Mild Elevation of N-Acetylaspartic Acid and Macrocephaly: Diagnostic Problem

Abstract

Patients with slightly increased excretion of N-acetylaspartic acid in urine, together with macrocephaly, present a dignostic dilemma for Canavan's disease. We describe a 13-year-old male patient with macrocephaly, mild developmental delay, increased signal intensity in the basal ganglia bilaterally, partial cortical blindness, and retinitis pigmentosa. Although the clinical course and magnetic resonance imaging findings did not resemble typical Canavan's disease, N-acetylaspartic acid excretion in the patient's urine was slightly elevated, 99.90 +/- 4.00 microg/mg creatinine, whereas the normal control range was < 83 microg/mg creatinine. Cultured skin fibroblasts from the patient showed no aspartoacylase activity. Cloning of genomic DNA isolated from the patient's fibroblasts showed an intronic mutation, specifically deletion of -2A and -3C at the acceptor site of exon 3 and disrupting the normal splicing of the gene. A second mutation was found in exon 6, 863 A-->G in aspartoacylase complementary DNA, causing a tyrosine-to-cysteine (Y288C) amino acid substitution. Expression of the mutation on exon 6 showed normal aspartoacylase activity. These data suggest that expression of the mutation may help to understand the enzyme defect in a patient with slightly increased N-acetylaspartic acid excretion.

Expression of aspartoacylase (ASPA) and Canavan disease

“…However, it is doubtful that COS cells represent a reliable tool for the evaluation of ASPA mutations. Notably, when Surendran et al overexpressed the ASPA mutation c.863A >G (p.Tyr288Cys) in COS cells they found no decrease of the enzyme activity compared with the ASPA WT enzyme (Surendran et al, 2003). Protein expression in COS cells can be difficult, namely because of the cytopathic effect exerted by high numbers of episomally replicating plasmids (Geisse and Henke, 2005).…”

“…Expression studies for ASPA WT have been carried out in Escherichia coli (Di Pietro et al, 2008;Kaul et al, 1993) but have resulted in low ASPA enzyme activity compared to experiments in eukaryotic COS cells (Hershfield et al, 2006(Hershfield et al, , 2007Janson et al, 2006;Kaul et al, 1994;Surendran et al, 2003). Posttranslational glycosylation is considered to stabilize the ASPA protein and its catalytic functions (Moore et .…”

“…A patient with the c.884 T > C [p.Phe295Ser] mutation has previously been identified in our laboratory; the lack of previous characterization of this ASPA mutant by expression analysis has prompted our interest. Mutation c.863A> G [p.Tyr288Cys] is remarkable because of contradicting opinions regarding its clinical relevance (Surendran et al, 2003;Tacke et al, 2005). Each mutation was inserted into the pcDNA3.1/+ ASPA wild-type (WT) cDNA clone using the Stratagene QuickChange Site-Directed Mutagenesis Kit.…”

“…Therefore, it is important that eukaryotic expression systems are used for the assessment of mutations in the ASPA gene. However, so far most of such expression analyses have been performed in Escherichia coli (Bitto et al, 2007;Di Pietro et al, 2008;Hershfield et al, 2007;Kaul et al, 1993;Namboodiri et al, 2000) only, just few authors have applied mammalian cells (COS cells) for overexpression of ASPA mutants (Hershfield et al, 2007;Janson et al, 2006;Kaul et al, 1996;Surendran et al, 2003;Zeng et al, 2002).…”

Mutational analysis of aspartoacylase: Implications for Canavan Disease

“…ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate (NAA). Typical CD pathology is marked by early onset macrocephaly, head-lag, ataxia, severe psychomotor retardation, brain vacuolization, and dysmyelination resulting in death during childhood, though there are several reports of clinically protracted disease courses (Elpeleg et al 1994,Janson et al 2006,Leone et al 1999,Matalon et al 1988,Shaag et al 1995,Surendran et al 2003a,Tacke et al 2005,Yalcinkaya et al 2005,Zafeiriou et al 1999,Zelnik et al 1993. Considerable effort has been devoted to understanding the basis of CD by elucidating the function of ASPA in the central nervous system (CNS).…”