Polymorphisms in Genes Involved in Homologous Recombination Repair Interact to Increase the Risk of Developing Acute Myeloid Leukemia

Vol. 10, 2675–2680, April 15, 2004 Clinical Cancer Research 2675

Claire Seedhouse, Rowena Faulkner, Conclusions: These results strongly suggest that DNA Nadia Ashraf, Emma Das-Gupta, and double-strand breaks and their repair are important in the pathogenesis of both de novo and t-AML. Nigel Russell Division of Haematology, School of Clinical Laboratory Sciences, INTRODUCTION University of Nottingham, and Nottingham City Hospital, Nottingham, United Kingdom. Acute myeloid leukemia (AML) is a clonal hemopoietic disorder that is frequently associated with genetic instability characterized by a diversity of chromosomal and molecular ABSTRACT changes. Most cases of AML arise de novo; however, ϳ10– Purpose: Double-strand break repair via homologous 20% of all cases of AML arise after exposure to chemotherapy recombination is essential in maintaining genetic integrity. or radiotherapy after the treatment of a primary malignancy RAD51 and XRCC3 are involved in the repair of DNA by [therapy-related AML (t-AML); reviewed by Pedersen- this pathway, and polymorphisms have been identified in Bjergaard et al. (1)]. both the RAD51 (RAD51-G135C) and XRCC3 (XRCC3- DNA is at constant risk from damage from both endogenous and exogenous sources. A large number of highly complex Thr241Met) genes. The object of this study was to examine mechanisms have evolved to protect DNA from damage includ- whether these polymorphisms may modulate susceptibility ing DNA repair pathways and systems that protect against to the development of acute myeloid leukemia (AML), a oxidative stress and other damaging agents. These pathways disease that is characterized by genetic instability. play a vital role in maintaining genetic integrity. The ability of Experimental Design: We studied the distribution of an individual to prevent and repair damage is genetically deter- polymorphisms in RAD51 and XRCC3 in 216 cases of de mined and is the result of combinations of multiple genes that novo AML, 51 cases of therapy-related AML (t-AML), and may display subtle differences in their activity (2–4). 186 control subjects using PCR followed by restriction en- Genetic polymorphisms have now been identified in a zyme digestion. The polymorphic deletion of the detoxifica- number of DNA repair genes and damage-detoxification genes. tion gene glutathione S-transferase M1 (GSTM1) was also Polymorphisms can affect protein function, promoter activity, examined by PCR. mRNA stability, and splice variants and, hence, can result in a Results: The risk of the development of AML was found change in the cellular ability to cope with DNA damage, which to be significantly increased when both variant RAD51-135C contributes to an altered disease susceptibility. The genotype and XRCC3-241Met alleles are present [odds ratio (OR), distributions of a number of these polymorphic genes have been 3.77; 95% confidence interval (CI), 1.39–10.24], whereas the shown to be associated with AML and/or t-AML including the risk of t-AML development is even higher (OR, 8.11; 95% base excision repair gene XRCC1 (5) and the detoxification CI, 2.22–29.68), presumably because of the large genotoxic genes NAD(P)H:quinine oxidoreductase (6, 7) and glutathione insult these patients receive after their exposure to radio- S-transferases (8, 9). therapy or chemotherapy. If we further divide the AML Double-strand breaks (DSBs) in DNA are arguably the group into patients in which the burden of DNA damage is most important class of DNA damage because they may lead to increased, because of the deletion of the GSTM1 gene, the either cell death or loss of genetic material resulting in chromo- risk of development of AML is further increased (OR, 15.26; somal aberrations. The balance of DSB repair activity appears to be critical to the genetic stability of cells. Too little repair leads 95% CI, 1.83–127.27). to the acquisition and persistence of mutations, whereas elevated levels of repair can inhibit the apoptotic pathway and can enable a cell with badly damaged DNA to attempt repair, potentially mis-repair, and survive. DSBs are predominantly repaired by either homologous recombination (HR) repair or nonhomolo- Received 10/8/03; revised 12/2/03; accepted 12/19/03. gous end-joining pathways in mammalian cells. Polymorphisms Grant support: From the Leukaemia Research Fund, United Kingdom. The costs of publication of this article were defrayed in part by the have now been identified in a number of genes involved in payment of page charges. This article must therefore be hereby marked protecting from, and repairing, DSBs and we have chosen to advertisement in accordance with 18 U.S.C. Section 1734 solely to study the distribution of several of these, in genes belonging to indicate this fact. the HR DNA DSB repair pathway. Requests for reprints: Claire Seedhouse, Department of Academic Haematology, Clinical Sciences Building, Nottingham City Hospital, HR uses the second, intact, copy of a chromosome as a Nottingham, NG5 1PB, United Kingdom. Phone: 44-(0)-115-8404722; template to copy the information lost at the DSB site on the first Fax: 44-(0)-115-8404721; E-mail: [email protected]. chromosome, and, hence, HR repair is a high-fidelity process.

One of the central proteins in the HR repair pathway is RAD51. MATERIALS AND METHODS RAD51 binds to DNA and promotes ATP-dependent homolo- Study Subjects. Blood or bone marrow samples were gous pairing and strand transfer reactions [reviewed in Baumann obtained at AML diagnosis after informed consent, according to and West (10)]. The RAD51 protein is essential to the viability the declaration of Helsinki, from 267 Caucasian patients, in- of a cell and gene knock-out models in mice are embryonically cluding 51 with t-AML. The diagnosis of AML was made using lethal (11, 12). Cells lacking RAD51 are characterized by an the French-American-British criteria after conventional cyto- accumulation of chromosomal breaks before cell death (13). chemical and surface marker analysis. The median ages of the Hence the role of RAD51 is vital in maintaining genetic stability AML patients were as follows; de novo AML, 64 years (range, within a cell. 11–96 years); and t-AML, 59 years (range, 27–88 years). Con- Ϫ Ј A G/C polymorphism at position 135 in the 5 untrans- trol peripheral blood samples were obtained from an equivalent lated region of the RAD51 gene has been identified (RAD51- number (n ϭ 186) of white Caucasian individuals, with no G135C; Ref. 14). Because of the interaction of RAD51 with the known malignancies, from the same small geographic area in breast/ovarian cancer susceptibility gene products BRCA1 and the United Kingdom. The median age of the controls was 50 BRCA2 (15–17), this polymorphism has been the focus of a years (range, 15–97 years). Genomic DNA was extracted from number of studies in breast and ovarian cancer (14, 18–20). The cells using QIAamp blood DNA isolation kits (Qiagen, Craw- polymorphism has been shown to be associated with breast ley, United Kingdom) according to the manufacturer’s protocol. cancer risk in BRCA2 mutation carriers (14, 18). However, Studies on the majority of these patients and controls have because of the importance of the HR DSB repair pathway and been described previously (5). the fact that AML is often characterized by a high degree of PCR RFLP Analysis. PCR followed by either direct genetic instability, we have decided to look at the frequency of this polymorphism in AML. analysis (GSTM1) or enzymatic digestion of the PCR products The XRCC3 protein also functions in the HR DNA DSB (RAD51 and XRCC3) was used for the genotyping of the three repair pathway and directly interacts with and stabilizes RAD51 polymorphisms. Approximately 50 ng of genomic DNA was ␮ and the closely related RAD51C (21–25). XRCC3 is a paralog used as template in each of the PCR amplifications. The 50- l ␮ of RAD51 and, similar to RAD51, it is essential for genetic reaction also consisted of each dNTP (at 150 M; Amersham stability (26–30). A polymorphism at codon 241 in the XRCC3 Biosciences United Kingdom Ltd, Little Chalfont, United King- ␮ gene results in a Thr-to-Met amino acid substitution (31). Pos- dom), each primer (at 1 M), 1.5–2.0 mM MgCl2, and 2 units of itive associations between XRCC3-241Met and malignancies Amplitaq Gold (PE Applied Biosystems, Warrington, United have been made by several groups, including studies on bladder Kingdom) in the manufacturer’s buffer. After an initial heat cancer (32) and melanoma skin cancer (33). The variant activation step at 95°C for 10 min, amplification was performed XRCC3-241Met allele has also been associated with a higher in a PTC-100TM Programmable Thermal Controller (MJ Re- level of DNA adducts compared with those individuals with the search, Inc, Watertown, MA) using the following conditions; wild-type gene (34). However, we have previously reported that XRCC3-Thr241Met, denaturation at 95°C for 1 min, annealing there is no association between XRCC3-241Met and AML or at 60°C for 1 min, extension at 72°C for 1 min for a total of 35 t-AML (5). cycles ending with a final extension at 72°C for 10 min. The A malignant phenotype is likely to result from the accu- conditions for the RAD51 PCR were as described except an mulation of many minor genotypes, and it is probable that annealing temperature of 52°C was used; an annealing temper- associations exist between polymorphisms in DNA repair genes ature of 57°C was used for the GSTM1 amplification. PCR and polymorphisms in genes encoding proteins involved in products were purified using a QIAquick PCR purification kit minimizing the effects of carcinogens. The glutathione S-trans- (Qiagen) before digestion and/or sequencing. ferases (GSTs) are a multigene family that detoxify reactive XRCC3-Thr241Met. XRCC3-Thr241Met was amplified electrophiles via conjugation to glutathione and, hence, prevent in a 415-bp product using primers described previously (31) damage to DNA. A deletion polymorphism in one isozyme of with 2 mM MgCl2. The PCR products were digested at 37°C this family [glutathione S-transferase M1(GSTM1)] occurs in overnight with 10 units of NlaIII [New England Biolabs (United ϫ ϳ50% of Caucasians (35) resulting in the absence of functional Kingdom) Ltd, Hitchin, United Kingdom] in 1 buffer supplied ␮ GSTM1 protein. GSTM1 detoxifies a variety of genotoxic with the enzyme and supplemented with 100 ng/ l BSA. All agents (reviewed in Hayes and Pulford; Ref. 36) and a reduction XRCC3 PCR products contain an internal NlaIII site, and, in in GSTM1 protein activity, due to the deletion polymorphism, addition, the presence of the Met polymorphism also generates has previously been shown to be associated with significantly an additional NlaIII site resulting in 104-bp, 141-bp, and 170-bp elevated levels of DNA adducts in WBCs (37) and with higher products for the polymorphic allele and 141-bp and 274-bp levels of sister-chromatid exchange (38) compared with indi- products for the wild-type threonine allele. viduals with wild-type GSTM1 protein levels. The digested products were resolved on 3% agarose gels We hypothesized that polymorphisms in the RAD51 and (Helena Biosciences, Sunderland, United Kingdom), stained XRCC3 HR DNA DSB repair genes may lead to aberrations with ethidium bromide and analyzed under UV light. within the pathway and contribute to genetic instability and RAD51-G135C. The RAD51-G135C polymorphism was subsequent AML. In addition, we wanted to establish whether amplified in a 157-bp fragment using primers described previ-

an increase in DNA damage caused by a deletion polymorphism ously (18) with 1 mM MgCl2. The PCR products were digested in GSTM1 would further contribute to the pathogenesis of AML. with 10 units of MvaI (Helena Biosciences) in the manufactur-

Table 1 Frequency of RAD51-135C, XRCC3-241Met and GSTM1 null polymorphisms in acute myeloid leukemia (AML) and control populations and the relative risk for AML associated with these genotypes AML t-AML Controls Genotype (n) n ORa (95% CI)b Pn OR (95% CI) P RAD51 186 206 51 g/g 166 171 1.0 (Ref) 39 1.0 (Ref) g/c 18 32 1.53 (0.81–2.90) 0.19 12 2.84 (1.24–6.51) 0.01 c/c 2 3 2.36 (0.38–14.53) 0.35 0 g/cϩc/c 20 35 1.60 (0.87–2.94) 0.13 12 2.66 (1.17–6.02) 0.02 XRCC3-241 175 216 44 Thr/Thr 92 99 1.0 (Ref) 20 1.0 (Ref) Thr/Met 64 87 1.32 (0.84–2.09) 0.23 16 1.29 (0.60–2.76) 0.51 Met/Met 19 30 1.56 (0.80–3.05) 0.19 8 1.90 (0.69–5.23) 0.21 Thr/Met ϩ Met/Met 83 117 1.38 (0.90–2.10) 0.14 24 1.44 (0.72–2.88) 0.30 GSTM1 177 200 42 Presentc 99 99 1.0 (Ref) 23 1.0 (Ref) Null 78 101 1.28 (0.84–1.95) 0.26 19 0.98 (0.48–1.97) 0.95 a OR, odds ratio; CI, confidence interval; t-AML, therapy-related AML; Ref, used as reference group. b Adjusted for age. c Carriers of at least one intact allele are used as reference.

er’s buffer. The polymorphic C allele eliminates the MvaI re- RESULTS striction site, and, therefore, the digestion of polymorphic sam- RAD51-G135C, XRCC3-Thr241Met, and GSTM1 ples resulted in a single band at 157 bp, whereas the wild-type Polymorphisms. We have examined the frequency of three allele resulted in two bands at 71 bp and 87 bp. All of the polymorphisms in 216 cases of de novo AML and 186 controls. polymorphic samples were sequenced. We also analyzed a subgroup of 51 patients with t-AML. Be- Two reviewers (C.S., E.D-G.) independently scored all of cause of an inadequate amount of DNA in some cases, several the genotypes, and samples that could not be scored were samples did not generate complete information on all three sequenced. Sequencing was also carried out on a 10% random polymorphisms. There was very little discrepancy in genotype sample population of control and AML PCR products. scores between the two reviewers; the PCR was repeated on GSTM1. The GSTM1 gene was amplified using previ- ambiguous samples. In all of the cases in which sequencing was ously described primers (39), ␤-globin was also amplified as an performed, the results confirmed the RFLP findings. Among the internal control using 2.5 mM MgCl2. The GSTM1 null genotype controls, the variant allele frequencies were: RAD51-135C, was indicated by the presence of the 268-bp ␤-globin gene and 0.06; and XRCC3 241Met, 0.29 ; 44.1% of the controls lacked lack of the 215-bp GSTM1 gene. both GSTM1 alleles. The RAD51-G135C and XRCC3- Sequencing Reactions. Sequencing reactions were set Thr241Met genotype frequencies among the control population up with ϳ200 ng of purified PCR product and 10 pmol primer were consistent with those expected from the Hardy-Weinberg using a Thermo Sequenase II dye terminator cycle sequencing equilibrium; RAD51-G135C, ␹2 ϭ 0.576, P ϭ 0.750; XRCC3 premix kit according to the manufacturer’s instructions (Amer- Thr241Met, ␹2 ϭ 1.030, P ϭ 0.597. sham Biosciences United Kingdom Ltd). The primers used for The distributions of the three genotypes in control individ- sequencing were the same as those used for the PCR amplifi- uals and AML and t-AML patients and the adjusted ORs for cations. The reactions were electrophoresed using an ABI 377 each genotype are shown in Table 1. The results for the RAD51- automated DNA sequencer (PE Applied Biosystems) as recom- G135C polymorphism demonstrate that the proportion of de mended by the manufacturer. novo AML and t-AML patients possessing at least one poly- Statistical Analysis. The observed genotype frequencies morphic C allele is higher than in the control group; this results of the XRCC3-Thr241Met and RAD51-G135C polymorphisms in a significant 2.66-fold increase in the risk of developing AML in the control cohorts were compared with those calculated by in the t-AML group. Neither the XRCC3-Thr241Met nor the the Hardy-Weinberg equilibrium (p2 ϩ q2 ϩ 2 pq ϭ 1; where p GSTM1 genotype distributions demonstrated statistical differ- is the variant allele frequency). The distribution of genotypes in ences in either de novo AML or t-AML compared with the AML populations compared with the control population were control group. The XRCC3-Thr241Met genotype results on a tested for significance using odds ratios (ORs) and their 95% section of this population have previously been reported (5). confidence intervals (95% CIs) calculated by logistic regression Combined Analysis of Polymorphisms. Table 2 shows analysis and adjusted for the effect of age. Values of P Յ 0.05 the combined analysis of the two polymorphisms present in were considered significant. genes that encode proteins involved in the HR DSB DNA repair All of the analyses were performed by the statistical pack- pathway in both AML and t-AML. Interactions between the two age SPSS for Windows, Version 9 (SPSS Inc, Chicago, Illinois). polymorphisms can be seen; this is associated with a significant

Table 2 Combined logistic regression analysis to study the to either repair the damage to the DNA or to stimulate apoptosis association between DNA double-strand break repair genotypes and so that the results of the damage cannot be inherited. The risk of acute myeloid leukemia (AML)/therapy-related AML (t-AML) risk developing de novo AML in the RAD51-135C/XRCC3-241Met RAD51 XRCC3-241 Control nn ORa (95% CI)b P variant group is still significant, although lower than for t-AML. AML We suggest that this risk is likely to be connected to DNA WT WT 78 74 1 (Ref) damage encountered via environmental and endogenous V WT 10 14 1.31 (0.53–3.24) 0.55 sources. This hypothesis is supported if we then take into WT V 74 83 1.19 (0.74–1.90) 0.48 consideration a deletion polymorphism in the detoxification V V 6 21 3.77 (1.39–10.24) 0.009 gene GSTM1. The GSTM1 protein detoxifies chemicals and t-AML metabolites that otherwise might damage DNA and cause, WT WT 78 16 1 (Ref) among other damage types, DNA DSBs. The absence of V WT 10 4 2.03 (0.54–7.69) 0.30 WT V 74 16 1.12 (0.50–2.50) 0.79 GSTM1 protein, due to the deletion polymorphism, results in an V V 6 7 8.11 (2.22–29.68) 0.002 increase of DNA damage. This has been demonstrated by an a OR, odds ratio; CI, confidence interval; WT, wild-type genotype; increase in DNA damage adducts (37) and higher levels of Ref, used as reference group; V, the presence of a variant allele (either sister-chromatid exchange (38). When we analyze the de novo heterozygous or homozygous). AML patients with respect to the three-variant genotype, we see b Adjusted for age. a substantial increase (15.26-fold) in the risk of developing the disease. Hence, as with the increase in DNA damage caused by previous therapy, an increase in DNA damage caused via deficient GSTM1 activity also significantly increases the risk of increase in the risk of development of both AML (OR, 3.77; AML when variant genotypes in the RAD51 and XRCC3 HR 95% CI, 1.39–10.24) and t-AML (OR, 8.11; 95% CI, 2.22– repair genes are present. An OR of 15.26 far exceeds the safety 29.68) in individuals with at least one variant RAD51-135C and “factor” of 10 generally accepted in polymorphism studies (2). at least one variant XRCC3-241Met allele. In addition to the We acknowledge that there is an age difference between analysis on the combined effects of the HR repair genes, the our control and case populations and the limitations this gener- control and AML groups have also been analyzed with respect ates, including the possibility that a small number of our con- to the presence or absence of the detoxification GSTM1 gene. trols may develop AML. In addition, the AML genotype anal- These results are shown in Table 3. The combination of a null ysis was performed on leukemic and not constitutional DNA; it GSTM1 genotype with the double HR gene variants results in a must, therefore, be kept in mind that somatic gene alterations, large increase in the risk of AML development (OR, 15.26; 95% although unlikely, may have influenced the genotype data. We CI, 1.83–127.27). have also considered that chromosomal loss of 14q32.3 (XRCC3), 15q15.1 (RAD51), or 1p13.3 (GSTM1) may have DISCUSSION affected our results. However, abnormalities of chromosomes 1, Our findings suggest that the HR DSB DNA repair path- 14, and 15 are not common in AML; and of the patients for way may be important in the pathogenesis of both de novo and whom we have cytogenetic data available, no patients had loss t-AML. We have demonstrated significant ORs in AML and of 1p13.3, only two patients had loss of chromosome 14, and t-AML associated with the presence of polymorphisms in two one patient had loss of chromosome 15. These patients were all HR genes, namely RAD51-135C and XRCC3-241Met. In addi- homozygous for the wild-type allele of the respective gene; and, tion, when a deletion polymorphism of the detoxification gene hence, if the missing allele was variant, this would have resulted GSTM1 is taken into consideration, there is a greater than in our underestimating the frequency of the variant allele in the 15-fold increase in the risk of development of AML when all AML group. Because of the strong significance of our results, three variant genotypes are present. we think that these limitations would be unlikely to have a The efficient repair of DSBs is essential for genetic stabil- substantial impact on our interpretations. We, therefore, suggest ity. The knock-out model of the HR repair gene RAD51 is that these genetic factors are expected to have an important embryonically lethal (11, 12) and XRCC3 is also essential for influence in AML risk in the population. genetic stability (26–30). Mutations in these genes are rare, demonstrating the absolute requirement of the encoded proteins. However subtle differences in protein levels or protein activity resulting from polymorphisms are likely to be tolerated by cells, Table 3 Combined logistic regression analysis to study the although these differences would be expected to have an effect association between DNA double-strand break repair genotypes, RAD51-135C and XRCC3-241Met, and GSTM1 genotype and acute when a high level of DNA damage is present. We have dem- myeloid leukemia (AML) risk onstrated that this may indeed be the case in AML. The presence Genotype Control AML ORa (95% CI)b P of variant RAD51-135C and XRCC3-241Met is associated with an increase in the risk of developing t-AML of more than 8-fold, All WT 38 29 1 (Ref) One variant 79 86 1.55 (0.85–2.82) 0.16 whereas the increase in risk for the development of de novo Two variants 41 48 1.57 (0.80–3.07) 0.19 AML is nearly 4-fold. The high risk associated with t-AML is Three variants 1 12 15.26 (1.83–127.27) 0.01 presumably because these patients have received a large geno- a OR, odds ratio; CI, confidence interval; WT, wild type; Ref, used toxic insult, either by chemotherapy or radiotherapy, and hence as reference group. the presence of a fully functional HR repair pathway is essential b Adjusted for age.

A deletion polymorphism also occurs at the GSTT1 locus observed in t-AML, because exposure to chemotherapy or ra- (40), and the distribution of the GSTT1 genotype was studied in diotherapy and the resulting high levels of DNA damage may our patient and control cohorts (data not shown). Although a mask the requirement of the GSTM1 protein. similar trend to that described among the GSTM1, RAD51, and Mismatch repair and base excision repair pathways have XRCC3 polymorphisms was also evident among GSTT1, already been demonstrated to contribute to the pathogenesis of RAD51 and XRCC3, because of the lower frequency of the AML and t-AML (5, 47–49); however, double- strand break GSTT1 deletion polymorphism (20% frequency of GSTT1 null repair has been, thus far, largely unstudied in AML. We have compared with 50% for GSTM1 null), the numbers of individ- shown that the efficient functioning of this pathway is signifi- uals required for statistical analysis are much greater than those cantly important in protecting individuals from both de novo available to us, and we were unable to draw any firm conclu- AML and t-AML. In addition, minimizing damage to DNA by sions from this work. However, our results encourage the es- harboring functional GSTM1 protein is also a vital requirement tablishment of large population analyses to study the combined for protecting against de novo AML. AML is characterized by effects of polymorphisms in DNA repair and detoxification genetic instability, and it is likely that defects in the HR repair genes. pathway will contribute to the chromosomal aberrations seen in Work is still required to determine the exact functional the majority of AML cases. Larger studies are now required to consequences of the DNA repair gene polymorphisms. Little is confirm these findings and to also further divide the patients into known about the effect of the RAD51–135C variant, although cytogenetic groups to establish whether the genotype described recent data suggest that this polymorphism may increase the here results in a particular pattern of chromosomal abnormality. levels of RAD51 mRNA in patients with breast cancer (41). If Studies involving greater numbers of individuals are also re- this also results in an increase in RAD51 protein expression, quired to demonstrate that none of our findings were achieved then it is possible that the fine balance of HR protein levels will by chance because of the large number of statistical analyses be disrupted. Overexpression of RAD51 or RAD52 have previ- that we performed for the work reported here. ously been demonstrated to have a dominant-negative effect on This report illustrates the importance of studying the com- HR initiation (42). In addition, it is possible that an increase in bined effects of genotypes, particularly those from more than the cellular levels of RAD51 protein may inhibit the stimulation one gene in a specific pathway. Our work demonstrates a of the apoptotic pathway and enable a cell with damaged DNA positive association between polymorphisms in genes involved to survive, acquire mutations and chromosomal aberrations, and in protecting the cells from DSBs and an increased risk of de subsequently convey the damage to daughter cells. This is a novo and t-AML susceptibility. plausible explanation as to why we found a significant increase in the risk of the development of t-AML when a variant RAD51- ACKNOWLEDGMENTS 135C allele was present (irrespective of other genotypes). We We thank Stephen Langabeer and Steve Chatters and the Kay suggest that the variant level of RAD51-135C alone is sufficient Kendall Leukemia Fund for t-AML DNA samples from the DNA/RNA to confer a significant increase in the risk of development of banking facilities at University College Hospital, London, and the t-AML (OR, 2.66; 95% CI, 1.17–6.02) because of the huge Medical Research Council Acute Leukemia Working Party for access to genotoxic insult that precedes the disease. samples from patients with AML entered into Medical Research Council Recent evidence indicates that the XRCC3–241Met variant trials. is functional in HR repair of DSBs (43). The variant protein was shown to be able to complement the HR defect in a XRCC3- REFERENCES deficient cell line; however, the investigators (Araujo et al.; Ref. 1. 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