Molecular basis of late onset ornithine transcarbamylase deficiency in male

我们报告了一群男性鸟氨酸转甲氨基酰基酶缺乏症（OTC）患者，发病时间从青春期晚期到老年期。在本研究中，我们发现了3例R4OH突变患者。我们在一名RFLP患者的肝脏标本中检测到R4OH突变，但在成纤维细胞和皮肤标本中，RFLP几乎正常。提示患者的R4OH突变可能为体细胞突变。在不同的族群中都发现了R4OH突变。这种突变可能是复发性突变的结果，因为突变涉及CpG岛。我们已经发现了与携带R4OH或Y55D突变的晚发型奥米汀转甲氨基酰基酶缺乏症患者相关的翻译后机制。本研究用Cos - 1细胞进行表达分析，结果表明，仅转染该质粒的细胞OTC活性为23.5 (nmol/min/ml)，含野生型cDNA的质粒为1955*140 （n=3）。另一方面，对R4OH突变体cDNA和Y55D突变体cDNA的OTC活性分别为670*130 （n=3）和627*124 （n=3）。R4OH突变体OTC和Y55D突变体OTC的β -半乳糖苷酶活性分别为正常OTC活性的28%和26%。当细胞裂解液经过5次冻融循环后，野生型OTC和Y55D突变体OTC的活性没有变化，而R4OH突变体OTC的活性下降到野生型处理前的6%。这些结果表明，R4OH突变体OTC在物理上不稳定，降解速度比野生型更快。因此，一旦施加代谢负荷，促进突变酶的失活和降解，R4OH OTC活性可能被抑制到不相容的水平。

We had reported a cluster of male ornithine transcarbamylase deficiency (OTC) patients with onset from late adolescence to the presenile period. In this study, we discovered three patients who have R4OH mutation. We detected R4OH mutation in liver specimens from one of the patients with RFLP, but in fibroblast and skin specimens the RFLP showed almost normal. This result indicated that the R4OH mutation in the patient may be somatic mutation. The R4OH mutation has been discovered in different ethnic groups. This mutation may arise as a result of a recurrent mutation because the mutation involves a CpG island.We have showed that posttranslational mechanisms related late onset omithine transcarbamylase deficiency patients bearing R4OH or Y55D mutations. In this study, expression analysis using Cos 1 cells indicated that the OTC activities of cells transfected with the plasmid only, and the plasmid containing wild type cDNA were 23.5 (nmol/min/ml) and 1955*140 (n=3) respectively. On the other hand OTC activities with R4OH mutant cDNA, and Y55D cDNA were 670*130 (n=3) and 627*124 (n=3) respectively. The activities of R4OH mutant OTC and Y55D mutant OTC, as normalized for beta-galactosidase activity were 28% and 26% of the normal OTC activity respectively. When the cell lysates were subjected to five cycles of freezing and thawing, the activities of the wild type OTC and Y55D mutant OTC did not change, whereas the activity of the R4OH mutant OTC decreased to 6% of wild type that before treatment. These results indicated that the R4OH mutant OTC was physically unstable and was degraded more rapidly than the wild type enzyme. Thus, the R4OH OTC activity may be depressed to an incompatible level once a metabolic burden that would facilitate inactivation and degradation of the mutant enzyme is applied.