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Methods

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform

¹ Division of Oncology, The Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA ² Orchid Biosciences, Inc., Princeton, New Jersey 08540, USA ³ Cardeza Foundation for Hematologic Research Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA ⁴ Center for Translational Medicine, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA ⁵ Dipartimento di Medicina Sperimentale e Patologia, Universita' degli Studi "La Sapienza," 00198 Roma, Italy

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.3865305.

⁶ Present address: Preanalytical Systems, Becton, Dickinson and Company, Franklin Lakes, New Jersey 07417, USA

⁷ Present address: Johnson & Johnson, Milltown, New Jersey 08850, USA.

⁸ Corresponding author.
E-mail paolo.fortina{at}jefferson.edu; fax (215) 955-6905.

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