Genome Wide DNA Methylation Analysis of Patients with Myelodysplastic Syndrome and Isolated Deletion (5q) Reveals Characteristic Methylation Profiles in Low and Intermediate-1 Risk Groups

Aberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q).

All patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25).

DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/).

Using a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p<0.0001).

Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score.

CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria.

We present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients.

Nowak:Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.