In this study we analyzed the effect of polymorphic variation of NAD(P)H: quinone oxidoreductase1 (NQO1) gene that
encode enzyme which detoxifies harmful quinines and protect hematopoietic stem cells against oxidative stress. C609T
polymorphism of NQO1 gene leads to loss of enzyme activity, which may be a risk factor in the etiology of specific types of
hematopoietic malignancies.We analyzed C609T polymorphism in NQO1 gene in the group of 82 patients (56 adult and
26 children) with different type of hematopoietic malignancies and 99 healthy participants (61 adult and 38 children)
using PCR and the RFLP method. We confirmed that the polymorphism C609T in NQO1 gene was more frequent in the
adult patients’ group with myeloid disorders, (p=0.0267) compared with adult controls.We could not confirm the association
C609T polymorphism with recurrent chromosome translocations (clonal karyotype changes) neither in the adult
nor in pediatric group of patients

Bernarda Lozić

Dragan Primorac

Radenka Kuzmanić Šamija

Robert Glavinić

Tatijana Zemunik

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Coll. Antropol. 35 (2011) 2: 385–388Original scientific paperAnalysis of the C609T Polymorphism of NQO1Gene in South Croatian Patients withHematological MalignanciesBernarda Lozi}1, Dragan Primorac2,3,4,5, Robert Glavini}6, Radenka Kuzmani} [amija1 andTatijana Zemunik71 University of Split, Split University Hospital Center, Department of Pediatrics, Split, Croatia2 University of Split, School of Medicine, Split, Croatia3 »J. J. Strossmayer« University, School of Medicine, Osijek, Croatia4 Penn State University, Eberly College of Science, University Park, USA5 University of New Haven, West Haven, USA6 University of Split, Split University Hospital Center, Split, Croatia7 University of Split, School of Medicine, Department of Medical Biology, Split, CroatiaA B S T R A C TIn this study we analyzed the effect of polymorphic variation of NAD(P)H: quinone oxidoreductase1 (NQO1) gene thatencode enzyme which detoxifies harmful quinines and protect hematopoietic stem cells against oxidative stress. C609Tpolymorphism of NQO1 gene leads to loss of enzyme activity, which may be a risk factor in the etiology of specific types ofhematopoietic malignancies.We analyzed C609T polymorphism in NQO1 gene in the group of 82 patients (56 adult and26 children) with different type of hematopoietic malignancies and 99 healthy participants (61 adult and 38 children)using PCR and the RFLP method. We confirmed that the polymorphism C609T in NQO1 gene was more frequent in theadult patients’ group with myeloid disorders, (p=0.0267) compared with adult controls. We could not confirm the associ-ation C609T polymorphism with recurrent chromosome translocations (clonal karyotype changes) neither in the adultnor in pediatric group of patients.Key words: hematological malignancies, polymorphism, NQO1, clonal chromosomal abnormalitiesIntroductionThe World Health Organization (WHO) classifies he-matological malignant diseases according to their lineage(myeloid, lymphoid, histiocytic/dendritic) and distingui-shes neoplasm of precursor cells from those comprised offunctionally mature cells. Within each category, distinctdiseases are defined according to a combination of mor-phology, immunophenotype, genetic futures and clinicalsyndromes1. Acute lymphoblastic leukemia (ALL) is themost common type of leukemia in early childhood whileprimary myeloid disorder (acute or chronic) is the mostcommon type of leukemia in the elderly2.From a clinical point of view it is very important tounderstand myeloid and lymphoid classification of neo-plasm3. Hematopoietic malignancies are clonal diseasesthat arise due to genetic changes in one hematopoieticstem cell in the bone marrow or lymph tissue4. Such al-tered cell divides forming abnormal clone of cells, whichcan be determined by cytogenetic findings of the same orsimilar changes in three metaphases5. Series of low--penetrance genes can be involved in the development ofhematological malignant diseases. An individual’s risk ofcomplex disease development presents a cumulative ef-fect of genetic and environmental factors. It seams thatpolymorphisms of certain genes play a role in leuke-mogenesis6.Therefore, in this study we analyzed the polymor-phism of the NQO1 gene that encode protein responsiblefor detoxification of endogenous and exogenous muta-gens and may influence susceptibility to leukemia deve-lopment6–11.385Received for publication March 11, 2011NAD(P)H: quinine oxidoreductase1 (NQO1) is a de-toxification enzyme that protects cells against oxidativestress and toxic quinines (11). NQO1 catalyze a 2-elec-tron reduction of quinines, causing prevention of theirparticipation in redox cycling and thus in oxidativestress6.Individuals with a lack of NQO1 activity are moresusceptible to the toxic and carcinogenic effects of qui-nines (reactive metabolites of benzene) and have in-creased risk of hematotoxicity and leukemia6,12. The hu-man NQO1 gene is located on chromosome 16q22.1 andhas six exons (13). Polymorphism C to T at position –609causes proline to serine substitution in codone 187 in theNQO1 protein. Homozygous T/T show the loss of enzymeactivity, and heterozygous C/T show low to intermediateNQO1 activity. It has been reported that children areparticularly more vulnerable than adults to oxidativestress and toxic quinines because their metabolism is lessdeveloped/matured, and they have a higher level of celldivision and growth6,12.In this case-control study, we analysed whether theNQO1 variation increase risk for an individual’s suscep-tibility to hematological malignancies. Our study wasconducted in Southern Croatian population, which canbe considered relatively homogenous from both geneticand socio-cultural points of view.Material and MethodsIn this study we investigated 82 patients (34 femalesand 48 males) and 99 healthy participants (45 femalesand 54 males). Investigated group of patients was dividedaccording to the age in two groups. Most of the patients(56 individuals) were of adult age (median age 58 years;range 34–82 years) with different types of myeloid neo-plasms. The rest of them were pediatric patients (26 indi-viduals) (median age 8.5 years; range 0.3–17 years) withprevalence of lymphoid neoplasms (20 individuals). Thegroup of healthy participants (61 adult and 38 pediatricindividuals) has been adjusted in age and sex distribu-tion with patient group. The cases had been diagnosed inthe Department of Pediatric and Internal Medicine ofSplit University Hospital between 2002 and 2007. Thediagnosis was established according to standard rules ofthe WHO classification. All patients were karyotyped us-ing previously described methods of cytogenetic analysison pretreatment bone marrow cells14.In our study we investigated C609T polymorphism ofthe NQO1 gene (rs1800566) using previously describedstandard methods15. For detection of C609T polymor-phism, we performed the polymerase chain reaction (PCR)and restriction fragment length polymorphism (RFLP)after digestion with restriction enzyme HifnI. The di-gested fragments were separated and visualized on aga-rose or polyacrylamide gels. Fragments of 151, 120, and33 bp on gels confirmed the presence of the polymorphicHinfI restriction site yields.NQO1 genotype frequencies in leukemia subgroupsand controls were compared by the c2-test. P-values lessthan 0.05 were considered nominally significant. Statis-tical analysis was performed using statistical packageMedCalc (Mariakerke, Belgium).ResultsThe distribution of C609T genotypes of the NQO1gene, in the adult patient group with myeloid disorderand adult controls are reported in Table 1. Genotype fre-quencies showed statistically significant difference be-tween those two groups (p=0.0267).A heterozygote (C/T) genotype was most frequent inthe group of unclassifiable myeloproliferative neoplasms(MPN) (7/15).Recurrent chromosome abnormalities (clonal abnor-malities) detected in the leukemia cells of the adult pa-tient group showed a wide spectrum of clonal karyotypeaberrations, and were found in 25/56 patients (12 ofthem were C609T heterozygous). The most frequentclonal rearrangement was reciprocate translocationt(9;22) observed in 14 patients exclusively with chronicmyelogenous leukemia BCR-ABL positive (CML), (5 het-erozygous). Heterozygote genotypes were also found inpatients with other different clonal abnormalities [dele-tion in chromosome 20q (2/2) and different abnormalitieswhich include chromosome 17q (3/3). However, no associ-ation was found between specific chromosomal abnor-malities and C609T genotypes of the NQO1 gene.The distribution of genotypes in the NQO1 gene, inthe pediatric leukemia patient group compared with pe-diatric control group is reported in Table 2 and statisticalsignificant difference was not found (p=0.1282).A heterozygote (C/T) genotype was most frequent inthe group of the acute lymphoblastic leukemia (8/20).The C609T genotypes of NQO1 gene also were not re-lated to specific clonal abnormalities in pediatric patientgroup.B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388386TABLE 1NQO1 C609T GENOTYPES AMONG ADULT MYELOID NEOPLASMSUBTYPES IN THE 56 INVESTIGATED AND 61 CONTROLSUBJECTSGROUPS C/C C/T T/T Chi square p valueControl group 48/61 12/61 1/61Patient group 31/56 23/56 2/56 7.25 0.026Subtypes of adult myeloid neoplasm No/TotalMPN (unclassifiable) 7/15 7/15 1/15 15/56CML 9/14 5/14 0/14 14/56PV 5/9 3/9 1/9 9/56MDS 5/8 3/8 0/8 8/56AML 7/10 3/10 0/10 10/56MPN=Myeloproliferative neoplasm; CML = Chronic myeloge-nous leukemia; BCR-ABL positive; PV = Polycythemia vera; MDS= Myelodysplastic syndromes; AML = Acute myeloid leukemia.DiscussionWe confirmed that homozygote T/T and heterozygoteC/T genotypes of NQO1 C609T polymorphism were sig-nificantly more frequent in the investigated group ofadult patients with different myeloid disorders. These re-sults suggest that presence of a polymorphic T allele rep-resent a risk factor for the development of primarymyeloid disorder in adults, which is associated with a re-duced amount or lack of enzyme involved in the detoxifi-cation of quinone carcinogens.NQO1 C609T allele frequency varies between differ-ent ethnic groups. The frequency of allele T of the NQO1C609T polymorphism in white populations of Europeand America is lower (17% in the United Kingdom, 19%in Germany and 16% USA) compared with Asia and Mex-ican Hispanic populations where it is significantly higher(42% in Korea, 47% in China, 38% in Japan, and 43% inMexico)12,16. The allele T frequency among healthy indi-viduals (adults and children), in our Southern Croatianpopulation was 11% what is the expected frequency forthe Caucasian population.Larson et al as well as Yamaguti et al in their studiesof different ethnic groups, mostly Caucasians, showed asignificant difference in frequency of heterozygous (C/T)and homozygous (T/T) between patients with myeloiddisorders and controls what is in concordance with ourresults15,17.Study performed in a Japanese population, showed asimilar frequency of C/T and T/T genotypes in patientgroup but they could not reach statistical significance be-cause of a higher frequency of the T allele in the healthyJapanese population18. An Israel study, conducted byMalik et al, showed that the frequency of C/T allele didnot differ between AML patients and control subjects be-cause of more frequent T allele in healthy population19.The study conducted in the Franch-Canadian prov-ince of Québec, Canada showed a significantly more fre-quent polymorphic T allele in the group of pediatric pa-tients with acute lymphoblastic leukemia (ALL)6. In con-trast to those, our results did not show association ofNQO1 C609T polymorphism with pediatric leukemia.Sirma et al in Turkish population also found that NQO1C609T polymorphism is not associated with de novo pe-diatric acute leukemia in their study group12. Recently ameta-analysis of seven studies that analyzed the risk ofde novo pediatric leukemia was conducted. Pooling datafrom seven studies, authors provided no evidence of a re-lationship between NQO1 C609T genotype and risk forpediatric leukemia20. Yeoh AE et al in Asians populationas well as Silveira Vda S et al in Brazilian populationshowed that carriers of NQO1 CT genotype have a lowerrisk of developing ALL compared to those carrying wild--type21,22. This finding was in contrast to nearly all previ-ous reports in the Caucasians. Probably racial and socio--cultural heterogeneity of populations can cause suchdivergent results.We could not confirm association of C609T polymor-phism with specific cytogenetic abnormalities in a kar-yotype. Karyotype was not found to be associated with aheterozygote (C/T) genotype in Israeli group of patientswhat is in concordance with our results19. However,Larson et al found that the T allele frequency was a 1.6fold higher in the patients with chromosomal aberrations(chromosome 5 and 7), while Smith et al showed thesame in patients with inversion of chromosome 1615,10.In summary, the frequency of an inactivating poly-morphism of NQO1 gene appears to be increased amongprimary myeloid disorders in adults, while we did notprovide evidence for the statistically significant effect ofthis polymorphism in pediatric leukemia. No evidencewas found in association of cytogenetic findings and inac-tivating polymorphism of NQO1 gene in both groups ofpatients. These results suggests that inactivating poly-morphism of functional genetic marker involved in themetabolic pathway of carcinogens may be a risk factor inleukemogenesis of specific types of hematopoietic malig-nancies in a certain age. Further research should includea larger number of genetically homogenous subjects.AcknowledgementsThis study was supported by the Croatian Ministry ofScience, Education and Sports (Project number 216--1080315-0293).R E F E R E N C E S1. VARADIMA JW, Chem Biol Interact, 184 (2010) 16. — 2. PUI CH,Childhood Leukemia (Cambridge University Press, New York, 1999). —3. HARRIS NL, JAFFE ES, DIEBOLD J, FLANDRIN G, MULLER-HER-MELINK HK, VARADIMA J, LISTER TA, BLOOMFIELD CD, HematolJ, 1 (2000) 53. — 4. DEWALD GW, MORRIS MA, LILLA VC, Chromo-some studies in neoplastic hematologic disorders. In: MCCLATCHEZ KD,(Eds): Clinical Laboratory Medicine (Williams and Wilkins, Baltimore,1994). — 5. MITELMAN F, KANEKO Y, BERGER R, Report of the Com-B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388387TABLE 2NQO1 C609T GENOTYPES AMONG CHILDHOOD LEUKEMIATYPES IN THE 26 INVESTIGATED AND 38 CONTROL SUBJECTSGROUPS C/C C/T T/T Chi square p valueControl group 30/38 8/38 0/38Patient group 16/26 10/26 0/26 2.31 0.1282Subtypes of childhood leukemia No/TotalALL 12/20 8/20 0/20 20/26AML 1/3 2/3 0/3 3/26CML juvenile 1/1 0/1 0/1 1/26MDS 2/2 0/2 0/2 2/26ALL = acute lymphoblastic leukemia; AML = acute myeloid leu-kemia; CML = chronic myelogenous leukemia juveniles BCR--ABL positive; MDS = myelodysplastic syndromes.mittee on Chromosome Changes in neoplasia. In: CUTICCHIA JA,PEARSON PL, (Eds): Human Gene Mapping (Johns University Press,Baltimore, 1994). — 6. KRAJNOVI] M, SINNETT H, RICHER C, LA-BUDA D, SINNETT D, Int J Cancer, 97 (2002) 230. — 7. VOSO MT, FA-BIANI E, D’ALO’ F, GUIDI F, DI RUSCIO, A SICA S, PAGANO L, GRE-CO M, HOHAUS S, LEONE G, Ann Oncol, 18 (2007) 1523. — 8. WIE-MELS JL, PANGAMENTA A, TAJLOR GM, EDEN OB, ALEXANDERFE, GREAVES MF, Cancer Res, 59 (1999) 4095. — 9. BOLUFER P, CO-LLADO M, BARRAGÀN E, CERVERA J, CALASANZ MJ, COLOMER D,ROMAN-GÓMEZ J, SANZ MA, Haematologica, 92 (2007) 308. — 10.SMITH MT, WANG Y, KANE E, ROLLINSON S, WIEMELS JL, ROMANE, RODDAM P, CARTWRIGHT R, MORGAN G, Blood, 97 (2001) 1422. —11. LANCIOTTI M, DUFOUR C, CORRAL L, DI MICHELE P, PIGULLOS, DE ROSSI G, BASSO G, LESZL A, LUCIANO M, LO NIGRO L, MI-CALIZZI C, VALSECCHI MG, BIONDI A, HAUPT R, Leukemia, 19 (2005)214. — 12. SIRMA S, AGAOGLU L, YILDIZ I, CAYLI D, HORGUSLUO-GLU E, ANAK S, YUKSEL L, UNUVAR A, CELKAN T, APAK H, KA-RAKAS Z, DEVECIOGLU O, OZBEK U, Pediatr Blood Cancer, 43 (2004)568. — 13. NEBERT DW, ROE AL, VANDALE SE, BINGHAM E, OAK-LEY GG, Genet Med, 4 (2002) 62. — 14. LE BEAU, MM, Cytogeneticanalysis of hematological malignancies. In: BARCH MJ (Eds): The ACTCytogenetic Laboratory Manual (New York, Raven, 1991). — 15. GAE-DIGK A, TYNDALE RF, JURIMA-ROMET M, SELLERS EM, GRANTDM, LEEDER JS, Pharmacogenetics, 8 (1998) 305. — 16. LARSON RA,WANG Y, BANERJEE M, WIMELS J, HARTFORD C, LE BEAU MM,SMITH MT, Blood, 94 (1999) 803. — 17. YAMAGUTI GG, LOURENCOGJ, COSTA FF, LIMA CS, Eur J Haematol, 83 (2009) 270. — 18. NAOE T,TAKEYAMA K, YOKOZAWA T, KIYOI H, SETO M, UIKE N, INO T, UT-SUNOMIYA A, MARUTA A, JIN-NAI I, KAMADA N, KUBOTA Y, NA-KAMURA H, SHIMAZAKI C, HORIIKE S, KODERA Y, SAITO H, UEDAR, WIEMELS J, OHNO R, Clin Cancer Res, 6 (2000) 4091. — 19. MALIKE, COHEN SB, SAHAR D, DANN EJ, RUND D, Haematologica, 91 (2006)956. — 20. GUHA N, CHANG JS, CHOKKALINGAM AP, WIEMELS JL,SMITH MT, BUFFLER PA, Am J Epidemiol, 168 (2008) 1221. — 21.YEOH AE, LU Y, CHAN JY, CHAN YH, ARIFFIN H, KHAM SK, QUAHTC, Leuk Res, 34 (2010) 276. — 22. SILVEIRA VDA S, CANALLE R,SCRIDELI CA, QUEIROZ RG, TONE LG, Environ Mol Mutagen, 51(2010) 48. — 23. SMITH MT, WANG Y, SKIBOLA CF, SLATER DJ, LONIGRO L, NOWELL PC, LANGE BJ, FELIX CA, Blood, 100 (2002) 4590.T. ZemunikUniversity of Split, School of Medicine, Department of Medical Biology, [oltanska 2, 21000 Split, Croatiae-mail: tzemunik@mefst.hrANALIZA POLIMORFIZMA C609T GENA ZA NQO1 U BOLESNIKA S HEMATOLO[KIM MALIGNIMBOLESTIMA U JU@NOJ HRVATSKOJS A @ E T A KU ovoj studiji analizirali smo utjecaj polimorfnih varijanti gena za NAD(P)H: kvinon oksidoreduktaza1 (NQO1) kojikodira enzim za detoksikaciju {tetnih kvinona i {titi hematopoetske mati~ne stanice od oksidativnog stresa. Polimor-fizam C609T gena za NQO1dovodi do gubitka enzimske aktivnosti {to mo`e biti rizi~ni ~imbenik u etiologiji specifi~nihtipova hematopoetskih malignih bolesti. Analizirali smo polimorfizam C609T gena za NQO1 u grupi od 82 bolesnika(56 odraslih i 26 djece) s razli~itim tipovima hematopoetskih malignih bolesti te u 99 zdravih ispitanika (61 odrasli i 38djece) koriste}i metode PCR i RFLP. Potvrdili smo da je polimorfizam C609T gena za NQO1 u~estaliji u skupini odraslihs mijeloidnim poreme}ajima, (p=0,0267) u usporedbi s kontrolnom skupinom odraslih ispitanika. Nismo mogli potvr-diti povezanost polimorfizma C609T s odre|enim kromosomskim translokacijama (promjene klonalnog kariotipa) ni uodrsloj kao ni u pedijatrijskoj skupini oboljelih.B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388388

English

Analysis of the C609T Polymorphism of NQO1 Gene in South Croatian Patients with Hematological Malignancies

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Coll. Antropol. 35 (2011) 2: 385–388Original scientific paperAnalysis of the C609T Polymorphism of NQO1Gene in South Croatian Patients withHematological MalignanciesBernarda Lozi}1, Dragan Primorac2,3,4,5, Robert Glavini}6, Radenka Kuzmani} [amija1 andTatijana Zemunik71 University of Split, Split University Hospital Center, Department of Pediatrics, Split, Croatia2 University of Split, School of Medicine, Split, Croatia3 »J. J. Strossmayer« University, School of Medicine, Osijek, Croatia4 Penn State University, Eberly College of Science, University Park, USA5 University of New Haven, West Haven, USA6 University of Split, Split University Hospital Center, Split, Croatia7 University of Split, School of Medicine, Department of Medical Biology, Split, CroatiaA B S T R A C TIn this study we analyzed the effect of polymorphic variation of NAD(P)H: quinone oxidoreductase1 (NQO1) gene thatencode enzyme which detoxifies harmful quinines and protect hematopoietic stem cells against oxidative stress. C609Tpolymorphism of NQO1 gene leads to loss of enzyme activity, which may be a risk factor in the etiology of specific types ofhematopoietic malignancies.We analyzed C609T polymorphism in NQO1 gene in the group of 82 patients (56 adult and26 children) with different type of hematopoietic malignancies and 99 healthy participants (61 adult and 38 children)using PCR and the RFLP method. We confirmed that the polymorphism C609T in NQO1 gene was more frequent in theadult patients’ group with myeloid disorders, (p=0.0267) compared with adult controls. We could not confirm the associ-ation C609T polymorphism with recurrent chromosome translocations (clonal karyotype changes) neither in the adultnor in pediatric group of patients.Key words: hematological malignancies, polymorphism, NQO1, clonal chromosomal abnormalitiesIntroductionThe World Health Organization (WHO) classifies he-matological malignant diseases according to their lineage(myeloid, lymphoid, histiocytic/dendritic) and distingui-shes neoplasm of precursor cells from those comprised offunctionally mature cells. Within each category, distinctdiseases are defined according to a combination of mor-phology, immunophenotype, genetic futures and clinicalsyndromes1. Acute lymphoblastic leukemia (ALL) is themost common type of leukemia in early childhood whileprimary myeloid disorder (acute or chronic) is the mostcommon type of leukemia in the elderly2.From a clinical point of view it is very important tounderstand myeloid and lymphoid classification of neo-plasm3. Hematopoietic malignancies are clonal diseasesthat arise due to genetic changes in one hematopoieticstem cell in the bone marrow or lymph tissue4. Such al-tered cell divides forming abnormal clone of cells, whichcan be determined by cytogenetic findings of the same orsimilar changes in three metaphases5. Series of low--penetrance genes can be involved in the development ofhematological malignant diseases. An individual’s risk ofcomplex disease development presents a cumulative ef-fect of genetic and environmental factors. It seams thatpolymorphisms of certain genes play a role in leuke-mogenesis6.Therefore, in this study we analyzed the polymor-phism of the NQO1 gene that encode protein responsiblefor detoxification of endogenous and exogenous muta-gens and may influence susceptibility to leukemia deve-lopment6–11.385Received for publication March 11, 2011NAD(P)H: quinine oxidoreductase1 (NQO1) is a de-toxification enzyme that protects cells against oxidativestress and toxic quinines (11). NQO1 catalyze a 2-elec-tron reduction of quinines, causing prevention of theirparticipation in redox cycling and thus in oxidativestress6.Individuals with a lack of NQO1 activity are moresusceptible to the toxic and carcinogenic effects of qui-nines (reactive metabolites of benzene) and have in-creased risk of hematotoxicity and leukemia6,12. The hu-man NQO1 gene is located on chromosome 16q22.1 andhas six exons (13). Polymorphism C to T at position –609causes proline to serine substitution in codone 187 in theNQO1 protein. Homozygous T/T show the loss of enzymeactivity, and heterozygous C/T show low to intermediateNQO1 activity. It has been reported that children areparticularly more vulnerable than adults to oxidativestress and toxic quinines because their metabolism is lessdeveloped/matured, and they have a higher level of celldivision and growth6,12.In this case-control study, we analysed whether theNQO1 variation increase risk for an individual’s suscep-tibility to hematological malignancies. Our study wasconducted in Southern Croatian population, which canbe considered relatively homogenous from both geneticand socio-cultural points of view.Material and MethodsIn this study we investigated 82 patients (34 femalesand 48 males) and 99 healthy participants (45 femalesand 54 males). Investigated group of patients was dividedaccording to the age in two groups. Most of the patients(56 individuals) were of adult age (median age 58 years;range 34–82 years) with different types of myeloid neo-plasms. The rest of them were pediatric patients (26 indi-viduals) (median age 8.5 years; range 0.3–17 years) withprevalence of lymphoid neoplasms (20 individuals). Thegroup of healthy participants (61 adult and 38 pediatricindividuals) has been adjusted in age and sex distribu-tion with patient group. The cases had been diagnosed inthe Department of Pediatric and Internal Medicine ofSplit University Hospital between 2002 and 2007. Thediagnosis was established according to standard rules ofthe WHO classification. All patients were karyotyped us-ing previously described methods of cytogenetic analysison pretreatment bone marrow cells14.In our study we investigated C609T polymorphism ofthe NQO1 gene (rs1800566) using previously describedstandard methods15. For detection of C609T polymor-phism, we performed the polymerase chain reaction (PCR)and restriction fragment length polymorphism (RFLP)after digestion with restriction enzyme HifnI. The di-gested fragments were separated and visualized on aga-rose or polyacrylamide gels. Fragments of 151, 120, and33 bp on gels confirmed the presence of the polymorphicHinfI restriction site yields.NQO1 genotype frequencies in leukemia subgroupsand controls were compared by the c2-test. P-values lessthan 0.05 were considered nominally significant. Statis-tical analysis was performed using statistical packageMedCalc (Mariakerke, Belgium).ResultsThe distribution of C609T genotypes of the NQO1gene, in the adult patient group with myeloid disorderand adult controls are reported in Table 1. Genotype fre-quencies showed statistically significant difference be-tween those two groups (p=0.0267).A heterozygote (C/T) genotype was most frequent inthe group of unclassifiable myeloproliferative neoplasms(MPN) (7/15).Recurrent chromosome abnormalities (clonal abnor-malities) detected in the leukemia cells of the adult pa-tient group showed a wide spectrum of clonal karyotypeaberrations, and were found in 25/56 patients (12 ofthem were C609T heterozygous). The most frequentclonal rearrangement was reciprocate translocationt(9;22) observed in 14 patients exclusively with chronicmyelogenous leukemia BCR-ABL positive (CML), (5 het-erozygous). Heterozygote genotypes were also found inpatients with other different clonal abnormalities [dele-tion in chromosome 20q (2/2) and different abnormalitieswhich include chromosome 17q (3/3). However, no associ-ation was found between specific chromosomal abnor-malities and C609T genotypes of the NQO1 gene.The distribution of genotypes in the NQO1 gene, inthe pediatric leukemia patient group compared with pe-diatric control group is reported in Table 2 and statisticalsignificant difference was not found (p=0.1282).A heterozygote (C/T) genotype was most frequent inthe group of the acute lymphoblastic leukemia (8/20).The C609T genotypes of NQO1 gene also were not re-lated to specific clonal abnormalities in pediatric patientgroup.B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388386TABLE 1NQO1 C609T GENOTYPES AMONG ADULT MYELOID NEOPLASMSUBTYPES IN THE 56 INVESTIGATED AND 61 CONTROLSUBJECTSGROUPS C/C C/T T/T Chi square p valueControl group 48/61 12/61 1/61Patient group 31/56 23/56 2/56 7.25 0.026Subtypes of adult myeloid neoplasm No/TotalMPN (unclassifiable) 7/15 7/15 1/15 15/56CML 9/14 5/14 0/14 14/56PV 5/9 3/9 1/9 9/56MDS 5/8 3/8 0/8 8/56AML 7/10 3/10 0/10 10/56MPN=Myeloproliferative neoplasm; CML = Chronic myeloge-nous leukemia; BCR-ABL positive; PV = Polycythemia vera; MDS=Myelodysplastic syndromes; AML=Acute myeloid leukemia.DiscussionWe confirmed that homozygote T/T and heterozygoteC/T genotypes of NQO1 C609T polymorphism were sig-nificantly more frequent in the investigated group ofadult patients with different myeloid disorders. These re-sults suggest that presence of a polymorphic T allele rep-resent a risk factor for the development of primarymyeloid disorder in adults, which is associated with a re-duced amount or lack of enzyme involved in the detoxifi-cation of quinone carcinogens.NQO1 C609T allele frequency varies between differ-ent ethnic groups. The frequency of allele T of the NQO1C609T polymorphism in white populations of Europeand America is lower (17% in the United Kingdom, 19%in Germany and 16% USA) compared with Asia and Mex-ican Hispanic populations where it is significantly higher(42% in Korea, 47% in China, 38% in Japan, and 43% inMexico)12,16. The allele T frequency among healthy indi-viduals (adults and children), in our Southern Croatianpopulation was 11% what is the expected frequency forthe Caucasian population.Larson et al as well as Yamaguti et al in their studiesof different ethnic groups, mostly Caucasians, showed asignificant difference in frequency of heterozygous (C/T)and homozygous (T/T) between patients with myeloiddisorders and controls what is in concordance with ourresults15,17.Study performed in a Japanese population, showed asimilar frequency of C/T and T/T genotypes in patientgroup but they could not reach statistical significance be-cause of a higher frequency of the T allele in the healthyJapanese population18. An Israel study, conducted byMalik et al, showed that the frequency of C/T allele didnot differ between AML patients and control subjects be-cause of more frequent T allele in healthy population19.The study conducted in the Franch-Canadian prov-ince of Québec, Canada showed a significantly more fre-quent polymorphic T allele in the group of pediatric pa-tients with acute lymphoblastic leukemia (ALL)6. In con-trast to those, our results did not show association ofNQO1 C609T polymorphism with pediatric leukemia.Sirma et al in Turkish population also found that NQO1C609T polymorphism is not associated with de novo pe-diatric acute leukemia in their study group12. Recently ameta-analysis of seven studies that analyzed the risk ofde novo pediatric leukemia was conducted. Pooling datafrom seven studies, authors provided no evidence of a re-lationship between NQO1 C609T genotype and risk forpediatric leukemia20. Yeoh AE et al in Asians populationas well as Silveira Vda S et al in Brazilian populationshowed that carriers of NQO1 CT genotype have a lowerrisk of developing ALL compared to those carrying wild--type21,22. This finding was in contrast to nearly all previ-ous reports in the Caucasians. Probably racial and socio--cultural heterogeneity of populations can cause suchdivergent results.We could not confirm association of C609T polymor-phism with specific cytogenetic abnormalities in a kar-yotype. Karyotype was not found to be associated with aheterozygote (C/T) genotype in Israeli group of patientswhat is in concordance with our results19. However,Larson et al found that the T allele frequency was a 1.6fold higher in the patients with chromosomal aberrations(chromosome 5 and 7), while Smith et al showed thesame in patients with inversion of chromosome 1615,10.In summary, the frequency of an inactivating poly-morphism of NQO1 gene appears to be increased amongprimary myeloid disorders in adults, while we did notprovide evidence for the statistically significant effect ofthis polymorphism in pediatric leukemia. No evidencewas found in association of cytogenetic findings and inac-tivating polymorphism of NQO1 gene in both groups ofpatients. These results suggests that inactivating poly-morphism of functional genetic marker involved in themetabolic pathway of carcinogens may be a risk factor inleukemogenesis of specific types of hematopoietic malig-nancies in a certain age. Further research should includea larger number of genetically homogenous subjects.AcknowledgementsThis study was supported by the Croatian Ministry ofScience, Education and Sports (Project number 216--1080315-0293).R E F E R E N C E S1. VARADIMA JW, Chem Biol Interact, 184 (2010) 16. — 2. PUI CH,Childhood Leukemia (Cambridge University Press, New York, 1999). —3. HARRIS NL, JAFFE ES, DIEBOLD J, FLANDRIN G, MULLER-HER-MELINK HK, VARADIMA J, LISTER TA, BLOOMFIELD CD, HematolJ, 1 (2000) 53. — 4. DEWALD GW, MORRIS MA, LILLA VC, Chromo-some studies in neoplastic hematologic disorders. In: MCCLATCHEZ KD,(Eds): Clinical Laboratory Medicine (Williams and Wilkins, Baltimore,1994). — 5. MITELMAN F, KANEKO Y, BERGER R, Report of the Com-B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388387TABLE 2NQO1 C609T GENOTYPES AMONG CHILDHOOD LEUKEMIATYPES IN THE 26 INVESTIGATED AND 38 CONTROL SUBJECTSGROUPS C/C C/T T/T Chi square p valueControl group 30/38 8/38 0/38Patient group 16/26 10/26 0/26 2.31 0.1282Subtypes of childhood leukemia No/TotalALL 12/20 8/20 0/20 20/26AML 1/3 2/3 0/3 3/26CML juvenile 1/1 0/1 0/1 1/26MDS 2/2 0/2 0/2 2/26ALL = acute lymphoblastic leukemia; AML = acute myeloid leu-kemia; CML = chronic myelogenous leukemia juveniles BCR--ABL positive; MDS = myelodysplastic syndromes.mittee on Chromosome Changes in neoplasia. In: CUTICCHIA JA,PEARSON PL, (Eds): Human Gene Mapping (Johns University Press,Baltimore, 1994). — 6. KRAJNOVI] M, SINNETT H, RICHER C, LA-BUDA D, SINNETT D, Int J Cancer, 97 (2002) 230. — 7. VOSO MT, FA-BIANI E, D’ALO’ F, GUIDI F, DI RUSCIO, A SICA S, PAGANO L, GRE-CO M, HOHAUS S, LEONE G, Ann Oncol, 18 (2007) 1523. — 8. WIE-MELS JL, PANGAMENTA A, TAJLOR GM, EDEN OB, ALEXANDERFE, GREAVES MF, Cancer Res, 59 (1999) 4095. — 9. BOLUFER P, CO-LLADO M, BARRAGÀN E, CERVERA J, CALASANZ MJ, COLOMER D,ROMAN-GÓMEZ J, SANZ MA, Haematologica, 92 (2007) 308. — 10.SMITHMT, WANG Y, KANE E, ROLLINSON S, WIEMELS JL, ROMANE, RODDAM P, CARTWRIGHT R, MORGANG, Blood, 97 (2001) 1422. —11. LANCIOTTI M, DUFOUR C, CORRAL L, DI MICHELE P, PIGULLOS, DE ROSSI G, BASSO G, LESZL A, LUCIANO M, LO NIGRO L, MI-CALIZZI C, VALSECCHI MG, BIONDI A, HAUPT R, Leukemia, 19 (2005)214. — 12. SIRMA S, AGAOGLU L, YILDIZ I, CAYLI D, HORGUSLUO-GLU E, ANAK S, YUKSEL L, UNUVAR A, CELKAN T, APAK H, KA-RAKAS Z, DEVECIOGLU O, OZBEK U, Pediatr Blood Cancer, 43 (2004)568. — 13. NEBERT DW, ROE AL, VANDALE SE, BINGHAM E, OAK-LEY GG, Genet Med, 4 (2002) 62. — 14. LE BEAU, MM, Cytogeneticanalysis of hematological malignancies. In: BARCH MJ (Eds): The ACTCytogenetic Laboratory Manual (New York, Raven, 1991). — 15. GAE-DIGK A, TYNDALE RF, JURIMA-ROMET M, SELLERS EM, GRANTDM, LEEDER JS, Pharmacogenetics, 8 (1998) 305. — 16. LARSON RA,WANG Y, BANERJEE M, WIMELS J, HARTFORD C, LE BEAU MM,SMITH MT, Blood, 94 (1999) 803. — 17. YAMAGUTI GG, LOURENCOGJ, COSTA FF, LIMA CS, Eur J Haematol, 83 (2009) 270. — 18. NAOE T,TAKEYAMA K, YOKOZAWA T, KIYOI H, SETO M, UIKE N, INO T, UT-SUNOMIYA A, MARUTA A, JIN-NAI I, KAMADA N, KUBOTA Y, NA-KAMURA H, SHIMAZAKI C, HORIIKE S, KODERA Y, SAITO H, UEDAR, WIEMELS J, OHNO R, Clin Cancer Res, 6 (2000) 4091. — 19. MALIKE, COHEN SB, SAHAR D, DANN EJ, RUND D, Haematologica, 91 (2006)956. — 20. GUHA N, CHANG JS, CHOKKALINGAM AP, WIEMELS JL,SMITH MT, BUFFLER PA, Am J Epidemiol, 168 (2008) 1221. — 21.YEOH AE, LU Y, CHAN JY, CHAN YH, ARIFFIN H, KHAM SK, QUAHTC, Leuk Res, 34 (2010) 276. — 22. SILVEIRA VDA S, CANALLE R,SCRIDELI CA, QUEIROZ RG, TONE LG, Environ Mol Mutagen, 51(2010) 48. — 23. SMITH MT, WANG Y, SKIBOLA CF, SLATER DJ, LONIGRO L, NOWELL PC, LANGE BJ, FELIX CA, Blood, 100 (2002) 4590.T. ZemunikUniversity of Split, School of Medicine, Department of Medical Biology, [oltanska 2, 21000 Split, Croatiae-mail: tzemunik@mefst.hrANALIZA POLIMORFIZMA C609T GENA ZA NQO1 U BOLESNIKA S HEMATOLO[KIM MALIGNIMBOLESTIMA U JU@NOJ HRVATSKOJS A @ E T A KU ovoj studiji analizirali smo utjecaj polimorfnih varijanti gena za NAD(P)H: kvinon oksidoreduktaza1 (NQO1) kojikodira enzim za detoksikaciju {tetnih kvinona i {titi hematopoetske mati~ne stanice od oksidativnog stresa. Polimor-fizam C609T gena za NQO1dovodi do gubitka enzimske aktivnosti {to mo`e biti rizi~ni ~imbenik u etiologiji specifi~nihtipova hematopoetskih malignih bolesti. Analizirali smo polimorfizam C609T gena za NQO1 u grupi od 82 bolesnika(56 odraslih i 26 djece) s razli~itim tipovima hematopoetskih malignih bolesti te u 99 zdravih ispitanika (61 odrasli i 38djece) koriste}i metode PCR i RFLP. Potvrdili smo da je polimorfizam C609T gena za NQO1 u~estaliji u skupini odraslihs mijeloidnim poreme}ajima, (p=0,0267) u usporedbi s kontrolnom skupinom odraslih ispitanika. Nismo mogli potvr-diti povezanost polimorfizma C609T s odre|enim kromosomskim translokacijama (promjene klonalnog kariotipa) ni uodrsloj kao ni u pedijatrijskoj skupini oboljelih.B. Lozi} et al.: The Role of NQO1 Gene in Leukemia, Coll. Antropol. 35 (2011) 2: 385–388388

Analysis of the C609T Polymorphism of NQO1 Gene in South Croatian Patients with Hematological Malignancies

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