Altered Expression of the Repair Genes in CD34+ Cells May be Responsible for Formation and Accumulation of Mutations in MDS Patients

Recent studies have demonstrated that in most cases of myelodysplastic syndrome (MDS) at least one mutation has been detected, suggesting that abnormal DNA repair may represent both cause and consequence of malignant transformation. In this study we investigated a possible role of different alterations in DNA repair genes in pathogenesis of MDS.

Methods

Gene expression of CD34+ cells was measured by RT² Profiler PCR Arrays (Qiagen). Polymorphisms and mutations were studied by targeting next generation sequencing (SeqCap EZ System, NimbleGen). Expression analysis of 84 DNA repair genes was performed in 18 MDS patients and subsequent analysis of selected genes was performed on a cohort of 80 patients. The enrichment resequencing of 84 genes was done in 16 patients.

Results

Five differentially expressed genes between CD34+ cells of patient and control samples were identified (p<0.05). The increased expression was detected in MPG and XPC genes and decreased expression in RAD51, RPA3 and XRCC2 genes. RAD51 gene showed significantly higher expression in the patients with low-risk MDS forms (RA, RARS, del5q) compared to control group (p=0.0005) and to contrast down-regulated expression was detected (p=0.0002) in high-risk MDS patients (RAEB-1 and -2, AML with myelodysplasia). The group of patients with RCMD showed the average expression at the level of the control group. The expression profile shown a gravity-related decreasing trend after dividing the patients according to IPSS-R, IPSS-R cytogenetic groups and blasts count. Expression profile of XRCC2 gene has similar characteristics as RAD51 (p<0.0001). The expression of RPA3 gene was generally decreased (p<0.0001) with a decreasing trend depending on the disease severity according to MDS forms, IPSS-R and cytogenetics. The RAD51, XRCC2 and RPA-3 genes are related to homologous recombination mechanism, with XRCC2 and RPA3as a supporting factors for RAD51. The survival curves for all the three genes shown significant differences between groups with over-, intermediate- and down-expressed gene (p=0.0001 for RAD51; 0.0022 for XRCC-2 and 0.0054 for RPA3).

MPG gene expression was up-regulated by all MDS types with no significant difference between each other (p=0.0015). Deeper testing of XPCgene expression demonstrated the up-regulation only in low-risk MDS group (p=0.0084).

Gene-based analysis showed seven candidate SNPs significantly associated with the disease susceptibility using the HapMAP-CEU population from NCBI PubMed database as control group. With the presence of MDS, these polymorphisms are significantly connected: rs4135113 (p=0.03), rs12917 (p=0.003), rs2230641 (p=0.01), rs2228529 and rs2228526 (p=0.04, respectively p=0.03) and rs1799977 (p=0.04). Within the distribution of tested patient groups according to cytogenetics, we observed significant increase of dependency of these polymorphisms (OR 4.1-9.7, p<0.0001) and the presence of MDS in the group of patients with very-high risk cytogenetics.

Conclusions

DNA repair mechanisms are responsible for correcting DNA damage and preserving genomic integrity. Our study demonstrates, that altered expression of the repair genes in CD34+ cells may be responsible for the formation and accumulation of mutations in hematological malignancies. Furthermore, we have identified genetic variants that might contribute to the pathogenesis of MDS by modifying individual risk for the disease.

Supported by grant (NT/13899, NT/14377, and NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic.