Expression of aspartoacylase (ASPA) and Canavan disease.

Canavan disease (CD) is a neurodegenerative disorder usually presenting in the first six months of life. CD patients can be identified via elevated levels of N-acetyl-l-aspartate in the pattern of urinary organic acids assessed by gas chromatography-mass spectrometry. They are characterized by deficiency of aspartoacylase (aminoacylase 2; ASPA) due to mutations in the ASPA gene. Information on the molecular basis of CD is rather sparse. A lack of expression studies of ASPA mutant proteins in appropriate expression systems has prompted this investigation. Studies with overexpressed ASPA mutant proteins were carried out in the HEK293 cell line, which provides the authentic human machinery for posttranslational modifications. All ASPA mutants tested (ASPA Arg168His, ASPA Pro181Thr, ASPA Tyr288Cys, ASPA Phe295Ser, and ASPA Ala305Glu) showed loss of ASPA activity, which can be explained by the intramolecular effects of the mutations in the enzyme. The mutation p.Phe295Ser even leads to absent ASPA mRNA expression, as revealed by quantitative real-time PCR. Using this approach, ASPA gene expression analysis yielded high levels of human ASPA gene expression not only in brain and kidney, but also in lung and liver. More information of ASPA localization in human organs and detailed characterization of mutations leading to a deficiency of ASPA can contribute to a better understanding of this inborn error of metabolism.

The molecular basis of aminoacylase 1 deficiency.

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.

Aminoacylase 1 deficiency associated with autistic behavior.

Aminoacylase 1 (ACY1) deficiency is a recently described inborn error of metabolism. Most of the patients reported so far have presented with rather heterogeneous neurologic symptoms. At this moment, it is not clear whether ACY1 deficiency represents a true metabolic disease with a causal relationship between the enzyme defect and the clinical phenotype or merely a biochemical abnormality. Here we present a patient identified in the course of selective screening for inborn errors of metabolism (IEM). The patient was diagnosed with autistic syndrome and admitted to the Children's Memorial Health Institute (CMHI) for metabolic evaluation. Organic acid analysis using gas chromatography-mass spectrometry (GC-MS) revealed increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, glycine, leucine, isoleucine, and valine. In Epstein-Barr virus (EBV)-transformed lymphoblasts, ACY1 activity was deficient. The mutation analysis showed a homozygous c.1057C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation and had normal results in the organic acid analysis using GC-MS. This article reports the findings of an ACY1-deficient patient presenting with autistic features.

Late lumen loss and follow-up percent diameter stenosis at different doses of oral valsartan six months after bare-metal stent implantation in type B2/C coronary lesions.

UNLABELLED: Higher doses of oral valsartan (160-320 mg) seem to reduce in-stent restenosis rate after bare-metal stent implantation. The value of 80, 160 or 320 mg valsartan should be analyzed by late lumen loss and follow-up percent diameter stenosis as surrogate parameters in a total of 60 patients with matched demographic, clinical and angiographic findings and continuous doses of valsartan. In each group 20 patients (14 males, 6 females) with a mean age of 62.1+/-9.1, 64.3+/-8.1 and 62.9+/-11.6 years after implantation of a total of 22, 33 and 27 stents in 21, 25 and 23 lesions were included. Quantitative coronary angiography was performed with an automated contour analysis system; reference diameter, minimum diameter, late lumen loss, follow-up percent diameter stenosis and restenosis rate were determined. RESULTS: In-stent restenosis rate including persistent area was n=5/21 (24%), n=4/25 (16%) and n=2/23 (8.7%) under 80, 160 and 320 mg valsartan. Late lumen loss was 0.79+/-0.49 mm, 0.60+/-0.43 mm and 0.37+/-0.25 mm, respectively, with significant differences between 80 and 320 mg (p<0.001) and 160 and 320 mg (p<0.05). Follow-up percent diameter stenosis was 31.8+/-18.6% under 80 mg, 25.2+/-17.5% under 160 mg and 13.8+/-9.4% with significant differences between 80 mg and 320 mg (p<0.0005) and 160 and 320 mg (p<0.01). CONCLUSIONS: Different doses of oral valsartan over six months after BMS implantation show a linear response with regard to late lumen loss and follow-up percent diameter stenosis.