Abstract

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. MDS is characterized by large mono-allelic deletions that lead to loss of heterozygosity. It is unclear how the loss of one allele affects the expression of genes on the remaining allele and the role epigenetic silencing plays in this process. To study this directly, we performed whole genome methylation analysis on 3 selected MDS cases with deletions of chromosomes 5q, 7q and 20q. DNA methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction enzyme digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of genes by calculating HpaII/MspI cut fragment intensity ratio. Array Comparative Genomic Hybridization (aCGH) was used to map the exact breakpoints in peripheral blood leucocytes obtained from these 3 MDS cases. Gene expression analysis of the cDNA from the same cells was performed using 37K oligo maskless arrays and integrated with methylation analysis. Promoter methylation status and gene expression of corresponding genes of six age-matched healthy and anemic controls were used as controls. Our results showed that the 5q14.2–31.3 deletion encoded for a total of 248 genes. On comparison with healthy controls, 73 gene promoters were hypomethylated and 143 were methylated. Promoters with lower methylation levels were associated with a greater proportion of genes with higher expression values, thus suggesting that epigenetic modification is a regulator of their transcription. On the other hand, hypermethylation of the remaining allele led to underexpression of important genes such as APC, Cyclin H, CD14 antigen, protein phosphatase 2 (PPP2CA), heat shock 70kDa protein 4, H2A histone family members and others. Similarly, the 7q11.2–36.3 deletion encoded for a total of 497 genes. Out of these, 130 promoters were methylated and resulted in lower expression of genes not previously implicated in MDS such as Alpha 2 Type I Collagen, Schwachman-Bodian-Diamond syndrome protein, MEF3 and others. Lastly, the 20q11.21–13.13 segment encoded for 173 genes out of which 67 were methylated when compared to controls. In these large deleted regions, only 8–25% of the genes had lower expression when compared to controls; suggesting that large segmental mono-allelic deletions themselves do not result in decreased gene expression. Most importantly, epigenetic modifications of the remaining allele seems to be a very important regulator of gene expression in these cases and can be a potential target for therapeutic strategies.

Author notes

Disclosure: No relevant conflicts of interest to declare.