Abstract 4849: Profiling of multiple gene mutations in myelodysplastic syndromes using high-throughput resequenceing combined with barcode labeling

Abstract

Abstract Myelodysplastic syndromes (MDS) are a highly heterogeneous group of myeloid neoplasms characterized by ineffective hematopoiesis and a predisposition to acute myeloid leukemia, where a model of multistep oncogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations, including mutations of RAS, RUNX1, TET2, CBL, and TP53, where multiple gene mutations frequently harbor in a single case. However, the entire profiles of these multiple gene mutations with their relationship with WHO classification, chromosomal alterations, and clinical pictures have not been fully explored in a large series of MDS cases. Screening possible gene mutations in dozens of candidate genes in a large number of samples using Sanger sequencing were a time-consuming and labor-intensive task. So in order to overcome this issue and to obtain comprehensive registries of gene mutations in known candidate genes in a total of 171 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 76 candidate genes using Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of multiple DNA samples. Briefly, each fragmented genomic DNA was first amplified by single-primer polymerase-chain reactions (PCR), from which target sequences were concentrated using the SureSelect system (Agilent). Captured targets were primed with 6-base barcode sequences to discriminate the sample, which were subjected to high-thoughput resequencing using Genome Analyzer IIx (Illumina). All 171 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS, KRAS, c-CBL, EZH2, and TET2 had been determined by Sanger sequencing, and thus were considered to an ideal sample set for this study, in which genome-wide copy numbers were characterized in detail and the known mutations works as a control to measure the performance of the barcode resequencing. The targeted sequences consisted of 1314 exons from 76 genes that comprised a total length of ∼0.5Mb, and included genes which were known to be mutated in MDS or related disorders, and also other candidate targets. We were able to analyze up to 96 samples per 1 run and efficiently detected mutations in targeted genes with high average coverage obtained from these sequences. On average, 52% of the total reads successfully captured the targets and 90% of targeted regions were covered with >20 depths of reading with 72.8× of mean coverage per sample from a single run. We identified more than 20 previously unpublished mutations in MDS, including those of NOTCH2 and ATM. In this meeting, we will present the result of our large-scale mutation study in MDS and related disorders and discuss the genetic basis of MDS in terms of multiple gene mutations as well as copy number alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2011-4849