New T530C mutation in the aspartoacylase gene caused Canavan disease with no correlation between severity and N-acetylaspartate excretion.

OBJECTIVE: Canavan disease (OMIM 271900) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). DESIGN AND METHODS: We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. RESULTS: MRI findings and quantification of high NAA excretion (1378.5 and 680.1µmolNAA/mmolcreatinine in urine of 4months and 4years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. CONCLUSIONS: The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression.

[Relationship between heroin spongiform leucoencephalopathy and respiratory chain complex I deficiency].

OBJECTIVE: To investigate the relationship between heroin spongiform leucoencephalopathy and respiratory chain complex I deficiency. METHODS: The activity of respiratory chain complex I in peripheral white blood cell mitochondria was compared between 36 cases of heroin spongiform leucoencephalopathy and 36 healthy subjects using enzyme-linked immunosorbent assay (ELISA). RESULTS: The activity of respiratory chain complex I was 5.6∓2.4 U/ml in patients with heroin spongiform leucoencephalopathy, significantly higher than that in the normal subjects (4.2∓2.1 U/ml, t=2.634, P<0.05). CONCLUSION: In patients with heroin spongiform leucoencephalopathy, mitochondrial dysfunction results in energy metabolism disorder to cause extensive demyelination of the cerebral white matter. Respiratory chain complex I deficiency of the mitochondria plays a significant role in the pathogenesis of heroin spongiform leucoencephalopathy.

Canavan disease, a rare early-onset human spongiform leukodystrophy: insights into its genesis and possible clinical interventions.

The brain contains high concentrations of the amino acid N-acetyl-l-aspartate (NAA) and its' glutamate adduct N-acetyl-l-aspartylglutamate (NAAG), both synthesized primarily by and stored in neurons. Upon depolarization both are exported to extracellular fluid (ECF) with NAA targeted to oligodendrocytes and NAAG targeted to astrocytes where they are hydrolyzed by specific enzymes. While the functions of these substances are incompletely known, their unique tri-cellular metabolism is apparently vital to normal brain function. Canavan disease (CD) is a globally occurring but rare early-onset human spongiform leukodystrophy associated with inborn genetic errors affecting the activity of aspartoacylase (ASPA), the enzyme highly expressed in oligodendrocytes that hydrolyzes NAA. Several hypotheses attempt to explain how the lack of ASPA activity results in the inability of oligodendrocytes to build or maintain axon-enveloping myelin sheaths, a failure reflected in the CD syndrome by profound neurological disturbances. Based on evidence provided by recent studies, as well as on descriptions of several atypical mild cases of CD and of a singular human case of an inborn error where NAA cannot be synthesized, we provide insights into the possible genesis of the CD syndrome and many of its phenotypic expressions. In this article we also evaluate current hypotheses, and discuss possible clinical interventions that may be of value in treatment of CD.

Reliable prenatal diagnosis of Canavan disease by measuring N-acetylaspartate in amniotic fluid using liquid chromatography tandem mass spectrometry.

OBJECTIVE: Prenatal diagnosis of Canavan disease by measuring N-acetylaspartic acid (NAA) in amniotic fluid is reliable and preferred over aspartoacylase enzyme assay especially in populations with unknown mutations. Typically based on GC-MS, existing methods are time-consuming and laborious. We developed a novel LC-MS/MS method for determination of NAA in amniotic fluid with minimal sample preparation. METHOD: NAA and d(3)-NAA were detected by negative-ion electrospray ionization-MS/MS. Quantification was achieved by standard addition using six 0.1 mL portions of each specimen enriched with increasing NAA amounts (0, 0.05, 0.1, 0.2, 0.3, and 0.4 microg) and endogenous NAA was calculated by extrapolation. RESULTS: Injection-to-injection time was 2 min whereas the turn around time from sample receipt was about 1 h. Intraday (n = 10) and interday (n = 10) variations were less than 9.4%. The reference range determined using gestation-matched controls (n = 12) of 1.1-2.7 micromol/L is in agreement with the literature. Specimens from at-risk pregnancies with established diagnosis (n = 4) were successfully analyzed. CONCLUSION: We developed a new method that enables reliable, sensitive, and selective determination of NAA in a small volume of amniotic fluid for the prenatal diagnosis of Canavan disease. The simple sample preparation adopted in this work precluded the necessity for extraction and derivatization.

[Pathological analysis of heroin spongiform leukoencephalopathy].

OBJECTIVE: To investigate the pathological characteristics of heroin spongiform leukoencephalopathy (HSLE). METHODS: Cerebral tissue specimens were obtained from 15 patients with HSLE and the histological observations under optical and electron microscopes were carried out by HE, Bielschowsky's, and chromotrope 2R-brilliant green staining. RESULTS: HSLE was characterized primarily by spongiform vacuolar degeneration of the cerebral white matter. Neurons in the gray matter, Purkinje and granular cells in the cerebella remain intact in all the cases. Numerous vacuoles, which merged to form larger cavities, appeared in the damaged white matter, and the axons survived in the deep white matter. The myelin sheath in the cerebellar white matter sustained more severe damages than those in the cerebral white matter. No vacuoles or lymphocyte infiltration occurred in the small peripheral vessels. CONCLUSION: HSLE is pathologically characterized by vacuolar degeneration due to primary damage of the myelin, and the spongiform vacuolar degeneration is closely associated with the severity of demyelination in the white matter.

N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology.

The brain is unique among organs in many respects, including its mechanisms of lipid synthesis and energy production. The nervous system-specific metabolite N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A in neurons, appears to be a key link in these distinct biochemical features of CNS metabolism. During early postnatal central nervous system (CNS) development, the expression of lipogenic enzymes in oligodendrocytes, including the NAA-degrading enzyme aspartoacylase (ASPA), is increased along with increased NAA production in neurons. NAA is transported from neurons to the cytoplasm of oligodendrocytes, where ASPA cleaves the acetate moiety for use in fatty acid and steroid synthesis. The fatty acids and steroids produced then go on to be used as building blocks for myelin lipid synthesis. Mutations in the gene for ASPA result in the fatal leukodystrophy Canavan disease, for which there is currently no effective treatment. Once postnatal myelination is completed, NAA may continue to be involved in myelin lipid turnover in adults, but it also appears to adopt other roles, including a bioenergetic role in neuronal mitochondria. NAA and ATP metabolism appear to be linked indirectly, whereby acetylation of aspartate may facilitate its removal from neuronal mitochondria, thus favoring conversion of glutamate to alpha ketoglutarate which can enter the tricarboxylic acid cycle for energy production. In its role as a mechanism for enhancing mitochondrial energy production from glutamate, NAA is in a key position to act as a magnetic resonance spectroscopy marker for neuronal health, viability and number. Evidence suggests that NAA is a direct precursor for the enzymatic synthesis of the neuron specific dipeptide N-acetylaspartylglutamate, the most concentrated neuropeptide in the human brain. Other proposed roles for NAA include neuronal osmoregulation and axon-glial signaling. We propose that NAA may also be involved in brain nitrogen balance. Further research will be required to more fully understand the biochemical functions served by NAA in CNS development and activity, and additional functions are likely to be discovered.

Brain N-acetylaspartate as a molecular water pump and its role in the etiology of Canavan disease: a mechanistic explanation.

N-acetyl-L-aspartate (NAA), an abundant amino acid present in the vertebrate brain, is synthesized and stored primarily in neurons. Its metabolism is also dynamic, with NAA turning over more than once each day by its regulated efflux into extracellular fluid (ECF), cycling between an anabolic L-aspartate acetylating compartment in neurons and a catabolic NAA deacetylating compartment in oligodendrocytes. An inborn error in NAA metabolism results in Canavan disease (CD), a rare and usually fatal early-onset autosomal recessive human central nervous system (CNS) disease, caused by failure of the catabolic metabolism of NAA resulting from a lack of sufficient amidohydrolase II activity in oligodendrocytes. Various hypotheses regarding the metabolism of NAA and its role have been considered, and although NAA may perform several functions in the CNS, an important role of NAA appears to be osmoregulatory. Based on this role, an osmotic-hydrostatic mechanism for the etiology of the CD phenotype is proposed. In CD, the daily addition of 13375 Pascals (0.132 atmospheres or 1.94 lbs per square inch) of hydrostatic pressure to brain ECF, on the brain cell side of brain-barrier epithelial membranes, resulting from the continuous synthesis and efflux of NAA, is considered to be responsible for the syndrome.