Biochemical Genetics of Acatalasemia

我们利用重组DNA技术分析了日本型无丙氨酸酶血症的分子缺陷。为了检查该疾病是由过氧化氢酶基因序列的大的缺失或重排引起的可能性，我们使用人过氧化氢酶cDNA的cDNA片段作为探针，对来自正常个体和患者的DNA进行Southern印迹。三种限制性内切酶与两种来源的DNA显示出相同的带型，从而排除了上述可能性。从患者的基因组DNA文库中分离过氧化氢酶基因。我们对该基因的外显子、外显子/内含子连接点和5'上游区域进行了序列分析。通过与正常过氧化氢酶基因序列的比较，我们确定了突变过氧化氢酶基因中的四个碱基替换。其中一个位于帽位点上游22个核苷酸处，另一个位于外显子4和内含子4之间的剪接点下游5个核苷酸处。前者可能对应于启动子突变，而后者可能对应于剪接信号的突变。前一种取代消除了在正常基因中发现的Hinf I限制性位点，因此如果它与疾病有关，则应该在患者的DNA印迹中产生独特的条带模式。因此，用Hinf I消化来自8个正常个体和患者的DNA样品，并通过Southern印迹分析。然而，在患者的DNA中没有发现独特的条带。在本分析中，发现了一个高频率的限制性片段长度多态性，这将是一个方便的标记，在疾病的筛选。目前正在进行进一步的结构和功能分析，以确定该疾病的致病突变。

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.