Results And Discussion

Our general strategy involved, first, a search for the three common mutations (E285A, Y231X, A305E) using site-specific restriction endonuclease digestion of PCR products from genomic DNA. Then PCR products from both cDNA and genomic DNA were sequenced. To confirm the alterations found, specific restriction endonuclease digestion analysis of genomic DNA was performed. When possible, the carrier status of the parents was also confirmed. In vitro mutagenesis and expression of mutant cDNA's was performed for many of the mutations reported in our earlier series (Zeng et al., 2002) but have not yet been done for the mutations identified more recently.

3.1. Mutations in 39 Non-Jewish Patients with Canavan Disease

The mutations found are shown in Tables 2 and 3. Novel mutations included 12 missense mutations, 2 nonsense mutations, 5 deletions, 1 insertion mutation, 1 case of two variations in a single allele, 1 elimination of a stop codon and 2 splice accepter site mutations. Table 4 list all mutations found by class.

The A305E mutation is known to be present primarily in patients of European origin and that was the case in our cohort; 16/17 alleles with this mutation were of European ancestry. The smaller percentage of our cases with the A305E mutation (21.7% 17/78) than in other case series (39.5-60%; Elpeleg & Shaag, 1999, Sistermans et al., 2000) may reflect the large number of non-European patients in our series.

3.2. Characteristics of 24 Novel Mutations

From 39 patients, we identified 24 novel mutations (tables 2-4). Within human ASPA are esterase-like sequences including amino acid motifs GGTHGNE, DCTV and VNEAAYY. Two of the mutations identified, G18R and E24G, produce substitutions of invariable amino acid residues within the first esterase catalytic domain consensus sequence GGTHGNE in the first and last residues. No ASPA activity was detected in COS-7 cells transfected with mutant cDNA containing the E24G mutation whereas the activity was markedly increased after transfection with normal ASPA cDNA. This suggested that the E24G mutation caused malfunction of ASPA.

Of the two splice accepter site mutations found, one (IVS 1-2A^T) caused retention of 40 nucleotides of intron 1 on the upstream side of exon 2 while the other (IVS 4-1G^C) resulted in skipping of exons 5 and 6. In the case of the IVS 1-2A^T, intron retention occurred due to the presence of a cryptic splice acceptor sequence within the intron introducing new amino acids (Zeng et al., 2002).

Table 2. Mutations in 22 non-Jewish CD Patients*

Patient

Mutation 1

Mutation 2

Ethnic origin

0 0

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