LC-MS/MS determination of dibasic amino acids for the diagnosis of cystinuria. Application in a family affected by a novel splice-acceptor site mutation in the SLC7A9 gene

Summary Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and the dibasic amino acids ornithine, lysine and arginine across cell membranes. Poor solubility of cystine in urine leads to kidney stones and associated symptoms and complications. Mutations of genes SLC3A1 and SLC7A9 encoding for amino acid transport systems are responsible for different types of cystinuria. In this study we describe a new LC-MS/MS assay for these amino acids in urine. Moreover, we report a novel splice-acceptor site mutation in the SLC7A9 gene that we believe is the cause of the phenotype observed in four siblings from a first-cousin marriage. Into the wells of a 96-well microtitre plate, 10 μl of urine was mixed with 90 μl of a solution containing [ 2 H 4 ]cystine, [ 2 H 2 ]ornithine, [ 13 C, 2 H 4 ]arginine and [ 2 H 5 ]glutamine that was used as an internal standard for lysine. Chromatographic separation was achieved isocratically and detection was in the selected-reaction monitoring mode. The injection-to-injection time was 8 min. Calibration curves were linear up to 1000 μmol/L. Intra-day ( n = 10) and inter-day ( n = 6) variations (750 and 10 μmol/L) were less than 11.4%. Urine samples from healthy individuals ( n = 135) were analysed and age-matched reference ranges were generated. The method was applied retrospectively and prospectively to analyse samples ( n = 13) from nine cystinuria patients. The mutation reported here was not found in 100 controls with similar ethnicity to the studied family and is believed to have consequences for the transcribed mature RNA and protein structure and function.