Molecular Mechanism of Late Onset Type of Ornithine Transcarbamylase Deficiency in Males

对青春期和成年男性OTC缺乏症患者进行鸟氨酸转氨酶(OTC)基因突变分析，发现5个家系中6例OTC缺乏症患者40位密码子上的精氨酸突变为组氨酸(R40H)，1例55位酪氨酸突变为天冬氨酸(Y55D)。用Cos-1细胞进行的表达实验表明，R40H和Y55D基因的表达水平与野生型OTC相似，1例R40H患者肝组织中的OTC基因表达水平与野生型OTC相似，表明至少R40H OTC是正常剪接的，与野生型OTC一样稳定。然而，R40H和Y55D OTC在Cos-1细胞中的活性降低到野生型OTC的28%，并且两种突变型OTC的交叉反应物数量都减少了。R40H OTC的活性下降到野生型OTC的5.5%，冻融5个周期后，R40H OTC的活性下降到野生型OTC的5.5%表明突变的OTC蛋白不稳定。而Y55D非处方药治疗后未见下降。本文研究了1例R40H患者肝组织的表观Km值和pH-活性分布。这些指标与正常酶的水平相当。这些结果提示，R40H突变中酶缺乏的机制(S)可能与OTC突变的不稳定或OTC前的线粒体定位不充分或两者兼而有之而加速降解有关。Y55D OTC缺失的机制尚不清楚。在一个家庭中，记录了R40H基因从父亲传递给女儿。R40HOTC基因的这种父性传播可能使其在普通人群中保留的频率高于与仅通过母系传播的早发性疾病相关的疾病。

Mutation analysis of ornithine transcarbamylase (OTC) gene in adolescent and adult male patients with OTC deficiency revealed a mutation that changes arginine at codon 40 to histidine (R40H) in 6 patients from 5 families and another one converting tyrosine at codon 55 to aspartate (Y55D) in one patient. Expression experiment using Cos-1 cells revealed that the R40H and Y55D mRNA levels were similar to that of wild-type OTCmRNA,so was OTC mRNA in liver tissue of one R40H patient, indicating that at least R40H OTC is normally spliced and as stable as wild-type mRNA.The activities of R40H and Y55D OTC's in the transfected Cos-1 cells were, however, reduced to 28% of that of wild-type OTC and the amounts of cross reactive material were reduced in both mutant OTC's. The activity of R40H OTC lecreased to 5.5% of the wild-type OTC when it was treated by five cycles of freezing and thawing, indicating instability of the mutant OTC protein. In contrast, Y55D OTC did not decrease after the treatment. Apparent Km values and pH-activity profile were studied in liver tissue of one R40H patient. These were comparable to those of normal enzyme. These results suggest that the mechanism (s) of enzyme deficiency in R40H mutation involve accelerated degradation due to instability of the mutant OTC,inadequate mitochondrial localization of the preOTC or both. The mechanism of deficiency of Y55D OTC remains to be elucidated. In one family, transmission of R40H gene from a father to a daughter was documented. This paternal transmission of R40HOTC gene may render it to be retained in general population more frequently than those associated with early onset disease which are exclusively transmitted throught maternal lineage.