Computational protocol: BRCA1 haplotype and clinical benefit of trabectedin in soft-tissue sarcoma patients

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Protocol publication

[…] Genomic DNA was extracted from paraffin-embedded tissues according to previously published procedures (). For Holland Bouin-fixed tissues, sections were washed with lithium carbonate to remove picric acid. Tissues were then washed with 1 × TNE (10 mM Tris, pH 8; 1 mM EDTA, pH 8; and 100 mM NaCl). DNA was then purified on GFX columns (GE Healthcare Europe GmbH, Saclay, France) following digestion with proteinase K (2 mgml−1, 55 °C for at least 2 h).For the first cohort, genotyping of the four BRCA1 SNPs (Gly742Glu, rs16941; Arg1183Lys, rs16942; Gly1613Ser, rs1799966 and Pro871Leu, and rs799917) was carried out using the GoldenGate technology from Illumina (San Diego, CA, USA) on the BeadArray platform following the manufacturer's recommendations using 150 ng of genomic DNA per patient as starting material. Raw hybridisation intensity data processing, clustering, and genotype calling were carried out using the genotyping module in the GenomeStudio package (Illumina).BRCA1 SNP genotyping was also carried out via pyrosequencing. Briefly, 100 ng of genomic DNA were amplified with specific primers. Pyrosequencing primers were designed using the Pyrosequencing SNP Primer Design (V 1.01) software (Qiagen, Courtaboeuf, France). For PCR, 10 pmol of each primer (forward 5′-biotinylated: 5′-CACCACTTTTTCCCATCAAGTC-3′, reverse primer: 5′-TGGCCCTCTGTTTCTACCTAGTT-3′, for rs16941;forward: 5′-AGCCTATGGGAAGTAGTCATGC-3′, reverse 5′-biotinylated primer: 5′ GCCAAATGTGTATGGGTGAAAG-3′, for rs16942; forward 5′-biotinylated: 5′-TGGCAACATACCATCTTCAACC-3′, reverse: 5′-GGCTTCTCCCTGCTCACACTTT-3′ for rs179966; forward: 5′-AAGGTTTCAAAGCGCCAGTC-3′, reverse 5′-biotinylated: 5′-CTCTTCTGCATTTCCTGGATTTGA-3′ for rs 799 917) were used. The reaction was completed in a buffer containing 3 mM MgCl2 and 0.5 U Taq polymerase Platinum (Invitrogen, Cergy Pontoise, France) in a 50 μl final volume. The mixture was denatured for 5 min at 95 °C and then 50 cycles were carried out: 30 s at 95 °C, 30 s at 60 °C, or 62 °C; and 30 s at 72 °C for rs16941 and rs16942, or rs179966 and rs799917, respectively. For pyrosequencing, ssDNA was prepared from 40 μl of biotinylated PCR product using streptavidin-coated sepharose beads (GE Healthcare), and 0.37 μM of the adequate sequencing probe (5′-CATTAATATTGCTTGAGCTG-3′ for rs16941, 5′-GCAAAAGCGTCCAGA-3′ for rs16942, 5′-GAGCAGCAGCTGGAC-3′ for rs179966 and 5′-CGCCAGTCATTTGCTC-3′ for rs799917) for analysis. Sequencing was performed with the SNP Gold reagent kit (Qiagen) according to the manufacturer's instructions.For BRCA1, haplotypes and frequency estimation were obtained using the Haploview software (). DNA extractions and SNP determination were centralised at the INSERM U916 research laboratory. […]

Pipeline specifications

Software tools GenomeStudio, Haploview
Application GWAS
Organisms Homo sapiens
Diseases Neoplasms, Sarcoma
Chemicals Doxorubicin