Exon regions 2–12 3–7 7–12 8–9 Intron regions 4–5 1,456 382 842 310 585 Fw forward, Rv reverse, a.t. annealing temperature, e.t. extension time Results This study was performed on three patients with a clinical diagnosis of NC who had been previously screened for the CTNS gene mutations that revealed only one or no mutated alleles. Patients 1 and 2 are siblings. Previous sequence analysis had shown a heterozygous 57-kb deletion. No mutation was found on the other allele. To identify the second CTNS mutation, gene transcripts were analyzed from total RNA extracted from PBMCs. The PCR reaction yielded a 1,456-bp fragment spanning exons 2–12 that contained a 85-bp deletion corresponding to the entire exon 5 (Fig. 1). To highlight this deletion, CTNS transcripts were amplified using primers spanning exons 3–7 and were resolved by agarose gel electrophoresis, demonstrating a shorter band compared with control cDNA (Fig. 1). Skipping of exon 5 in these two patients causes a frameshift at position 141 bp, with a premature stop codon at position 1265 Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv 5′–gagacgctgagagaacctttgc–3′ 5′–gagtggtctcctctgtccgc–3′ 5′–ccctgaagctcgtagagaaatg–3′ 5′–gatggaccaggccacaaagtag–3′ 5′–ggatctactttgtggcctggt–3′ 5′–gagtggtctcctctgtccgc–3′ 5′–ctgattaagacaggaaggcc–3′ 5′–acctagcatttccctaccc–3′ 5′–gccttgcctgccctacatctc–3′ 5′–acttcactcatccttggagccc–3′ a.t.: 64°C e.t.: 90 s a.t.: 60°C e.t.: 30 s a.t.: 60°C e.t.: 60 s a.t.: 56°C e.t.: 30 s a.t.: 63°C e.t.: 60 s 160 bp. This splicing defect was further analyzed by sequencing the flanking intronic regions up to 300 bp upstream and downstream of exon 5. The analysis showed a point mutation at position –24 bp (c.141–24 T > C) of intron 4 that was not detected in 100 control chromosomes. The predicted protein generated by exon 5 skipping was truncated at position 53 after introducing six modified amino acids at the carboxy terminal end (wild-type cystinosin: 367 amino acids). Genomic DNA analysis failed to reveal any CTNS gene mutations in patient 3. CTNS transcripts were amplified from skin fibroblasts and demonstrated a homozygous duplication located in the exon 9 region (Fig. 1). Specifi- cally, the messenger RNA (mRNA) sequence of exon 9 was followed by a 75-bp sequence corresponding to a portion of intron 9, located 118 bp downstream to the exon donor splicing site and by a repeat sequence of the last 73 bp of exon 9. At the genomic level, this transcript corresponded Pediatr Nephrol (2010) 25:1263–1267 Table 2 Primer sequences and polymerase chain reaction (PCR) amplification conditions Amplified products Product length (bp) Primer sequences PCR conditions Fig. 1 Detected DNA and messenger RNA (mRNA) changes. a Patients 1 and 2 had a T>C substitution at position –24, upstream of exon 5 (c.141–24 T>C) that was not detected by standard sequencing. This mutation causes a skipping of exon 5 in the CTNS gene transcripts, as demonstrated by direct sequencing and agarose gel electrophoresis after reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of the region spanning exons 3–7. The smaller band (297 bp) corresponds to the amplicon lacking exon 5. b Patient 3 had a duplication of the genomic DNA sequence encompassing the last 73 bp of exon 9 and the first 193 bp of intron 9, shown in dark gray. This duplication causes an additional splicing of the mRNA transcript, with the incorporation of a portion of intron 9 followed by a repeat sequence corresponding to the duplicated portion of intron 9. By agarose gel electrophoresis using primers spanning exons 7 to 12, a larger 990 bp band is observed