Biochemical Genetics of Acatalasemia

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We analyzed the molecular defect in Japanese-type acatalasemia using recombinant DNA techniques. To check the possibility that the disease resulted from a large deletion or rearrangement of the catalase gene sequence, we performed a Southern blotting for the DNA from a normal individual and a patient, using a cDNA fragment of human catalase cDNA as a probe. Three restriction enzymes exhibited identical band patterns with the DNA from both origins, hereby eliminating the above possibility. Catalase gene was isolated from a genomic DNA library of the patient. We have been carrying out sequence analysis for the exons, exon/intron junctions, and the 5' upstream region of the gene. We have identified four base substitutions in the mutant catalase gene, by comparing the sequence with that of the normal catalase gene. Particularly, one of them was at 22 nucleotides upstream of the cap site, and another was located at five nucleotides downstream of the splice junction between exon 4 and intron 4. The former might correspond to a promoter mutation, whereas the latter a mutation of a splicing signal. The former substitution eliminates a Hinf I restriction site which is found in the normal gene, and thus should produce a unique band pattern in the DNA blot of the patient, if it is linked to the disease. Hence, DNA samples from eight normal individuals and the patient were digested with Hinf I, and analyzed by Southern blotting. However, no unique band was found with the DNA of the patient. In this analysis, a highly frequent restriction-fragment length polymorphism was found. This will be a convenient marker in the screening of the disease. Further structural and functional analyses are now in progress to identify the causal mutation of the disease.