1264 regions of the gene, as well as in the CTNS promoter, in patients with cystinosis. Of these, approximately 70 mutations cause NC. These include small deletions or insertions, missense or nonsense mutations, and splicing mutations [3, 6–13]. In clinical practice, the diagnosis of NC is sustained by the demonstration of increased intraleucocyte cystine levels. Genetic tests have primarily a confirmatory value and allow for genetic counseling. Evidence suggests that NC is a monogenic disease, although the mutation detection rate is not 100%. Shotelersuk et al., for example, sequenced the CTNS gene in 108 American patients with NC and failed to detect mutations in 19% of affected individuals [6]. The CTNS promoter was, however, not analyzed in their study. Similar studies that also included the promoter region showed only heterozygous or no mutations in 18% of 46 Italian patients and in 6% of 108 French patients [9, 11]. In this work, we studies CTNS gene transcripts in three patients with NC from two families to search for mutations that were not detected at the genomic level. Patients and methods Patients Among 49 Dutch patients with NC from 44 different families, we selected three patients in whom sequence analysis failed to show homozygous or double heterozy- gous mutations in the CTNS gene (coding regions, intron/ exon boundaries, or promoter region). Clinical data of these patients are summarized in Table 1. The diagnosis of NC was established in all patients by demonstration of increased intraleucocyte cystine levels and corneal cystine crystal depositions. Informed consent was obtained to perform the additional genetic studies included in this work. Peripheral blood mononuclear cell isolation and fibroblasts cell lines Anticoagulated whole blood samples were diluted with phosphate-buffered saline (PBS) (1:1), layered over Ficoll Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA), Table 1 Patient characteristics M male, F female, ESRF end-stage renal failure Gender Age of diagnosis (months) Age at ESRF (years) Intraleucocyte cystine (nmol ½ cystine/mg protein) Ocular symptoms Pediatr Nephrol (2010) 25:1263–1267 and centrifuged at 1,800 g for 20 min at room temperature. The peripheral blood mononuclear cell (PBMC) layer was removed and washed twice with PBS following two centrifugation steps at 250 g for 10 min. RNA studies were performed in patient 3 on a previously established fibroblast cell line from that patient [14]. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from PBMCs or fibroblasts using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was reverse-transcribed into complementary DNA (cDNA) using a first-strand cDNA synthesis kit (Roche Diagnostics, Mannheim, Germany). CTNS transcripts were amplified by PCR adding Taq DNA polymerase, PCR buffer with magnesium chloride (MgCl2), deoxyribonucleotide triphosphate (dNTP) (Invitrogen Life Technologies), and primers (Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturers’ instructions. Primers and PCR conditions for CTNS amplification are described in Table 2. PCR products were analyzed using a 1.5% agarose gel with GelRed™ staining (Biotium Inc., Hayward, CA, USA). PCR fragments were extracted and purified with gel extraction kit (QIAGEN, Milan, Italy). Transcript sequencing was performed using primers detailed in Table 2 and dye terminator cycles sequencing (DTCS) Quick Start Mix (Beckman Coulter, Brea, CA, USA) at following conditions: 96°C for 30 s, 53°C for 30 s, and 60°C for 240 s for 37 cycles. After purification, samples were sequenced on a CEQ2000XL DNA Analysis System (Beckman Coulter). Genomic DNA analysis Genomic DNA was extracted from anticoagulated whole peripheral blood by QIAamp DNA blood kit (QIAGEN) in accordance with the manufacturer’s instructions. The CTNS gene was amplified using primers and PCR conditions detailed in Table 2. Direct sequencing was performed with the primers described in Table 2 and DTCS Quick Start Mix (Beckman Coulter) at conditions detailed above. Samples were sequenced on a CEQ2000XL DNA Analysis System (Beckman Coulter). Patient 1 F 41 12 5.2 Patient 2 M 48 10.3 7.8 Patient 3 F 18 14.2 9 Corneal cystine crystals Corneal cystine crystals Corneal cystine crystals, band keratopathy