Detailed Genome-Wide DNA-Mapping of CD34+ Cells Purified from Patients with MDS Using High-Resolution SNP Arrays Identifies Significant Regions of Genomic Alterations.

Identification of common genomic lesions in progenitor cells of MDS Patients could lead to the discovery of new target genes in this disease and may be of prognostic value. Therefore, we carried out a detailed genome-wide mapping of genomic DNA from highly purified CD34+ progenitor cells from MDS patients and healthy individuals with high-resolution single nucleotide polymorphism (SNP) microarrays which scan 500,000 SNPs with a median inter-SNP distance of approximately 2.5 kb. Bone marrow aspirates were obtained from 14 MDS patients (IPSS low risk n=6, high risk n=8) and 6 healthy individuals after informed consent. CD34+ cells were purified by high gradient magnetic cell separation. Genomic DNA and RNA were extracted with standard TRIZOL technique and quality controlled with the Agilent Bioanalyzer 2100 and Nanodrop ND-1000 systems. 500 ng of each of the genomic DNA were processed according to the protocol of the Affymetrix 500 k NspI and StyI genomic mapping protocol, hybridized to 500 k NspI/StyI chip sets and scanned on an Affymetrix GeneChip scanner 3000. The median SNP call rate of analysed samples was 88.6% and ranged from 76.3% to 95.4%. One sample from the MDS patients and two samples from the healthy donors were excluded from analysis due to insufficient call rates. Raw signal intensity data was generated by the GCOS 4.0 software and imported into Partek Genomics 6.2 software. The control samples of healthy individuals were assigned a copy number of two and used as a reference baseline to calculate copy numbers in MDS samples. On the calculated values genomic smoothing was performed with a window width of 0.5 Mbps and a Gaussian width at half maximum 50% of window width. Significant regions of copy number alterations were calculated with a test region width of 0.5 Mbp and contiguous regions set to contain at least 1 Mbp (p<0.01). In addition, gene expression profiling (HG-U133 plus 2.0) was performed by standard Affymetrix technique. Numerous so far unknown significant regions of putative deletion or amplification which are not detectable by standard genomic analysis were discovered in MDS samples. Commonly deleted or amplificated regions appeared on chromosomes 1, 2, 3, 4, 5, 6, 11, 17, 19, 21 and 22. Gene lists of significant regions were created and subsequently used to perform a supervised analysis of gene expression data generated from the same bone marrow samples. This integration of genomic copy number analysis with global gene expression data showed that alterations of copy number directly affects gene expression patterns. In conclusion, this is the first high-density genomic mapping of CD34+ bone marrow cells from patients with MDS which could identify a number of so far unknown DNA-deletions/amplifications. These data contribute substantially to the understanding of the pathophysiology of MDS in greater detail and furthermore can be used to identify genes/regions which could resemble targets of new specific treatment options.