Molecular studies of Canavan Disease

Canavan disease (CD), an autosomal recessive leukodystrophy, is caused by the deficiency of aspartoacylase {ASPA}. The human ASPA cDNA spanning 1,435 bp has been isolated and characterized. The single uninterrupted ORF in the cDNA predicted a 313 amino acid long protein. The authenticity of the cDNA has been established by its expression in E. coli and Cosl-cells. Human ASPA gene was also cloned and found to span 29 kb of the human genome. Human ASPA is coded by 6 exons intervened by 5 introns. The exon/intron splice junction sites follow the \u27gt\u27/\u27ag\u27 consensus sequence rule. The human ASPA gene was assigned to the 17pl3-ter region. Human ASPA coding sequences were demonstrated to be conserved in yeast, chicken, rabbit, cow, dog, mouse, rat and monkey. Sixty-four probands (or 128 chromosomes) with CD were analyzed for mutations in the ASPA gene. Four point mutations have been identified in Canavan alleles. The 693OA and 914C\u3eA base changes result in non-sense tyr231\u3eter and missense ala305\u3eglu mutations respectively, that lead to complete loss of ASPA activity. The 854A\u3eC transversion resulted in a glu285\u3eala missense mutation, and the mutant ASPA has 2.5% of the activity expressed by the wild type enzyme. The 433-2(A\u3eG) transition at the splice acceptor site in intron 2 would lead to skipping of exon III, accompanied by a frameshift in the final ASPA transcript. Of the 128 unrelated Canavan chromosomes analyzed; 88 were from probands of Ashkenazi Jewish descent and 40 were from non-Jewish probands. The glu285\u3eala, tyr231\u3eter and 433-2(A\u3eG) mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305\u3eglu mutation was found exclusively in non-Jewish probands and constituted 60% of the 40 mutant chromosomes. These results provide the basis for studying epidemiology of CD in at-risk populations; offer a DNA-based pre- and postnatal diagnosis of CD; provide the possibility to create an animal model of CD for understanding its pathophysiology and to develop strategies for possible enzyme and gene therapy