Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

São Paulo State University, San Paulo, São Paulo, Brazil
Journal of Inherited Metabolic Disease (Impact Factor: 3.37). 11/2002; 25(7):557-70. DOI: 10.1023/A:1022091223498
Source: PubMed

ABSTRACT Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. Of these, 14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frame shift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

3 Reads
  • Source
    • "For this reason, if a nonsynonymous single nucleotide variation (nsSNV) occurs at the active site, thereby changing an active site amino acid residue, it is highly likely that enzyme activity will be lost or altered [1-7]. It follows that pathway activity should also be affected, with a potential end result of disease [8-10] or lethality. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background We have previously suggested a method for proteome wide analysis of variation at functional residues wherein we identified the set of all human genes with nonsynonymous single nucleotide variation (nsSNV) in the active site residue of the corresponding proteins. 34 of these proteins were shown to have a 1:1:1 enzyme:pathway:reaction relationship, making these proteins ideal candidates for laboratory validation through creation and observation of specific yeast active site knock-outs and downstream targeted metabolomics experiments. Here we present the next step in the workflow toward using yeast metabolic modeling to predict human metabolic behavior resulting from nsSNV. Results For the previously identified candidate proteins, we used the reciprocal best BLAST hits method followed by manual alignment and pathway comparison to identify 6 human proteins with yeast orthologs which were suitable for flux balance analysis (FBA). 5 of these proteins are known to be associated with diseases, including ribose 5-phosphate isomerase deficiency, myopathy with lactic acidosis and sideroblastic anaemia, anemia due to disorders of glutathione metabolism, and two porphyrias, and we suspect the sixth enzyme to have disease associations which are not yet classified or understood based on the work described herein. Conclusions Preliminary findings using the Yeast 7.0 FBA model show lack of growth for only one enzyme, but augmentation of the Yeast 7.0 biomass function to better simulate knockout of certain genes suggested physiological relevance of variations in three additional proteins. Thus, we suggest the following four proteins for laboratory validation: delta-aminolevulinic acid dehydratase, ferrochelatase, ribose-5 phosphate isomerase and mitochondrial tyrosyl-tRNA synthetase. This study indicates that the predictive ability of this method will improve as more advanced, comprehensive models are developed. Moreover, these findings will be useful in the development of simple downstream biochemical or mass-spectrometric assays to corroborate these predictions and detect presence of certain known nsSNVs with deleterious outcomes. Results may also be useful in predicting as yet unknown outcomes of active site nsSNVs for enzymes that are not yet well classified or annotated. Reviewers This article was reviewed by Daniel Haft and Igor B. Rogozin.
    Biology Direct 06/2014; 9(1):9. DOI:10.1186/1745-6150-9-9 · 4.66 Impact Factor
  • Source
    • "DNA variation within CCR5 receptors might affect gene expression and/or function of the receptor (Dean et al., 1996). Many of these polymorphisms are rare variants and were restricted to some ethnic groups (Shioda and Nakayama, 2000; Zeng et al., 2002). To date, several CCR5 polymorphisms have been identified in any particular population. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The chemokine receptors components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5∆32) confer high degree of resistance to HIV infection, Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5∆32 variant is present in 10-15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians). The prevalence of CCR5 gene variants including CCR5∆32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5∆32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing. Among Emiratis, the allele frequency of the CCR5∆32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5∆32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans. We conclude that the allele frequency of the most critical CCR5 ploymorphism (∆32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.
    Gene 08/2013; 529(1). DOI:10.1016/j.gene.2013.07.062 · 2.14 Impact Factor
  • Source
    • "ASPA, also known as aminoacylase 2, acts to deacetylate only one acetylated amino acid, namely N-acetylaspartate (NAA; D'Adamo et al., 1977; Madhavarao et al., 2003). ASPA hydrolyzes NAA into free acetate and aspartate (Kaul et al., 1993; Zeng et al., 2002) and is expressed strongly in a number of tissues including the brain and kidney (Birnbaum, 1955; Hershfield et al., 2006; Madhavarao et al., 2004). In peripheral tissues, ASPA may function like other aminoacylases to recycle NAA derived from the breakdown of proteins such as actin, which is acetylated at its N-terminal aspartate (Alving et al., 1966; Gaetjens et al., 1966). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aspartoacylase (ASPA) catalyzes deacetylation of N-acetylaspartate (NAA) to generate acetate and aspartate. Mutations in the gene for ASPA lead to reduced acetate availability in the CNS during development resulting in the fatal leukodystrophy Canavan disease. Highly specific polyclonal antibodies to ASPA were used to examine CNS expression in adult rats. In white matter, ASPA expression was associated with oligodendrocyte cell bodies, nuclei, and some processes, but showed a dissimilar distribution pattern to myelin basic protein and oligodendrocyte specific protein. Microglia expressed ASPA in all CNS regions examined, as did epiplexus cells of the choroid plexus. Pial and ependymal cells and some endothelial cells were ASPA positive, as were unidentified cellular nuclei throughout the CNS. Astrocytes did not express ASPA in their cytoplasm. In some fiber pathways and nerves, particularly in the brainstem and spinal cord, the axoplasm of many neuronal fibers expressed ASPA, as did some neurons. Acetyl coenzyme A synthase immunoreactivity was also observed in the axoplasm of many of the same fiber pathways and nerves. All ASPA-immunoreactive elements were unstained in brain sections from tremor rats, an ASPA-null mutant. The strong expression of ASPA in oligodendrocyte cell bodies is consistent with a lipogenic role in myelination. Strong ASPA expression in cell nuclei is consistent with a role for NAA-derived acetate in nuclear acetylation reactions, including histone acetylation. Expression of ASPA in microglia may indicate a role in lipid synthesis in these cells, whereas expression in axons suggests that some neurons can both synthesize and catabolize NAA.
    Glia 10/2011; 59(10):1414-34. DOI:10.1002/glia.21186 · 6.03 Impact Factor
Show more