Differential Expression of the Homologous Recombination DNA Repair Genes in Early and Advanced Stages of Myelodysplastic Syndrome

BACKGROUND AND AIMS

The high incidence of mutations in patients with myelodysplastic syndrome (MDS) strongly suggests an implication of defective DNA repair mechanisms in the MDS pathogenesis. Based on the hypothesis that genetic changes are amplified during evolution of MDS-cell clone and disease progression; we investigated abnormalities in DNA repair gene expressions and monitored their possible development during MDS progression. Further, we focused on sequencing of selected DNA repair genes and searched for their mutations associated with the disease.

METHODS

First, gene expression of 84 DNA repair genes in bone marrow (BM) CD34+ cells of 18 MDS patients was measured by RT² Profiler PCR Arrays (Qiagen). Validation of expression data in selected genes was performed on a cohort of 100 MDS patients. Moreover, paired samples from 15 patients with disease progression were used for monitoring of RAD51 and XRCC2 gene expressions in the course of disease. Mutational analysis was performed on 84 DNA repair genes in 16 patients by targeting next generation sequencing (NGS) (SeqCap EZ System, NimbleGen). Detected mutation was confirmed by Sanger sequencing. Multivariate analysis using a Cox regression model to determine the independent impact of each variable (BM blasts, hemoglobin, neutrophils, platelet count, karyotype, and gene expressions of RAD51 and XRCC2) examined for overall survival (OS) was done.

RESULTS

RAD51 (p<0.0001) and XRCC2 (p<0.0001) genes showed differential expression between low-risk, high-risk patients and control samples. Patients with high expression level of RAD51 gene had significantly longer OS to those with low level (median: 108.4 vs. 21.9 months; p<0.0001; HR=0.26). The expression of RPA3 gene was decreased (FC= -2.65) in all MDS patients (p<0.0001). Down-regulated expression of XRCC2 gene, located on 7q36.1, correlated with monosomy 7 or partial deletion of the long arm of chromosome 7 (7q-) which are associated with a poor prognostic category. Thus, XRCC2 gene may be a candidate leukemogenic gene. Furthermore, expressions of RAD51 and XRCC2 genes were measured in paired samples of patients with disease progression; the expressions were gradually decreased along the stepwise progression (from initial to advanced stages of MDS). Multivariate analysis identified high expression level of RAD51 gene (HR 0.49; p=0.01) and cytogenetic category (HR 1.68; p=0.002) as significant prognostic factors for OS. Interestingly, overexpression of RAD51 and XRCC2was observed in patients with advanced disease progression to AML with myelodysplasia with 35-40% of BM blasts. This might indicate the evolution of highly resistant clone with an increased function of DNA repair.

A heterozygous frameshift mutation caused by a deletion of two base pair in codon 263 of XRCC2 gene (XRCC2: c.789_790delCA) was detected in one patient. CD3+ T-cells were used as control to distinguish germline mutation from somatic one. The result indicated the germline mutation. XRCC2 expression was significantly down-regulated in this case (FC= -5.04).

CONCLUSIONS

Our study demonstrates that an alteration of DNA repair factors, mainly RAD51 and XRCC2, key mediators in the homologous recombination repair of DNA double strand breaks, may contribute to the pathogenesis of MDS. The gene expression of RAD51 and cytogenetic category were shown to be the strongest independent prognostic factors for OS. The inappropriate function of DNA repair in CD34+ cells seems to be progressive in the course of disease, allowing formation and accumulation of new mutations during MDS clone evolution resulting in development of highly resistant leukemic cells. Notably, we described the new germinal mutation in XRCC2 repair gene associated with MDS.

Supported by AZV grants (16-33485A and 16-31689A) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic.