Agilent cytogenomics 4

DNA was extracted from the fibroblasts using QIAamp^® DNA Blood kit (Qiagen, Milan, Italy) according to the manufacturer’s protocol. PCR amplifications were performed using platinum-Taq DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA, USA) and specific primers for the 23 different NSD1 exons (see Table S3). The PCR conditions were a single denaturation step at 95 °C for 3 min followed by 40 cycles of 95 °C/30 s, 58 °C/30 s and 72 °C (1 min/kb), with a final extension step at 72 °C for 5 min. PCR products were sequenced using the ABI BigDye Terminator Ready Reaction Mix (Thermo Fisher Scientific, Foster City, CA, USA) and analyzed on an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The sequences were aligned with Seqscape analysis software V.2.5 (Thermo Fisher Scientific, Foster City, CA, USA). To verify variants with deletions of the 5q35.3 region, array-CGH was performed with CGH 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. The data were analyzed with the Agilent Cytogenomics 4.0.3.12 software (Agilent Technologies, Santa Clara, CA, USA). All genomic positions were reported according to the human genome assembly (GRCh37/hg19).

MLPA analysis, unlike FISH, recognizes both the deletions of the entire NSD1 gene and microdeletions of one or more exons.
The analysis was carried out on genomic DNA with the MLPA SALSA P026 Kit (MRC-Holland, Amsterdam, The Netherlands). All reactions (denaturation, ligation, and PCR) were performed following the manufacturer’s instructions. PCR products were run on a 3130xl automated sequencer (Applied Biosystems, Foster City, CA, USA) and data were analyzed using Genemapper v 3.2 and Coffalyser v.140721.1958 software (Applied Biosystems, Foster City). In selected cases, Array-CGH analysis was carried out to define the size and the breaking point of the deletions.
Array-CGH was performed using Superprint G3 CGH 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturers’ protocol. Data were analyzed by Agilent Cytogenomics 4.0.3.12 software (Agilent Technologies, Santa Clara, CA, USA). All genomic positions were reported according to the human genome assembly (GRCh37/hg19).

Array-CGH was performed using an Agilent Sureprint G3 CGH+SNP 4 × 180 K microarray (G4890A; Agilent Technologies) following the manufacturer’s protocols. Briefly, 500 ng of purified DNA of a patient and a control (Agilent Technologies) were double-digested with RsaI and AluI enzymes (Agilent Technologies) for 2 h at 37°C. Each digested sample was labeled for 2 h with the Agilent Genomic DNA Labeling Kit, using Cy5-dUTP for the patient DNA and Cy3-dUTP for the control DNA. Labeled products were purified and prepared according to the Agilent protocol. After probe denaturation and pre-annealing with 50 ng of human Cot-1 DNA (Thermo Fisher Scientific, Yokohama, Japan), hybridization was performed at 65°C for 24 h in a rotating oven. After washing steps, the array slide was scanned with an Agilent Microarray Scanner (G2565CA). The spot intensities were measured and the image files quantified using the Agilent Feature Extraction 11.0.1.1 software. Text outputs from the quantitative analyses were imported into Agilent CytoGenomics 4.0 software (Agilent Technologies).