Studies on the Mechanism of Oxalogenesis and Primary Hyperoxaluria Type 1

本研究的目的是（1）阐明哺乳动物体内草酸生成的机制;（2）克隆人肝脏丝氨酸丙酮酸转氨酶（SPT）cDNA和基因序列，为原发性高氧血症1型（PH 1）的基因分析奠定基础。由于一名PH 1患者在本研究期间死亡，因此也从患者的肝脏中克隆了SPT-cDNA，并测定了核苷酸序列。关于体内草酸盐的产生，已知在体外催化乙醛酸氧化为草酸盐的三种酶中没有一种起实质性作用。相反，大鼠口服噻唑烷-2，4-二羧酸酯（乙醛酸与半胱氨酸的加合物）显示出略微增加草酸盐的尿排泄。目前正在研究噻唑烷-2，4-二羧酸酯可能参与乙醛酸盐形成草酸盐的过程。关于PH 1情况下的遗传缺陷，对来自患者肝脏的SPT-cDNA的核苷酸序列分析揭示了在位置613处T至C的点突变（相对于起始Met的ATG三联体的第一个核苷酸A），其编码残基205处的Ser至Pro取代。T到C的转换产生了一个新的SmaI位点，这使我们能够证明点突变发生在患者的SPT基因中。SmaI酶切基因组DNA可能有助于这类PH 1的诊断基因分析。在该患者的肝脏中，SPT在Western印迹和免疫细胞化学分析中不仅活性非常低，而且蛋白质也非常低，但在RNA印迹分析中发现SPT-mRNA的水平甚至高于正常水平，这表明由于突变，SPT在患者肝脏中的降解加速。

In this study, I aimed at (1) elucidating the mechanism of oxalogenesis in mammals, and (2) cloning and sequencing the serine : pyruvate aminotransferase (SPT) cDNA and gene from human liver for the gene analysis in the future of primary hyperozaluria type 1 (PH1). Since a patient with PH1 died during the period of this study, SPT-cDNA was also cloned from the patient's liver and the nucleotide sequence was determined.With respect to the oxalate production in vivo, none of the three enzymes known to catalyze in vitro the oxidation of glyoxylate to oxalate appeared to play substantial role. Instead, oral administration to rat of thiazolidine-2, 4-dicarboxylate, an adduct of glyoxylate with cysteine, was shown to increase slightly the urinary excretion of oxalate. Studies on the possible involvement of thiazolidine-2, 4-dicarboxylate in the oxalate formation from glyoxylate are currently under way. With respect to the genetic defect in the case of PH1, nucleotide sequence analysis of SPT-cDNA from the patient's liver revealed a point mutation of T to C at position 613 (relative to the first nucleotide, A, of the ATG triplet for the initiation Met) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1. In the liver of this patient, SPT was very low with respect to not only the activity but also the protein detectable on Western blot and immunocytochemical analyses, but the level of SPT-mRNA was found on RNA blot analysis to be even higher than normal, suggesting that the degradation of SPT is accelerated in the patient's liver due to the mutation.