Profiling of Multiple Gene Mutations In Myelodysplastic Syndromes Using High-Throughput Resequenceing Combined with Barcode Labeling

Abstract 4011

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 multisteponcogenesis has been implicated in their pathogenesis. On the other hand, recent advances in cancer genome analysis disclosed a number of gene mutations involved in the development of MDS, including mutations of RAS, RUNX1, CEBPA, TET2, CBL, EZH2 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 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 170 cases with MDS and related disorders, we performed high throughput mutation analyses of more than 80 candidate genes using Genome Solexa-based next-generation resequencing technology combined with target gene capture and barcode labeling of individual samples. Briefly, each fragmented genomic DNA was frist 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 Analizer®(Illumina). All 170 cases were already analyzed by Affymetrix SNP arrays, and their mutation status regarding RUNX1, p53, NRAS and KRAS, c-CBL 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. Targeted 80 genes consisted of exons with total length of ∼500Kb, and included genes which were known to be mutated in MDS and related disorders, and other candidate targets of mutations. We were able to analyze up to 80 samples per 1 run and efficiently detected mutations in targeted genes by the high average coverage obtained from these sequences. On average 80% of targeted regions were covered with >20 depths of reading. 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.