Development of DNA diagnosis of urea cycle diseases and diabetes due to insulin receptor abnormality

尿素循环是氨基酸代谢中形成的氨解毒的主要途径。尿素循环涉及氨基甲酰磷酸合成酶I(CPS I)、鸟氨酸转氨甲基酶(OTC)、精氨酸琥珀酸合成酶(AS)、精氨酸琥珀酸裂解酶(AL)和精氨酸酶5种酶，这些酶都存在酶缺陷。如果这些酶中的一种缺失，氨到尿素的转化就会受到损害，从而发生高氨血症。作为开展这些疾病的DNA诊断的第一步，分离了土霉素和精氨酸酶的cDNA克隆，并测定了它们的结构。基因组克隆的分离表明，OTC基因是X连锁的，长约73kb，由10个外显子组成，精氨酸酶基因长14kb，由8个外显子组成。MSPI存在限制性片段长度多态(RFLP)。等位基因频率为0.33/0.67。我们对4个OTC缺乏症家系进行了MspI-RFLP分析。1例或4例母亲为RFLP杂合子，DNA诊断适用。针对精氨酸酶基因，用限制性内切酶PvuII和HincII鉴定了两个RFLP。PvuII-RFLP等位基因频率为0.07/0.97，HincII-RFLP等位基因频率为0.09/0.91。我们现在正在使用其他限制性内切酶和基因片段寻找新的RFLP。已有研究表明，2型糖尿病的部分原因是胰岛素受体异常。利用我们最近分离的胰岛素受体基因作为探针，在带有StuI的基因中发现了一个RFLP。对56例患者和70例健康人的等位基因频率进行了测定。这种RFLP与糖尿病之间没有明显的联系。

The urea cycle is the major pathway for detoxication of ammonia formed in amino acid metabolism. The urea cycle involves five enzymes, carbamyl phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate synthetase (AS), argininosuccinate lyase (AL) and arginase, There are enzyme deficiencies in all these enzymes. If one of these enzymes is missing, conversion of ammonia to urea is impaired and hyperammonemia occurs. As the first step to develop DNA diagnosis of these diseases, cDNA clones for OTC and arginase were isolated and their structures were determined. Isolation of genomic clones showed that OTC gene is X-linked, is about 73 kb long and consists of 10 exones, and that arginase gene is 14 kb long and consists of 8 exones.Genomic DNAs from 22 unrelated Japanese (33 alleles) were digested with several restriction enzymes and hybridized with OTC CDNA. Restriction fragment length polymorphism (RFLP) was found for MSPI. Allele frequency was 0.33/0.67. We performed an MspI-RFLP analysis in 4 families with an OTC deficiency. The mother in 1 or the 4was heterozygous for RFLP and DNA diagnosis is applicable. With respect to arginase gene, two RFLPs were identified, using restriction enzymes PvuII and HincII. Allele frequency of the PvuII-RFLP is 0.07/0.97 and that of the HincII-RFLP is 0.09/0.91. We are now searching for new RFLPs using other restriction enzymes and gene fragments.It has been shown that a part of type 2 diabetes is due to insulin receptor abnormalities. Using, as a probe, insulin receptor cDNA that we recently isolated, an RFLP was found in the gene with StuI. Allele frequencies were determined for 56 patients and 70 healthy individuals. No significant linkage between this RFLP and the diabetes.