© 2007 European Society for Medical Oncology
hematologic malignancies |
Increased risk of acute myeloid leukaemia due to polymorphisms in detoxification and DNA repair enzymes
Istituto di Ematologia, Universita’ Cattolica del Sacro Cuore, Roma, Italy
* Correspondence to: Dr M. Teresa Voso, Istituto di Ematologia, Universita’ Cattolica del Sacro Cuore, L.go A. Gemelli, 1, 00168 Roma, Italy. Tel: +39-0630154180; Fax: +39-0635503777; E-mail: mtvoso{at}rm.unicatt.it
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Abstract |
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Background: Polymorphisms in genes involved in detoxification and DNA-repair pathways may modify the individual's risk for genomic damage, and, as a consequence, the risk of developing malignant diseases.
Patients and methods: We performed a case-control study including 160 cases of acute myeloid leukaemia (AML) and 162 matched controls to test the impact of six genomic polymorphisms on the risk to develop AML and/or therapy-related AML.
Results: We found a significantly higher prevalence of the polymorphic variants RAD51-G135C and CYP3A4-A-290G genes in AML cases, when compared with controls (P = 0.02 and P = 0.04), increasing the risk of AML 2.1-folds (95% CI: 1.1–4.0) and 3.2-fold (95% CI: 1.1–11.5), respectively. Carriers of both the RAD51-G135C and CYP3A4-A-290G variants were at highest AML risk (P = 0.003; OR:13,6; 95% CI: 2.0–585.5), suggesting a synergistic effect between these polymorphisms.
Conclusions: These results suggest that polymorphic variants in DNA-repair and detoxification enzymes may co-operate in modulating the individual's risk of AML.
Key words: acute myeloid leukaemia, detoxification, DNA-repair, polymorphisms
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introduction |
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Acute myeloid leukaemia (AML) is a clonal disorder characterized by the acquisition of somatic mutations in haematopoietic progenitors leading to disruption of differentiation. Exposition to DNA damaging agents may play an important role in the pathogenesis of AML, as 10–20% of AML are therapy-related (t-AML), developing in patients who received chemo- and/or radiotherapy for a primary malignancy.
Detoxification and DNA repair enzymes protect DNA from damage, due to both endogenous and exogenous sources. When detoxification or repair are ineffective, the DNA damage can cause chromosomal instability leading to severe failure of cell functions, and either apoptosis or oncogenesis. Genetic differences defined by polymorphisms altering the enzymatic activities in detoxification and DNA repair pathways are prime candidates for studies to explain variation in individual susceptibility to develop AML.
Phase I detoxification enzymes catalyse the transformation of xenobiotics to reactive intermediates, which are then eliminated through phase II enzymes. Human cytochrome P450 (CYP) enzymes play a key role as phase I enzymes in the metabolism of drugs and environmental chemicals, and are important for carcinogenesis and cancer treatment. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. CYP3A is the most abundant component of the CYP system in the human liver. The A(-290)G polymorphism in the 5’ promoter region of CYP3A4, also designated as CYP3A4-V or CYP3A4*1B was shown to be under-represented in therapy-related AML [1, 2].
Glutathione S-transferases (GST) are the most important family of phase II enzymes. They inactivate several carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress. We have previously shown that GSTT1 and M1 deletions may influence AML risk and modify patients’ prognosis [3, 4], and now focused on GSTA1 and GSTP1 polymorphisms. The GSTA1-C-69T polymorphism in the 5’-UTR region, named allele *B, has been associated with a significantly lower enzyme expression [5]. The GSTP1 Ile/Val variant at position 105 reduces enzymatic activity for several electrophilic substrates [6, 7], and was shown to be associated to increased risk of therapy-related AML [8].
Another detoxification enzyme, with a role in the inactivation of free radicals due to hydroquinone, a product of benzene metabolism, is the NADPH-quinone oxidoreductase 1 (NQO1). This gene exhibits a polymorphism, the NQO1-Pro187Ser allele, which leads to protein instability and loss of function, with increase of oxidative stress damages. This enzymatic variant has been associated with acute leukaemia, t-AML/MDS and clonal haematopoiesis following chemotherapy [9–12].
When undetoxified, carcinogens induce the formation of DNA adducts, but genomic integrity may still be restored through DNA repair. Homologus recombination (HR) and non-homologus end-joining (NHEJ) are the two major repair pathways for DNA double-strand breaks (DBS), the most severe lesions in the genome. In particular, homologous recombination (HR) is active during the S-phase of the cell cycle, particularly in rapidly growing cells, such as haematopoietic cells [13].
The RAD51 gene plays a crucial role in homologous recombination and in maintaining the genetic stability of the cell. The RAD51-G-135C polymorphism at position -135 in the 5’ UTR region is associated to RAD51 protein over-expression and to increased DNA repair [14–16]. In HR, RAD51 interacts with and is stabilized by XRCC3, during strand invasion and cross-strand resolution. The RAD51-G-135C variant and the XRCC3-Thr241Met polymorphism at codon 241 have been associated to the development of t-AML [17].
The aim of our study was to assess the role of polymorphisms of six genes, involved in xenobiotic detoxification and DNA DSB repair, as genetic risk factors for AML, and in particular, we were interested whether combinations of polymorphic variants between the different pathways could interact, further increasing the AML risk.
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materials and methods |
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study population and DNA preparation
Bone marrow or blood samples were obtained from 160 AML patients (130 de novo and 30 therapy-related), diagnosed according to WHO classification [18] (Table 1). Control peripheral blood samples were obtained from 162 Caucasians, with a negative history for previous malignancies, and matched for sex and age (79 females, 81 males, median age 59, range 19–87 years). Informed consent was obtained according to Institutional guidelines.
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Mononuclear cells (MNCs) were separated using Ficoll gradient centrifugation. DNA was extracted using DNAzol (Gibco BRL, Eggenstein, Germany), following the manufacturer's instructions.
genotyping by PCR-RFLP
The polymorphisms RAD51-G-135C, XRCC3-Thr241Met, NQO1-Pro187Ser, GSTA1-C-69T and GSTP1-Ile105Val were detected using PCR-RFLP techniques as described [5, 9, 17, 19]. Primer sequences, annealing temperatures and restriction enzymes are shown in Table 2.
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PCR products were generated using 100 ng of genomic DNA, 0.5 µM of each primer (Table 2) and the HotMasterMix (Eppendorf, Hamburg, Germany). All amplifications were carried out in a T3 Thermocycler (Biometra GmbH, Goettingen, Germany) and PCR products were visualized on ethidium bromide-stained 3% agarose gels.
genotyping by mismatch PCR-RFLP
We established a mismatch PCR-RFLP technique to detect the CYP3A4-A-290G polymorphic variant. The forward primer contains a mismatch, which in the presence of the A to G polymorphism introduces a restriction site (5’ CCGG 3’) recognized by the Msp1 restriction enzyme (New England Biolabs Inc., Ipswich, MA). Digestion of the PCR product with 10 U of Msp1 restriction enzyme in a final volume of 20 µl results either in retention of the 165-base pair (bp) product or complete digestion to 142-bp and 23-bp fragments corresponding to individuals homozygous for the A (wild-type) or G (variant) alleles respectively. The presence of both the 165 and 142 bp fragments corresponded to heterozygous individuals. The polymorphism was detected on a 3% NuSieve 3:1 gel (Cambrex srl, Italy). The specificity of this technique was confirmed by sequencing 10 ‘wild-type’ and 10 ‘variant’ samples (data not shown).
statistical analysis
Hardy–Weinberg equilibrium was calculated for each enzyme using the Pearson 
2 test. Differences in genotypes distribution between AML (de novo and/or therapy-related) cases and control subjects were evaluated using 2 test (Yates corrected). Odds ratios (OR) with 95% confidence intervals (CI) were also calculated for the genotypes. When expected cell values were less than 5, the Fisher exact test and the exact limits for confidence intervals were preferred. The combined analysis of polymorphisms and stratifications for sex, age and cytogenetics were performed by logistic regression using the Stata 6.0 software (Stata Corp., College Station, TX).
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results |
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genotype frequencies
We examined the frequencies of six polymorphisms in 160 AML cases and 162 control subjects, matched for age and sex. The genotype distributions and the adjusted ORs are shown in Table 3. Genotype distributions were in Hardy–Weinberg equilibrium, with the exception of XRCC3, whose distribution in controls slightly deviated from expected (P = 0.02).
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When looking at phase I detoxification enzyme polymorphisms, a higher prevalence of the variant G allele at CYP3A4-290 was found in cases, when compared with controls (9.5% vs 3.1%, P = 0.037). This translated into a 3.21-fold risk for AML in carriers of CYP3A4-A-290G. On the other hand, the frequency of the homozygous variant phase II GSTA1*B genotype was significantly lower in AML patients, than in controls (5.7 vs 17.7%, P = 0.003, Table 3), reducing the AML risk. No differences between patients and controls were found when looking at NQO1-Pro187Ser and GSTP1-Val105Ile polymorphisms.
When analysing polymorphisms in DNA repair enzymes, we found that carriers of the variant C allele at position -135 in the RAD51 gene were more frequent among AML patients in comparison to the control group (21.9 vs 11.8%, P = 0.024). This translated in to a 2.09 fold increase in the AML risk for carriers of RAD51-G-135C allele. The XRCC3 Met allele increased the AML risk only when enzymatic activity was absent due to homozygous mutated genotype, when compared with heterozygous and wild-type genotypes (24.4% in AML cases versus 12.4% in the controls, P = 0.009, OR 2.27, 95% CI 1.21–4.29).
We did not find significant differences in polymorphism frequencies between de novo versus therapy-related AML and between different cytogenetic risk groups (data not shown). The NQO1-Pro187Ser homozygous variant was over-represented in t-AML, when compared with AML de novo (10.34 vs 3.94%), as previously reported [12, 20], although this difference did not reach statistical significance (P = 0.161).
interaction between detoxification and DNA repair polymorphisms
Since major genetic instability may result from combined minor defects, we performed a logistic regression analysis to test whether combinations of different polymorphisms may modulate the AML risk. Subjects with at least one variant allele in both RAD51 and CYP3A4 genes had a significantly increased AML risk (P = 0.003, OR 13.96, 95% CI 1.95–585.84) (Table 4).
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Other two-way combinations of polymorphisms did not show any further impact on AML risk (data not shown). As most of the polymorphisms tested have been previously described as risk factors for AML/t-AML, we assumed a high probability of association and, therefore, adjusted the P-value for multiple testing to 0.01.
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discussion |
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We studied the effects of DNA repair and detoxification enzyme polymorphisms on AML and t-AML risk. We found an increased frequency of the RAD51 -135C allele in AML, and particularly in de novo AML, when compared with controls, but not between t-AML and controls (data not shown). Over-expression of RAD51 has been reported in immortalized cell lines and primary tumours, and may lead to altered recombination and disrupted stoichiometry of repair complexes, contributing to chromosomal rearrangements [16]. Previous reports found increased frequency of the RAD51 -135C allele in t-AML patients compared with controls [17], suggesting an effect of RAD51 over-expression during leukemogenesis induced by chemo/radiotherapy. Our negative result in the t-AML subgroup may be due to the small number of t-AML cases. Thus, increased homologous recombination activity may protect the damaged cell from apoptosis, but if carried out erroneously may allow survival of damaged cells and increase the risk of malignant transformation.
RAD51 interacts with several other proteins, including XRCC3, whose polymorphisms have been associated to altered DNA double-strand break-induced HR [21]. Cell lines defective in the XRCC3 gene had a 25-fold decrease in homology-directed repair of DNA DSB and high frequencies of spontaneous chromosomal aberrations [22, 23]. The C-to-T substitution at position 18067 of XRCC3, resulting in a Thr-to-Met amino acid substitution at codon 241 was identified by Shen et al. [25], and this polymorphic variant has been associated with increased risks for t-AML [17], melanoma [25] and bladder carcinoma [26]. In our patients, absence of enzymatic activity due to the homozygous XRCC3-241Met genotype was associated to an increased leukaemia risk, with no differences between de novo and therapy-related AML.
CYP3A4 is a phase I enzyme involved in detoxification and activation of several drugs, including epipodophyllotoxins and alkylating agents. CYP3A4 converts epipodophyllotoxin to catechol metabolites, readily oxidized to quinones, which are genotoxins and increase the formation of topoisomerase II cleavage complexes over the native enzyme. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. The functional role of the CYP3A4 A-290G variant is controversial. It was initially suggested that it could reduce transcription based on the low incidence of the polymorphism in patients with t-AML/t-MDS [2]. Felix et al. reported 3% CYP3A4-A-290G variant in t-AML versus 19% in de novo AML [2]. Similarly, in our AML group, we found a significantly increased frequency of CYP3A4-A-290G variants in AML, compared with controls (9.4 vs 3.1%), but, only 3.4% of t-AML were carriers of the variant allele. Recent studies using the hepatoblastoma cell line HepG2 transfected with the variant promoter sequence indicate that, contrary to expectations, transcription of the variant allele is increased [1]. In summary, patients with de novo AML may have increased activity of CYP3A4 due to the variant allele.
We found no differences in the frequency of the NQO1 Pro187Ser polymorphism between AML patients and controls, although there was a trend for NQO1 ser/ser variants to be over-represented in t-AML, when compared with de novo AML (10.3 vs 3.9%) in line with other studies reporting a higher incidence of NQO1-variant in t-AML [12, 20].
Surprisingly, the GSTA1 *B/*B genotype was less frequent in AML, than in controls (P = 0.003, OR 0.28, 95% CI 0.11–0.68), indicating a protective role on AML risk. This is the first report on the GSTA1-C-69T polymorphism in AML. GST enzymes influence the intracellular levels of glutathione [4] and interact with other proteins involved in proliferation and signal transduction control [27, 28]. In vitro studies have shown that among human GSTs, GSTA1 has the highest catalytic activity for glutathione conjugation and the GSTA1*B allele, leads to reduced levels of GSTA1 enzyme [5, 29].
We were then interested on interactions between DNA repair and detoxification polymorphisms in determining the AML and/or t-AML risk. Seedhouse et al. showed that the RAD51-G-135C variant, associated with the GSTM1 null genotype and XRCC3-241Met allele, increases the risk of AML 15-fold [17]. We found a significantly increased AML risk associated to the combination of at least one variant allele of RAD51 and CYP3A4 genes. This is, to our knowledge, the first report on the role of combined phase I and DNA repair polymorphisms on AML risk. This association was independent of the effects of single polymorphisms. We hypothesize that intermediate species, due to modified CYP3A4 metabolism, may induce increased DNA toxicity, and when the DNA damage is defectively repaired by polymorphic RAD51, this will increase the individual's risk to develop AML.
Other DNA repair pathways, which have been associated to AML risk, and are therefore prime candidates for studies on the association to detoxification polymorphisms, are nucleotide and base excision repair (NER and BER) [30, 31]. Among these, of interest are polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), important for BER, and of Xeroderma pigmentosum genes (XPD, XPF, XPG), which encode DNA helicases necessary for the DNA unwinding during NER.
In conclusion, polymorphic variants of enzymes involved in xenobiotic detoxification and DNA repair via HR may synergistically increase individual susceptibly to AML.
Received for publication January 17, 2007. Revision received April 3, 2007. Accepted for publication April 11, 2007.
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