doi:10.1016/j.clinbiochem.2008.01.021 
Copyright © 2008 The Canadian Society of Clinical Chemists Published by Elsevier Inc.
A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity
Valentina Di Pietroa, Alessandra Gambacurtab, Angela Maria Amorinic, Antonino Finocchiaroa, Serena D'Ursoc, Lia Ceccarellia, Barbara Tavazzia, Bruno Giardinaa and Giuseppe Lazzarinoc, 
, 
aInstitute of Biochemistry and Clinical Biochemistry, Catholic University of Rome “Sacro Cuore”, Rome, Italy
bDepartment of Experimental Medicine and Biochemical Sciences, University of Rome “Tor Vergata”, Rome, Italy
cDepartment of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
Received 26 September 2007;
revised 17 January 2008;
accepted 22 January 2008.
Available online 7 February 2008.
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Abstract
Objective
To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme.
Design and methods
Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations.
Results
Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity.
Conclusion
Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease.
Keywords: Canavan disease; N-acetylaspartate; N-acetylaspartoacylase; T677C mutation; Site-directed mutagenesis
Fig. 1. Nucleotide sequence of the exon 5 of the hASPA gene of the CD-affected patient carrying in homozygosis the A731G mutation.
Fig. 2. Nucleotide sequence of the exon 5 of the hASPA gene of the consanguineous aunt of the CD-affected patient carrying in heterozygosis both the T677C mutation and the silent C693T polymorphism.
Fig. 3. Initial rate (V0) of the reaction of NAA hydrolysis catalyzed by the wild type and I226T-mutated hASPA, as a function of increasing substrate concentration. No residual activity of the I226T-mutated hASPA was detectable at any NAA tested. Sigmoidal curve of the wild type hASPA indicates the dimeric structure of the mature protein (Hill's coefficient n = 1.8, data not shown).
Fig. 4. The familial tree of the CD-affected patient showing the new mutation T677C in the consanguineous aunt and the anomalous frequency of the silent C693T polymorphism in three non-consanguineous subjects. Black and white square and circle = father (I, 1; A731G/WT, C693T/WT; 35 years old) and mother (I, 2; A731G/WT; 32 years old) of the CD patient; dotted and white circle = consanguineous aunt (I, 3; T677C/WT, C693T/WT; 28 years old) of the CD patient; white square = uncle-in-law (I, 4; C693T/WT; 33 years old) of the CD patient. Black square = CD-affected patient (II, 1; A731G/A731G; 4 years old). Asterisks indicate heterozygotic carriers of the C693T silent polymorphism.
Table 1.
Kinetic parameters of the E. coli expressed, purified hASPA of both wild type and I226T mutated proteins
Protocol for hASPA expression and conditions to evaluate hASPA activity are fully described in Methods section.
Abstract
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