Computational protocol: Mutation frequencies of GNAQ, GNA11, BAP1, SF3B1, EIF1AX and TERT in uveal melanoma: detection of an activating mutation in the TERT gene promoter in a single case of uveal melanoma

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Protocol publication

[…] DNA was extracted from macrodissected tumour material conserved in RNAlater (Ambion, Monza, Italy) or from archival formalin-fixed, paraffin-embedded (FFPE) blocks using QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). For FFPE samples, two 10-μm-thick sections were cut from each paraffin block and subjected to de-paraffinisation using scalar concentrations (100% to 40%) of Histoclear (Sigma Aldrich, Milan, Italy), followed by re-hydration and overnight digestion with proteinase K (100 μg ml−l, Sigma Aldrich) at 37 °C. DNA concentration and quality were checked in the Nanodrop ND-1000 spectrophotometer and integrity of DNA was tested by agarose gel electrophoresis. Processing of genomic DNA was performed using the GeneChip Mapping 250 K Assay Kit (Affymetrix, Santa Clara, CA, USA) as recently described (). Briefly, 250 ng of DNA sample were digested with the restriction enzyme NspI. Adapters were ligated using T4 DNA Ligase. Whole genome amplification was performed on a GeneAmp PCR System 9700 (Applied Biosystems–Life Technologies Corporation, Monza, Italy). Ninety micrograms of amplified and normalised PCR product were fragmented and labelled. Hybridisation, washing, staining and scanning of SNP arrays were performed on the Affymetrix fluidic station. Quality of the samples was assessed on agarose gels before the hybridisation step. Affymetrix Genotyping Console (GTC4.1.2) was used to perform genotype call and quality control assessments. Copy number analysis was performed using CNAG3.0 ().Multiplex ligation-dependent probe amplification (MLPA) was performed according to the manufacturer's instructions (MRC-Holland, Amsterdam, The Netherlands). In short, 5 μl of genomic DNA diluted in TE at a concentration of 20 ng ml−1 were denatured at 98 °C for 5 min, cooled to 25 °C and 3 μl of a 1 : 1 mixture of MLPA buffer and SALSA P027.C1 probe-mix was added. After hybridisation for 16 h at 60 °C, 32 μl ligation mix were added, and the reaction was incubated for 15 min at 54 °C followed by 5 min at 98 °C. Subsequently, 10 μl of the SALSA PCR-mix (FAM label) were added to 40 μl of ligation product and this was amplified by PCR in 35 cycles (30 s, 95 °C; 30 s 60 °C; 60 s 72 °C). PCR products were quantified using the ABI 3130XL (Applied Biosystems-Life Technologies Corporation, Milan, Italy) and coffalyser.net software (MRC-Holland). Raw data were expressed as peak heights, as a measure of peak intensity, for each of the 50 probes (38 test probes and 12 control probes). For each probe, the relative ratio was calculated using population normalisation and using the internal control probe normalisation. The MLPA data were considered reliable if six or more control probes were within the normal range. Loss was identified as MLPA relative ratio <0.70 and gain as MLPA relative ratio >1.3.For RNA extraction, tumour samples were homogenised in the tissue lyserMixer Mill (Qiagen) in total RNA extraction lysis buffer using RNeasy (Qiagen). RNA quality was assessed in the BioAnalyser (Agilent, St Clara, CA, USA). Gene expression profiling was performed using Affymetrix HGU133plus2 arrays following standard procedures as described (). Assignment to prognostic gene expression classes was performed by nearest neighbour classification using the gene expression values for the discriminator genes described ().PCRs were carried out using 25 ng of genomic DNA in a 25 μl reaction mix including 10 × Platinum PCR Supermix, 1.5 mM MgCl2, 200 μM dNTPs, 1 μM primers and 0.5U Taq Platinum (Invitrogen–Life Technologies Corporation, Monza, Italy) on a Veriti 96 wells Thermal Cycler (Applied Biosystems–Life Technologies Corporation). Thermal cycling conditions were as follows: 94 °C for 4 min, followed by 25 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 7 min. PCR conditions were slightly modified for EIF1AX gene (2 mM MgCl2 were used for exon 1 and the annealing temperature was 54 °C for exon 1 and 64 °C for exon 2).Telomerase reverse transcriptase promoter region oligonucleotide primers were synthesised according to the Eukaryotic Promoter Database genomic reference sequence of TERT and designed to amplify a portion of the TERT core promoter (−144 to +43). The amplicon of 187 bp includes the two major recurrent mutations at coordinates chr 5 : 1 295 228 and chr 5: 1 295 250. PCR were performed by using 100–200 ng of genomic DNA in a 50 μl reaction mix with 400 μM dNTPs (Fermentas-Thermo Scientific Life Science, Milan, Italy), 0.75 μM primers, 2 mM MgCl2, 1X Buffer and 2.5 U AmpliTaq Gold 360 DNA Polymerase (Applied Biosystems-Life Technologies Corporation) on a Veriti 96 wells Thermal Cycler (Applied Biosystems, Life Technologies Corporation). Thermal cycling conditions were as follows: 95 °C for 7 min, followed by 42 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min.All PCR primers (see ) except for TERT were designed with a universal sequence at 5′-end (universal forward primer 5′-GTTGTAAAACGACGGCCAGT-3′ and M13 (−48) reverse primer 5′-GTGTGAAATTGTTATCCGCT-3′) to perform single-pass sequencing. Additional primers were synthesised for FFPE samples that did not yield any amplicon using the first set of primers. Mutational screening was carried out by direct sequencing of fragments obtained by PCR using an ABI3130xl and ABI3730 Genetic Analyzer (Applied Biosystems–Life Technologies Corporation). Sequencing data were analysed using SeqScape v2.5 software (Applied Biosystems–Life Technologies Corporation) and MacVector V11 software (MacVector Inc., Cary, NC, USA).Transcription factor binding analysis was performed using TFSEARCH based on the TRANSFAC database (). […]

Pipeline specifications

Software tools SeqScape, MacVector, TFSearch
Databases TRANSFAC EPD
Application Sanger sequencing
Organisms Homo sapiens
Diseases Melanoma, Neoplasms
Chemicals Cytidine, Thymidine