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AML with trisomy 8
Leukemia Research 37 (2013) 742–746
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High frequency of NAD(P)H:quinone oxidoreductase 1 (NQO1) C609 T germline polymorphism in MDS/AML with trisomy 8 Sophia Zachaki a,b , Chrysa Stavropoulou a , Theodora Koromila c , Kalliopi N. Manola a , Marina Kalomoiraki a , Aggeliki Daraki a , Daphne Koumbi a , Anastasia Athanasiadou d , Emmanuel Kanavakis b,e , Panagoula Kollia c , Constantina Sambani a,∗ a
Laboratory of Health Physics, Radiobiology and Cytogenetics, NCSR “Demokritos”, Athens, Greece Department of Medical Genetics, Faculty of Medicine, University of Athens, Greece c Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Greece d Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece e Research Institute for the Study of Genetic and Malignant Disorders in Childhood, St. Sophia’s Children’s Hospital, Athens, Greece b
a r t i c l e
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Article history: Received 26 August 2012 Received in revised form 6 March 2013 Accepted 9 April 2013 Available online 1 May 2013 Keywords: MDS AML Genetic susceptibility Single nucleotide polymorphism (SNP) NAD(P)H:quinone oxidoreductase 1 (NQO1) Trisomy 8 (+8)
a b s t r a c t The NQO1 C609 T germline polymorphism resulting in a lowering of enzyme activity may confer susceptibility to MDS. To assess this association, we performed a case–control study including 330 Greek patients with de novo MDS and 416 healthy donors, using a Real-Time PCR genotyping method. Focusing on cytogenetic aberrations most commonly found in MDS, we retrospectively genotyped 566 MDS/AML patients carrying −5/del(5q), −7/del(7q), +8, del(20q) and −Y. The case–control analysis revealed no differences in NQO1 genotype distribution. Interestingly, a 6-fold increased frequency of the homozygous variant genotype was observed among patients with isolated trisomy 8 (p < 0.0001), suggesting that null NQO1 activity may influence the occurrence of +8 in MDS/AML. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) comprise a heterogeneous group of acquired clonal hematopoietic stem cell disorders, characterized by bone marrow hypercellularity, dysplasia, various degrees of cytopenia and an advanced risk of progression to acute myeloid leukemia (AML) [1–3]. Acquired clonal chromosomal abnormalities are found in about 30–50% of de novo MDS and approximately 80% of secondary or therapy-related MDS cases [4]. These abnormalities are predominantly characterized by total/partial chromosomal losses or gains and rarely by balanced structural aberrations. The most frequently observed chromosomal abnormalities include long-arm deletions of chromosomes 5, 7 and 20, whereas, trisomy 8 represents the most common chromosomal gain [5,6]. The etiology is unknown in the majority of cases; however, models for the development of sporadic MDS and
∗ Corresponding author at: Laboratory of Health Physics, Radiobiology and Cytogenetics, NCSR “Demokritos”, 153 41 Agia Paraskevi, Athens, Greece. Tel.: +30 2106503866; fax: +30 210 6534710. E-mail address: [email protected] (C. Sambani). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.04.015
AML suggest the role of cumulative genetic and toxic environmental factors in genetically predisposed individuals [7]. Clues to the etiology of myelodysplasia are expected to be gained through the study of genetic susceptibility in candidate genes. The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that protects cells against oxidative damage. It plays an important role in detoxification of quinones derived from the oxidation of phenolic metabolites of benzene, by catalyzing two or four electron reductions of these substrates [8]. These reductions are beneficial to the cell by preventing redox cycling and the generation of free radicals [9,10]. The NQO1 is ubiquitously present in all tissues, including bone marrow [10–13]. As it has been reported, bone marrow cells including CD34+ progenitors with negligible NQO1 production show an increased susceptibility to benzene toxicity [8]. The pivotal role of NQO1 activity is reinforced by studies demonstrating that loss of NQO1 gene expression in NQO1-null mice causes myeloid hyperplasia of bone marrow and significant increase in blood neutrophils [14]. The NQO1 enzyme activity strongly depends on a singlenucleotide polymorphism (SNP) at the NQO1 locus. Specifically, a cytosine-to-thymine substitution (C→T) at position 609 of the cDNA (C609 T) produces a proline to serine substitution at amino
S. Zachaki et al. / Leukemia Research 37 (2013) 742–746
acid 187 (P187S) of the mature protein that destabilizes and inactivates the enzyme [15,16]. Individuals homozygous for the mutant allele (T/T) completely lack NQO1 activity, whereas heterozygotes (C/T) present approximately threefold decreased enzyme activity [17]. The frequency of the NQO1 C609 T polymorphism exhibits ethnic variation with the highest prevalence of the homozygous variant genotype (T/T) occurring in Asian populations (19% Korean; 22% Chinese) and the lowest prevalence in Caucasians (4%) [18]. The NQO1 C609 T gene polymorphism has been previously investigated as a predisposing element in various types of hematological malignancies. Several results suggest that individuals carrying NQO1 variant genotypes may be at increased risk for the development of de novo MDS and AML [19,20], t-AML [16,17,21,22], childhood acute lymphoblastic leukemia (ALL) [23] and ALL with MLL rearrangements in infants [24]. However, other reports have come to contradictory results [25–29], possibly due to the heterogeneity of the diseases, insufficient sample sizes and ethnic variations [30]. Thus far, the role of the NQO1 polymorphism in de novo MDS susceptibility remains elusive. Considering the critical role of the NQO1 enzyme in detoxification mechanisms implicated in MDS pathogenesis, we hypothesize that the NQO1 germline polymorphism influencing NQO1 activity, may lead to an additive effect on bone marrow toxicity and thus, may confer susceptibility to MDS development and/or promote certain chromosomal changes. To this end, we conducted a case–control study to evaluate the NQO1 genotype in a large series of 330 Greek patients with MDS and 416 healthy donors. The NQO1 gene status was also evaluated in relation to patients’ characteristics. Focusing on most common cytogenetic aberrations found in MDS, we retrospectively analyzed the NQO1 genotypic distribution in 566 patients with MDS/AML carrying −5/del(5q), −7/del(7q), trisomy 8, del(20q) and −Y. 2. Materials and methods 2.1. Patients The case–control study included 330 patients with confirmed diagnosis of primary MDS (217 males, 113 females, median age 68 years) and 416 gender and age matched healthy individuals (270 males, 146 females, median age 66.5 years). Diagnosis of MDS was established in the major Greek hospitals. Patients with MDS secondary to a previous malignancy or a history of chemo/radiotherapy were excluded from the study. Healthy donors were unrelated individuals with a negative history for previous malignancies and normal peripheral blood cell counts. Both cases and controls enrolled in the study came from different areas of Greece, thus having a homogeneous ethnic background. Leftover bone marrow (BM) specimens from patients and peripheral blood (PB) samples from healthy donors were used for DNA extraction and subsequent genotypic analysis. Genomic DNA was also extracted from 566 MDS/AML patients with known specific cytogenetic abnormalities. Among them, 199 cases showed −5/del(5q), 153 had −7/del(7q), 202 presented trisomy 8 (+8), 51 cases had del(20q) and 65 cases exhibited loss of the Y chromosome (−Y). As a sole change, Y loss was considered as disease-associated clonal abnormality when found in more than 75% of metaphase cells [31]. A total of 84 of 566 patients carrying more than one of the mentioned recurrent aberrations were included in more than one corresponding cytogenetic category. The study was approved by the Ethical Scientific Committee of the National and Kapodistrian University of Athens and written informed consent was provided from all sample donors included in the study.
2.2. Cytogenetic analysis BM samples at diagnosis were submitted for chromosome studies at the Cytogenetics Unit, NCSR “Demokritos”, for centralized karyotyping. The cytogenetic analysis was performed using unstimulated short-term cultures. Karyotypes were described in accordance to the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN 2009) [32]. Enrolment in the study required feasibility of karyotyping 15 or more metaphases per patient. Patients were stratified according to the IPSS cytogenetic classification into three risk groups; good: normal karyotype, del(5q), del(20q) or −Y as sole aberrations, intermediate: trisomy 8, single miscellaneous, double abnormalities and poor: complex karyotype (i.e., ≥3 anomalies) or chromosome 7 abnormalities [33].
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2.3. Genotype analysis Genomic DNA was extracted from PB cells of healthy donors or BM samples of MDS patients using the QIAamp DNA-extraction midikit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Isolated DNA was resuspended in TrisEDTA buffer, pH 8.0, and stored at −20 ◦ C until use. The NQO1 C609 T SNP genotyping was performed for all samples using both polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (PCR-RFLP) and a Real-Time PCR method. Both methods revealed the same NQO1 genotype result for all samples. The conventional PCR-RFLP assay for NQO1 genotyping was performed as described by Park SJ, 2003 [34]. The Real-Time PCR was performed on a LightCycler 1.0 using LightCycler DNA Master Hybridization Probes Kit (Roche, Mannheim, Germany). The PCR reaction was carried out according to the manufacturer’s instructions and sense and antisense primers as well as anchor hybridization probe sequences have previously described [35]. PCR primers and the hybridization probes were synthesized by Olfert Landt (TIB MOLBIOL, Berlin, Germany). The two NQO1 alleles (wild type C and mutant T) were distinguished by determination of melting curves using the LightCycler Software, version 3. 2.4. Statistical analysis Allele frequencies were determined by allele-counting method. Pearson Chi-square test with continuity correction were employed to investigate the case–control differences in the distribution of the genotypes or other parameters under study. Odds ratios (ORs) are given with 95% confidence interval (CI). All tests were two-sided and statistical significance was set at 5%. Statistical analysis was performed using SPSS (Statistical Package for the Social Sciences) version 12 software.
3. Results 3.1. Clinical characteristics, cytogenetic results and NQO1 genotypes Table 1 summarizes the clinical and cytogenetic characteristics of the 330 patients with primary MDS studied for the NQO1 genotypic distribution. In the present series of patients, 217 men and 113 women (male:female ratio 2:1) were included. The median age was 68 years (range 19–92 years). Two hundred eighty seven patients (287/330, 87%) were older than 60 years. A successful karyotypic result was achieved in 290 out of 330 patients (88%). Of the 290 patients, 108 (37.2%) showed clonal karyotypic abnormalities. None of them showed abnormalities of chromosome 16q22.1 (gene location of NQO1). The most commonly detected cytogenetic abnormalities found as sole abnormalities were +8 (9.7%), −7/del(7q) (3.5%), −Y (2.8%), del(5q) (1.8%), and del(20q) (0.7%), with trisomy 8 being the most frequently observed single change. Regarding NQO1 gene status, the distribution of the variant genotypes (heterozygotes C/T and homozygotes T/T) did not differ significantly between male and female patients (36.4 vs 26.5% and 1.4 vs 2.7%, respectively). According to age, a higher frequency of the homozygous variant genotype (T/T) was observed in patients ≤60 years, as compared to older patients (4.7% vs 1.4%, respectively). As for IPSS prognostic groups, higher frequencies of the C/T and T/T variant genotypes were observed in patients belonging to the intermediate and the poor risk groups, respectively; however, the above trends did not reach statistical significance. Higher incidences of C/T and T/T mutant genotypes were also observed in patients with trisomy 8 and −7/del(7q), as compared to the other cytogenetic categories. 3.2. NQO1 genotypes and MDS susceptibility Table 2 summarizes the distribution of NQO1 genotypes and allele frequencies among MDS patients and controls. In the control population, the NQO1 genotype was distributed as follows: 62.3% wild type (C/C), 35.3% heterozygous (C/T) and 2.4% homozygous mutants (T/T). These frequencies observed within the sample of healthy Greek donors for the C/T and T/T variant NQO1 genotypes
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Table 1 Distribution of NQO1 genotype in MDS patients according to patients’ characteristics and cytogenetic subgroups. No. (%)
NQO1 genotype frequency (%)
n = 330
C/C
C/T
T/T
Gender Male Female
217 (65.8) 113 (34.2)
135 (62.2) 80 (70.8)
79 (36.4) 30 (26.5)
3 (1.4) 3 (2.7)
ns*
Age (years) ≤60 ≥61
43 (13) 287 (87)
26 (60.4) 188 (65.5)
15 (34.9) 95 (33.1)
2 (4.7) 4 (1.4)
ns
91 (36.7) 34 (13.7) 65 (26.2) 29 (11.7) 29 (11.7)
62 (68.1) 24 (70.6) 42 (64.6) 15 (51.7) 22 (75.8)
28 (30.8) 9 (26.5) 22 (33.9) 14 (48.3) 6 (20.7)
1 (1.1) 1 (2.9) 1 (1.5) 0 (0.0) 1 (3.5)
ns
Karyotypeb Normal Abnormal +8 −7/del(7q) −Y Del(5q) Del(20q) Other (single or double) Complex
182 (62.8) 108 (37.2) 28 (9.7) 10 (3.5) 8 (2.8) 5 (1.8) 2 (0.7) 22 (7.6) 33 (11.4)
108 (59.3) 65 (60.2) 11 (39.3) 5 (50.0) 6 (75.0) 4 (80.0) 1 (50.0) 16 (72.7) 22 (66.7)
71 (39.0) 40 (37.0) 16 (57.1) 4 (40.0) 2 (25.0) 1 (20.0) 1 (50.0) 6 (27.3) 10 (30.3)
3 (1.7) 3 (2.8) 1 (3.6) 1 (10.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.0)
ns
IPSS risk groupsc Good Intermediate Poor
196 (67.5) 61 (21.0) 33 (11.7)
126 (64.3) 27 (44.3) 20 (60.6)
67 (34.2) 33 (54.1) 11 (33.3)
3 (1.5) 1 (1.6) 2 (6.1)
ns
FABa RA RARS RAEB RAEB-T CMML
p
a,b,c Percentages calculated on the number of patients with known FAB classification (248 out of 330)a and with available cytogenetic data (290/330)b,c . Cytogenetic risk groups were defined according to Greenberg et al. [33]. *ns: not significant.
del(20q) (p = 0.001, 2 = 27.503, d.f. = 8). Allele frequencies distribution analysis confirmed these differences, showing a higher T allele frequency among patients with +8 and del(20q), as compared to the other cytogenetic subgroups (p = 0.032, 2 = 10.526, d.f. = 4). Comparison between patients and controls revealed a 5-fold increased frequency of the NQO1 homozygous T/T genotype in patients with +8 (11.9% vs 2.4%, respectively) (p* < 0.0001, 2 = 24.182, d.f. = 2) (Table 3). Among the 202 patients with trisomy 8, 159 presented sole +8, of which 22 patients carried the T/T variant genotype (13.8%). Also, comparison between patients with sole +8 and controls showed an even higher frequency of T/T genotype (6-fold increase). A higher incidence of heterozygotes C/T among patients with del(20q) was observed as compared to the controls (58.8% vs 35.3%, respectively) (p* = 0.004, 2 = 11.206, d.f. = 2). Allele frequencies distribution analysis between patients and controls showed that patients with +8 exhibited a 1.8-fold increased risk of carrying at least one variant T allele, as compared to the expected values based on the control population (p* < 0.0001, 2 = 16.652, d.f. = 1, OR = 1.77, 95%CI = [2.33 − 1.34]). Similarly, patients with del(20q) showed a 1.8-fold increased risk of carrying at least one variant T allele (p* = 0.021, 2 = 5.356, d.f. = 1, OR = 1.76, 95%CI = [2.79 − 1.11]) (Table 3).
are in accordance with the Hardy–Weinberg laws of equilibrium (2 = 4.269, d.f. = 1, p < 0.01) and fell into the range of values registered in other Caucasian populations [18]. Comparison of the NQO1 genotype and allele frequencies distribution between MDS cases and controls revealed no significant differences of the variant genotypes (C/T and T/T) (34.2 vs 35.3% and 1.8 vs 2.4%, respectively) (p > 0.05) (Table 2). 3.3. NQO1 genotypes in patients with MDS/AML and specific chromosome aberrations The higher incidences of variant NQO1 genotypes observed in MDS patients with +8 and −7/del(7q) (Table 1), prompted us to analyze the NQO1 gene status in a large number of patients carrying certain specific abnormalities in their karyotype. For this purpose, we retrospectively analyzed a cohort of 566 patients with MDS/AML. Among them, 199 cases showed −5/del(5q), 153 had −7/del(7q), 202 showed +8, 51 cases had del(20q) and 65 cases exhibited Y loss. Table 3 summarizes the distribution of the NQO1 genotype and the allele frequencies among the different cytogenetic groups. Interestingly, we observed significantly higher incidences of NQO1 homozygotes for the T allele (T/T) into the group of patients with +8 and heterozygotes (C/T) among patients with
Table 2 Genotype distribution and allele frequencies of the NQO1 C609 T polymorphism in MDS cases and healthy controls. Group
No.
MDS patients Controls
330 416
NQO1 genotype frequency (%)
Allele frequency
C/C
C/T
T/T
C
T
211 (64.0) 259 (62.3) 2 = 0.440, d.f. = 2 p = 0.803
113 (34.2) 147 (35.3)
6 (1.8) 10 (2.4)
535 (0.811) 665 (0.799) 2 = 0.006, d.f. = 1 p = 0.991
125 (0.189) 157 (0.201)
The p*-value was calculated by comparison of NQO1 genotypic distribution between each cytogenetic group and controls; ns: not significant; OR: odds ratio; CI: confidence interval.
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Table 3 NQO1 genotype distribution and allele frequencies in 566 MDS/AML patients with certain karyotypic abnormalities. Pts No.
NQO1 genotype frequency (%) C/C
C/T
T/T
+8
202
107 (53.0)
71 (35.2)
24 (11.9)
−7/del(7q) −5/del(5q) −Y Del(20q)
153 199 65 51
88 (57.5) 125 (62.8) 36 (55.3) 21 (41.2)
56 (36.6) 68 (34.2) 25 (38.5) 30 (58.8)
9 (5.9) 6 (3.0) 4 (6.2) 0 (0.0)
p
0.001
p*
Allele frequency C
T
<0.0001
285 (0.705)
119 (0.295)
ns ns ns 0.004
232 (0.758) 318 (0.799) 97 (0.746) 72 (0.706)
74 (0.242) 80 (0.201) 33 (0.254) 30 (0.294)
p
p*
0.032
<0.0001 OR = 1.77, 95% CI = [2.33 − 1.34] ns ns ns 0.021 OR = 1.76, 95% CI = [2.79 − 1.11]
The p*-value was calculated by comparison of NQO1 genotypic distribution between each cytogenetic group and controls; ns: not significant; OR: odds ratio; CI: confidence interval.
4. Discussion In this study we investigated whether NQO1 C609 T gene polymorphism might have an impact on MDS pathogenesis and we examined its potential relationship with patients’ characteristics and cytogenetic findings. The cytogenetic features of our study group of MDS patients were found to be comparable to those of prior reports from Europe comprising large series of patients [5,6]. Similarly, the observed frequencies of homozygous variant genotype among the Greek healthy donors fell into the range previously reported in Caucasian populations [18]. Our case–control study, which represents the largest series of patients with primary MDS ever evaluated for the NQO1 C609 T gene polymorphism, showed no differences in the frequencies of the NQO1 variant genotypes between patients and healthy donors. The result suggests that NQO1 polymorphism does not correlate with susceptibility to de novo MDS. The finding is consistent with a prior investigation on patients from Italy in which similar incidences of NQO1 genotypes have been reported in 114 de novo MDS patients and 159 controls [29]. Given that MDS more commonly affects men that women, we examined a possible association between gender and NQO1 polymorphism. The results revealed similar allele frequencies and genotypic distributions in males and females in both patient and control populations, suggesting that the NQO1 polymorphism do not modulate MDS risk in a gender-dependent manner. Nevertheless, male patients harboring the NQO1 T/T variant genotype show a higher risk of developing acute promyelocytic leukemia than their female counterparts [36]. Higher incidences of variant genotypes were observed in MDS patients with +8, −7/del(7q) and del(20q). Admittedly, the number of cases in each cytogenetic group was too small to draw safe conclusions. Thereby, this observation prompted us to investigate the persistence of this association by studying larger series of patients. To this end, we retrospectively analyzed the NQO1 gene status in 566 MDS/AML patients with −5/del(5q), −7/del(7q), +8, del(20q) and −Y. Interestingly, the results showed that patients with either +8 or del(20q) exhibited a 1.8-fold increased risk of carrying at least one variant T allele, as compared to the allele frequencies of the control population. Particularly, genotypic analysis revealed significantly increased frequencies of heterozygotes (C/T) among patients with del(20q) and homozygous variant individuals (T/T) among patients with +8, as compared to either healthy donors or other cytogenetic groups. The latter finding showing a 5-fold increased frequency of homozygotes T/T among patients with +8 might suggest a potentially causative role of lack of NQO1 activity in the occurrence of trisomy 8 in MDS/AML. NQO1 induction by hematotoxic metabolites in the bone marrow comprises a protective mechanism against quinone-induced damage that could lead to leukemogenic effects [12,13]. Individuals carrying the T/T genotype lack NQO1 protein and therefore present
increased susceptibility to various types of leukemia [9]. Taken the above together with the increased frequency of variant homozygotes observed in patients with +8, it could be suggested that NQO1 enzyme deficiency may affect individual’s vulnerability to hematotoxicity and may contribute to an increased risk of trisomy 8 in MDS/AML. This is strengthened by the increased chromosome 8 aneusomy observed in human CD34+ blood progenitor cells after exposure to benzene metabolites [37]. Previous occupational and leisure-time exposure to benzene and other organic solvents has been suggested to increase the risk for AML with trisomy 8 as sole aberration [38,39]. Until now, the mechanism of trisomy 8 occurrence remains unclear. It has been proposed that hematotoxic metabolites induce aneuploidy through errors in chromosome segregation as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint [40]. Trisomy 8, arising from genetic instability, provides proliferative advantage to the cells which present clonal expansion over cells of normal karyotype and fail to undergo apoptosis [41,42]. Based on the case–control study, our results do not provide evidence for a pathogenetic role of the NQO1 polymorphism on de novo MDS susceptibility. Nevertheless, the 6-fold increased frequency of homozygous variant T/T genotype found in patients with isolated trisomy 8 is noteworthy and may indicate a strong association between lack of NQO1 activity and trisomy 8 occurrence in MDS/AML. Further association studies on this functional polymorphism and disease progression might confer a subgroup with defined prognostic significance within the heterogeneous population of patients with trisomy 8. Role of the funding source This work has been co-financed by the European Union (European Social Fund, ESF) and Greek National funds through the Operational Program “Education and Lifelong Learning” of the NSRF-Research Funding Program “Heracleitus II”. Study Sponsor had no involvement in the study design, collection, analysis and interpretation of data, writing of manuscript or submitting for publication. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The authors thank E.M. Delicha for statistical analysis and P. Diamantopoulou, K. Stavropoulou for excellent technical assistance. Contributions. SZ and ChS contributed equally to this manuscript. SZ, ChS, TK, MK, AD and DK performed laboratory work and
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[21] Naoe T, Takeyama K, Yokozama T, Kiyoi H, Seto M, Uike N, et al. Analysis of genetic polymorphism in NQO1, GST-M1, GST-T1, and CYP3A4 in 469 Japanese patients with therapy-related leukemia/Myelodysplastic syndrome and de novo acute myeloid leukemia. Clin Cancer Res 2000;6:4091–5. [22] Bolufer P, Collado M, Barragan E, Calasanz MJ, Colomer D, Tormo M, et al. Profile of polymorphisms of drug-metabolising enzymes and the risk of therapyrelated leukaemia. Br J Haematol 2007;136(4):590–6. [23] Krajinovic M, Sinnett H, Richer C, Labuda D, Sinnett D. Role of NQO1, MPO and CYP2E1 genetic polymorphisms in the susceptibility to childhood acute lymphoblastic leukemia. Int J Cancer 2002;10;97(2):230–6. [24] Guha N, Chang JS, Chokkalingam AP, Wiemels JL, Smith MT, Buffler PA. NQO1 polymorphisms and de novo childhood leukemia: a HuGE review and metaanalysis. Am J Epidemiol 2008;168:1221–32. [25] Seedhouse C, Bainton R, Lewis M, Harding A, Russell N, Das-Gupta E. The genotypic distribution of the XRCC1 gene indicates a role for base excition repair in the development of therapy-related acute myeloblastic leukemia. Blood 2002;100:3761–6. [26] Kracht T, Schrappe M, Strehl S, Reiter A, Elsner HA, Trka J, et al. NQO1 C609T polymorphism in distinct entities of pediatric hematologic neoplasms. Haematologica 2004;89:1492–7. [27] Rund D, Krichevsky S, Bar-Cohen S, Goldschmidt N, Kedmi M, Malik E, et al. Therapy-related leukemia: clinical characteristics and analysis of new molecular risk factors in 96 adult patients. Leukemia 2005;19:1919–28. [28] Malik E, Cohen SB, Sahar D, Dann EJ, Rund D. The frequencies of NAD(P)H quinone oxidoreductase (NQO1) variant allele in Israeli ethnic groups and the relationship of NQO1*2 to adult acute myeloid leukemia in Israeli patients. Haematologica 2006;91:956–9. [29] Voso MT, Fagiani E, D’Alo F, Guidi F, Di Ruscio A, Sica S, et al. Increased risk of acute myeloid leukaemia due to polymorphisms in detoxification and DNA repair enzymes. Ann Oncol 2007;18:1523–8. [30] Stavropoulou C, Sambani C, Rigana H, Georgakakos VN, Voutsinas G, Manola KN, et al. Low frequency of the glutathione-S-transferase T1-null genotype in patients with primary Myelodysplastic syndrome and 5q deletion. Leukemia 2008;22:1643–6. [31] Wiktor A, Rybicki BA, Piao ZS, Shurafa M, Barthel B, et al. Clinical significance of Y chromosome loss in hematologic disease. Genes Chromosomes Cancer 2000;27(1):11–6. [32] Shaffer LG, Slova KML, Campell LJ, editors. ISCN 2009: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland: S. Karger; 2009. [33] Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, et al. International scoring system for evaluating prognosis in Myelodysplastic syndromes. Blood 1997;89:2079–88. [34] Park SJ, Zhao H, Spitz MR, Grossman HB, Xifeng W. An association between NQO1 genetic polymorphism and risk of bladder cancer. Mutat Res 2003;536:131–7. [35] Harth V, Donat S, Ko Y, Abel J, Vetter H, Brünning T. NAD(P)H quinone oxidoreductase 1 codon 609 polymorphism and its association to colorectal cancer. Arch Toxicol 2000;73:528–31. [36] Bolufer P, Collado M, Barragán E, Cervera J, Calasanz MJ, Colomer D, et al. The potential effect of gender in combination with common genetic polymorphisms of drug-metabolizing enzymes on the risk of developing acute leukemia. Haematologica 2007;92:308–14. [37] Smith MT, Zhang L, Jeng M, Wang Y, Guo W, Duramad P, et al. Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells. Carcinogenesis 2000;21:1485–90. [38] Albin M, Björk J, Welinder H, Tinnerberg H, Mauritzson N, Johansson B, et al. Acute myeloid leukemia and clonal chromosome aberrations in relation to past exposure to organic solvents. Scand J Work Environ Health 2000;26:482–91. [39] Davico L, Sacerdote C, Ciccone G, Pegoraro L, Kerim S, Ponzio G, et al. Chromosome 8, occupational exposures, smoking, and acute nonlymphocytic leukemias: a population-based study. Cancer Epidemiol Biomarkers Prev 1998;7:1123–5. [40] King RW. When 2 + 2 = 5: the origins and fates of aneuploid and tetraploid cells. Biochim Biophys Acta 2008;1786:4–14. [41] Sloand EM, Pfannes L, Chen G, Shah S, Solomou EE, Barrett J, et al. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins. Blood 2007;109:2399–405. [42] Zhang L, Lan Q, Guo W, Hubbard AE, Li G, Rappaport SM, et al. Chromosomewide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene. Carcinogenesis 2011;32:605–12.
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High frequency of NAD(P)H:quinone oxidoreductase 1 (NQO1) C609 T germline polymorphism in MDS/AML with trisomy 8 Sophia Zachaki a,b , Chrysa Stavropoulou a , Theodora Koromila c , Kalliopi N. Manola a , Marina Kalomoiraki a , Aggeliki Daraki a , Daphne Koumbi a , Anastasia Athanasiadou d , Emmanuel Kanavakis b,e , Panagoula Kollia c , Constantina Sambani a,∗ a
Laboratory of Health Physics, Radiobiology and Cytogenetics, NCSR “Demokritos”, Athens, Greece Department of Medical Genetics, Faculty of Medicine, University of Athens, Greece c Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Greece d Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece e Research Institute for the Study of Genetic and Malignant Disorders in Childhood, St. Sophia’s Children’s Hospital, Athens, Greece b
a r t i c l e
i n f o
Article history: Received 26 August 2012 Received in revised form 6 March 2013 Accepted 9 April 2013 Available online 1 May 2013 Keywords: MDS AML Genetic susceptibility Single nucleotide polymorphism (SNP) NAD(P)H:quinone oxidoreductase 1 (NQO1) Trisomy 8 (+8)
a b s t r a c t The NQO1 C609 T germline polymorphism resulting in a lowering of enzyme activity may confer susceptibility to MDS. To assess this association, we performed a case–control study including 330 Greek patients with de novo MDS and 416 healthy donors, using a Real-Time PCR genotyping method. Focusing on cytogenetic aberrations most commonly found in MDS, we retrospectively genotyped 566 MDS/AML patients carrying −5/del(5q), −7/del(7q), +8, del(20q) and −Y. The case–control analysis revealed no differences in NQO1 genotype distribution. Interestingly, a 6-fold increased frequency of the homozygous variant genotype was observed among patients with isolated trisomy 8 (p < 0.0001), suggesting that null NQO1 activity may influence the occurrence of +8 in MDS/AML. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Myelodysplastic syndromes (MDS) comprise a heterogeneous group of acquired clonal hematopoietic stem cell disorders, characterized by bone marrow hypercellularity, dysplasia, various degrees of cytopenia and an advanced risk of progression to acute myeloid leukemia (AML) [1–3]. Acquired clonal chromosomal abnormalities are found in about 30–50% of de novo MDS and approximately 80% of secondary or therapy-related MDS cases [4]. These abnormalities are predominantly characterized by total/partial chromosomal losses or gains and rarely by balanced structural aberrations. The most frequently observed chromosomal abnormalities include long-arm deletions of chromosomes 5, 7 and 20, whereas, trisomy 8 represents the most common chromosomal gain [5,6]. The etiology is unknown in the majority of cases; however, models for the development of sporadic MDS and
∗ Corresponding author at: Laboratory of Health Physics, Radiobiology and Cytogenetics, NCSR “Demokritos”, 153 41 Agia Paraskevi, Athens, Greece. Tel.: +30 2106503866; fax: +30 210 6534710. E-mail address: [email protected] (C. Sambani). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.04.015
AML suggest the role of cumulative genetic and toxic environmental factors in genetically predisposed individuals [7]. Clues to the etiology of myelodysplasia are expected to be gained through the study of genetic susceptibility in candidate genes. The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that protects cells against oxidative damage. It plays an important role in detoxification of quinones derived from the oxidation of phenolic metabolites of benzene, by catalyzing two or four electron reductions of these substrates [8]. These reductions are beneficial to the cell by preventing redox cycling and the generation of free radicals [9,10]. The NQO1 is ubiquitously present in all tissues, including bone marrow [10–13]. As it has been reported, bone marrow cells including CD34+ progenitors with negligible NQO1 production show an increased susceptibility to benzene toxicity [8]. The pivotal role of NQO1 activity is reinforced by studies demonstrating that loss of NQO1 gene expression in NQO1-null mice causes myeloid hyperplasia of bone marrow and significant increase in blood neutrophils [14]. The NQO1 enzyme activity strongly depends on a singlenucleotide polymorphism (SNP) at the NQO1 locus. Specifically, a cytosine-to-thymine substitution (C→T) at position 609 of the cDNA (C609 T) produces a proline to serine substitution at amino
S. Zachaki et al. / Leukemia Research 37 (2013) 742–746
acid 187 (P187S) of the mature protein that destabilizes and inactivates the enzyme [15,16]. Individuals homozygous for the mutant allele (T/T) completely lack NQO1 activity, whereas heterozygotes (C/T) present approximately threefold decreased enzyme activity [17]. The frequency of the NQO1 C609 T polymorphism exhibits ethnic variation with the highest prevalence of the homozygous variant genotype (T/T) occurring in Asian populations (19% Korean; 22% Chinese) and the lowest prevalence in Caucasians (4%) [18]. The NQO1 C609 T gene polymorphism has been previously investigated as a predisposing element in various types of hematological malignancies. Several results suggest that individuals carrying NQO1 variant genotypes may be at increased risk for the development of de novo MDS and AML [19,20], t-AML [16,17,21,22], childhood acute lymphoblastic leukemia (ALL) [23] and ALL with MLL rearrangements in infants [24]. However, other reports have come to contradictory results [25–29], possibly due to the heterogeneity of the diseases, insufficient sample sizes and ethnic variations [30]. Thus far, the role of the NQO1 polymorphism in de novo MDS susceptibility remains elusive. Considering the critical role of the NQO1 enzyme in detoxification mechanisms implicated in MDS pathogenesis, we hypothesize that the NQO1 germline polymorphism influencing NQO1 activity, may lead to an additive effect on bone marrow toxicity and thus, may confer susceptibility to MDS development and/or promote certain chromosomal changes. To this end, we conducted a case–control study to evaluate the NQO1 genotype in a large series of 330 Greek patients with MDS and 416 healthy donors. The NQO1 gene status was also evaluated in relation to patients’ characteristics. Focusing on most common cytogenetic aberrations found in MDS, we retrospectively analyzed the NQO1 genotypic distribution in 566 patients with MDS/AML carrying −5/del(5q), −7/del(7q), trisomy 8, del(20q) and −Y. 2. Materials and methods 2.1. Patients The case–control study included 330 patients with confirmed diagnosis of primary MDS (217 males, 113 females, median age 68 years) and 416 gender and age matched healthy individuals (270 males, 146 females, median age 66.5 years). Diagnosis of MDS was established in the major Greek hospitals. Patients with MDS secondary to a previous malignancy or a history of chemo/radiotherapy were excluded from the study. Healthy donors were unrelated individuals with a negative history for previous malignancies and normal peripheral blood cell counts. Both cases and controls enrolled in the study came from different areas of Greece, thus having a homogeneous ethnic background. Leftover bone marrow (BM) specimens from patients and peripheral blood (PB) samples from healthy donors were used for DNA extraction and subsequent genotypic analysis. Genomic DNA was also extracted from 566 MDS/AML patients with known specific cytogenetic abnormalities. Among them, 199 cases showed −5/del(5q), 153 had −7/del(7q), 202 presented trisomy 8 (+8), 51 cases had del(20q) and 65 cases exhibited loss of the Y chromosome (−Y). As a sole change, Y loss was considered as disease-associated clonal abnormality when found in more than 75% of metaphase cells [31]. A total of 84 of 566 patients carrying more than one of the mentioned recurrent aberrations were included in more than one corresponding cytogenetic category. The study was approved by the Ethical Scientific Committee of the National and Kapodistrian University of Athens and written informed consent was provided from all sample donors included in the study.
2.2. Cytogenetic analysis BM samples at diagnosis were submitted for chromosome studies at the Cytogenetics Unit, NCSR “Demokritos”, for centralized karyotyping. The cytogenetic analysis was performed using unstimulated short-term cultures. Karyotypes were described in accordance to the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN 2009) [32]. Enrolment in the study required feasibility of karyotyping 15 or more metaphases per patient. Patients were stratified according to the IPSS cytogenetic classification into three risk groups; good: normal karyotype, del(5q), del(20q) or −Y as sole aberrations, intermediate: trisomy 8, single miscellaneous, double abnormalities and poor: complex karyotype (i.e., ≥3 anomalies) or chromosome 7 abnormalities [33].
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2.3. Genotype analysis Genomic DNA was extracted from PB cells of healthy donors or BM samples of MDS patients using the QIAamp DNA-extraction midikit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Isolated DNA was resuspended in TrisEDTA buffer, pH 8.0, and stored at −20 ◦ C until use. The NQO1 C609 T SNP genotyping was performed for all samples using both polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (PCR-RFLP) and a Real-Time PCR method. Both methods revealed the same NQO1 genotype result for all samples. The conventional PCR-RFLP assay for NQO1 genotyping was performed as described by Park SJ, 2003 [34]. The Real-Time PCR was performed on a LightCycler 1.0 using LightCycler DNA Master Hybridization Probes Kit (Roche, Mannheim, Germany). The PCR reaction was carried out according to the manufacturer’s instructions and sense and antisense primers as well as anchor hybridization probe sequences have previously described [35]. PCR primers and the hybridization probes were synthesized by Olfert Landt (TIB MOLBIOL, Berlin, Germany). The two NQO1 alleles (wild type C and mutant T) were distinguished by determination of melting curves using the LightCycler Software, version 3. 2.4. Statistical analysis Allele frequencies were determined by allele-counting method. Pearson Chi-square test with continuity correction were employed to investigate the case–control differences in the distribution of the genotypes or other parameters under study. Odds ratios (ORs) are given with 95% confidence interval (CI). All tests were two-sided and statistical significance was set at 5%. Statistical analysis was performed using SPSS (Statistical Package for the Social Sciences) version 12 software.
3. Results 3.1. Clinical characteristics, cytogenetic results and NQO1 genotypes Table 1 summarizes the clinical and cytogenetic characteristics of the 330 patients with primary MDS studied for the NQO1 genotypic distribution. In the present series of patients, 217 men and 113 women (male:female ratio 2:1) were included. The median age was 68 years (range 19–92 years). Two hundred eighty seven patients (287/330, 87%) were older than 60 years. A successful karyotypic result was achieved in 290 out of 330 patients (88%). Of the 290 patients, 108 (37.2%) showed clonal karyotypic abnormalities. None of them showed abnormalities of chromosome 16q22.1 (gene location of NQO1). The most commonly detected cytogenetic abnormalities found as sole abnormalities were +8 (9.7%), −7/del(7q) (3.5%), −Y (2.8%), del(5q) (1.8%), and del(20q) (0.7%), with trisomy 8 being the most frequently observed single change. Regarding NQO1 gene status, the distribution of the variant genotypes (heterozygotes C/T and homozygotes T/T) did not differ significantly between male and female patients (36.4 vs 26.5% and 1.4 vs 2.7%, respectively). According to age, a higher frequency of the homozygous variant genotype (T/T) was observed in patients ≤60 years, as compared to older patients (4.7% vs 1.4%, respectively). As for IPSS prognostic groups, higher frequencies of the C/T and T/T variant genotypes were observed in patients belonging to the intermediate and the poor risk groups, respectively; however, the above trends did not reach statistical significance. Higher incidences of C/T and T/T mutant genotypes were also observed in patients with trisomy 8 and −7/del(7q), as compared to the other cytogenetic categories. 3.2. NQO1 genotypes and MDS susceptibility Table 2 summarizes the distribution of NQO1 genotypes and allele frequencies among MDS patients and controls. In the control population, the NQO1 genotype was distributed as follows: 62.3% wild type (C/C), 35.3% heterozygous (C/T) and 2.4% homozygous mutants (T/T). These frequencies observed within the sample of healthy Greek donors for the C/T and T/T variant NQO1 genotypes
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S. Zachaki et al. / Leukemia Research 37 (2013) 742–746
Table 1 Distribution of NQO1 genotype in MDS patients according to patients’ characteristics and cytogenetic subgroups. No. (%)
NQO1 genotype frequency (%)
n = 330
C/C
C/T
T/T
Gender Male Female
217 (65.8) 113 (34.2)
135 (62.2) 80 (70.8)
79 (36.4) 30 (26.5)
3 (1.4) 3 (2.7)
ns*
Age (years) ≤60 ≥61
43 (13) 287 (87)
26 (60.4) 188 (65.5)
15 (34.9) 95 (33.1)
2 (4.7) 4 (1.4)
ns
91 (36.7) 34 (13.7) 65 (26.2) 29 (11.7) 29 (11.7)
62 (68.1) 24 (70.6) 42 (64.6) 15 (51.7) 22 (75.8)
28 (30.8) 9 (26.5) 22 (33.9) 14 (48.3) 6 (20.7)
1 (1.1) 1 (2.9) 1 (1.5) 0 (0.0) 1 (3.5)
ns
Karyotypeb Normal Abnormal +8 −7/del(7q) −Y Del(5q) Del(20q) Other (single or double) Complex
182 (62.8) 108 (37.2) 28 (9.7) 10 (3.5) 8 (2.8) 5 (1.8) 2 (0.7) 22 (7.6) 33 (11.4)
108 (59.3) 65 (60.2) 11 (39.3) 5 (50.0) 6 (75.0) 4 (80.0) 1 (50.0) 16 (72.7) 22 (66.7)
71 (39.0) 40 (37.0) 16 (57.1) 4 (40.0) 2 (25.0) 1 (20.0) 1 (50.0) 6 (27.3) 10 (30.3)
3 (1.7) 3 (2.8) 1 (3.6) 1 (10.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (3.0)
ns
IPSS risk groupsc Good Intermediate Poor
196 (67.5) 61 (21.0) 33 (11.7)
126 (64.3) 27 (44.3) 20 (60.6)
67 (34.2) 33 (54.1) 11 (33.3)
3 (1.5) 1 (1.6) 2 (6.1)
ns
FABa RA RARS RAEB RAEB-T CMML
p
a,b,c Percentages calculated on the number of patients with known FAB classification (248 out of 330)a and with available cytogenetic data (290/330)b,c . Cytogenetic risk groups were defined according to Greenberg et al. [33]. *ns: not significant.
del(20q) (p = 0.001, 2 = 27.503, d.f. = 8). Allele frequencies distribution analysis confirmed these differences, showing a higher T allele frequency among patients with +8 and del(20q), as compared to the other cytogenetic subgroups (p = 0.032, 2 = 10.526, d.f. = 4). Comparison between patients and controls revealed a 5-fold increased frequency of the NQO1 homozygous T/T genotype in patients with +8 (11.9% vs 2.4%, respectively) (p* < 0.0001, 2 = 24.182, d.f. = 2) (Table 3). Among the 202 patients with trisomy 8, 159 presented sole +8, of which 22 patients carried the T/T variant genotype (13.8%). Also, comparison between patients with sole +8 and controls showed an even higher frequency of T/T genotype (6-fold increase). A higher incidence of heterozygotes C/T among patients with del(20q) was observed as compared to the controls (58.8% vs 35.3%, respectively) (p* = 0.004, 2 = 11.206, d.f. = 2). Allele frequencies distribution analysis between patients and controls showed that patients with +8 exhibited a 1.8-fold increased risk of carrying at least one variant T allele, as compared to the expected values based on the control population (p* < 0.0001, 2 = 16.652, d.f. = 1, OR = 1.77, 95%CI = [2.33 − 1.34]). Similarly, patients with del(20q) showed a 1.8-fold increased risk of carrying at least one variant T allele (p* = 0.021, 2 = 5.356, d.f. = 1, OR = 1.76, 95%CI = [2.79 − 1.11]) (Table 3).
are in accordance with the Hardy–Weinberg laws of equilibrium (2 = 4.269, d.f. = 1, p < 0.01) and fell into the range of values registered in other Caucasian populations [18]. Comparison of the NQO1 genotype and allele frequencies distribution between MDS cases and controls revealed no significant differences of the variant genotypes (C/T and T/T) (34.2 vs 35.3% and 1.8 vs 2.4%, respectively) (p > 0.05) (Table 2). 3.3. NQO1 genotypes in patients with MDS/AML and specific chromosome aberrations The higher incidences of variant NQO1 genotypes observed in MDS patients with +8 and −7/del(7q) (Table 1), prompted us to analyze the NQO1 gene status in a large number of patients carrying certain specific abnormalities in their karyotype. For this purpose, we retrospectively analyzed a cohort of 566 patients with MDS/AML. Among them, 199 cases showed −5/del(5q), 153 had −7/del(7q), 202 showed +8, 51 cases had del(20q) and 65 cases exhibited Y loss. Table 3 summarizes the distribution of the NQO1 genotype and the allele frequencies among the different cytogenetic groups. Interestingly, we observed significantly higher incidences of NQO1 homozygotes for the T allele (T/T) into the group of patients with +8 and heterozygotes (C/T) among patients with
Table 2 Genotype distribution and allele frequencies of the NQO1 C609 T polymorphism in MDS cases and healthy controls. Group
No.
MDS patients Controls
330 416
NQO1 genotype frequency (%)
Allele frequency
C/C
C/T
T/T
C
T
211 (64.0) 259 (62.3) 2 = 0.440, d.f. = 2 p = 0.803
113 (34.2) 147 (35.3)
6 (1.8) 10 (2.4)
535 (0.811) 665 (0.799) 2 = 0.006, d.f. = 1 p = 0.991
125 (0.189) 157 (0.201)
The p*-value was calculated by comparison of NQO1 genotypic distribution between each cytogenetic group and controls; ns: not significant; OR: odds ratio; CI: confidence interval.
S. Zachaki et al. / Leukemia Research 37 (2013) 742–746
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Table 3 NQO1 genotype distribution and allele frequencies in 566 MDS/AML patients with certain karyotypic abnormalities. Pts No.
NQO1 genotype frequency (%) C/C
C/T
T/T
+8
202
107 (53.0)
71 (35.2)
24 (11.9)
−7/del(7q) −5/del(5q) −Y Del(20q)
153 199 65 51
88 (57.5) 125 (62.8) 36 (55.3) 21 (41.2)
56 (36.6) 68 (34.2) 25 (38.5) 30 (58.8)
9 (5.9) 6 (3.0) 4 (6.2) 0 (0.0)
p
0.001
p*
Allele frequency C
T
<0.0001
285 (0.705)
119 (0.295)
ns ns ns 0.004
232 (0.758) 318 (0.799) 97 (0.746) 72 (0.706)
74 (0.242) 80 (0.201) 33 (0.254) 30 (0.294)
p
p*
0.032
<0.0001 OR = 1.77, 95% CI = [2.33 − 1.34] ns ns ns 0.021 OR = 1.76, 95% CI = [2.79 − 1.11]
The p*-value was calculated by comparison of NQO1 genotypic distribution between each cytogenetic group and controls; ns: not significant; OR: odds ratio; CI: confidence interval.
4. Discussion In this study we investigated whether NQO1 C609 T gene polymorphism might have an impact on MDS pathogenesis and we examined its potential relationship with patients’ characteristics and cytogenetic findings. The cytogenetic features of our study group of MDS patients were found to be comparable to those of prior reports from Europe comprising large series of patients [5,6]. Similarly, the observed frequencies of homozygous variant genotype among the Greek healthy donors fell into the range previously reported in Caucasian populations [18]. Our case–control study, which represents the largest series of patients with primary MDS ever evaluated for the NQO1 C609 T gene polymorphism, showed no differences in the frequencies of the NQO1 variant genotypes between patients and healthy donors. The result suggests that NQO1 polymorphism does not correlate with susceptibility to de novo MDS. The finding is consistent with a prior investigation on patients from Italy in which similar incidences of NQO1 genotypes have been reported in 114 de novo MDS patients and 159 controls [29]. Given that MDS more commonly affects men that women, we examined a possible association between gender and NQO1 polymorphism. The results revealed similar allele frequencies and genotypic distributions in males and females in both patient and control populations, suggesting that the NQO1 polymorphism do not modulate MDS risk in a gender-dependent manner. Nevertheless, male patients harboring the NQO1 T/T variant genotype show a higher risk of developing acute promyelocytic leukemia than their female counterparts [36]. Higher incidences of variant genotypes were observed in MDS patients with +8, −7/del(7q) and del(20q). Admittedly, the number of cases in each cytogenetic group was too small to draw safe conclusions. Thereby, this observation prompted us to investigate the persistence of this association by studying larger series of patients. To this end, we retrospectively analyzed the NQO1 gene status in 566 MDS/AML patients with −5/del(5q), −7/del(7q), +8, del(20q) and −Y. Interestingly, the results showed that patients with either +8 or del(20q) exhibited a 1.8-fold increased risk of carrying at least one variant T allele, as compared to the allele frequencies of the control population. Particularly, genotypic analysis revealed significantly increased frequencies of heterozygotes (C/T) among patients with del(20q) and homozygous variant individuals (T/T) among patients with +8, as compared to either healthy donors or other cytogenetic groups. The latter finding showing a 5-fold increased frequency of homozygotes T/T among patients with +8 might suggest a potentially causative role of lack of NQO1 activity in the occurrence of trisomy 8 in MDS/AML. NQO1 induction by hematotoxic metabolites in the bone marrow comprises a protective mechanism against quinone-induced damage that could lead to leukemogenic effects [12,13]. Individuals carrying the T/T genotype lack NQO1 protein and therefore present
increased susceptibility to various types of leukemia [9]. Taken the above together with the increased frequency of variant homozygotes observed in patients with +8, it could be suggested that NQO1 enzyme deficiency may affect individual’s vulnerability to hematotoxicity and may contribute to an increased risk of trisomy 8 in MDS/AML. This is strengthened by the increased chromosome 8 aneusomy observed in human CD34+ blood progenitor cells after exposure to benzene metabolites [37]. Previous occupational and leisure-time exposure to benzene and other organic solvents has been suggested to increase the risk for AML with trisomy 8 as sole aberration [38,39]. Until now, the mechanism of trisomy 8 occurrence remains unclear. It has been proposed that hematotoxic metabolites induce aneuploidy through errors in chromosome segregation as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint [40]. Trisomy 8, arising from genetic instability, provides proliferative advantage to the cells which present clonal expansion over cells of normal karyotype and fail to undergo apoptosis [41,42]. Based on the case–control study, our results do not provide evidence for a pathogenetic role of the NQO1 polymorphism on de novo MDS susceptibility. Nevertheless, the 6-fold increased frequency of homozygous variant T/T genotype found in patients with isolated trisomy 8 is noteworthy and may indicate a strong association between lack of NQO1 activity and trisomy 8 occurrence in MDS/AML. Further association studies on this functional polymorphism and disease progression might confer a subgroup with defined prognostic significance within the heterogeneous population of patients with trisomy 8. Role of the funding source This work has been co-financed by the European Union (European Social Fund, ESF) and Greek National funds through the Operational Program “Education and Lifelong Learning” of the NSRF-Research Funding Program “Heracleitus II”. Study Sponsor had no involvement in the study design, collection, analysis and interpretation of data, writing of manuscript or submitting for publication. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The authors thank E.M. Delicha for statistical analysis and P. Diamantopoulou, K. Stavropoulou for excellent technical assistance. Contributions. SZ and ChS contributed equally to this manuscript. SZ, ChS, TK, MK, AD and DK performed laboratory work and
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