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

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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.