Combined analysis of loss of heterozygosity and DNA copy number by high-resolution SNP arrays identifies minimal genomic deletions in CD34+ cells purified from patients with MDS Free

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Genomic lesions in progenitor cells of MDS patients may play a key role in the pathophysiology of hematopoiesis in this disease but the identification of such alterations remains still difficult. 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. The median SNP call rate of analysed samples was 88.6% and ranged from 76.3% to 95.4%. 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, the probability of loss of heterozygosity was calculated using the MDS data compared to data from normal CD34+ cells. Furthermore, gene expression profiling (HG-U133 plus 2.0) was performed for all CD34+ cells 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. The integration of genomic copy number analysis with global gene expression data showed that alterations of copy number directly affects gene expression patterns. These data are highly valuable for understanding 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. In conclusion, this is the first combined high-resolution genomic and gene-expression analysis of CD34+ bone marrow cells from normal individuals and patients with MDS which could identify a number of so far unknown deleted DNA-regions in MDS.