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 (type I and III). Previously, nine missense mutations, four mutations associated with an absence of an exon in mRNA, and one splicing mutation have been identified in human neonatal citrullinemia (Kobayashi et al. J Biol Chem 1990 and Mol Biol Med 1991). These fourteen patients were mainly American except three alleles from Japan., Furthermore, reverse transcription of mRNA, amplification of cDNA and sequencing of cDNA clones were used to characterize mutations in eleven Japanese citrullinemic patients. In this paper, we describe five new missense mutations (A118T, A192V, R273C, G280R and R363L) and one new insertion mutation in mRNA.Three alleles have R304W mutation and eight alleles have DELTAEx7 mutation deleted exon 7 in mRNA.In order … More to identify the abnormality in ASS gene causing DELTAEx7 mutation and to establish DNA diagnosis, we isolated and sequenced a phage clone which involves intron 6 and 7 flanking regions of exon7. The DELTAEx7 mutation results in a transition from ccagGT to ccggGT at the 3'-side and within the splice site of intron 6 (IVS6^<-2> mutation), and results in the creation of an MspI restriction site. The DNA diagnoses of 29 Japanese alleles with classical citrullinemia show that 15 alleles have IVS6^<-2> mutation and 4 alleles have R304W mitation and that these two mutations appear in 66% of the mutated alleles in Japanese patients (Kobayashi et al.manuscript in preparation).We also describe a different type of citrullinemia. Type II citrullinemia is found in most patients with adult-onset citrullinemia in Japan, and ASS deficiency is found specifically in the liver. Previous studies have shown that the decrease of hepatic ASS activity is caused by a decrease in enzyme protein with normal kinetic properties and that there were no apparent abnormalities in the amount, translational activity, and gross structure of hepatic ASS mRNA.In the present work, we show by sequencing analysis that there was no mutation in the ASS mRNA from two patients with type II citrullinemia. We also report RFLP analysisi of a consanguineous family with type II citrullinemia, by using three DNA polymorphisms located within the ASS gene locus. In spite of having consanguineous parents, the patient was not a homozygous haplotype for the ASS gene. The RFLP analysisi of 16 affected patients from consanguineous parents showed that 5 of 16 patients had the heterozygous pattern for one of the three DNA probes and that the frequency of the heterozygous haplotype was not different from the control frequency. These results suggest that the primary defect of type II citrullinemia is not within the ASS gene locus (Kobayashi et al. Am J Hum Genet 1993). Less