Original Article
Brief Report
AGC1 Deficiency Associated with Global Cerebral Hypomyelination
N Engl J Med 2009; 361:489-495July 30, 2009
- Abstract
The mitochondrial aspartate–glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate–aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.
Media in This Article
Figure 1
MRI Scans in the Patient.Figure 2
ATP Production in Isolated Mitochondria from the Patient's Muscle Tissue and Functional Characterization of Mutant Mitochondrial Aspartate–Glutamate Carrier Isoform 1 (AGC1).Article Activity
- Article
The mitochondrial aspartate–glutamate carrier isoform 1 (AGC1, or aralar), encoded by the solute carrier family 25, member 12, gene SLC25A12, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate, plus a proton.1 As a component of the malate–aspartate shuttle, AGC1 also has a role in the transfer of reducing equivalents from NADH from the cytosol to the mitochondria. In humans, there are two AGC isoforms: AGC1 and AGC2. AGC1 is the only isoform expressed in the adult central nervous system and skeletal muscle.2,3 Mutations in the SLC25A13 gene, encoding AGC2, cause type 2 citrullinemia,4 characterized by episodes of hyperammonemia5 and a liver-specific deficiency of argininosuccinate synthetase and of one of its substrates, aspartate (which is produced in the mitochondrion and in healthy individuals is transported to the cytosol by the liver-specific isoform AGC21). Here, we describe a case of AGC1 deficiency in a child who showed severe hypotonia, arrested psychomotor development, and seizures beginning at a few months of age.
Case Report
Our patient was a 3-year-old girl, the firstborn child of distantly related Swedish parents, delivered after an uncomplicated pregnancy. Development was normal during the first months of life. Delayed psychomotor development was noted at approximately 5 months of age. At the age of 7 months, the girl began having seizures. She presented with episodes of apnea and later, sporadic tonic seizures developed, with focal clonic jerks. Physical examination revealed severe muscular hypotonia and psychomotor retardation affecting mainly motor skills. She had poor head control and could not roll over or grasp objects. Eye contact and smiling response could be elicited only with difficulty. The plasma lactate level was increased, at 6 mmol per liter, and there was one episode of a slightly elevated lactate level in the cerebrospinal fluid, at 2.6 mmol per liter (normal range, 0.5 to 2.3), in relation to an episode of apnea, but levels were otherwise within the normal range. Plasma amino acid levels were normal; the glutamate level was 70 μmol per liter (1.0 mg per deciliter) (normal range, 0 to 200 [0 to 2.9]), and the aspartate level was 5 μmol per liter (0.7 mg per deciliter) (normal range, 0 to 25 [0 to 0.3]). Repeated electroencephalography, beginning at 9 months of age, showed generalized slowing of background activity but no interictal epileptiform discharges. As of the last examination, at 3 years 8 months of age, there has been essentially no further progress in psychomotor development. The patient cannot sit without support, crawl, or be pulled to a standing position. Severe spasticity has developed, with generalized hyperreflexia. The epilepsy is treated with carbamazepine and levetiracetam. Her height, weight, and head circumference are within the normal ranges.
The Regional Ethics Committee at Karolinska Institutet approved this study. We obtained written informed consent from both parents.
Methods and Results
Magnetic Resonance Investigations
We carried out magnetic resonance imaging (MRI) of the brain when the patient was 8 months of age, 1 year 4 months of age, and 2 years 9 months of age (Figure 1A and 1BFigure 1
MRI Scans in the Patient.). We observed a global lack of myelination in the cerebral hemispheres, with reduced supratentorial cerebral volume. This was obvious at 8 months of age and more pronounced at 1 year 4 months of age. There was a slight improvement in myelination in the centrum semiovale and pyramidal tracts at 2 years 9 months of age, but there was no corresponding low signal in the white matter (as compared with the cortex), typical of normal development, on T2-weighted imaging. Myelination remained lacking, over time, in the periphery of the frontal, occipital, and temporal lobes. In contrast, the cerebellum, brainstem, and thalami were essentially normal with regard to the MRI signal, configuration, and volume. The putamen and globus pallidus were slightly smaller than the normal size. On MRI performed when the patient was 2 years 9 months of age, we did not observe focal lesions in the cortical gray matter, disturbance in circulation of the cerebrospinal fluid, pathologic signal in the basal ganglia, or diffusion abnormalities.We carried out magnetic resonance single-volume spectroscopy of the patient's left basal ganglia, in the occipital midline and the frontal lobe, when she was 2 years 9 months of age (Figure 1C). We used a 1.5-T clinical magnetic resonance scanner and recorded 1H spectra with a point-resolved spectroscopy sequence at an echo time of 35 msec and a repetition time of 1500 msec. Voxel size was 8 cm3 in each case. All spectra showed a severely reduced peak corresponding with N-acetyl aspartate, an essentially normal choline peak, and an elevated myo-inositol peak. The ratio of N-acetyl aspartate to creatine was 0.7. Lactate and lipid peaks were not substantially elevated.
ATP Production and Activities of Respiratory-Chain Enzymes
Because we suspected a mitochondrial disorder, we obtained two muscle-tissue–biopsy specimens from the tibial anterior muscle and determined mitochondrial ATP production and activities of respiratory-chain enzymes in isolated mitochondria.6 Histologic and histochemical analysis showed some fat accumulation, but otherwise the tissue was normal. Activities of complexes I, I and III, II, II and III, and IV in the respiratory chain were normal (data not shown), in contrast with mitochondrial ATP production, which was drastically reduced when glutamate and succinate and glutamate and malate were used as substrates (Figure 2AFigure 2
ATP Production in Isolated Mitochondria from the Patient's Muscle Tissue and Functional Characterization of Mutant Mitochondrial Aspartate–Glutamate Carrier Isoform 1 (AGC1).). ATP production using all other substrate combinations was normal or only slightly reduced. We therefore suspected impaired function of AGC1 in muscle.DNA Sequence Analysis
We sequenced all 18 exons and exon–intron junctions of SLC25A12 (National Center for Biotechnology Information GeneID 8604) from amplified genomic DNA, using a kit (Big Dye Terminator v3.1 Cycle Sequencing kit, Applied Biosystems) and a DNA analyzer (model 3730, Applied Biosystems). We found a homozygous c.1769A→G transition, causing a Q590R missense mutation, in exon 17 (Figure 3AFigure 3
The Q590R Mutation in the SLC25A12 Gene Encoding Mitochondrial Aspartate–Glutamate Carrier Isoform 1 (AGC1).). Both parents were heterozygous for the mutation, which was absent in 100 unaffected Swedish controls, each of whom had four Swedish grandparents.Function and Expression of Mutant AGC1
Wild-type and mutant (Q590R) AGC1 were produced in Escherichia coli CO214(DE3), a mutant strain of E.coli BL21(DE3), and liposomes were reconstituted with recombinant proteins in the presence of aspartate or glutamate, as described previously.1,7 We removed external substrate from the proteoliposomes by means of extrusion chromatography, initiated transport at 25°C by adding [14C]aspartate or [14C]glutamate to proteoliposomes, and terminated transport by adding pyridoxal 5′-phosphate for a final concentration of 15 mM and bathophenanthroline for a concentration of 10 mM.1 The amount of wild-type and mutant proteins incorporated into liposomes was about 20% of that added to the reconstitution mixture. The mutant form of AGC1 was unable to transport aspartate or glutamate, even after 60 minutes of incubation (Figure 2B and 2C).
We observed that AGC1 colocalized with mitochondria in fibroblasts from both the patient and controls, and we found normal levels of AGC1 and porin in total and mitochondrial fractions of the patient's lymphocytes. Moreover, mutant AGC1 coprecipitated with mitochondrial membranes to the same extent as did wild-type AGC1, indicating its ability to integrate in the inner mitochondrial membrane (see the Supplementary Appendix, available with the full text of this article at NEJM.org).
Q590 is a highly conserved residue in the aspartate–glutamate mitochondrial carrier subfamily (Figure 3B). It protrudes into the internal cavity of the transporter just above the proposed substrate-binding site.8 To examine the consequences of the Q590R mutation, we modeled the docking of aspartate, as well as glutamate (not shown), by determining the configurations and orientations of R586, R583 and R490 residues of the AGC1-binding site (Figure 3C and 3D). Our models of AGC1 suggest that both aspartate and glutamate sit directly on top of the salt-bridge network that closes the cavity at the “matrix” side of the molecule (Figure 3C). Models of docking between substrate and mutant AGC1 suggest that the substrate is positioned far from the salt-bridge network and is bound to R590 and F494 in addition to R586, R583, and R490 (Figure 3D). It seems likely, therefore, that arginine in position 590 results in the trapping of the substrate at the binding site, impeding its movement through the protein.
Discussion
This study sheds light on the role of AGC1 in the central nervous system. AGC1 expression in the central nervous system is restricted to neurons9 and is absent in white matter.3 AGC1 is a component of the malate–aspartate shuttle, which is thought to be required for mitochondrial oxidation of cytosolic NADH in the brain (Figure 2D).10
Magnetic resonance findings are heterogeneous in patients with mitochondrial encephalopathy due to impaired oxidative phosphorylation, but there are some commonalities,11 including symmetrical focal lesions in the basal ganglia and brainstem nuclei, sometimes together with cortical infarct–like lesions. In addition, lactate accumulation detected with the use of magnetic resonance spectroscopy is a metabolic marker of impaired oxidative phosphorylation.11,12 However, our patient had no substantial accumulation of lactate detected on magnetic resonance spectroscopy. Furthermore, MRI showed global cerebral hypomyelination with relatively spared gray matter and without obvious signal changes in the basal ganglia and brainstem, despite the fact that AGC1 expression is restricted to neurons.
These findings do not support compromised neuronal energy production by the respiratory chain as a major pathogenetic mechanism in AGC1 deficiency, suggesting that the malate–aspartate shuttle may not be bioenergetically essential in the central nervous system. The glycerol-3-phosphate cycle, known to be active in the brain, is an alternative pathway of transfer for reducing equivalents into the mitochondria.13 It could not compensate for inhibition of the malate–aspartate shuttle in isolated synaptosomes,10 but it may compensate for a compromised malate–aspartate shuttle in vivo.
Neuronal aspartate is a substrate for the formation of N-acetyl aspartate by aspartate-N-acetyltransferase in mitochondria14 and microsomes.15 N-acetyl aspartate produced in neurons undergoes transaxonal transfer to oligodendrocytes, where it supplies acetyl groups for the synthesis of myelin lipids16,17 (Figure 2D). The phenotype of Slc25a12-knockout mice suggests that myelin formation depends on aspartate transport from mitochondria to the cytosol. Motor-coordination deficits developed in these mice, from day 12 onward, along with impaired myelination in the central nervous system.18 In parallel, there was a striking deficit in levels of aspartate and N-acetyl aspartate in the brain and in cultured neurons, suggesting that the major site of aspartate production in the central nervous system is the mitochondrion, that AGC1 affects aspartate efflux into the cytosol, and that this efflux is essential for N-acetyl aspartate formation. The knockout mice had normal numbers of neurons, suggesting that the malate–aspartate shuttle is not essential for the survival of neurons in these animals. MRI revealed a global lack of myelination in the cerebral hemispheres of our patient, and N-acetyl aspartate levels were drastically reduced, as determined by proton magnetic resonance spectroscopy. These findings are concordant with the phenotype of the Slc25a12-knockout mouse. Although N-acetyl aspartate reduction is considered a marker of neuronal viability and number,19 the patient had relatively conserved gray matter, supporting our hypothesis that the major pathogenetic mechanism of her syndrome is impaired neuronal aspartate transport, which prevents myelin formation by failing to provide N-acetyl aspartate to oligodendrocytes. The Slc25a12-knockout mouse thus seems to represent a model of the human disease.
The hypomyelination in our patient was confined to the cerebral hemispheres, with findings essentially normal in the cerebellum and brainstem. In the mouse, AGC1 and AGC2 have overlapping expression patterns during embryonic development, and full tissue-specific expression is attained postnatally.2,10 The ontogeny of tissue-specific expression of AGC1 in the human central nervous system is unknown, and residual expression of AGC2 may explain the regional differences seen in our patient.
The difference in phenotypes associated with AGC1 deficiency and AGC2 deficiency is consistent with the different tissue-expression patterns of these two isoforms, resulting in metabolic disturbances in the central nervous system and the liver, respectively. In both cases, the major pathogenetic mechanism is reduced mitochondrial aspartate efflux. In the liver, aspartate is needed for the urea cycle, whereas in the brain, it is required for N-acetyl aspartate and myelin formation. In conclusion, we describe a phenotype associated with AGC1 deficiency, with a presentation of severe, early postnatal psychomotor developmental arrest, as well as hypotonia and seizures. A dominating feature of the syndrome is global cerebral hypomyelination.
Supported by grants from the Swedish Research Council (12198), the Karolinska Institutet, and the Stockholm County Council (to Dr. Wedell) and from Ministero dell'Università e della Ricerca, the Italian Human ProteomeNet (project RBRN07BMCT_009), and Apulia Region Neurobiotech (Progetto Strategico 124) (to Dr. Palmieri).
No potential conflict of interest relevant to this article was reported.
Source Information
From the Center for Inherited Metabolic Diseases (R.W., M.B., K.N., A.W.) and the Department of Neuroradiology (T.K.), Karolinska University Hospital; and the Department of Molecular Medicine and Surgery, Center for Molecular Medicine (V.T., M.B., A.W.), the Department of Laboratory Medicine (F.H.S.), and the Department of Clinical Neuroscience (T.K.), Karolinska Institutet — both in Stockholm; the Department of Pharmaco-Biology, University of Bari, Bari, Italy (F.M.L., C.L.P., F.P.); and the Center of Child Neurology and Habilitation, Östersund Hospital, Östersund, Sweden (M.J.).
Address reprint requests to Dr. Wedell at the Department of Molecular Medicine and Surgery, Center for Molecular Medicine 02, Karolinska Institutet–Karolinska University Hospital, SE-171 76 Stockholm, Sweden, or at anna.wedell@ki.se.
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