Abstract LB-175: Methylome analysis by the combination of methylated DNA enrichment and next-generation sequencing in multiple myeloma
Authors: Nojima Masanori , Aoki Yuka , Yasui Hiroshi , Suzuki Hiromu , Takatsuka Shintaro , Shinomura Yasuhisa , Toyota Minoru 
Published: July 2012

[Aim] To attempt methylome analysis in multiple myeloma (MM) cells using high-throuput next-generation sequencer, and assess the association between the methylome status and aneuploidy status detected by array CGH analysis in MM cells.

[Methods] After the extraction of DNA from 2 MM cell lines and 9 primary MM samples, we enriched sheared fragments of methylated DNA using MethylminerTM Methylated DNA Enrichment Kit (invitrogen).

Fragment libraries were then prepared followed by emulsion PCR and templated beads preparation.

Sequencing analysis using SOLiDTM3 system (life technologies) was performed for these fragment libraries from enriched methylated DNA. In this study, using adoptors with individual barcodes when library preration, we could analyse at 12 samples (including input samples) at one run. Analyses after sequencing were the following. We counted tag numbers in promoter region of each gene (determined by sequences between +/− 100 bp from transcription start site (TSS)) read by sequencing. The number of the tags quantitatively reflects methylation levels of corresponding DNA sequences. We also performed array CGH analysis in the 82 primary MM samples. Combining these methods, we can assess the association between DNA methylation and chromosomal instability.

[Results and comments] Of all RefSeq genes which have CpG islands in their promoter region (18592/30638 genes, determined by unique accession number in NCBI36/hg18), the number of genes which showed at least one tag was 6549 (35.2%). Referring to the results of mRNA expression analysis using cDNA microarray (Agilent, matched by the accession number), we found a significant reduction of expression levels according to the tag number increase. Also, a very high correlation between tag numbers and mRNA expression levels (Rs = −0.562. We also analysed the genome-wide association between expression up-regulation by decitabine (DAC) and DNA methylation. A correlation between tag numbers and fold-up regulation by DAC was significant, although correlation coefficient was not that high (Rs=0.239). This result means DAC could affect gene expression by a mechanism not depending on demethylation effect. We have also performed the same analysis with genes in which there are no CpG islands. Without CpG islands, only almost no correlations were found between mRNA expression levels (Rs=−0.087), fold up-regulation by DAC (Rs=0.067) and tag numbers. Interestingly, in genes without promoter methylation, gene ontology (GO) analysis demonstrated that genes related to immune response were significantly up-regulated by DAC. This could reflect the direct cytotoxic effect of DAC or some other mechanism.

We are now performing the array CGH analysis in primary MM samples to assess the association between methylome status and aneuploidy status in MM.

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 LB-175. doi:10.1158/1538-7445.AM2011-LB-175