Overexpression of MPG Is Associated with Distinct Defects in Base Excision Repair in a Subgroup of Patients with MDS.

Dysfunction of the DNA repair machinery may be one of the potential mechanisms leading to DNA damage and subsequent evolution of chromosomal abnormalities in MDS. However, the complexity of the possible mechanisms that could result in increased propensity to chromosomal breaks constitutes one of the obstacles in the rational targeting of deficient repair pathways for future investigations. Our study involved 37 patients with MDS (19 RA/RARS, 16 RAEB/AML, 2 CMML) and 10 controls. As expression arrays constitute a suitable hypothesis-forming tool, we have applied a specialized expression array to interrogate the most important DNA repair mechanisms in highly purified CD34+ progenitor cells from controls (N=10) and MDS patients (N=12). Based on the expression cut off value of (>1.5 x background) we detected 22/113 genes present on the array in all samples tested. 16/22 DNA repair genes were concordantly upregulated (> 2 fold above the control values established in pooled control reference). As a defective base pair excision repair (BER) pathway could play a role in the generation of chromosomal breaks, we focused our investigation on enzymes involved in this pathway. Expression array analysis showed that the MPG gene was significantly overexpressed, and we have validated this finding using TaqMan PCR in original samples and additional cohort of patients; 21/37 cases showed significantly elevated MPG transcripts. Overexpression of MPG may be in response to increased alkylation of DNA or may serve as a marker of CpG island methylation. While the latter alternative has been the subject of intense study, increased level of MPG is consistent with the presence of chemically modified bases incorporated into DNA during a toxic insult. Polymerase b (POLB) is a downstream enzyme in BER pathway, and its upregulation could indicate increased activity of this repair mechanism. Consequently, to test the integrity of the BER pathway, we set out to determine POLB expression levels by TaqMan PCR; two distinct subsets of patients (>2 and ≤ 2 fold of the expression in controls) were identified (5/21 and 16/21). While 4/5 vs. 7/16 patients had chromosomal abnormalities in high vs. low POLB group respectively, these patient groups were otherwise clinically indistinguishable. Hence, we devised our next set of experiments that would discern the differences between these patients at a molecular level. Increased POLB would be consistent with the adequate BER activity. Here, we chose to further investigate the consequences of POLB upregulation in order to distinguish whether BER function results in appropriate DNA repair (upregulation of POLB and MPG) or whether it constitutes response to the deficiency of the AP endonuclease (upregulation of MPG and normal or downregulation of POLB) located immediately downstream of MPG. The content of apurinic sites per genome constitutes a suitable parameter to distinguish the aforementioned possibilities. An apurinic site-specific genomic ELISA assay clearly identified patients (6/19) in whom low POLB expression coincided with an elevated number of apurinic sites (>0.50 apurinic sites per 1x10 5 bp). We conclude that such a constellation is reflective of altered function of AP endonuclease or inadequate expression of MPG that could predispose to single and double-stranded breaks, and consequently lead to evolution of abnormal clone.