Gene expression analysis
All the samples were collected according to the current version of the Declaration of Helsinki and written informed consent for publication of this manuscript and accompanying images was obtained from the patients’ parents and relatives. All experiments were ethically approved by the ‘Bambino Gesù’ Children’s Hospital Scientific Board.
Skin fibroblasts of the patient were obtained from a biopsy of a hypochromic cutaneous lesion using standard methods. Skin fibroblasts of two healthy children were also collected and used as calibrator samples (reference). Total RNA was isolated from fibroblasts with TRIzol (Ambion, Life Technologies, Paisley, UK) according to the manufacturer’s protocol. For qPCR analysis, 1 μg of total RNA was reverse-transcribed to cDNA using a high-capacity cDNA archive kit (Applied Biosystems, Life Technologies, Paisley, UK). The expression levels of 26 selected genes and one housekeeping gene (GAPDH) of the ring17 patient were measured in each sample by real-time qPCR using pre-designed TaqMan gene expression assays on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) according to the manufacturer’s protocol, and compared with controls.
The investigations involved 15 genes (ACTG1, GCGR, ARHGDIA, NPB, SIRT7, SECTM1, UTS2R, NARF, FOXK2, RAB40B, FN3KRP, FN3K, TBCD, ZNF750, METRNL) and 9 genes (DOC2B, RPH3AL, FAM57A, GEMIN4, RNMTL1, NXN, TUSC5, YWHAE, MYO1C) located on the sub-telomere region of the long and short arms, respectively, of chromosome 17 encompassing a 1.5 Mb region, starting from each telomere. Two other genes (NF1 and LIS1) placed outside the 1.5 Mb region (17q11.2 and 17p13.3, respectively), were also investigated.
For each sample, three replicates were run for each gene in a 96-well plate. Real-time qPCR reactions were carried out following the manufacturer’s instructions. Gene expression values were determined as ΔCt (Ct(GAPDH) − Ct(gene)) and relative quantities between different samples were determined as ΔΔCt (ΔCt(patient) − ΔCt(calibrator sample)) . All data are expressed as mean ± standard deviation. The statistical comparison between the ring 17 patient and control individuals was performed using Student’s t test at a level of significance of 0.05 (P < 0.05).
Classical and molecular cytogenetic investigations
Karyotyping with G-banding was performed using standard methods on lymphocytes obtained from peripheral blood and fibroblasts from a biopsy of skin with or without hypochromic cutaneous lesions. Chromosomes were also examined on lymphocytes of both of the patient’s parents, as well as her sister, her maternal grandparents and her aunt. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2009).
To search for possible deletions at telomeric regions, FISH was performed with human pan-telomeric probes, P1 artificial chromosome (CTD-2348K1, 17p13.3) and fosmid (WI-837H17, 17q25.3) clones. The probes were directly labeled with Cy3-dUTP and fluorescein-dUTP (Perkin Elmer Life Sciences, Boston, MA, USA).
Array-CGH was performed using Agilent Technologies Array-CGH Kits (Santa Clara, CA, USA), as described previously . The platform is a 60-mer oligonucleotide-based microarray with an overall median probe spatial resolution of 13 kb. To evaluate whether the copy number variations, detected by array-CGH, were potentially correlated with the clinical phenotype of our patient, bioinformatic analysis was carried out, consulting the Database of Genomic Variants BioXRT (http://projects.tcag.ca/variation/).
DNA was extracted from peripheral blood of the patient and her relatives with a high pure PCR template preparation kit (Roche, Mannheim, Germany), according to the producer’s instructions. To assess the familiar origin of the ring 17, a panel of STRs was assembled using multiple primer pairs obtained from the UniSTS database (http://www.ncbi.nlm.nih.gov/unists/). The STRs analyzed, listed starting from 17pter to 17qter, were: D17S578, D17S1875, D17S969, NF1, D17S788, D17S1306, D17S1819, D17S789, D17S1839, D17S1292. The first five STRs were the informative ones. DNA was amplified by means of a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA) following the standard protocol. One primer from each pair was fluorescently labeled and PCR products were run on an ABI Prism 310 (Applied Biosystems), using GeneMapper v 3.0 software.
Additional studies were performed also on café au lait skin spots and a normal skin biopsy of the patient to investigate the parental transmission of the chromosome 17.
Q-FISH staining was performed as previously described  with minor modifications. Briefly, 48 hours after the seeding, slides were rinsed with PBS at pH 7.5, and fixed in 4% formaldehyde for 2 min. After two rinses in PBS, the slides were incubated in acidified pepsin solution for 10 min, rinsed, and dehydrated through graded alcohols. Slides and probes (Cy3 linked telomeric and chromosome 2 centromeric PNA probe, DAKO Cytomatation, Denmark) were co-denatured at 80°C for 3 min and hybridized for 2 hours at room temperature in a humidified chamber. After hybridization, slides were washed twice for 15 min in 70% formamide, 10 mM Tris at pH 7.2, and 0.1% BSA, followed by three 5-minute washes in 0.1 M Tris at pH 7.5, 0.15 M NaCl, and 0.08% Tween 20. Slides were then dehydrated with an ethanol series and air dried. Finally, slides were counterstained with 4,6-diamidino-2-phenylindole (Sigma Aldrich, St. Louis, MO) in Vectashield (Vector Laboratories, Burlingame, CA). Images were captured at 63× magnification with an Axio Imager M1 (Carl Zeiss, Germany) equipped with a charge-coupled device camera, and the telomere size was analyzed with ISIS software (MetaSystems, Germany). The software calculates telomere lengths as the ratio between the fluorescence of each telomere signal and the fluorescence of the centromere of chromosome 2, used as the internal reference in each metaphase analyzed. The centromere 2 DNA sequence, which the probe recognizes, has a stable length and can be used as a reference. Data were expressed as a percentage (T/C%) . For each individual, at least 20 metaphases have been analyzed.