Molecular analysis of human acatalasemia. Identification of a splicing mutation.

To search for the molecular defect of Japanese-type acatalasemia, we cloned the mutant catalase gene from a person with this deficiency. The nucleotide sequence of the mutant gene was determined for all exons, exon/intron junctions, and 5' and 3' flanking regions, and the findings were compared with the sequence from the normal gene. Seven base differences were found between the two genes. Among them, a G to A substitution at the fifth position of intron 4 (a splicing mutation) seemed most likely to be responsible for the defective catalase synthesis in the subject. To obtain suggestive evidence, we constructed chimeric genes that contained a segment of either the normal or mutant catalase gene, encompassing a 3' part of exon 4, the entire intron 4 and a 5' portion of exon 5, within the third exon of the human alpha-globin gene. When this chimeric gene construct was introduced into simian virus 40-transformed simian cells (COS-7), the transcript of the normal catalase/alpha-globin chimeric gene was spliced correctly, as revealed by Northern blotting and RNase mapping techniques. In contrast, the splicing of the mutant chimeric pre-mRNA occurred between the 5' donor site of the preceding intron and the 3' acceptor site of the intron containing the substitution, thereby skipping one entire exon sequence. Thus, the G to A transition at the fifth position of intron 4 of the catalase gene indeed severely limits the correct splicing of the RNA product. The same splice site mutation was found in the genomic DNA of another acatalasemic individual from an unrelated family. We suggest that this base substitution is the causal mutation of these cases of Japanese-type acatalasemia.