disease, aging, and development,
Cell 125 (2006) 1241–1252.

[26] M. Zeviani, V. Carelli, Mitochondrial disorders, Curr. Opin. Neurol. 20 (2007)
564–571.

[27] E. Trushina, C.T. McMurray, Oxidative stress and mitochondrial dysfunction in
neurodegenerative diseases, Neuroscience 145 (2007) 1233–1248.

[28] F. Palmieri, The mitochondrial transporter family (SLC25): physiological and
pathological implications, in: M.A. Hediger (Ed.), The ABC of solute carriers,
Pflugers Arch. — Eur. J. Physiol., 447, 2004, pp. 689–709.

[29] F. Palmieri, M. Klingenberg, Mitochondrial metabolite transporter family, in: W.J.
Lennarz, M.D. Lane (Eds.), Encyclopedia of Biological Chemistry, vol. 2, Elsevier,
Oxford, 2004, pp. 725–732.

[30] E. Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trézéguet, G.J. Lauquin, G.
Brandolin, Structure of mitochondrial ADP/ATP carrier in complex with carbox-
yatractyloside, Nature 426 (2003) 39–44.

[31] E.R. Kunji, M. Harding, Projection structure of the atractyloside-inhibited
mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae, J. Biol. Chem. 278
(2003) 36985–36988.

[32] A.J. Robinson, E.R. Kunji, Mitochondrial carriers in the cytoplasmic state have a
common substrate binding site, Proc. Natl. Acad. Sci. U. S. A.103 (2006) 2617–2622.

[33] A.R. Cappello, D.V. Miniero, R. Curcio, A. Ludovico, L. Daddabbo, I. Stipani, A.J.
Robinson, E.R.S. Kunji, F. Palmieri, Functional and structural role of amino acid
residues in the odd-numbered transmembrane α-helices of the bovine mitochon-
drial oxoglutarate carrier, J. Mol. Biol. 369 (2007) 400–412.

[34] G. Fiermonte, F. De Leonardis, S. Todisco, L. Palmieri, F.M. Lasorsa, F. Palmieri,
Identification of the mitochondrial ATP-Mg/Pi transporter. Bacterial expression,
reconstitution, functional characterization and tissue distribution, J. Biol. Chem.
279 (2004) 30722–30730.

[35] G. Agrimi, M.A. Di Noia, C.M.T. Marobbio, G. Fiermonte, F.M. Lasorsa, F. Palmieri,
Identification of the human mitochondrial S-adenosylmethionine transporter:
bacterial expression, reconstitution, functional characterization and tissue
distribution, Biochem. J. 379 (2004) 183–190.

[36] V. Dolce, P. Scarcia, D. Iacopetta, F. Palmieri, A fourth ADP/ATP carrier isoform in
man: identification, bacterial expression, functional characterization and tissue
distribution, FEBS Lett. 579 (2005) 633–637.

[37] S. Floyd, C. Favre, F.M. Lasorsa, M. Leahy, G. Trigiante, P. Stroebel, A. Marx, G.
Loughran, K. O'Callaghan, C.M.T. Marobbio, D.J. Slotboom, E.R.S. Kunji, F. Palmieri,
R. O'Connor, The insulin-like growth factor-I-mTOR signaling pathway induces the
mitochondrial pyrimidine nucleotide carrier to promote cell growth, Mol. Biol. Cell
18 (2007) 3545–3555.

[38] L. Palmieri, S. Alberio, I. Pisano, T. Lodi, M. Meznaric Petrusa, J. Zidar, A. Santoro, P.
Scarcia, F. Fontanesi, E. Lamantea, I. Ferrero, M. Zeviani, Complete loss-of-function

of the heart-/muscle-specific adenine nucleotide translocator is associated with
mitochondrial myopathy and cardiomyopathy, Hum. Mol. Genet. 14 (2005)
3079–3088.

[39] A.R. Curran, D.M. Engelman, Sequence motifs, polar interactions and conforma-
tional changes in helical membrane proteins, Curr. Opin. Struct. Biol. 13 (2003)
412–417.

[40] B.H. Graham, K.G. Waymire, B. Cottrell, I.A. Trounce, G.R. MacGregor, D.C. Wallace,
A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a
deficiency in the heart/muscle isoform of the adenine nucleotide translocator, Nat.
Genet. 16 (1997) 226–234.

[41] E.Z. Jordens, L. Palmieri, M. Huizing, L.P. van den Heuvel, R.C.A. Sengers, A. Doërner,
W. Ruitenbeek, J.M.F. Trijbels, J. Valsson, G. Sigfusson, F. Palmieri, J.A.M. Smeitink,
Adenine nucleotide translocator 1 deficiency associated with Sengers syndrome,
Ann. Neurol. 52 (2002) 95–99.

[42] J.A. Mayr, O. Merkel, S.D. Kohlwein, B.R. Gebhardt, H. Böhles, U. Fötschl, J. Koch, M.
Jaksch, H. Lochmüller, R. Horváth, P. Freisinger, W. Sperl, Mitochondrial
phosphate-carrier deficiency: a novel disorder of oxidative phosphorylation, Am.
J. Hum. Genet. 80 (2007) 478–484.

[43] V. Dolce, V. Iacobazzi, F. Palmieri, J.E. Walker, The sequences of human and bovine
genes of the phosphate carrier from mitochondria contain evidence of
alternatively spliced forms, J. Biol. Chem. 269 (1994) 10451–10460.

[44] G. Fiermonte, V. Dolce, F. Palmieri, Expression in Escherichia coli, functional
characterization, and tissue distribution of isoforms A and B of the phosphate
carrier from bovine mitochondria, J. Biol. Chem. 273 (1998) 22782–22787.

[45] V. Dolce, G. Fiermonte, F. Palmieri, Tissue-specific expression of the two isoforms of
the mitochondrial phosphate carrier in bovine tissues, FEBS Lett. 399 (1996) 95–98.

[46] J. Kaukonen, J.K. Juselius, V. Tiranti, A. Kyttälä, M. Zeviani, G.P. Comi, S. Keränen, L.
Peltonen, A. Suomalainen, Role of adenine nucleotide translocator 1 in mtDNA
maintenance, Science 289 (2000) 782–785.

[47] G. Van Goethem, B. Dermaut, A. Löfgren, J.J. Martin, C. Van Broeckhoven, Mutation
of POLG is associated with progressive external ophthalmoplegia characterized by
mtDNA deletions, Nat. Genet. 28 (2001) 211–212.

[48] J.N. Spelbrink, F.Y. Li, V. Tiranti, K. Nikali, Q.P. Yuan, M. Tariq, S. Wanrooij, N.
Garrido, G. Comi, L. Morandi, L. Santoro, A. Toscano, G.M. Fabrizi, H. Somer, R.
Croxen, D. Beeson, J. Poulton, A. Suomalainen, H.T. Jacobs, M. Zeviani, C. Larsson,
Human mitochondrial DNA deletions associated with mutations in the gene
encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria, Nat.
Genet. 28 (2001) 223–231.

[49] F. Fontanesi, L. Palmieri, P. Scarcia, T. Lodi, C. Donnini, A. Limongelli, V. Tiranti, M.
Zeviani, I. Ferrero, A.M. Viola, Mutations in AAC2, equivalent to human adPEO-
associated ANT1 mutations, lead to defective oxidative phosphorylation in
Saccharomyces cerevisiae and affect mitochondrial DNA stability, Hum. Mol.
Genet. 13 (2004) 923–934.

[50] M. Huizing, V. Iacobazzi, L. Ijlst, P. Savelkoul, W. Ruitenbeek, L.P. van den Heuvel, C.
Indiveri, J. Smeitink, F.J.M. Trijbels, R.J.A. Wanders, F. Palmieri, Cloning of the
human carnitine-acylcarnitine carrier cDNA, and identification of the molecular
defect in a patient, Am. J. Hum. Genet. 61 (1997) 1239–1245.

[51] V. Iacobazzi, M.A. Naglieri, C.A. Stanley, R.J.A. Wanders, F. Palmieri, The structure
and organization of the human carnitine/acylcarnitine translocase (CACT) gene,
Biochem. Biophys. Res. Commun. 252 (1998) 770–774.

[52] C. Indiveri, V. Iacobazzi, N. Giangregorio, F. Palmieri, The mitochondrial carnitine
carrier protein: cDNA cloning, primary structure, and comparison with other
mitochondrial transport proteins, Biochem. J. 321 (1997) 713–719.

[53] J.R. de Lucas, C. Indiveri, A. Tonazzi, P. Perez, N. Giangregorio, V. Iacobazzi, F.
Palmieri, Functional characterisation of residues within the carnitine/acylcarnitine
translocase RX2PANAAXF distinct motif, Mol. Membr. Biol. 25 (2008) 152–163.

[54] C.A. Stanley, D.E. Hale, G.T. Berry, S. Deleeuw, J. Boxer, J.P. Bonnefont, Brief report: a
deficiency of carnitine-acylcarnitine translocase in the inner mitochondrial
membrane, New Engl. J. Med. 327 (1992) 19–23.

[55] S.V. Pande, M. Brivet, A. Slama, F. Demaugre, C. Aufrant, J.M. Saudubray, Carnitine-
acylcarnitine translocase deficiency with severe hypoglycemia and auriculo
ventricular block. Translocase assay in permeabilized fibroblasts, J. Clin. Invest.
91 (1993) 1247–1252.

[56] V. Iacobazzi, F. Invernizzi, S. Baratta, R. Pons, W. Chung, B. Garavaglia, C. Dionisi-
Vici, A. Ribes, R. Parini, M.D. Huertas, S. Roldan, G. Lauria, F. Palmieri, F. Taroni,
Molecular and functional analysis of SLC25A20 mutations causing carnitine-
acylcarnitine translocase deficiency, Hum. Mutat. 24 (2004) 312–320.

[57] L. IJlst, C.W. van Roermund, V. Iacobazzi, W. Oostheim, J.P. Ruiter, J.C. Williams, F.
Palmieri, R.J. Wanders, Functional analysis of mutant human carnitine acylcarni-
tine translocases in yeast, Biochem. Biophys. Res. Commun. 280 (2001) 700–706.

[58] P. Pérez, O. Martínez, B. Romero, I. Olivas, A.M. Pedregosa, F. Palmieri, F. Laborda, J.R.
De Lucas, Functional analysis of mutations in the human carnitine/acylcarnitine
translocase in Aspergillus nidulans, Fungal Genet. Biol. 39 (2003) 211–220.

[59] J.A. Camacho, C. Obie, B. Biery, B.K. Goodman, C.A. Hu, S. Almashanu, G. Steel, R.
Casey, M. Lambert, G.A. Mitchell, D. Valle, Hyperornithinaemia–hyperammonae-
mia–homocitrullinuria syndrome is caused by mutations in a gene encoding a
mitochondrial ornithine transporter, Nat. Genet. 22 (1999) 151–158.

[60] C. Indiveri, A. Tonazzi, F. Palmieri, Identification and purification of the ornithine/
citrulline carrier from rat liver mitochondria, Eur. J. Biochem. 207 (1992) 449–454.

[61] L. Palmieri, V. De Marco, V. Iacobazzi, F. Palmieri, M.J. Runswick, J.E. Walker,
Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier
involved in arginine biosynthesis, FEBS Lett. 410 (1997) 447–451.

[62] G. Fiermonte, V. Dolce, L. David, F.M. Santorelli, C. Dionisi-Vici, F. Palmieri, J.E.
Walker, The mitochondrial ornithine transporter. Bacterial expression, reconstitu-
tion, functional characterization, and tissue distribution of two human isoforms,
J. Biol. Chem. 278 (2003) 32778–32783.

577F. Palmieri / Biochimica et Biophysica Acta 1777 (2008) 564–578



Author's personal copy

[63] F.C. Kuo, W.L. Hwu, D. Valle, J.E. Darnell Jr., Colocalization in pericentral
hepatocytes in adult mice and similarity in developmental expression pattern of
ornithine aminotransferase and glutamine synthetase mRNA, Proc. Natl. Acad. Sci.
U. S. A. 88 (1991) 9468–9472.

[64] C. Indiveri, A. Tonazzi, I. Stipani, F. Palmieri, The purified and reconstituted
ornithine/citrulline carrier from rat liver mitochondria: electrical nature and
coupling of the exchange reaction with H+ translocation, Biochem. J. 327 (1997)
349–355.

[65] S. Salvi, F.M. Santorelli, E. Bertini, R. Boldrini, C. Meli, A. Donati, A.B. Burlina, C.
Rizzo, M. Di Capua, G. Fariello, C. Dionisi-Vici, Clinical and molecular findings in
hyperornithinemia–hyperammonemia–homocitrullinuria syndrome, Neurology
57 (2001) 911–914.

[66] J. Camacho, R. Mardach, N. Rioseco-Camacho, E. Ruiz-Pesini, O. Derbeneva, D.
Andrade, F. Zaldivar, Y. Qu, S.D. Cederbaum, Clinical and functional characteriza-
tion of a human ORNT1 mutation (T32R) in the hyperornithinemia–hyperammo-
nemia–homocitrullinuria (HHH) syndrome, Pediatr. Res. 60 (2006) 423–429.

[67] V.E. Shih, M.L. Efron, H.W. Moser, Hyperornithinemia, hyperammonemia, and
homocitrullinuria. A new disorder of amino acid metabolism associated with
myoclonic seizures and mental retardation, Am. J. Dis. Child. 117 (1969) 83–92.

[68] A. Tessa, G. Fiermonte, C. Dionisi-Vici, E. Paradies, M.R. Baumgartner, Y.H. Chien, S.
Fecarotta, C. Loguercio, H. Ogier de Baulny, M.C. Nassogne, F. Deodato, G. Parenti, S.L.
Rutledge, M.A. Villaseca, G. Scarano, C.L. Pierri, F. Palmieri, F.M. Santorelli, Identi-
fication of novel mutations in the SLC25A15 gene in hyperornithinemia, hyper-
ammonemia, and homocitrullinuria (HHH) syndrome: a clinical, molecular and
functional study. Under submission.

[69] Y.B. Lu, K. Kobayashi, M. Ushikai, A. Tabata, M. Iijima, M.X. Li, L. Lei, K. Kawabe, S.
Taura, Y. Yang, T.T. Liu, S.H. Chiang, K.J. Hsiao, Y.L. Lau, L.C. Tsui, D.H. Lee, T. Saheki,
Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13
gene of Japanese patients with citrin deficiency, J. Hum. Genet. 50 (2005) 338–346.

[70] K. Kobayashi, D.S. Sinasac, M. Iijima, A.P. Boright, L. Begum, J.R. Lee, T. Yasuda, S.
Ikeda, R. Hirano, H. Terazono, M.A. Crackower, I. Kondo, L.C. Tsui, S.W. Scherer, T.
Saheki, The gene mutated in adult-onset type II citrullinaemia encodes a putative
mitochondrial carrier protein, Nat. Genet. 22 (1999) 159–163.

[71] L. Palmieri, B. Pardo, F.M. Lasorsa, A. del Arco, K. Kobayashi, M. Iijima, M.J. Runswick,
J.E. Walker, T. Saheki, J. Satrústegui, F. Palmieri, Citrin and aralar1 are Ca2+-
stimulated aspartate/glutamate transporters in mitochondria, EMBO J. 20 (2001)
5060–5069.

[72] A. del Arco, M. Agudo, J. Satrústegui, Characterization of a second member of the
subfamily of calcium-binding mitochondrial carriers expressed in human non-
excitable tissues, Biochem. J. 345 (2000) 725–732.

[73] M. Iijima, A. Jalil, L. Begum, T. Yasuda, N. Yamaguchi, M. Xian Li, N. Kawada, H.
Endou, K. Kobayashi, T. Saheki, Pathogenesis of adult-onset type II citrullinemia
caused by deficiency of citrin, a mitochondrial solute carrier protein: tissue and
subcellular localization of citrin, Adv. Enzyme Regul. 41 (2001) 325–342.

[74] F.M. Lasorsa, P. Pinton, L. Palmieri, G. Fiermonte, R. Rizzuto, F. Palmieri,
Recombinant expression of the Ca2+-sensitive aspartate/glutamate carrier
increases mitochondrial ATP production in agonist-stimulated Chinese hamster
ovary cells, J. Biol. Chem. 278 (2003) 38686–38692.

[75] K. Kobayashi, N. Shaheen, R. Kumashiro, K. Tanikawa, W.E. O'Brien, A.L. Beaudet, T.
Saheki, A search for the primary abnormality in adult-onset type II citrullinemia,
Am. J. Hum. Genet. 53 (1993) 1024–1030.

[76] K. Kobayashi, H. Kakinoki, T. Fukushige, N. Shaheen, H. Terazono, T. Saheki, Nature
and frequency of mutations in the argininosuccinate synthetase gene that cause
classical citrullinemia, Hum. Genet. 96 (1995) 454–463.

[77] T. Ohura, K. Kobayashi, Y. Tazawa, I. Nishi, D. Abukawa, O. Sakamoto, K. Iinuma, T.
Saheki, Neonatal presentation of adult-onset type II citrullinemia, Hum. Genet.108
(2001) 87–90.

[78] D. Dimmock, K. Kobayashi, M. Iijima, A. Tabata, L.J. Wong, T. Saheki, B. Lee, F.
Scaglia, Citrin deficiency: a novel cause of failure to thrive that responds to a high-
protein, low-carbohydrate diet, Pediatrics 119 (2007) 773–777.

[79] M. Moriyama, M.X. Li, K. Kobayashi, D.S. Sinasac, Y. Kannan, M. Iijima, M. Horiuchi,
L.C. Tsui, M. Tanaka, Y. Nakamura, T. Saheki, Pyruvate ameliorates the defect in
ureogenesis from ammonia in citrin-deficient mice, J. Hepatol. 44 (2006) 930–938.

[80] T. Yasuda, N. Yamaguchi, K. Kobayashi, I. Nishi, H. Horinouchi, M.A. Jalil, M.X. Li, M.
Ushikai, M. Iijima, I. Kondo, T. Saheki, Identification of two novel mutations in the
SLC25A13 gene and detection of seven mutations in 102 patients with adult-onset
type II citrullinemia, Hum. Genet. 107 (2000) 537–545.

[81] G. Fiermonte, D. Soon, A. Chaudhuri, E. Paradies, P.J. Lee, S. Krywawych, F. Palmieri,
R.H. Lachmann, An adult with type II citrullinemia presenting in Europe, New Engl.
J. Med. 338 (2008) 1408–1409.

[82] A.R. Cappello, R. Curcio, D.V. Miniero, I. Stipani, A.J. Robinson, E.R.S. Kunji, F.
Palmieri, Functional and structural role of amino acid residues in the even-
numbered transmembrane α-helices of the bovine mitochondrial oxoglutarate
carrier, J. Mol. Biol. 363 (2006) 51–62.

[83] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Improved protein-
ligand docking using GOLD, Proteins 52 (2003) 609–623.

[84] D.S. Sinasac, M. Moriyama, M.A. Jalil, L. Begum, M.X. Li, M. Iijima, M. Horiuchi, B.H.
Robinson, K. Kobayashi, T. Saheki, L.C. Tsui, Slc25a13-knockout mice harbor
metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia,
Mol. Cell. Biol. 24 (2004) 527–536.

[85] T. Saheki, M. Iijima, M.X. Li, K. Kobayashi, M. Horiuchi, M. Ushikai, F. Okumura, X.J.
Meng, I. Inoue, A. Tajima, M. Moriyama, K. Eto, T. Kadowaki, D.S. Sinasac, L.C. Tsui,
M. Tsuji, A. Okano, T. Kobayashi, Citrin/mitochondrial glycerol-3-phosphate
dehydrogenase double-knockout mice recapitulate features of human citrin
deficiency, J. Biol. Chem. 282 (2007) 25041–25052.

[86] R.I. Kelley, D. Robinson, E.G. Puffenberger, K.A. Strauss, D.H. Morton, Amish lethal
microcephaly: a new metabolic disorder with severe congenital microcephaly and
2-ketoglutaric aciduria, Am. J. Med. Genet. 112 (2002) 318–326.

[87] M.J. Rosenberg, R. Agarwala, G. Bouffard, J. Davis, G. Fiermonte, M.S. Hilliard, T.
Koch, L.M. Kalikin, I. Makalowska, D.H. Morton, E.M. Petty, J.L. Weber, F. Palmieri, R.I.
Kelley, A.A. Schäffer, L.G. Biesecker, Mutant deoxynucleotide carrier is associated
with congenital microcephaly, Nat. Genet. 32 (2002) 175–179.

[88] V. Dolce, F. Fiermonte, M.J. Runswick, F. Palmieri, J.E. Walker, The human
mitochondrial deoxynucleotide carrier and its role in toxicity of nucleoside
antivirals, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 2284–2288.

[89] M.J. Lindhurst, G. Fiermonte, S. Song, E. Struys, F. De Leonardis, P.L. Schwartzberg,
A. Chen, A. Castegna, N. Verhoeven, C.K. Mathews, F. Palmieri, L.G. Biesecker,
Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion,
embryonic lethality, CNS malformations, and anemia, Proc. Natl. Acad. Sci. U. S. A.
103 (2006) 15927–15932.

[90] V. Iacobazzi, M. Ventura, G. Fiermonte, G. Prezioso, M. Rocchi, F. Palmieri, Geno-
mic organization and mapping of the gene (SLC25A19) encoding the human
mitochondrial deoxynucleotide carrier (DNC), Cytogenet. Cell Genet. 93 (2001)
40–42.

[91] F. Molinari, A. Raas-Rothschild, M. Rio, G. Fiermonte, F. Encha-Razavi, L.
Palmieri, F. Palmieri, Z. Ben-Neriah, N. Khadom, M. Vekemans, T. Attié-Bitach,
A. Munnich, P. Rustin, L. Colleaux, Impaired mitochondrial glutamate transport
in autosomal recessive neonatal myoclonic epilepsy, Am. J. Hum. Genet. 76
(2005) 334–339.

[92] G. Fiermonte, L. Palmieri, S. Todisco, G. Agrimi, F. Palmieri, J.E. Walker,
Identification of the mitochondrial glutamate transporter. Bacterial expression,
reconstitution, functional characterization, and tissue distribution of two human
isoforms, J. Biol. Chem. 277 (2002) 19289–19294.

[93] D.A. Berkich, M.S. Ola, J. Cole, A.J. Sweatt, S.M. Hutson, K.F. LaNoue, Mitochondrial
transport proteins of the brain, J. Neurosci. Res. 85 (2007) 3367–3377.

[94] M. Ramos, A. del Arco, B. Pardo, A. Martínez-Serrano, J.R. Martínez-Morales, K.
Kobayashi, T. Yasuda, E. Bogónez, P. Bovolenta, T. Saheki, J. Satrústegui,
Developmental changes in the Ca2+-regulated mitochondrial aspartate-glutamate
carrier aralar1 in brain and prominent expression in the spinal cord, Brain Res.
Dev. Brain Res. 143 (2003) 33–46.

578 F. Palmieri / Biochimica et Biophysica Acta 1777 (2008) 564–578