Mutational analyses were carried out on 45 unrelated patients by direct sequencing of the exons of the SLC3A1 and SLC7A9 genes. We identified 17 different mutations, 10 in the SLC3A1 gene (T216M, C242R, R365L, 1136+ 2T→C, G398R, R456C, M467T, M467K, S547W, L573X), and seven in the SLC7A9 gene (G73R, G105R, A331V, 1048-1051del ACTC, V375I, S379R, 1265-1266delTG). The most frequent mutations were T216M (21.6%) and M467T (14.8%) in the SLC3A1 gene and G105R (22.7%) in the SLC7A9 gene (manuscript in preparation).

Here we describe five novel mutations: two in the SLC3A1 gene (one missense and one nonsense) and three in the SLC7A9 gene (two missense and one deletion). A summary of the phenotypes of cystinuria patients with novel mutations, and their relatives is shown in Table 1.

Identification of Novel Mutations in the SLC3A1 Gene. The missense mutation C242R, in exon 3, involves substitution of T by C at nucleotide position 724. It changes the cysteine TGT codon to CGT for arginine (Figure 1A). This is a conserved amino acid residue in a part of a subunit that is located extracellulary, and the effect of the C242R mutation suggest that it may be critical for proper function [13]. This mutation was found in two Serbian patients of whom one was classified as type I cystinuria since his parents exhibited a normal urinary excretion profile (Table 1), and he is a compound heterozygote for C242R and R456C (patient CY-13). The R456C mutation involves a conserved residue in a part of the subunit that is located intracellulary [9].

The second patient with C242R (CY-15) was classified as type I/unclassified (Table 1) because the urinary excretion profile of his father was normal, while his mother was unavailable for analysis. The second mutation in patient CY-15 was also a new one, the nonsense L573X mutation in exon 10. This mutation leads to a stop codon instead of a leucine residue at codon 573 (Figure 1B). The leucine residue is not conserved and the mutation leads to truncation of the subunit by 16.7%, including the fourth transmembrane domain.

Identification of Novel Mutations in the SLC7A9 Gene. The G73R mutation in exon 3 of the SLC7A9 gene was found in a Serbian patient (CY-8). The mutation includes a substitution of G by A at nucleotide position 402 (Figure 2A), which leads to the replacement of conserved glycine (GGG) by arginine (AGG). According to the model for the light subunit of this amino acid transporter suggested by Font et al. and the International Cystinuria Consortium [11], glycine at position 73 is a conserved amino acid residue that is located in the second transmembrane domain. The mother, father, and sister were also examined. The patient’s urinary excretion profile was non type I/non type I (Table 1). The patient inherited the G73R mutation from her father, who presented with high values for urinary cystine and dibasic amino acids. CY-8 inherited a G105R mutation from her mother, who also had increased urinary cystine and dibasic amino acids.

The second novel mutation found in the SLC7A9 gene was V375I in exon 11 (Figure 2B) that involves a substitution of G by A at position 1123. This mutation involves the substitution of valine by isoleucine and is located on the boundary between the fifth extracellular loop and tenth transmembrane domain. The patient inherited this mutation from his mother, and a second mutation (G105R) from his father. The urinary amino acid profile of patient CY 3 was type I/non type I, since the mother exhibited elevated levels of cystine and ornithine, consistent with non type I cystinuria, while the father showed normal urinary amino acid levels, consistent with type I cystinuria. Although G105R affects a highly conserved amino acid residue, a small percentage of the heterozygotes carrying this mutation exhibit elevated levels of urinary cystine and dibasic amino acids [8,9,11].

A deletion of four nucleotides (ACTC) at position 1048-1051 in exon 10 in the SLC7A9 gene (Figure 2C) was detected in the homozygous state, in a Turkish patient (CY-36). This mutation was most probably generated by a mechanism of "slipped misspairing" between the two direct repeats (TC), followed by a deletion of one of the direct repeats and the intervening AC sequence [14]. Because of the urinary excretion profile of the parents, the patient was classified as non type I/non type I (Table 1). Consanguinity between the parents was confirmed.

In conclusion, this report describes two novel mutations in the SLC3A1 gene (C242R and L573X) and three in the SLC7A9 gene (G73R, V375I, 1048-1051delACTC) and expands the spectrum of cystinuria mutations.