molecular basis of argininosuccinate synthetase deficiency in citrullinemia.

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Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase(ASS)which functions as a member of urea cycle in the liver.This enzyme defect is found in all tissues or cells of the classic neonatal citrullinemia. Fourteen mutations were identified by sequencing amplified CDNA from 14 neonatal patients. Mutations causing neonatal citrullinemia are extremely heterogeneous, and all non-consanguineous individuals studied to date are compound heterozygotes. The nucleotide coding sequence and deduced amino acid analysis are available for four mammalian species, yeast and three bacterial species. Six of nine missense mutations in humans occur in amino acid positions that are completely conserved in these organisms. To confirm these missense and deletion mutations, we established the in vitro expression system for ASS protein using PCAGGS vector in BMT-10 cells. Five mutant CDNA were tested in this expression system and were confirmed that all mutant had no ASS activity and no or low level of ASS-CRM.We also describe a different type of citrullinemia(adult onset form)which occurs in association with decreased ASS activity in the liver but normal activity in the kidney and other cells. Decreased hepatic ASS with normal kinetics properties and heat stability is accompanied by normal levels of ASSmRNA in the liver, no gross structural abnormality, and translational activity. No mutations were found in the whole sequence of ASSmRNA(except 20 base of 5'-end). We have analyzed about 80 patients with adult citrullinemia to date, and the proportion of patients with consanguinity were approximately 20%. RFLP analysis of 11 patients from consanguineous marriage suggested the possibility that the primary defect is not located on the ASS gene. We are now performing further homozygosity mapping.