High-Resolution Analyses of Genetic and Epigenetic Aberrations in Infant Leukemia with MLL Rearrangement.

Abstract

MLL rearrangement-positive leukemia is one of the most aggressive types of leukemia. It is diagnosed predominantly in infants and typically shows a multilineage phenotype. Since current chemotherapy fails in more than 50% of infantile leukemia with MLL rearrangement, a better understanding of biological features of the disease is importantly in order to develop more specific and successful treatment strategies. Thus, to explore both genetic and epigenetic lesions associated with MLL rearrangement-positive infantile leukemia, we performed genome-wide analyses of copy number alterations/allelic imbalances as well as methylation analysis using Affymetrix GeneChip SNP genotyping microarrays and promoter tiling array combined with methylated DNA immunoprecipitation (MeDIP). Combined with newly developed algorithm, CNAG/AsCNAR, SNP array analysis enables accurate copy number analysis at extremely high resolutions. In addition, by sensing subtle distortions in allele-specific signals caused by allelic imbalance using anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers even in the face of up to 70–80% normal cell contamination. In total, 25 specimens from MLL rearrangement-positive leukemia were analyzed using high-density SNP-genotyping microarrays (Affymetrix GeneChip 100K/500K arrays). While unbalanced translocation involving chromosome 11q23 is the most frequent genetic alterations, uniparental disomy of 17q is also common genetic alterations in MLL rearrangement-positive leukemia. A number of other genetic changes were also identified but these were mostly found in a single case. On the other hand, epigenetic abnormalities have been implicated in MLL rearrangement-positive leukemia, because MLL is known to be involved in epigenetic regulations. In our assay, fragmented genomic DNA from leukemia specimens was immunoprecipitated with anti-methylcytosine antibody (MeDIP). The immunoprecipitated DNA was amplified by PCR and subjected to hybridization to the promoter tiling array, in which regulatory regions of more than 25,000 genes are tiled by 6.5 millions of oligonucleotide probes to enable sensitive detection of target sequences. This tiling array covers approximate 59% of CpG islands in the human genomes. In addition to the previously described genes, such as FHIT and HOX family, a number of tumor specific methylated sites were identified in the leukemic cells and which were subsequently verified by bisulfate sequencing. These results indicated that high-resolution analyses of genetic and epigenetic aberrations using microarray techniques are powerful and useful for detection of new findings in the pathogenesis of infant leukemia with MLL rearrangement.