Carstens R P, Fenton W A, Rosenberg L R
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Am J Hum Genet. 1991 Jun;48(6):1105-14.
Ornithine transcarbamylase (OTC) is an X-linked, liver-specific enzyme that catalyzes the second step of the urea cycle. In humans, inherited deficiency of OTC in hemizygous affected males usually results in severe ammonia intoxication and early death. To characterize mutations responsible for OTC deficiency, we used the PCR to amplify cDNAs prepared from patient livers which demonstrated no OTC enzyme activity and no OTC cross-reacting material on western blots. In three of seven cases, smaller than normal products were observed. Sequencing of these cDNAs revealed that two were missing exon 7 of the OTC gene and that the other was missing the first 12 bp of exon 5. Sequencing of genomic DNA from these three patients revealed that one mutant missing exon 7 had a T-to-C substitution in the 5' splice donor site of intron 7. The other mutant missing exon 7 had an A-to-G change in the third position of intron 7. It is interesting that both of these mutations resulted in skipping the preceding exon rather than in inclusion of some or all of the affected intron. In the third mutant, an A-to-T substitution was found in the 3' splice acceptor site at the end of intron 4. Here, a cryptic splice acceptor site within exon 5 was used. Northern blotting of liver RNA from these patients demonstrated (a) reduced, but significant, amounts of OTC mRNA in one of the patients who had a deleted exon 7 but (b) very little OTC mRNA in the other two patients. We propose that these point mutations, which result in aberrant splicing of the OTC pre-mRNAs, lead to OTC deficiency through either decreased efficiency of mRNA export from the nucleus to the cytosol or synthesis of enzyme subunits that are unstable and rapidly degraded. We speculate that abnormal mRNA splicing may represent a relatively common mechanism in the pathogenesis of this disease.
鸟氨酸转氨甲酰酶（OTC）是一种X连锁的肝脏特异性酶，它催化尿素循环的第二步。在人类中，半合子受影响男性中OTC的遗传性缺乏通常会导致严重的氨中毒和早期死亡。为了鉴定导致OTC缺乏的突变，我们使用聚合酶链反应（PCR）扩增从患者肝脏制备的互补DNA（cDNA），这些肝脏在蛋白质免疫印迹上显示没有OTC酶活性且没有OTC交叉反应物质。在7例患者中的3例中，观察到比正常产物小的片段。对这些cDNA进行测序发现，其中两个缺失OTC基因的外显子7，另一个缺失外显子5的前12个碱基对。对这3名患者的基因组DNA进行测序发现，一个缺失外显子7的突变体在内含子7的5'剪接供体位点有一个T到C的替换。另一个缺失外显子7的突变体在内含子7的第三个位置有一个A到G的变化。有趣的是，这两个突变都导致跳过前面的外显子，而不是包含部分或全部受影响的内含子。在第三个突变体中，在内含子4末端的3'剪接受体位点发现了一个A到T的替换。在这里，使用了外显子5内的一个隐蔽剪接受体位点。对这些患者肝脏RNA进行的Northern印迹分析表明：（a）在一名缺失外显子7的患者中，OTC信使核糖核酸（mRNA）的量减少但仍有显著水平；（b）在另外两名患者中，OTC mRNA的量很少。我们认为，这些导致OTC前体mRNA异常剪接的点突变，通过降低mRNA从细胞核输出到细胞质的效率或合成不稳定且迅速降解的酶亚基，导致OTC缺乏。我们推测，异常的mRNA剪接可能是这种疾病发病机制中一种相对常见的机制。
