1266 to a duplication of the last 73 bp of exon 9 and the first 193 bp of intron 9, which introduces an additional splicing site that removes the first 118 bp of intron 9. When resolved on an agarose gel, the amplicon of patient 3 produced a larger band that included 148 additional nucleotides (Fig. 1). The predicted protein was truncated at position 232 by a frameshift that generated 5 mutated amino acids, followed by a stop codon. Discussion In this work, two novel mutations in the CTNS gene were detected by studying CTNS mRNA transcripts in patients in whom traditional genomic sequencing failed to detect mutations in one or both alleles. Specifically, a splicing defect and a DNA duplication were identified. Analysis of gene transcripts may be limited by tissue-specific expression patterns. Fortunately, the CTNS gene is ubiquitously expressed, and accumulation of increased cystine has been demonstrated in various tissues and cell lines, including fibroblasts, proximal tubular cells, and PBMCs [15–17]. In all our patients, the clinical diagnosis of NC was confirmed by measuring intraleucocyte cystine levels. The splicing defect in patients 1 and 2 was located in an intronic region that is not routinely investigated (T > C substitution 24 bp upstream of the splicing acceptor site of intron 4). This mutation is located at a hypothetical lariat branch site and probably plays an important role in the splicing of exon 5. Lariat branch sites are crucial for appropriate splicing of pre-mRNAs, and point mutations in these regions have been shown to cause abnormal mRNA splicing [18–20]. The 266-bp DNA duplication in patient 3 could not be detected using standard sequencing primers spanning exon 9 and its immediate neighboring intronic regions. In fact, this is the first DNA duplication described in patients with NC. Apart from point mutations and small insertion/ deletions, all other mutations involve larger deletions [3, 6]. With this study, mutations in the CTNS gene were identified in all investigated Dutch patients, with the exception of one individual from whom RNA material was not available. Because the diagnosis of NC is substantiated by very specific findings, such as increased intraleucocyte cystine levels and corneal cystine crystals depositions, failure to discover mutations in these patients can be reasonably explained only by detection failures or by the existence of a second cystinotic gene. Our findings support the hypothesis that cystinosis is a monogenic disorder and suggest that CTNS transcripts should be studied when traditional genomic sequencing does not allow mutation detection. 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