Abstract
RAD51 (Rec A homolog of E.coli) is a polymorphic gene and one of the central proteins in homologous recombination-DNA-double-stand breaks (HR-DNA-DSB) repair pathway, which is vital in maintaining genetic stability within a cell. The x-ray repair cross complementing (XRCC3) protein also functions in HR-DNA-DSB repair pathway and directly interacts with and stabilizes RAD51 and the closely related RAD51C. The aim of this study was to determine the prevalence of the RAD51 and XRCC3 repair gene polymorphisms among acute myeloid leukemia (AML) patients and to define their role in development of AML and its correlation with the clinical presentation, laboratory data as well as treatment outcome using polymerase chain reaction-restriction fragment length polymorphism assay in 50 de novo AML patients as well as 30 healthy subjects as a control group. Our study revealed that RAD51 G135C and XRCC3 Thr241Met alleles were associated with increased risk of AML with odds ratio (OR) of 2.833 and 2.909 and 95% confidence interval (CI) of 1.527 to 8.983 and 1.761 to 9.788, respectively. Moreover, when combining the 2 genes polymorphisms, a significant elevation of the risk of AML was found with OR of 3.124 and 95% CI of 1.872 to 11.243. As regards treatment outcome, a highly statistical significant difference was found between XRCC3 genotypes with P value of 0.001, whereas no significant difference was present between RAD51 genotypes with P value of 0.29. This clarifies that XRCC3 gene polymorphisms was found to have a significant impact on the risk of treatment failure with OR of 3.560 and 95% CI of 1.167 to 10.875; however, RAD51 gene polymorphism was not found to have an equivalent effect with OR of 2.813 and 95% CI of 0.933 to 10.828. So XRCC3 gene polymorphism might be considered as a prognostic marker in AML. In conclusion, RAD51 and XRCC3 genes polymorphisms may play an important role in the development of AML.
Leukemias are complex diseases with a wide range of clinical, morphological, biological, cytogenetic, molecular, and immunophenotypic features.1The World Health Organization classification subdivides AML predominantly according to cytogenetic analysis because recurrent chromosomal abnormalities identify distinct leukemia entities and have a major impact on prognosis.2
DNA is at constant risk from damage from both endogenous and exogenous sources. A large number of highly complex mechanisms have evolved to protect DNA from damage including DNA repair pathways and systems that protect against oxidative stress and other damaging agents.3The main DNA repair pathways in mammalian cells are nucleotide excision repair, base excision repair, mismatch repair, and double-strand break (DSB) repair.4
Double-strand breaks in DNA are the most important class of DNA damage because they may lead to either cell death or loss of genetic material resulting in chromosomal aberrations. The balance of DSB repair activity seems to be critical to the genetic stability of cells. DSBs are predominantly repaired by either homologous recombination (HR) repair or nonhomologous end joining pathways in mammalian cells.4
One of the central proteins in the HR repair pathway is RAD51. RAD51 binds to DNA and promotes ATP-dependent homologous pairing and strand transfer reactions. The RAD51 protein is essential to the viability of a cell, and cells lacking RAD51 are characterized by an accumulation of chromosomal breaks before cell death. Hence, the role of RAD51 is vital in maintaining genetic stability within a cell.5
The XRCC3 protein also functions in HR-DNA-DSB repair pathway and directly interacts with and stabilizes RAD51 and the closely related RAD51C.6XRCC3 is a paralog of RAD51, and similar to RAD51, it is essential for genetic stability.7
Genetic polymorphisms have now been identified in a number of genes involved in protecting from and repairing DSBs and including genes belonging to the HR-DNA-DSB repair pathway. A malignant phenotype is likely to result from the accumulation of many minor genotypes, and it is probable that associations exist between polymorphisms in DNA repair genes including RAD51 and XRCC3 HR-DNA-DSB repair genes and the risk of acute leukemia.4Moreover, the effect of these genetic polymorphisms on the risk of AML may vary from one population to the other because of the differences in frequencies and types of polymorphisms as well as the exposed carcinogens in a studied population.8
The aim of this work was to study the prevalence of the polymorphisms in the RAD51 and XRCC3 HR-DNA-DSB repair genes among AML patients using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and to define their role in modulating susceptibility to development of AML and its correlation with the clinical presentation, laboratory data, and treatment outcome.
SUBJECTS AND METHODS
Subjects
The present study was conducted on 50 patients with de novo AML; their ages ranged between 14 and 65 years, with a mean of 42.58 ± 15.16 years and a median of 38 years. There were 29 male (58%) and 21 female subjects (42%). Patients were studied before chemotherapy and followed up for the disease outcome after induction chemotherapy. Patients were diagnosed and selected among cases referred to National Cancer Institute, Cairo University. Thirty age- and sex-matched healthy individuals also were included as a control group.
The diagnosis of leukemia was based on complete history taking, clinical examination for organomegaly and lymphadenopathy, and laboratory investigations for diagnosis of AML including complete blood count, cytochemistry and immunophenotyping of leukemic blast cells, and special laboratory investigations (for patients and controls) for the detection of RAD51 and XRCC3 HR-DNA-DSB repair gene polymorphisms using PCR-RFLP technique according to the method described by Seedhouse et al.5
METHODS
Sample Collection
Three milliliters of venous blood were collected by sterile venipuncture under complete aseptic conditions on 1.5 mg/mL Potassium EDTA sterile vacutainer from patients and control subjects and divided as follows: 1 mL of venous blood for performing complete blood picture and 2 mL for the study of genotyping for the polymorphisms of the RAD51and XRCC3 genes by PCR-RFLP assay.
DETECTION OF RAD51 AND XRCC3 GENE POLYMORPHISMS BY PCR-RFLP
DNA Extraction
Genomic DNA was extracted from cells using QIAamp blood DNA isolation kits (Qiagen, Crawley, UK) according to the manufacturer's protocol.
PCR Reaction for Amplification of RAD51 and XRCC3 Genes
A mixture of 25-μL reaction consisting of 3 μL genomic DNA, 12.5 μL of PCR master mix, 2 μl of each primer, and 5.5 μL DW was prepared.
Primers Used for RAD51-G135C Polymorphism
Forward primer 5'-TGGGAACTGCAACTCATCTG-3' and reverse primer 5'-GCGCTCCTCTCTCCAGCAG-3'. The following cycles were used: an initial heat-activation step at 95°C for 5 minutes and 35 cycles of: denaturation at 95°C for 1 minute, annealing at 56°C for 1 minute, and extension at 72°C for 1 minute, then final extension at 72°C for 10 minutes.
Primers Used for XRCC3 Thr 241Met Polymorphism
Forward primer 5'-GGTCGAGTGACAGTCCAAACG-3' and reverse primer 5'-CTACCCGCAGGAGCCGGAG-3'. The following cycles were used: An initial heat-activation step at 95°C for 5 minutes and 35 cycles of: denaturation at 95°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute, then final extension at 72°C for 10 minutes.
The samples were then run in parallel on 2% agarose gel using gel electrophoresis (electro-4, Thermal Hybaid, from Promega) and visualized on a UV transilluminator (wave length, 312) to detect the presence or absence of DNA bands. For the RAD51-G135C and XRCC3-Thr241Met polymorphisms, 157-bp and 415-bp fragments were amplified, respectively.
Digestion of PCR Products by Specific Restriction Enzymes
For RAD51 gene polymorphism, the amplified PCR products (157 bp) were digested at 37°C overnight with 1 μl MvaI conventional restriction enzyme in the manufacturer's buffer (Helena Biosciences, Sunderland, UK), generating 2 fragments of 71 bp and 87 bp after digestion in the presence of the G allele, and it is designated G/G (ie, wild type). If only polymorphic C allele exists, it eliminates the MvaI restriction site, and no digestion occurs; only one 157-bp band will emerge, and it is designated C/C (ie, homozygous), if the 3 bands 71, 87, and 157 bp are present, it is designated G/C (ie, heterozygous) as shown in Figure 1.
For XRCC3 gene polymorphism, the amplified PCR products (415 bp) were digested at 37°C overnight with 1 μl NIaIII conventional restriction enzyme in the manufacturer's buffer (Helena Biosciences), generating 2 fragments of 141 bp and 274 bp after digestion in the presence of the Thr allele, and it is designated Thr/Thr (ie, wild type). If only the polymorphic Met allele exists, it generates an additional NIaIII restriction site, resulting in 3 fragments 104, 141, and 170 bp will emerge, and it is designated Met/Met (ie, homozygous), if the 4 bands 104, 141, 170, and 274 bp are present it is designated Thr/Met (ie, heterozygous) as shown in Figure 2.
Detection of PCR Products
Finally, the digested products of RAD51 and XRCC3 genes were separated, identified, and visualized using the QIAxecl System (Qiagen).
DECLARATION OF ETHICS
A written informed consent was obtained from all the patients according to Helsinki guidelines of research ethics.
STATISTICAL ANALYSIS
Data was analyzed using SPSS win statistical package version 15. Numerical data were expressed as a mean, SD, and range as appropriate. Qualitative data were expressed as frequency and percentage; χ2 test or Fisher exact test was used to examine the relation between qualitative variables. Odds ratio (OR) and 95% confidence interval (CI) were calculated for risk estimation. For quantitative data, comparison between 2 groups was done using the Mann-Whitney test (non-parametric t test). Comparison between 3 groups was done using Kruskal-Wallis test (non-parametric analysis of variance test). Relation between numerical variables was tested using Pearson product-moment correlation coefficient. A P value less than 0.05 was considered significant, and less than 0.01 was considered highly significant.
RESULTS
The patients' and the control group characteristics are summarized in Table 1.
Table 2 displays statistical comparison of genotypes distributions of RAD51 and XRCC3 gene polymorphisms in AML patients and control group subjects. Comparison revealed a statistical significant difference between the 2 groups with higher expression of mutant alleles among AML patients with P value 0.03 and 0.01, respectively.
Correlation of RAD51 and XRCC3 genes polymorphism with age, sex, and clinical and laboratory data in AML patients revealed a statistical significant negative correlation between RAD51 gene polymorphism and hemoglobin level with r value of −0.426 and P value of 0.014 and a highly statistical significant negative correlation between XRCC3 polymorphism and hemoglobin level and platelet count with r value of −0.846 and P value of 0.001 and r value of −0.790 and P value of 0.001, respectively. On the other hand, a highly statistical significant positive correlation was found between XRCC3 polymorphism and the percentage of peripheral blood and bone marrow blast cells with r value of 0.670 and P value of 0.001 and r value of 0.627 and P value of 0.001, respectively. Meanwhile, there was no statistical significant correlation found between RAD51 or XRCC3 gene polymorphism and the age, sex, and clinical and other laboratory data including total leukocyte count, French-American-British subtypes, and immunophenotyping and cytogenetic abnormalities with P value greater than 0.05.
Statistical comparison of different RAD51 and XRCC3 genotypes as regards age, sex, and clinical and laboratory data in AML patients is studied. Regarding RAD51 genotypes, there was a statistically significant difference between different RAD51 genotypes in AML patients as regards the hemoglobin level with P value of 0.04, whereas there was no statistical significant difference as regards age, sex, and clinical and laboratory data with P value greater than 0.05. For XRCC3 genotypes, there was a highly statistically significant difference between patients with different XRCC3 genotypes as regards hepatomegaly, lymphadenopathy, hemoglobin level, total leukocytic count, platelets count, PB and BM blast cells, and CD14+ phenotype with P value of 0.006, 0.001, 0.001, 0.001, 0.001, 0.001, 0.001, and 0.014, respectively, whereas there was no statistically significant difference between XRCC3 genotypes as regards age, sex, splenomegaly, French-American-British subtypes, and immunophenotypic markers other than CD14 and cytogenetic abnormalities with P value greater than 0.05.
Statistical comparison between AML patients and the control group as regards the risk of AML is shown in Table 3. RAD51 and XRCC3 gene polymorphism were associated with increased risk of AML (OR, 2.833 and 2.909, respectively; 95% CI, 1.527-8.983 and 1.761-9.788, respectively).
Statistical comparison between AML patients and the control group as regards single and combined genes polymorphism is illustrated in Table 4. Comparison revealed a statistically significant difference between the 2 groups with P value of 0.04. Combined RAD51 and XRCC3 gene polymorphism was associated with increased risk of AML with OR of 3.124 and 95% CI of 1.872 to 11.243.
Statistical comparison between RAD51 and XRCC3 genotypes as regards treatment outcome in AML patients is shown in Tables 5 and 6, respectively. Regarding RAD51 gene, comparison revealed no statistically significant difference between different genotypes with P value of 0.29. Thus, RAD51 gene polymorphism was found to have a nonsignificant impact on the risk of treatment failure with OR of 2.813 and 95% CI of 0.933 to 10.828. However, comparison revealed a highly statistically significant difference between XRCC3 genotypes with P value of 0.001. This suggests that XRCC3 gene polymorphism has a significant impact on the risk of treatment failure with OR of 3.560 and 95% CI of 1.167 to 10.875.
DISCUSSION
Acute myeloid leukemia is a cancer of a clonal hemopoietic disorder of the myeloid line of blood cells, which is frequently associated with genetic instability characterized by a diversity of chromosomal and molecular changes.9Genetic polymorphisms have been identified in a number of DNA repair genes and damage-detoxification genes. Polymorphisms can affect protein function, promoter activity, messenger RNA stability, and splice variants and, hence, can result in a change in the cellular ability to cope with DNA damage, which contributes to altered disease susceptibility. The genotype distributions of a number of these polymorphic genes have been shown to be associated with AML including RAD51 and XRCC3 HR-DNA-DSB repair genes.10
The RAD51 family of genes, including RAD51 and the 5 human RAD51 (hRAD51) paralogs, RAD51B (RAD51L1), RAD51C (RAD51L2), RAD51D (RAD51L3), XRCC2, and XRCC3, have been identified. Each of these genes shows only a limited degree of sequence similarity to hRAD51.11The RAD51 family of genes are known to have crucial nonredundant role, as well as the evidence for extensive genetic instability arising from loss of their activity.12
A G/C polymorphism at position -135 in 5' untranslated region of the RAD51 gene has been identified (RAD51-G135C).5The DNA damage is defectively repaired by this polymorphic RAD51.13This polymorphism may increase the levels of RAD51 messenger RNA in patients with AML. If this also results in an increase in RAD51 protein expression, then it is possible that the fine balance of the HR protein levels will be disrupted, and it may inhibit the stimulation of the apoptotic pathway and enable a cell with damaged DNA to survive, acquire mutations and chromosomal aberrations, and subsequently convey the damage to daughter cells. This is a possible explanation as to why there is a significant increase in the risk of the development of t-AML when a variant level of RAD51- G135C allele was present.5
The XRCC genes are a diverse set of human genes that complement hamster-cell mutants having the common feature of sensitivity to ionizing radiation and other DNA damages.11The most frequent polymorphism in XRCC3 is XRCC3 C18067T, which results in a Thr to Met amino acid substitution at codon 241. Carriers of the variant T-allele of XRCC3 T241M have higher DNA adduct levels in lymphocyte DNA compared with homozygous C-allele carriers, indicating that the polymorphism is associated with lowered DNA repair capacity.14
The polymorphic forms of RAD51 and XRCC3 genes have a role in susceptibility to cancer and leukemia.15The risk of the development of AML was found to be significantly increased when both variant RAD51-135C and XRCC3-241Met alleles are present, whereas the risk of t-AML development is even higher, presumably because of the large genotoxic insult these patients receive after their exposure to radiotherapy or chemotherapy. These results strongly suggest that DNA DSBs and their repair are important in the pathogenesis of both de novo and t-AML.5
In the present study, genotypic analysis of the RAD51 gene in AML patients showed that 39 patients (78%) expressed the wild type (G/G), and 11 patients (22%) expressed the mutant type; 9 (18%) were heterozygous (G/C), and 2 (4%) were homozygous (C/C). Whereas in the control group, 26 individuals (87%) expressed the wild type (G/G), and 4 (13%) expressed the mutant type; 3 of them (10%) were heterozygous (G/C), and only 1 individual (3%) was homozygous (C/C). These results were nearly similar to the results of Voso et al.13who studied the RAD51 gene polymorphism in AML patients and reported that G/G, G/C, and C/C genotypes were 78%, 21%, and 1%, respectively, for their patients, whereas in the control group, G/G, G/C, and C/C genotypes were 88%, 11%, and 1%, respectively. Also, this comes in consistency with the findings of Seedhouse et al.,5who found that the frequency of G/G, G/C, and C/C genotypes were 84%, 14%, and 2%, respectively, for their patients, whereas in the control group, G/G, G/C, and C/C genotypes were 89%, 10%, and 1%, respectively.
As regards genotypic analysis of the XRCC3 gene in AML patients, 22 patients (44%) expressed the wild type (Thr/Thr), whereas 28 patients (56%) expressed the mutant type; 20 (40%) were heterozygous (Thr/Met), and 8 (16%) were homozygous (Met/Met). Whereas in the control group, 18 individuals (60%) expressed the wild type (Thr/Thr), and 12 (40%) expressed the mutant type; 9 (30%) were heterozygous (Thr/Met), and 3 (10%) were homozygous (Met/Met). These results are in accordance with that recorded by Seedhouse et al.,5who studied the XRCC3 gene polymorphism in AML patients and reported that Thr/Thr, Thr/Met, and Met/Met genotypes were 46%, 40%, and 14%, respectively, for their patients, whereas in the control group Thr/Thr, Thr/Met, and Met/Met genotypes were 47%, 41%, and 12%, respectively. Also, Voso et al.13found that the frequency of Thr/Thr, Thr/Met, and Met/Met genotypes were 34%, 42%, and 24%, respectively, for their AML patients, whereas in the control group, Thr/Thr, Thr/Met, and Met/Met genotypes were 30%, 58%, and 12%, respectively.
Comparison between AML patients and the control subjects regarding RAD51 gene polymorphism revealed a statistically significant difference between the 2 groups, and RAD51 gene polymorphism was associated with a significant increased risk of AML with OR of 2.833 and 95% CI of 1.527 to 8.983. In approval with these results, Seedhouse et al.5documented that RAD51 gene polymorphism was a risk factor of AML and stated that it increases the risk of AML by 2.6 folds independently. Also, Voso et al.13found that the RAD51 gene polymorphism was associated with an increase in the risk for developing de novo AML by 2.1 folds. Also, these results come in consistency with Jawad et al.,16who studied the polymorphism in human homeobox HLX1 and RAD51 genes and stated that when the DNA repair gene RAD51 polymorphism was combined with HLX1 variant alleles, a synergistic 9.5 fold increase in the risk of t-AML was observed with 95% CI of 2.22 to 40.64. On the contrary, Rollinson et al.17stated that RAD51 gene polymorphism was not associated with the risk of AML.
Comparison between AML patients and the control subjectsregarding XRCC3 gene polymorphism revealed a statistically significant difference between the 2 groups, and XRCC3 gene polymorphism was associated with significant increased risk of AML with OR of 2.909 and 95% CI of 1.761 to 9.788. In agreement with these results, Seedhouse et al.5reported that XRCC3-241 Met allele increase the risk of AML by 3.8 folds. On the contrary to our results, Seedhouse et al.10found that the XRCC3 gene polymorphism did not show a statistically significant difference between the 2 groups, and it was not associated with increased risk of AML because, as explained by him, the sample was too small to allow to conduct combine logistic regression analysis of this group. This difference between studies could indicate difference between populations in the influence of these genetic polymorphisms on genetic susceptibility to leukemia or in exposures involved in leukemogenesis.18
Knowing that the malignant phenotype is likely to result from the accumulation of many minor genotypes, in the present work, we tried to assess whether there was an association between the combined presence of the 2 polymorphisms and increased risk of AML development. When we analyzed de novo AML patients with respect to the 2 polymorphisms, a substantial increase in the risk of AML development of the disease was observed.
An increase in DNA damage significantly increased the risk of development of AML when RAD51 and XRCC3 polymorphism (mutant type) are present. Thus, a significant association was observed, suggesting that the polymorphisms interact to increase the risk of AML with OR of 3.124 and 95% CI of 1.872 to 11.243 for combined RAD51 and XRCC3 gene polymorphism. This comes in consistency with Seedhouse et al.,10who reported the relationship between RAD51 and XRCC3 gene polymorphism and cancer risk and found that increase risk of AML was linked to several gene polymorphisms in base excision repair (XRCC1 Arg399Gln), nucleotide excision repair (XPR Lys751Gln), and DSB repair (RAD51 G135C and XRCC3 Thr241Met) pathways. Yin et al.19suggested that several gene polymorphisms may be linked to secondary AML etiology through failure to recognize or excise accumulated DNA lesions. Also, Bhatla et al.20found that XRCC3 and RAD51 gene polymorphisms show similar genotype frequencies in control and patient populations, suggesting that these variants, when assessed singly, do not play a role in the etiology of AML. In contrast, when XRCC3 and RAD51 genotypes were examined together, a significant increase in susceptibility to AML was seen. Also, Seedhouse et al.5stated that when XRCC3 and RAD51 genotypes were examined together, a significant increase in susceptibility to AML was seen.
As regards correlation of RAD51 and XRCC3 genotypes with age, sex, and clinical and laboratory data in AML patients, there was a statistically significant negative correlation between RAD51 gene polymorphism and hemoglobin level with r value of -0.426 and a highly statistically significant negative correlation between XRCC3 polymorphism and hemoglobin level and platelet count with r value of −0.846 and −0.790, respectively. On the other hand, there was a highly statistically significant positive correlation found between XRCC3 polymorphism and the percentage of peripheral blood and bone marrow blast cells with r value of 0.670 and 0.627, respectively. Although there was no statistically significant correlation found between RAD51 or XRCC3 gene polymorphism and the age, sex, and clinical and laboratory data including total leukocyte count, French-American-British subtypes, immunophenotyping, and cytogenetic abnormalities.
As studies of the effect of RAD51 and XRCC3 gene polymorphism on the outcome of AML are scarce, the current study tried to evaluate the association between these gene polymorphisms and the disease outcome. No statistically significant difference was found between patients with different RAD51 genotypes as regards the treatment outcome; thus, RAD51 G135C polymorphism was found to have a nonsignificant impact on the risk of treatment failure with OR of 2.813 and 95% CI of 0.933 to 10.828, which might be explained by the small sample size. Yet, in the present study, the percentage of patients with unfavorable outcome (relapse and death) among patients expressing polymorphic RAD51 G135C allele (78% and 100% for heterozygous [G/C] and homozygous [C/C] types. respectively) was higher than that of the patients with favorable outcome (remission). Thus, determining an individual's RAD51 genotype is important for the prediction of both the risk of developing acute leukemia and treatment outcome. Bhatla et al.20found that RAD51 gene polymorphism did not influence the outcome of AML therapy in the study of de novo AML patients. On the contrary, Liu et al.21concluded that RAD51 gene polymorphism was significantly related to response to therapy, adverse effects, and prognosis of AML and reported that the detection of the RAD51 gene polymorphism genotypes may be useful in selecting individual chemotherapy regimens for patients with AML. Also, Bolufer et al.22reported that the RAD51 gene polymorphism showed significant unfavorable outcome among AML patients.
There was a highly statistically significant difference found between patients with different XRCC3 genotypes as regards treatment outcome. The percentage of patients with unfavorable outcome was higher than that of the patients with favorable outcome among those expressing the polymorphic XRCC3 Thr241Met genotype (95% and 100% for heterozygous [Thr/Met] and homozygous [Met/Met] types, respectively). Thus, XRCC3 gene polymorphism was found to have a significant impact on the risk of treatment failure with OR of 3.560 and 95% CI of 1.167 to 10.875. This comes in consistency with Liu et al.,21who found that XRCC3 gene polymorphism was significantly related to response to therapy and prognosis of AML and reported that detection of the XRCC3 gene polymorphism genotypes may be useful in selecting individual chemotherapy regimens for AML patients. In another study, Bhatla et al.20found that analysis of outcome of therapy of their study population showed that patients heterozygous for the XRCC3 Thr241Met allele had improved postinduction disease-free survival compared with children homozygous for the major or minor allele, each of whom had similar outcomes. Improved survival was due to reduced relapse in the heterozygous children, and this effect was most marked in children randomized to therapy likely to generate DNA DSB (etoposide and daunomycin), compared with antimetabolite (fludarabine and cytarabine)-based therapy.
In conclusion, RAD51 and XRCC3 gene polymorphism may play an important role in the pathophysiology, development, and progression of AML. Their expression may have clinical relevance and important role as prognostic factors in AML. They may be useful as predictive tests for treatment outcome in AML patients.
ACKNOWLEDGMENT
The authors thank El-Kasr El-Aini Hospital, Cairo University, Cairo, Egypt, for the help in performing this study and also our patients for participating in our research.