Studies on the Mechanism of Oxalogenesis and Primary Hyperoxaluria Type 1

在本研究中，我旨在(1)阐明哺乳动物中草酸生成的机制，(2)克隆和测序人肝脏丝氨酸：丙酮酸氨基转移酶（SPT） cDNA和基因，为将来原发性高氮尿症1型（PH1）的基因分析奠定基础。由于本研究期间有一名PH1患者死亡，我们也从该患者的肝脏中克隆了SPT-cDNA，并确定了其核苷酸序列。关于体内草酸的生成，已知的三种酶在体外催化乙醛酸盐氧化成草酸似乎都没有发挥实质性作用。相反，大鼠口服噻唑烷- 2,4 -二羧酸盐（一种乙醛酸盐与半胱氨酸的加合物）可略微增加尿中草酸盐的排泄。目前正在进行关于噻唑烷- 2,4 -二羧酸盐可能参与乙醛酸盐生成草酸盐的研究。对于PH1病例中的遗传缺陷，对患者肝脏的SPT-cDNA的核苷酸序列分析显示，在第613位（相对于起始Met的ATG三重体的第一个核苷酸a）出现T到C的点突变，编码残基205处Ser到Pro的取代。T到C的转换产生了一个新的smi位点，这使我们能够证明在患者的SPT基因中发生了点突变。基因组DNA的smi消化可能对这种类型的PH1的诊断基因分析有用。在该患者的肝脏中，SPT不仅活性很低，而且在Western blot和免疫细胞化学分析中检测到的蛋白质含量也很低，但在RNA blot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，患者肝脏中SPT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.