Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients.

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.

Orotic aciduria and plasma urea cycle-related amino acid alterations in short bowel syndrome, evoked by an arginine-free diet.

BACKGROUND: The small bowel is believed to play a crucial role in endogenous arginine synthesis. Therefore, an insufficient arginine supply in the situation of massive intestinal resection might impede normal arginine metabolism. This study sought to determine the clinical and metabolic effects of an arginine-free diet in stable short-bowel patients. METHODS: Four patients, mean age 49 years (range: 26-67), mean time from intestinal resection 46 m (range: 15-97), and remnant small bowel of 30 to 100 cm consumed an L-amino acid arginine-free diet (egg pattern) for 5 days (0.9 g protein equivalent/kg/d plus malabsorption adjustments). Fasting plasma amino acids, ammonium, and blood chemistries were assessed at days 0, 3, and 5. Urinary orotate, orotidine, uric acid, urea, creatinine, and total nitrogen were evaluated daily. RESULTS: Significant decreases in plasma levels of arginine, ornithine, and hydroxyproline occurred at day 5. A decreasing trend in plasma citrulline and a significant plasma glutamine increase were also observed in the same period. Conversely, ammonium concentrations remained normal. Regarding urine compounds, striking orotic aciduria with a peak at day 4 (14-fold vs baseline) and significant decreases in uric acid and urea excretion were found. There were no relevant clinical events. CONCLUSIONS: Despite the limited number of patients in our work and their relative heterogeneity, our results support the idea of the indispensability of an exogenous arginine supply in humans under short bowel syndrome conditions. Studies in larger series are needed to further investigate these findings.