Studies on the Mechanism of Oxalogenesis and Primary Hyperoxaluria Type 1

本研究旨在(1)阐明哺乳动物草酸发生的机制，(2)从人肝脏中克隆和测序丝氨酸：丙酮酸转氨酶(SPT)基因，为将来进行原发性高锌尿症1型(PH1)的基因分析奠定基础。由于一名PH1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。关于体内草酸的产生，已知的三种在体外催化乙醛酸氧化为草酸的酶似乎都没有起到实质性的作用。相反，大鼠口服乙醛酸与半胱氨酸的加合物-2，4-二羧酸噻唑烷后，尿草酸排泄量略有增加。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目前正在进行中。对于PH1的遗传缺陷，从患者的肝脏中提取的SPT-cDNA的核苷酸序列分析显示，在第613位(相对于起始蛋氨酸的ATG三联体的第一个核苷酸A)发生T到C的点突变，编码205位的Ser到Pro的替换。T到C的转换创建了一个新的SmaI位点，这使我们能够证明患者的SPT基因发生了点突变。基因组DNA的SMAI消化可能有助于该类型PH1的诊断基因分析。在这名患者的肝脏中，无论是蛋白质印迹还是免疫细胞化学分析，SPT的活性和蛋白含量都很低，但RNA印迹分析发现，SPT-mRNA的水平甚至高于正常水平，这表明该突变加速了患者肝脏中SPT的降解。

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.