Background: Cystinuria is an inherited disorder of defective renal reabsorption of cystine and the dibasic amino acids. Recently, SLC3A1 and SLC7A9 have been identified as responsible genes. While point mutations in the two genes are well known to cause cystinuria, only a few studies are aimed on the identification of gross genomic alterations. Here, we report our results of a systematic screening for deletions and duplications in SLC3A1 and SLC7A9 by quantitative real-time polymerase chain reaction (PCR).
Methods: We screened a cohort of 49 cystinurics for copy number deviations in the genes SLC3A1 and SLC7A9 by quantitative real-time PCR assays using fluorogenic 5' nuclease chemistry. The detected duplication in SLC3A1 was analyzed in detail by further real-time assays, reverse transcription (RT)-PCR and direct sequencing.
Results: In seven patients, we could identify a large duplication in SLC3A1 spanning from intron 4 to intron 9. This tandem duplication was accompanied by a small inversion of 25 bp and a 2 bp deletion in intron 9. As a formation mechanism, we presume that the inversion in intron 9 and several Alu sequences neighbored to the affected region provoke a chromatin structure that stimulates the duplication event. In addition to the SLC3A1 duplication, we observed deletions in SLC7A9 in three patients.
Conclusion: The frequency of genomic rearrangements in our patient population illustrates the significant contribution of large genomic alterations to the mutation spectrum in cystinuria. As we could show, quantitative real-time PCR is a reliable and effective tool for the identification of unbalanced genomic rearrangements.